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WO2025072196A1 - Methods for culturing and assaying b cell lymphocytes - Google Patents

Methods for culturing and assaying b cell lymphocytes Download PDF

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Publication number
WO2025072196A1
WO2025072196A1 PCT/US2024/048204 US2024048204W WO2025072196A1 WO 2025072196 A1 WO2025072196 A1 WO 2025072196A1 US 2024048204 W US2024048204 W US 2024048204W WO 2025072196 A1 WO2025072196 A1 WO 2025072196A1
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Prior art keywords
composition
microns
cell
microfluidic
cells
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PCT/US2024/048204
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French (fr)
Inventor
Samuel S. HINMAN
Di Wu
Katherine Marie PARTINGTON
John Junyeon KIM
Minha Park
Jason M. MCEWEN
Vincent Haw Tien PAI
Lily CHAO
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Bruker Cellular Analysis Inc
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Bruker Cellular Analysis Inc
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Publication of WO2025072196A1 publication Critical patent/WO2025072196A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0635—B lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • Single B cell screening approaches provide attractive strategies for discovery of monoclonal antibodies, including human monoclonal antibodies, as compared with hybridoma and phage display owing to the low fusion efficiency and loss of cognate heavy/light chain pairing in the latter two approaches, respectively.
  • a central challenge to single B cell screening Attorney Docket No.01149-0025-00PCT in humans and most other animals is the lack of robust B cell media to maintain durable B cell survival and antibody secretion for the duration of the assay and down selection processes.
  • compositions for culturing B cells including memory B cells, which may be human B cells (e.g., human memory B cells).
  • memory B cells which may be human B cells (e.g., human memory B cells).
  • the B cells can be activated, resulting in proliferation and/or immunoglobulin (e.g., antibody) secretion, and/or maintained in a state of sustained immunoglobulin (e.g., antibody) expression/secretion.
  • compositions disclosed herein are methods of culturing, activating, and/or screening B cells that involve contacting the B cells with one or more compositions disclosed herein.
  • a composition for culturing B cell lymphocytes the composition comprising: a protein kinase C (PKC) agonist (e.g., a PKC-specific agonist), wherein the composition lacks feeder cells.
  • PKC protein kinase C
  • PMA phorbol 12-myristate 13-acetate
  • PPD phorbol 12,13-dibutyrate
  • MPMA 4-O-methyl ether
  • concentration of at least about 1.0 ng/mL e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL.
  • 0.5 ng/mL e.g., at least about 1.0 ng/mL, Attorney Docket No.01149-0025-00PCT at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL.
  • concentration of at least about 1.0 ng/mL e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL.
  • IL-21 is human IL- 21.
  • concentration of at least about 10 ng/mL e.g., at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL.
  • BAFF is human BAFF.
  • concentration of less than about 1.0 ng/mL e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks IL-2).
  • concentration of less than about 1.0 ng/mL e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks TNF-alpha.
  • nM nanomolar
  • a PI3K agonist e.g., a PI3K-specific agonist
  • composition of embodiment 20, wherein the PI3K agonist comprises naringenin, naringin, butin, eriodictyol, homoeriodictyol, hesperetin, hesperidin, and/or isosakuranetin.
  • a STAT3 agonist e.g., a STAT3-specific agonist
  • the STAT3 agonist comprises leukemia inhibitory factor (LIF) (e.g., human LIF (hLIF)), as well as fragments and variants that retain STAT3 agonist activity.
  • LIF leukemia inhibitory factor
  • the composition comprises a CD40-clustering agent at a concentration of less than about 1.0 ng/mL (e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks CD40-clustering agent).
  • PKC agonist is phorbol 12,13-dibutyrate (PBD).
  • the composition comprises the PKC agonist at a concentration of at least about 10 ng/mL (e.g., at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, at least about 100 ng/mL, at least about 150 ng/mL, at least about 200 ng/mL, at least about 250 ng/mL, at least about 500 ng/mL, or at least about 750 ng/mL).
  • 10 ng/mL e.g., at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, at least about 100 ng/mL, at least about 150 ng/mL, at least about 200 ng/mL, at least about 250
  • concentration from about 10 ng/mL to about 1000 ng/mL (e.g.
  • serum e.g., any serum suitable for culturing B cell lymphocytes.
  • compositions for culturing B cell lymphocytes comprising: a Toll-like receptor 9 (TLR9) agonist; Attorney Docket No.01149-0025-00PCT IL-6; and IL-4, wherein the composition lacks feeder cells.
  • TLR9 Toll-like receptor 9
  • the composition comprises IL-6 at a concentration of at least about 1.0 ng/mL (e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL).
  • composition of embodiment 36 wherein the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL (e.g., from about 5.0 ng/mL to about 25 ng/mL, about 10 ng/mL to about 50 ng/mL, about 15 ng/mL to about 75 ng/mL, about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints).
  • IL-6 is human IL- 6.
  • concentration of at least about 0.5 ng/mL e.g., at least about 1.0 ng/mL, at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL.
  • IL-4 is human IL- 4.
  • concentration of at least about 1.0 ng/mL e.g., at least about 10 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, or at least about 100 ng/mL.
  • a concentration from about 1.0 ng/mL to about 100 ng/mL (e.g., from about 10 ng/mL to about 50 ng/mL, about 25 ng/mL to about 75 ng/mL, about 50 ng/
  • 1.0 ng/mL e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL.
  • IL-21 is human IL-21.
  • ng/mL e.g., at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL.
  • BAFF is human BAFF.
  • PKC protein kinase C
  • TLR9 agonist is a CpG oligodeoxynucleotide (ODN).
  • composition of embodiment 54 wherein the CpG ODN is a B-class CpG ODN and/or has a linear structure.
  • composition of embodiment 54 or 55, wherein the composition comprises CpG ODN at a concentration of at least about 0.25 micromolar ( ⁇ M) (e.g., at least about 0.5 ⁇ M, at least about 1.0 ⁇ M, at least about 1.5 ⁇ M, at least about 2.0 ⁇ M, or at least about 2.5 ⁇ M).
  • ⁇ M micromolar
  • 5.0 ⁇ M e.g., from about 0.25 ⁇ M to about 4.0 ⁇ M, from about 0.5 ⁇ M to about 3.0 ⁇ M, from about 1.0 ⁇ M to about 2.0 ⁇ M, from about 0.25 ⁇ M to about 1.0
  • composition of any one of embodiments 36 to 57 further comprising: serum (e.g., any serum suitable for culturing B cell lymphocytes).
  • serum e.g., any serum suitable for culturing B cell lymphocytes.
  • the composition of embodiment 58, wherein the serum is present at an amount of about 1% to about 20% (v/v) (e.g., about 2% to about 15% (v/v), or about 5% to about 10% v/v) 60)
  • the composition of embodiment 58 or 59, wherein the serum is fetal bovine serum.
  • a method of culturing a B cell lymphocyte comprising: incubating the B cell lymphocyte in a composition of any one of embodiments 1 to 35, wherein the incubating maintains the expression of antibodies by the B cell lymphocyte, optionally wherein a population of B cell lymphocytes incubated in the composition exhibit at least a 60% (e.g., at least 65%, at least 70%, at least 75%, or at least 80%) maintenance of antibody expression after 24 hours of culture.
  • the B cell lymphocyte is a mammalian B cell.
  • 64 The method of embodiment 62, wherein the B cell lymphocyte is a human B cell.
  • the B cell lymphocyte is a memory B cell or a derivative thereof (e.g., an activated memory B cell).
  • the incubating is performed for at least 12 hours (e.g., at least 15 hours, at least 18 hours, at least 21 hours, at least 24 hours, at least 27 hours, at least 30 hours, at least 33 hours, or at least 36 hours).
  • the incubating is performed continuously.
  • a method of culturing a B cell lymphocyte comprising: incubating the B cell lymphocyte in a first composition, wherein the first composition is a composition of embodiment 61; and incubating the B cell lymphocyte in a second composition, wherein the second composition is a composition of embodiment 35; wherein the first composition stimulates activation and/or expression of antibody by the B cell lymphocyte, and wherein the second composition maintains continued expression of antibody by the B cell lymphocyte.
  • the method of embodiment 70, wherein the B cell lymphocyte is a mammalian B cell.
  • the incubation in the first composition is performed in about 200 microliters or less of the first composition (e.g., 150 microliters or less, 120 microliters or less, 100 microliters or less, 80 micro
  • any one of embodiments 70 to 77 and 79 wherein the incubation in the second composition is performed in about 200 microliters or less of the second composition (e.g., 150 microliters or less, 120 microliters or less, 100 microliters or less, 80 microliters or less, 60 microliters or less, or 50 microliters or less).
  • the incubation in the second composition is performed for at least 12 hours (e.g., at least 15 hours, at least 18 hours, at least 21 hours, at least 24 hours, at least 27 hours, at least 30 hours, at least 33 hours, or at least 36 hours).
  • a method of inducing proliferation of a B cell lymphocyte comprising: incubating the B cell lymphocyte in a composition of any one of embodiments 36 to 61, wherein the incubating is performed for a time sufficient to induce proliferation of the B cell lymphocyte.
  • a method of maintaining immunoglobulin (e.g., antibody) expression by a B cell lymphocyte comprising: incubating the B cell lymphocyte in a composition of any one of embodiments 1 to 35, wherein the incubating is performed for a time sufficient to induce and/or maintain immunoglobulin expression by the B cell lymphocyte.
  • the method of embodiment 83, wherein the immunoglobulin is IgG.
  • a method for screening a B cell lymphocyte for expression of an antibody that specifically binds to an antigen of interest comprising: culturing the B cell lymphocyte according to the method of any one of embodiments 62 to 69; providing the antigen of interest; and detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte.
  • the method of embodiment 88, wherein detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte is performed using a sandwich assay (e.g., an ELISA-type sandwich assay).
  • FIG.1A illustrates a microfluidic device and a system with associated control equipment according to some embodiments of the disclosure.
  • FIG.1B illustrates a microfluidic device with sequestration pens according to an embodiment of the disclosure.
  • FIGS. 2A to 2B illustrate a microfluidic device having sequestration pens according to some embodiments of the disclosure.
  • FIG. 2C illustrates a sequestration pen of a microfluidic device according to some embodiments of the disclosure.
  • FIG.3 illustrates a sequestration pen of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 4A to 4B illustrate electrokinetic features of a microfluidic device according to some embodiments of the disclosure.
  • FIG.5A illustrates a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure.
  • FIG. 5B illustrates an imaging device according to some embodiments of the disclosure.
  • Attorney Docket No.01149-0025-00PCT [0017]
  • FIGS.6A to 6B illustrate workflows for antibody discovery according to some embodiments of the disclosure.
  • FIG.6C illustrates a workflow for activating a memory B cell according to some embodiments of the disclosure. [0019] FIGS.
  • FIG. 7A-7C summarize data regarding the frequency of memory B cells in human PBMCs (7A) and the extent of proliferation observed upon contacting the memory B cells with an activating composition (7B), and the viability of memory B cells following such activation (7C), as further described in the Example 1, according to embodiments of the present disclosure.
  • FIG.8 summarizes data regarding the pre- and post-activation expression levels of CD20 and CD138, showing differentiation of memory B cells into antibody-secreting plasma cells, according to embodiments of the present disclosure.
  • FIG.9 summarizes data regarding the loading of single, activated human B cells into chambers of OptoSelect® 11k and 20k microfluidic chips, according to embodiments of the present disclosure.
  • FIG.10 summarizes data regarding the number of human B cells secreting IgG antibodies (two left-most columns) and SARS-CoV-2-specific antibodies (two right-most columns), activated according to embodiments of the present disclosure.
  • FIG. 11 summarizes data regarding the percentage of activated B cells that maintained immunoglobulin (i.e., antibody) secretion after being cultured in a maintaining composition according to embodiments of the present disclosure.
  • FIGS.12A to 12B illustrate approaches for amplification of antibody heavy and light chain sequences according to embodiments of the present disclosure.
  • FIG. 13 summarizes data regarding the recovery of antigen-specific antibody sequences from single B cells according to embodiments of the present disclosure.
  • FIG.14 summarizes data showing a comparison of B-cell proliferation after 5 days of culture using the activation medium described compared to two different commercial B-cell expansion medias.
  • FIGS. 15A and 15B summarize data showing a comparison of the addition of PMA to the activation media (18A) and the addition of PMA and/or TNF- ⁇ to the activation media (18B).
  • FIG.16 summarizes data showing dose-dependency of PMA on the retention of IgG secretion over time by the activated B cells.
  • Attorney Docket No.01149-0025-00PCT [0029]
  • FIG. 17 summarizes data showing a comparison of PMA addition to the load and culture media of activated B cells.
  • FIG.18 summarizes data showing a comparison of PKC agonists on activated B cells.
  • DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0031] This specification describes exemplary embodiments and applications of the disclosure. The disclosure, however, is not limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Moreover, the figures may show simplified or partial views, and the dimensions of elements in the figures may be exaggerated or otherwise not in proportion.
  • one element e.g., a material, a layer, a substrate, etc.
  • one element can be “on,” “attached to,” “connected to,” or “coupled to” another element regardless of whether the one element is directly on, attached to, connected to, or coupled to the other element or there are one or more intervening elements between the one element and the other element.
  • directions e.g., above, below, top, bottom, side, up, down, under, over, upper, lower, horizontal, vertical, “x,” “y,” “z,” etc.
  • directions are relative and provided solely by way of example and for ease of illustration and discussion and not by way of limitation.
  • elements e.g., elements a, b, c
  • such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or a combination of all of the listed elements. Section divisions in the specification are for ease of review only and do not limit any combination of elements discussed.
  • microfluidic features are described as having a width or an area, the dimension typically is described relative to an x-axial and/or y-axial dimension, both of which lie within a plane that is parallel to the substrate and/or cover of the microfluidic device.
  • the height of a microfluidic feature may be described relative to a z-axial direction, which is perpendicular to a plane that is parallel to the substrate and/or cover of the microfluidic device.
  • a cross sectional area of a microfluidic feature such as a channel or a passageway, may be in reference to a x-axial/z-axial, a y-axial/z-axial, or an x-axial/y-axial area.
  • first and second may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.
  • a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc.
  • Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points.
  • substantially thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance.
  • “substantially” means within ten percent.
  • the term “ones” means more than one.
  • the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • ⁇ m means micrometer
  • ⁇ m 3 means cubic micrometer
  • pL means picoliter
  • nL means nanoliter
  • ⁇ L (or uL) means microliter.
  • air refers to the composition of gases predominating in the atmosphere of the earth.
  • gases typically include nitrogen (typically present at a concentration of about 78% by volume, e.g., in a range from about 70-80%), oxygen (typically present at about 20.95% by volume at sea level, e.g. in a range from about 10% to about 25%), argon (typically present at about 1.0% by volume, e.g. in a range from about 0.1% to about 3%), and carbon dioxide (typically present at about 0.04%, e.g., in a range from about 0.01% to about 0.07%).
  • Air may have other trace gases such as methane, nitrous oxide or ozone, trace pollutants and organic materials such as pollen, diesel particulates and the like. Air may include water vapor (typically present at about 0.25%, or may be present in a range from about 10ppm to about 5% by volume). Air may be provided for use in culturing experiments as a filtered, controlled composition and may be conditioned as described herein.
  • trace gases such as methane, nitrous oxide or ozone
  • trace pollutants and organic materials such as pollen, diesel particulates and the like.
  • Air may include water vapor (typically present at about 0.25%, or may be present in a range from about 10ppm to about 5% by volume). Air may be provided for use in culturing experiments as a filtered, controlled composition and may be conditioned as described herein.
  • a “microfluidic device” or “microfluidic apparatus” is a device that includes one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprised of fluidically interconnected circuit elements, including but not limited to region(s), flow path(s), channel(s), chamber(s), and/or pen(s), and at least one port configured to allow the fluid (and, optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device.
  • a microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel, and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 ⁇ L.
  • the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 ⁇ L.
  • the microfluidic circuit may be configured to have a first end fluidically connected with a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected with a second port (e.g., an outlet) in the microfluidic device.
  • a “nanofluidic device” or “nanofluidic apparatus” is a type of microfluidic device having a microfluidic circuit that contains at least one circuit element configured to hold a volume of fluid of less than about 1 ⁇ L, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less.
  • a nanofluidic device may comprise a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more).
  • circuit elements e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more).
  • one or more (e.g., all) of the at least one circuit elements is configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL.
  • one or more (e.g., all) of the at least one circuit elements are configured to hold a volume of fluid of about 20 nL to 200nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.
  • a microfluidic device or a nanofluidic device may be referred to herein as a “microfluidic chip” or a “chip”; or “nanofluidic chip” or “chip”.
  • a “microfluidic channel” or “flow channel” as used herein refers to flow region of a microfluidic device having a length that is significantly longer than both the horizontal and vertical dimensions.
  • the length of the channel is generally defined by the flow path of the channel. In the case of a straight channel, the length would be the “longitudinal axis” of the channel.
  • the “horizontal dimension” or “width” of the channel is the horizontal dimension as observed in a transverse section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangential to the flow path of the channel at the plane of the transverse section).
  • the “vertical dimension” or “height” of the channel is the vertical dimension as observed in a transverse section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangential to the flow path of the channel at the plane of the transverse section).
  • Attorney Docket No.01149-0025-00PCT [0045]
  • the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer.
  • the length of a flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween.
  • the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns, (e.g., from about 40 to about 150 microns).
  • a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element.
  • a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof.
  • a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein.
  • the flow channel may include valves, and the valves may be of any type known in the art of microfluidics. Examples of microfluidic channels that include valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, each of which is herein incorporated by reference in its entirety.
  • the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer.
  • the length of a flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween.
  • the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns, (e.g., from about 40 to about 150 microns).
  • a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element.
  • a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof.
  • a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein.
  • the flow channel may include valves, and the valves may be of any type known in the art of microfluidics. Examples of microfluidic channels that include valves are disclosed in U.S.
  • Patents 6,408,878 and 9,227,200 each of which is herein incorporated by reference in its entirety.
  • Attorney Docket No.01149-0025-00PCT [0047]
  • the term “transparent” refers to a material which allows visible light to pass through without substantially altering the light as is passes through.
  • “brightfield” illumination and/or image refers to white light illumination of the microfluidic field of view from a broad-spectrum light source, where contrast is formed by absorbance of light by objects in the field of view.
  • structured light is projected light that is modulated to provide one or more illumination effects.
  • a first illumination effect may be projected light illuminating a portion of a surface of a device without illuminating (or at least minimizing illumination of) an adjacent portion of the surface, e.g., a projected light pattern, as described more fully below, used to activate DEP forces within a DEP substrate.
  • the intensity e.g., variation in duty cycle of a structured light modulator such as a DMD, may be used to change the optical power applied to the light activated DEP actuators, and thus change DEP force without changing the nominal voltage or frequency.
  • structured light includes projected light that may be corrected for surface irregularities and for irregularities associated with the light projection itself, e.g., fall-off at the edge of an illuminated field.
  • Structured light is typically generated by a structured light modulator, such as a digital mirror device (DMD), a microshutter array system (MSA), a liquid crystal display (LCD), or the like.
  • a structured light modulator such as a digital mirror device (DMD), a microshutter array system (MSA), a liquid crystal display (LCD), or the like.
  • Illumination of a small area of the surface, e.g., a selected area of interest with structured light improves the signal-to-noise-ratio (SNR), as illumination of only the selected area of interest reduces stray/scattered light, thereby lowering the dark level of the image.
  • SNR signal-to-noise-ratio
  • An important aspect of structured light is that it may be changed quickly over time.
  • a light pattern from the structured light modulator may be used to autofocus on difficult targets such as clean mirrors or surfaces that are far out of focus.
  • a clean mirror a number of self-test features may be replicated such as measurement of modulation transfer function and field curvature/tilt, without requiring a more expensive Shack-Hartmann sensor.
  • spatial power distribution may be measured at the sample surface with a simple power meter, in place of a camera.
  • Structured light patterns may also be used as a reference feature Attorney Docket No.01149-0025-00PCT for optical module/system component alignment as well used as a manual readout for manual focus.
  • micro-object refers generally to any microscopic object that may be isolated and/or manipulated in accordance with the present disclosure.
  • micro-objects include: inanimate micro-objects such as microparticles; microbeads (e.g., polystyrene beads, glass beads, amorphous solid substrates, LuminexTM beads, or the like); magnetic beads; microrods; microwires; quantum dots, and the like; biological micro-objects such as cells; biological organelles; vesicles, or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts, and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated micro-beads, liposome-coated magnetic beads, or the like).
  • inanimate micro-objects such as microparticles
  • microbeads e.g., polystyrene beads, glass beads, amorphous solid substrates, LuminexTM beads, or the like
  • magnetic beads
  • Beads may include moieties/molecules covalently or non-covalently attached, such as fluorescent labels, proteins (including receptor molecules), carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species capable of use in an assay.
  • beads/solid substrates including moieties/molecules may be capture beads, e.g., configured to bind molecules including small molecules, peptides, proteins or nucleic acids present in proximity either selectively or non-selectively.
  • a capture bead may include a nucleic acid sequence configured to bind nucleic acids having a specific nucleic acid sequence or the nucleic acid sequence of the capture bead may be configured to bind a set of nucleic acids having related nucleic acid sequences. Either type of binding may be understood to be selective.
  • Capture beads containing moieties/molecules may bind non-selectively when binding of structurally different but physico-chemically similar molecules is performed, for example, size exclusion beads or zeolites configured to capture molecules of selected size or charge. Lipid nanorafts have been described, for example, in Ritchie et al.
  • biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, or the like, prokaryotic cells, bacterial cells, fungal cells, protozoan cells, or the like, cells dissociated from a tissue, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like, immunological cells, such as T cells, B cells, natural killer cells, macrophages, and the like, embryos (e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells, cells from a cell line, cancer cells, Attorney Docket No.01149-0025-00PCT infected cells, trans
  • a mammalian cell can be, for example, from a human, a mouse, a rat, a rabbit, a horse, a goat, a sheep, camelid (including llama and alpaca), a cow, a pig, a primate (e.g., monkey), a bird (e.g., chicken), or the like.
  • B Cell equates to “B Cell Lymphocyte.”
  • a colony of biological cells is “clonal” if all of the living cells in the colony that are capable of reproducing are daughter cells derived from a single parent cell.
  • all the daughter cells in a clonal colony are derived from the single parent cell by no more than 10 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 14 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 17 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 20 divisions.
  • the term “clonal cells” refers to cells of the same clonal colony. [0055] As used herein, a “colony” of biological cells refers to 2 or more cells (e.g.
  • the term “maintaining (a) cell(s)” refers to providing an environment comprising both fluidic and gaseous components and, optionally a surface, that provides the conditions necessary to keep the cells viable and/or expanding and/or expressing immunoglobulin (i.e., antibody).
  • the term “expanding” when referring to cells refers to increasing in cell number.
  • gas permeable means that the material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, the gas permeable material or structure is permeable to more than one of oxygen, carbon dioxide and nitrogen and may further be permeable to all three of these gases.
  • a “component” of a fluidic medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, or the like.
  • a composition “substantially lacks” a component means that the composition is completely devoid of the component (e.g. undetectable) or has a Attorney Docket No.01149-0025-00PCT substantially reduced amount of the component, (e.g., less than 1%, 0.5%, or 0.1%), or that the component is not added to the composition. If any one or more of the conditions, undetectable, less than 1%, 0.5%, or 0.1%, or not added to the composition, are met, the composition substantially lacks a component.
  • a composition comprises a “non-effective” concentration or amount of a component means that the concentration or amount does not produce an intended or desired effect.
  • certain components of the composition may include one or more of IL-6, IL-4, IL-21, BAFF, IL-2, which include known wild-type sequences and functionally-active fragments and variants thereof (e.g., at least 90%, 95%, 98% or greater identity to wild-type sequence and/or conservative amino acid substitutions).
  • an “agonist” includes any component that directly binds and activates a receptor or which indirectly activates a receptor by forming a complex with another component that binds the receptor or by causing the modification of another component that thereupon directly binds and activates the receptor.
  • an “agonist” that is a component of a composition means that the agonist is not made by the cells in culture (e.g., a PKC agonist or a TLR9 agonist).
  • a PKC agonist or a TLR9 agonist e.g., a PKC agonist or a TLR9 agonist.
  • diffusion refers to thermodynamic movement of a component of the fluidic medium down a concentration gradient.
  • flow of a medium means bulk movement of a fluidic medium primarily due to any mechanism other than diffusion, and may encompass perfusion. For example, flow of a medium can involve movement of the fluidic medium from one point to another point due to a pressure differential between the points.
  • Such flow can include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of the liquid, or any combination thereof.
  • Flowing can comprise pulling solution through and out of the microfluidic channel (e.g., aspirating) or pushing fluid into and through a microfluidic channel (e.g. perfusing).
  • substantially no flow refers to a rate of flow of a fluidic medium that, when averaged over time, is less than the rate of diffusion of components of a material (e.g., an analyte of interest) into or within the fluidic medium.
  • the ratio of a rate of flow of a component in a fluidic medium (i.e., advection) divided by the rate of diffusion of such component can be expressed by a dimensionless Peclet number.
  • the Peclet number associated with a particular region within the microfluidic device can vary with the component or components of the fluidic medium being considered (e.g., the analyte of interest), as the rate of diffusion of a component or components in a fluidic medium can depend on, for example, temperature, the size, mass, and/or shape of the component(s), and the strength of interactions between the component(s) and the fluidic medium.
  • the Peclet number associated with a particular region of the microfluidic device and a component located therein can be 0.95 or less, 0.9 or less, 0.85 or less, 0.8 or less, 0.75 or less, 0.7 or less, 0.65 or less, 0.6 or less, 0.55 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less.
  • fluidically connected means that, when the different regions are substantially filled with fluid, such as fluidic media, the fluid in each of the regions is connected so as to form a single body of fluid. This does not mean that the fluids (or fluidic media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) which are in flux as solutes move down their respective concentration gradients and/or fluids flow through the device.
  • solutes such as proteins, carbohydrates, ions, or other molecules
  • a “flow path” refers to one or more fluidically connected circuit elements (e.g., channel(s), region(s), chamber(s) and the like) that define, and are subject to, the trajectory of a flow of medium.
  • a flow path is thus an example of a swept region of a microfluidic device.
  • Other circuit elements e.g., unswept regions
  • isolated a micro-object confines a micro-object to a defined area within the microfluidic device.
  • the defined area can be, for example, a chamber.
  • a “chamber” is a region within a microfluidic device (e.g., a circuit element) that allows one or more micro- object(s) to be isolated from other micro-objects located within the microfluidic device.
  • chambers include microwells, which may be regions etched out of a substrate (e.g., a planar substrate), as described in U.S. Patent Application Publication Nos. 2013/0130232 (Weibel et al.) and 2013/0204076 (Han et al.), or a region formed in a multi- layer device, such as the microfluidic devices described in WO 2010/040851 (Dimov et al.) or U.S. Patent Application No.
  • chambers Attorney Docket No.01149-0025-00PCT include valved chambers, such as described in WO 2004/089810 (McBride et al.) and U.S. Patent Application Publication No.2012/0015347 (Singhal et al.).
  • Other examples of chambers include the chambers described in: Somaweera et al. (2013), “Generation of a Chemical Gradient Across an Array of 256 Cell Cultures in a Single Chip”, Analyst., Vol. 138(19), pp 5566-5571; U.S. Patent Application Publication No. 2011/0053151 (Hansen et al.); and U.S.
  • the chamber can be configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL.
  • the chamber can be configured to hold a volume of fluid of about 20 nL to 200nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.
  • pen or “penning” specifically refers to disposing micro- objects within a sequestration pen within the microfluidic device.
  • Forces used to pen a micro- object may be any suitable force as described herein such as dielectrophoresis (DEP), e.g., an optically actuated dielectrophoretic force (OEP); gravity; magnetic forces; locally actuated fluid flow; or tilting.
  • DEP dielectrophoresis
  • OEP optically actuated dielectrophoretic force
  • gravity magnetic forces
  • locally actuated fluid flow or tilting.
  • penning a plurality of micro-objects may reposition substantially all the micro-objects.
  • a selected number of the plurality of micro-objects may be penned, and the remainder of the plurality may not be penned.
  • a DEP force e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects.
  • micro-objects may be introduced to a flow region, e.g., a microfluidic channel, of the microfluidic device and thereafter introduced into a chamber by penning.
  • unpen or “unpenning” refers to repositioning micro-objects from within a sequestration pen to a new location within a flow region, e.g., a microfluidic channel, of the microfluidic device.
  • Forces used to unpen a micro-object may be any suitable force as described herein such as dielectrophoresis, e.g., an optically actuated dielectrophoretic force; gravity; magnetic forces; locally actuated fluid flow; or tilting.
  • unpenning a plurality of micro-objects may reposition substantially all the micro-objects.
  • a selected number of the plurality of micro-objects may be unpenned, and the remainder of the plurality may not be unpenned.
  • a DEP force e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects.
  • export or “exporting” can include, consist of, or consist essentially of repositioning micro-objects from a location within a microfluidic device, e.g., a flow region, a microfluidic channel, a chamber, etc., to a location outside of the microfluidic device, such as a well plate, a tube, or other receiving vessel.
  • exporting a micro-object comprises withdrawing (e.g., micro-pipetting) a volume of medium containing the micro-object from within the microfluidic device and depositing the volume of medium in or upon the location outside of the microfluidic device.
  • withdrawing the volume of medium is preceded by disassembling the microfluidic device (e.g., removing an upper layer, such as a cover or lid, of the microfluidic device from a lower layer , such as a base or substrate, of the microfluidic device) to facilitate access (e.g., of a micro- pipetted) to the internal regions of the microfluidic device.
  • exporting a micro-object comprises flowing a volume of fluid containing the micro-object through the flow region (including, e.g., a microfluidic channel) of the microfluidic device, out through an outlet of the microfluidic device, and depositing the volume of medium in or upon the location outside of the microfluidic device.
  • micro-object(s) within the microfluidic channel may be exported without requiring disassembly (e.g., removal of the cover of the device) or insertion of a tool into an interior region of the microfluidic device to remove micro- objects for further processing.
  • Export or “exporting” may further comprise repositioning micro-objects from within a chamber, which may include a sequestration pen, to a new location within a flow region, such as a microfluidic channel, as described above with regard to “unpenning”.
  • a microfluidic (or nanofluidic) device can comprise “swept” regions and “unswept” regions.
  • a “swept” region is comprised of one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which experiences a flow of medium when fluid is flowing through the microfluidic circuit.
  • the circuit elements of a swept region can include, for example, regions, channels, and all or parts of chambers.
  • an “unswept” region is comprised of one or more fluidically interconnected circuit element of a microfluidic circuit, each of which experiences substantially no flux of fluid when fluid is flowing through the microfluidic circuit.
  • An unswept region can be fluidically connected to a Attorney Docket No.01149-0025-00PCT swept region, provided the fluidic connections are structured to enable diffusion but substantially no flow of media between the swept region and the unswept region.
  • the microfluidic device can thus be structured to substantially isolate an unswept region from a flow of medium in a swept region, while enabling substantially only diffusive fluidic communication between the swept region and the unswept region.
  • a flow channel of a micro-fluidic device is an example of a swept region while an isolation region (described in further detail below) of a microfluidic device is an example of an unswept region.
  • a “non-sweeping” rate of fluidic medium flow means a rate of flow sufficient to permit components of a second fluidic medium in an isolation region of the sequestration pen to diffuse into the first fluidic medium in the flow region and/or components of the first fluidic medium to diffuse into the second fluidic medium in the isolation region; and further wherein the first medium does not substantially flow into the isolation region.
  • an “isolation region” refers to a region within a microfluidic device that is configured to hold a micro-object such that the micro-object is not drawn away from the region as a result of fluid flowing through the microfluidic device.
  • isolation region can further refer to the structures that define the region, which can include a base/substrate, walls (e.g., made from microfluidic circuit material), and a cover.
  • antibody refers to an immunoglobulin (Ig) and includes both polyclonal and monoclonal antibodies; multichain antibodies, such as IgG, IgM, IgA, IgE, and IgD antibodies; single chain antibodies, such as camelid antibodies; mammalian antibodies, including primate antibodies (e.g., human), rodent antibodies (e.g., mouse, rat, guinea pig, hamster, and the like), lagomorph antibodies (e.g., rabbit), ungulate antibodies (e.g., cow, pig, horse, donkey, camel, and the like), and canidae antibodies (e.g., dog); primatized (e.g., humanized) antibodies; chimeric antibodies, such
  • antibody fragment refers to fragments, derived from or related to an antibody, which bind antigen.
  • antibody fragments may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues.
  • biological micro-objects e.g., biological cells
  • specific biological materials e.g., proteins, such as antibodies
  • sample material comprising biological micro-objects (e.g., cells) to be assayed for production of an analyte of interest can be loaded into a swept region of the microfluidic device.
  • biological micro-objects e.g., mammalian cells, such as human cells
  • Ones of the biological micro-objects e.g., mammalian cells, such as human cells
  • the remaining sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Because the selected biological micro-objects are in unswept regions, the selected biological micro-objects are not substantially affected by the flowing out of the remaining sample material or the flowing in of the assay material.
  • the selected biological micro-objects can be allowed to produce the analyte of interest, which can diffuse from the unswept regions into the swept region, where the analyte of interest can react with the assay material to produce localized detectable reactions, each of which can be correlated to a particular unswept region. Any unswept region associated with a detected reaction can be analyzed to determine which, if any, of the biological micro-objects in the unswept region are sufficient producers of the analyte of interest.
  • An antigen as referred to herein, is a molecule or portion thereof that can bind with specificity to another molecule, such as an Ag-specific receptor.
  • An antigen may be any portion of a molecule, such as a conformational epitope or a linear molecular fragment, and often can be recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system.
  • An antigen may include a peptide, polysaccharide, or lipid.
  • An antigen may be characterized by its ability to bind to an antibody's variable Fab region. Different antibodies have the potential to discriminate among different epitopes present on the antigen surface, the structure of which may be modulated by the presence of a hapten, which may be a small molecule.
  • an antigen is a cancer cell- associated antigen.
  • the cancer cell-associated antigen can be simple or complex; the antigen can be an epitope on a protein, a carbohydrate group or chain, a biological or chemical agent other than a protein or carbohydrate, or any combination thereof; the epitope may be linear or conformational.
  • the cancer cell-associated antigen can be an antigen that uniquely identifies cancer cells (e.g., one or more particular types of cancer cells) or is upregulated on cancer cells as compared to its expression on normal cells. Typically, the cancer cell-associated antigen is present on the surface of the cancer cell, thus ensuring that it can be recognized by an antibody.
  • the antigen can be associated with any type of cancer cell, including any type of cancer cell that can be found in a tumor known in the art or described herein.
  • the antigen can be associated with lung cancer, breast cancer, melanoma, and the like.
  • the term Attorney Docket No.01149-0025-00PCT “associated with a cancer cells,” when used in reference to an antigen, means that the antigen is produced directly by the cancer cell or results from an interaction between the cancer cell and normal cells.
  • the terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA.
  • Nucleic acid sequence refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.
  • B used to denote a single nucleotide, is a nucleotide selected from G (guanosine), C (cytidine) and T (thymidine) nucleotides but does not include A (adenine).
  • H used to denote a single nucleotide, is a nucleotide selected from A, C and T, but does not include G.
  • D used to denote a single nucleotide
  • K used to denote a single nucleotide
  • M used to denote a single nucleotide
  • N used to denote a single nucleotide, is a nucleotide selected from A, C, G, and T.
  • R used to denote a single nucleotide
  • S used to denote a single nucleotide
  • V used to denote a single nucleotide
  • Y used to denote a single nucleotide, is a nucleotide selected from C and T. II.
  • compositions for Culturing and/or Maintaining B Cells including memory B cells, which may be human B cells (e.g., human memory B cells) are provided.
  • the B cells can be activated, resulting in proliferation and/or immunoglobulin (e.g., antibody) secretion, and/or maintained in a state of sustained Attorney Docket No.01149-0025-00PCT immunoglobulin (e.g., antibody) expression/secretion.
  • Methods of culturing, activating, and/or screening B cells that involve contacting the B cells with one or more compositions disclosed herein are also provided.
  • the composition may be an activation composition that promotes B cell activation and/or expansion.
  • the composition results in proliferation of B cells, and/or increasing the size of B cells, and/or initiating and/or increasing immunoglobulin expression in B cells, for example by differentiating memory B cells into plasmablasts.
  • the activation composition can comprise a Toll-like receptor 9 (TLR9) agonist (e.g., a TLR9-specific agonist), IL-6, and IL-4, and lack feeder cells.
  • TLR9 Toll-like receptor 9
  • the TLR9 agonist is a CpG oligodeoxynucleotide (ODN), which may be a B-class CpG ODN and/or have a linear structure.
  • ODN CpG oligodeoxynucleotide
  • the CpG ODN is present at a concentration of at least about 0.25 micromolar ( ⁇ M), at least about 0.5 ⁇ M, at least about 1.0 ⁇ M, at least about 1.5 ⁇ M, at least about 2.0 ⁇ M, or at least about 2.5 ⁇ M, or in some embodiments at least about 5 ⁇ M.
  • the CpG ODN is present at a concentration from about 0.25 ⁇ M to about 5.0 ⁇ M, from about 0.25 ⁇ M to about 4.0 ⁇ M, from about 0.5 ⁇ M to about 3.0 ⁇ M, from about 1.0 ⁇ M to about 2.0 ⁇ M, from about 0.25 ⁇ M to about 1.0 ⁇ M, from about 0.5 ⁇ M to about 1.5 ⁇ M, from about 1.0 ⁇ M to about 2.5 ⁇ M, from about 1.5 ⁇ M to about 3.0 ⁇ M, from about 2.0 ⁇ M to about 4.0 ⁇ M, from about 2.5 ⁇ M to about 5.0 ⁇ M, or any range defined by two of the foregoing endpoints.
  • the composition comprises IL-6 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL.
  • the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 5.0 ng/mL to about 25 ng/mL, from about 10 ng/mL to about 50 ng/mL, from about 15 ng/mL to about 75 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints
  • the IL-6 is human IL-6.
  • the composition comprises IL-4 at a concentration of at least about 0.5 ng/mL, at least about 1.0 ng/mL, at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL.
  • the composition comprises IL- 4 at a concentration from about 0.5 ng/mL to about 10 ng/mL, from about 1.0 ng/mL to about 2.5 ng/mL, from about 1.5 ng/mL to about 5.0 ng/mL, from about 2.0 ng/mL to about 7.5 ng/mL, from about 2.5 ng/mL to about 10 ng/mL, or any range defined by two of the foregoing endpoints.
  • the IL-4 is human IL-4. Attorney Docket No.01149-0025-00PCT [0094]
  • the activation composition further comprises IL-2, IL- 21, BAFF, or any combination thereof.
  • the composition comprises IL-2, IL-21, and/or BAFF.
  • the IL-2, IL-21, and BAFF can each, independently, be a human protein.
  • the composition comprises IL-2.
  • the composition comprises IL-2 at a concentration of at least about 1.0 ng/mL, at least about 10 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, or at least about 100 ng/mL.
  • the composition comprises IL-2 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 10 ng/mL to about 50 ng/mL, from about 25 ng/mL to about 75 ng/mL, from about 50 ng/mL to about 100 ng/mL, from about 5 ng/mL to about 25 ng/mL, from about 25 ng/mL to about 50 ng/mL, from about 50 ng/mL to about 75 ng/mL, from about 75 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints.
  • the IL-2 is human IL-2.
  • the composition comprises IL-21. In some embodiments, the composition comprises IL-21 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL.
  • the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 2.0 ng/mL to about 20 ng/mL, from about 5.0 ng/mL to about 40 ng/mL, from about 10 ng/mL to about 60 ng/mL, from about 15 ng/mL to about 80 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints.
  • the IL-21 is human IL-21.
  • the composition comprises BAFF.
  • the composition comprises BAFF at a concentration of at least about 10 ng/mL, at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL.
  • the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL, from about 20 ng/mL to about 50 ng/mL, from about 30 ng/mL to about 60 ng/mL, from about 40 ng/mL to about 70 ng/mL, from about 50 ng/mL to about 80 ng/mL, from about 60 ng/mL to about 90 ng/mL, from about 70 ng/mL to about 100 ng/mL, or any range defined by two of the forgoing endpoints.
  • the BAFF is human BAFF.
  • the activation composition comprises a non-effective concentration of a protein kinase C (PKC) agonist (e.g., less than about 10 ng/mL) or substantially lacks PKC agonist.
  • PKC protein kinase C
  • the composition comprises a PKC agonist at a concentration of less than about 10 ng/mL, less than about 5.0 ng/mL, less than Attorney Docket No.01149-0025-00PCT about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, or substantially lacks PKC agonist.
  • the activation composition can further comprise serum (e.g., any serum suitable for culturing B cell lymphocytes), which can be present at an amount of about 1% to about 20% v/v, about 2% to about 15%, or about 5% to about 10%.
  • the serum can be, for example, fetal bovine serum.
  • the composition is a liquid medium.
  • the composition may be a maintaining composition for maintaining immunoglobulin expression in the B cells.
  • the maintaining composition can comprise a protein kinase C (PKC) agonist (e.g., a PKC-specific agonist) and lack feeder cells.
  • PKC protein kinase C
  • the PKC agonist is a phorbol compound, which may be a phorbol ester.
  • the phorbol ester may be phorbol 12-myristate 13- acetate (PMA), phorbol 12,13-dibutyrate (PBD), 4-alpha-phorbol 12-myristate 13-acetate (4- alpha-PMA), dPPA, 4-alpha-phorbol (isophorbol), phorbol 12-myristate 13-acetate 4-O- methyl ether (MPMA), or the like. Examples of non-limiting phorbol esters are shown in Table 1 below.
  • the PKC agonist is present at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, at least about 100 ng/mL, at least about 150 ng/mL, at least about 200 ng/mL, at least about 250 ng/mL, at least about 500 ng/mL, or at least about 750 ng/mL.
  • the PKC agonist is present at a concentration from about 10 ng/mL to about 1000 ng/mL, from about 25 ng/mL to about 200 ng/mL, from about 50 ng/mL to about 250 ng/mL, from about 75 ng/mL to about 300 ng/mL, from about 100 ng/mL to about 400 ng/mL, from about 150 ng/mL to about 500 ng/mL, from about 200 ng/mL to about 600 ng/mL, from about 250 ng/mL to about 700 ng/mL, from about 300 ng/mL to about 800 ng/mL, from about 400 ng/mL to about 900 ng/mL, from about 500 ng/mL to about 1000 ng/mL, or any range defined by two of the foregoing endpoints.
  • the maintaining composition further comprises IL-6, IL-4, IL-21, BAFF, or any combination thereof.
  • the Attorney Docket No.01149-0025-00PCT composition comprises IL-6 and/or IL-4.
  • the IL-6, IL-4, IL-21, and BAFF can each, independently, be a human protein.
  • the composition comprises IL-6 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL.
  • the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 5.0 ng/mL to about 25 ng/mL, from about 10 ng/mL to about 50 ng/mL, from about 15 ng/mL to about 75 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints.
  • the IL-6 is human IL-6.
  • the composition comprises IL-4 at a concentration of at least about 0.5 ng/mL, at least about 1.0 ng/mL, at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL.
  • the composition comprises IL- 4 at a concentration from about 0.5 ng/mL to about 10 ng/mL, from about 1.0 ng/mL to about 2.5 ng/mL, from about 1.5 ng/mL to about 5.0 ng/mL, from about 2.0 ng/mL to about 7.5 ng/mL, from about 2.5 ng/mL to about 10 ng/mL, or any range defined by two of the foregoing endpoints.
  • the IL- 4 is human IL-4.
  • the composition comprises IL-21.
  • the composition comprises IL-21 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL.
  • the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 2.0 ng/mL to about 20 ng/mL, from about 5.0 ng/mL to about 40 ng/mL, from about 10 ng/mL to about 60 ng/mL, from about 15 ng/mL to about 80 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints.
  • the IL-21 is human IL-21.
  • the composition comprises BAFF.
  • the composition comprises BAFF at a concentration of at least about 10 ng/mL, at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL.
  • the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL, from about 20 ng/mL to about 50 ng/mL, from about 30 ng/mL to about 60 ng/mL, from about 40 ng/mL to about 70 ng/mL, from about 50 ng/mL to about 80 ng/mL, from about 60 ng/mL to about 90 ng/mL, from about 70 ng/mL to about 100 ng/mL, or any range defined by two of the forgoing endpoints.
  • the BAFF is human BAFF.
  • the maintaining composition comprises a non-effective concentration of IL-2.
  • the non-effective concentration of IL-2 comprises a concentration of less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL or substantially lacks IL-2.
  • the maintaining composition comprises a non-effective concentration of TNF-alpha.
  • the non-effective concentration of TNF-alpha comprises a concentration of less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks TNF-alpha.
  • the maintaining composition comprises a non-effective concentration of CpG oligodeoxynucleotides (ODNs).
  • the non-effective concentration of CpG oligodeoxynucleotides comprises a concentration of less than about 250 nanomolar (nM), less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than about 1 nM, or substantially lacks CpG ODNs.
  • the ODNs may include class B/linear ODNs, and in particular ODN 2006.
  • the maintaining composition comprises a non-effective concentration of a PI3K agonist.
  • the non-effective concentration of a PI3K agonist comprises less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks PI3K agonist.
  • the PI3K agonist may include naringenin, naringin, butin, eriodictyol, homoeriodictyol, hesperetin, hesperidin, and isosakuranetin.
  • the maintaining composition comprises a non- effective amount of a STAT3 agonist.
  • the non-effective amount of a STAT3 agonist comprises a concentration of less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks STAT3 agonist.
  • the STAT3 agonist may include leukemia inhibitory factor (LIF) (e.g., human LIF (hLIF)), as well as fragments and variants that retain STAT3 agonist activity.
  • LIF leukemia inhibitory factor
  • the non-effective amount of LIF comprises a concentration of less than about 10 ng/ml, less than about 1.0 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks LIF.
  • the maintaining composition comprises a non-effective amount of a CD40-clustering agent.
  • the non-effective amount of a Attorney Docket No.01149-0025-00PCT CD40-clustering agent comprises a concentration of less than about 1.0 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks CD40-clustering agent.
  • the CD40- clustering agent may include CD40 ligand (i.e., CD40L), active fragments and variants thereof, and anti-CD40 agonist antibodies, and in some embodiments, the CD40 clustering agent may be attached to a bead that contacts the cell.
  • the maintaining composition can further comprise serum (e.g., any serum suitable for culturing B cell lymphocytes), which can be present at an amount of about 1% to about 20% v/v, or about 2% to about 15%, or about 5% to about 10%.
  • the serum can be, for example, fetal bovine serum.
  • the composition is a liquid medium.
  • the B cells may be cultured in the activating composition followed by culture in the maintaining composition in accordance with any of the embodiments described herein. III. Methods for Antibody Discovery [0102] As mentioned above, the time needed in screening cells for lead candidates using macroscale workflows that are typically currently used, significantly adds to the drug development timeline.
  • FIG. 6A shows a general workflow which is directed to providing acceleration of antibody discovery campaigns.
  • the method includes isolating B cells and importing the cells in a microfluidic device, preferably the microfluidic device as disclosed in the following sections.
  • the cells can be loaded into the channel or chamber of the microfluidic device and cultured individually. In some embodiments, up to 50k single B cells may be loaded.
  • cells that are determined to be healthy (e.g., viable), substantially healthy, or enriched in a proportion of cells that are healthy may be introduced preferentially to the chamber(s) of the microfluidic device.
  • the method may also include conducting binding or functional assays, which can be, but is not limited to bead-based analyses for testing the IgG-antigen specificity of the antibodies secreted in each pen.
  • the method may further include loading nucleic acid capture objects, which may be any nucleic acid capture object at described herein, and performing on- chip lysis, nucleic acid capture and reverse transcription.
  • nucleic acid capture objects are generated through these steps by using the Attorney Docket No.01149-0025-00PCT capture objects of the present disclosure.
  • the nucleic acid capture objects additionally are labelled, to permit correlation of the binding/functional assay results with the specific nucleic acid isolated from the cell(s) responsible for the assay results.
  • Detection of the labels may be performed at any point during the workflow to identify the label for each capture object in each chamber.
  • the barcoded cDNA sequences which are captured on the capture objects and comprise the BCR sequence (i.e., barcoded BCR beads)
  • the capture objects enable the identification of the origin of the barcoded BCR beads on the 96-well plate.
  • the method of the present disclosure aims at increasing screening throughput to up to 50k single plasma B cells and over 1000 exports of target B cell receptor (BCR) sequences. Overall, this workflow provides for high throughput antibody discovery methods.
  • a healthy cell is a cell demonstrating characteristics of viability, e.g., is a viable cell and has the ability to continue to grow and optionally, produce either biomolecules of interest and/or produce daughter cells having the same capabilities. Disposing into the chambers, e.g., sequestration pens, of the microfluidic device only, substantially only or an increased proportion of healthy cells out of an imported population can increase the likelihood of identifying useful cells/clonal populations thereof. Further, resources used during a biomolecule production development/identification campaign are not expended upon non-viable cells, reducing waste and preserving the use of the pre-defined number of chambers for cells that have some possibility of expressing the biomolecule of interest.
  • another aspect of the present disclosure is to identify healthy cells before importing them into the chamber of the microfluidic device.
  • identifying healthy cells within a microfluidic device can be difficult, due to the very nature of the small scale of the microfluidic device.
  • only a relatively small Attorney Docket No.01149-0025-00PCT number of cells may be imported into the device, and staining of such small number of cells may not be able to generate a fluorescent intensity sufficient for meaningful detection.
  • a staining method can be combined with a brightfield image observation for the purposes of identifying healthy cells.
  • identifying a healthy cell can involve the use of a machine learning algorithm to process image data.
  • the machine learning algorithm is capable of identifying healthy cells without staining.
  • the machine learning algorithm can include a neural network, such as a convolutional neural network.
  • a convolutional neural network generally accomplishes an advanced form of image processing and classification/detection by first looking for low level features such as, for example, edges and curves, and then advancing to more abstract (e.g., unique to the type of images being classified) concepts through a series of convolutional layers.
  • a CNN can do this by passing an image through a series of convolutional, nonlinear, pooling (or downsampling, as will be discussed in more detail below), and fully connected layers, and get an output.
  • the output can be a single class or a probability of classes that best describes the image or detects objects on the image.
  • a training data used in establishing the CNN model of the present disclosure may include a fluorescent image having the cells of interest stained, a brightfield image having the cells of interest annotated, or combinations thereof.
  • the dyes suitable in the present disclosure may include but are not limited to calcein, zombie violet stain, annexin, acridine orange, propidium iodide, or combinations thereof. Any suitable stain that discriminates between a healthy cell and a dead/dying and/or non-viable cell, as is known to one of skill in the art may be used.
  • other dyes that are specific to a marker of interest can also be used, for instance, Alexa Fluor® 647 anti-mouse CD138 (Syndecan-1) Antibody (BioLegend), which is highly specific for terminally differentiated live Attorney Docket No.01149-0025-00PCT plasma cells and stains CD138 presented on the surface.
  • a training data includes images of cells stained with a fluorescent dye in combination with images of the cells under brightfield.
  • a healthy cell can be identified, for example, by observing the morphologies of the cells under brightfield.
  • a healthy cell e.g., a viable cell
  • a healthy cell can be characterized as having a clear cell boundary, good contrast, round shape, or combinations thereof.
  • a healthy cell can be determined by identifying the unhealthy ones. For instance, an unhealthy cell can be characterized as having debris-like appearance, unclear or different contrast, or combinations thereof.
  • assessment of viability can be made in a relative manner by comparing cells in a sample. For instance, a healthy cell can have a larger diameter while other cells having a smaller diameter are more likely to be unhealthy/dead or merely cell debris.
  • cells may be first detected under brightfield and then labeled as live/dead based on the fluorescent intensity. In some embodiment, labeling of live/dead cells is based on a cutoff value of the fluorescent intensity, which can be selected in accordance with the user’s likings or needs.
  • the method of penning healthy cells using the trained machine learning algorithm may be employed to increase the penning efficiency of healthy cells, and decrease the numbers of non-viable cells penned.
  • the percentage of healthy cells relative to non-viable cells imported into the chambers, e.g., sequestration pens, after identification by the algorithm may be improved by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. V.
  • Binding interactions between a first molecule and a second molecule can be measured in a chamber of a microfluidic chip.
  • the chamber can be any of the chambers described or referenced herein, including a microwell or a sequestration pen, and the assay formats can vary widely.
  • the assay can be a “sandwich” assay in which a surface, such as a bead or an internal surface of a wall of the microfluidic device, is configured to capture and/or present the first molecule; binding of the second molecule is detected via a third molecule that is labeled and capable of binding to the complex formed by the second molecule binding to the first molecule, thereby associating the label of the third molecule with the surface Attorney Docket No.01149-0025-00PCT in a detectable manner.
  • the second molecule can be produced by a biological cell.
  • the assay surface can be in the chamber (e.g., as described in U.S. Patent Application Publication No.
  • the assay can be a diffusion gradient assay in which the second molecule has a label (which can be linked to the second molecule, or can be an intrinsic property of the second molecule, such as auto- fluorescence) and the diffusive properties of the labeled second molecule in the presence of the first molecule can be monitored, e.g., as described in PCT International Publication No. WO 2017/181135.
  • the first molecule can be produced by a biological cell.
  • Still other assays can feature a blocking interaction in which a molecule of interest binds to the first molecule and thereby blocks an interaction of the first molecule with the second molecule.
  • the molecule of interest can be produced by a biological cell, and the second molecule can comprise a label.
  • the blocking assays can feature the first molecule bound to a surface. The surface can be located, for example, in the chamber or a region proximal to the chamber, such as a channel. Examples of blocking assays are described below and elsewhere herein, including in the examples and in the claims. [0115] In-channel Binding Assay.
  • a method of assay for a specific binding interaction between a first molecule and a second molecule is provided.
  • the method can be performed within a microfluidic device having a channel and a chamber, such as a microwell or a sequestration pen, fluidically connecting to the channel.
  • the method can include: introducing each of a plurality of biological cells into a respective one of a plurality of chambers; incubating the biological cells and allowing the biological cell to produce and/or secrete a molecule of interest; introducing a micro-object including a plurality of first molecules into the channel; and monitoring an accumulation of the molecule of interest on the micro-object.
  • monitoring an accumulating of the molecule of interest on the micro-object including introducing a third molecule that is labeled and capable of binding to the complex formed by the molecule of interest binding to the first molecule, thereby associating the label of the third molecule with the accumulation of the molecule of interest on the micro-object.
  • introducing a micro-object including a plurality of first molecules, e.g., a reporter cell, into the channel including introducing a plurality of the micro- objects and allowing the plurality of the micro-objects fill the channel at a density.
  • an optimal density is such that nearly the entire channel is filled with the micro- objects. A density that is below optimal might result in a sparse number of micro-objects in the channel and an under-sampling of the secreted molecule of interest, making unambiguous identification of the secreting chamber difficult.
  • the optimal density can vary depending on the size of the micro-objects introduced.
  • the micro-objects are biological cells, and the density can be from about 10 7 to 10 9 , or about 10 8 to 2x10 8 cells/mL.
  • the micro-object including the plurality of first molecules, e.g., reporter cells may be cells that may be cells that culture in suspension. In other embodiments, adherent cell types may be used as reporter cells when detachment protocols are used.
  • adherent CHO cells may be successfully used when a detachment protocol may include: culturing to confluence prior to importation, e.g. not exceeding confluence; and treating with a detachment reagent such as Accutase (ThermoFisher Scientific, A1110501), TrypLE or the like, e.g. treatment at about 22°C for 10 min with no agitation.
  • a detachment reagent such as Accutase (ThermoFisher Scientific, A1110501), TrypLE or the like, e.g. treatment at about 22°C for 10 min with no agitation.
  • the adherent CHO cells were then successfully importable as monodisperse cells and the target cell densities were achieved.
  • Specific detachment protocols may be determined as needed for other cell types.
  • the preparatory culture density may be varied, e.g., less than about 100% confluent, less than about 90% confluent, less than about 80% confluent, less than about 60% confluent, or less than about 50% confluent.
  • the detachment reagent may be varied.
  • the duration of the detachment treatment may be varied, e.g., from about 5 min to about 1h, about 10 min, about 15 min, about 20 min, about 30 min, about 45 min, about 60 min or any value therebetween.
  • agitation is not employed.
  • the cells may be agitated during the detachment treatment.
  • Temperature may be varied in order to successfully detach the cells, and may be varied from about 15°C to about 36°C, about 10°C to about 40°C, or any temperature therebetween. Filtration through a cell strainer may be useful to remove cell clumps or other large debris, and may be performed before concentrating the cells to target import concentration.
  • the cells may be concentrated by centrifuging at 400 x g for 5 min, and resuspended to the desired concentration.
  • the third molecule which is labeled and capable of binding to the complex formed by the molecule of Attorney Docket No.01149-0025-00PCT interest binding to the first molecule, e.g., a labelled antibody, may be added to the media when resuspending the cells.
  • the first molecule and/or the molecule of interest can be a protein.
  • the protein can be, for example, a cell surface protein or an extracellular protein.
  • the protein can be a modified protein, such as a glycosylated protein, a lipid-anchored protein, or the like.
  • the molecule of interest can specifically bind to the first molecule.
  • the first molecule and molecule of interest can be an antigen-antibody pair.
  • the biological cell can be a B cell producing an antibody of interest (i.e., molecule of interest) and the first molecule presented on the surface of the micro-objects can be an antigen or epitope of the produced antibody.
  • the third molecule can be a secondary antibody binding to the produced antibodies (i.e., the secreted second molecule), and the detection thereof is associated with the binding of the first molecule and the molecule of interest.
  • the micro-object can be one or more beads or cells that express the first molecule. If a cell, the cell can express the first molecule naturally or can be genetically modified (e.g., stably or transiently transfected) to express the first molecule. If a bead, the bead can be created by conjugating the first molecule on its surface. VI.
  • Methods of Assembling Full Length V(D)J sequences from fragmented NGS Next Generation Sequencing, massively parallel sequencing
  • Methods for assembly of complete V(D)J sequences from fragmented NGS data originating from a single antibody producing cell e.g., a B-Cell
  • a single antibody producing cell e.g., a B-Cell
  • Microfluidic device/system feature cross- applicability. It should be appreciated that various features of microfluidic devices, systems, and motive technologies described herein may be combinable or interchangeable.
  • FIG.1A illustrates an example of a microfluidic device 100.
  • a perspective view of the microfluidic device 100 is shown having a partial cut-away of its cover 110 to provide a partial view into the microfluidic device 100.
  • the microfluidic device 100 generally comprises a microfluidic circuit 120 comprising a flow path 106 through Attorney Docket No.01149-0025-00PCT which a fluidic medium 180 can flow, optionally carrying one or more micro-objects (not shown) into and/or through the microfluidic circuit 120.
  • the microfluidic circuit 120 is defined by an enclosure 102.
  • the enclosure 102 can be physically structured in different configurations, in the example shown in FIG.1A the enclosure 102 is depicted as comprising a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110.
  • the support structure 104, microfluidic circuit structure 108, and cover 110 can be attached to each other.
  • the microfluidic circuit structure 108 can be disposed on an inner surface 109 of the support structure 104, and the cover 110 can be disposed over the microfluidic circuit structure 108.
  • the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120, forming a three-layer structure.
  • the support structure 104 can be at the bottom and the cover 110 at the top of the microfluidic circuit 120 as illustrated in FIG.1A.
  • the support structure 104 and the cover 110 can be configured in other orientations.
  • the support structure 104 can be at the top and the cover 110 at the bottom of the microfluidic circuit 120.
  • a passage include a valve, a gate, a pass-through hole, or the like.
  • port 107 is a pass-through hole created by a gap in the microfluidic circuit structure 108.
  • the port 107 can be situated in other components of the enclosure 102, such as the cover 110. Only one port 107 is illustrated in FIG.1A but the microfluidic circuit 120 can have two or more ports 107.
  • the support structure 104 can comprise one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates.
  • the support structure 104 can comprise one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates can be electrically connected to a single electrode).
  • the support structure 104 can further comprise a printed circuit board assembly (“PCBA”).
  • PCBA printed circuit board assembly
  • the semiconductor substrate(s) can be mounted on a PCBA.
  • the microfluidic circuit structure 108 can define circuit elements of the microfluidic circuit 120.
  • Such circuit elements can comprise spaces or regions that can be Attorney Docket No.01149-0025-00PCT fluidly interconnected when microfluidic circuit 120 is filled with fluid, such as flow regions (which may include or be one or more flow channels), chambers (which class of circuit elements may also include sub-classes including sequestration pens), traps, and the like.
  • Circuit elements can also include barriers, and the like. In the microfluidic circuit 120 illustrated in FIG.
  • the microfluidic circuit structure 108 comprises a frame 114 and a microfluidic circuit material 116.
  • the frame 114 can partially or completely enclose the microfluidic circuit material 116.
  • the frame 114 can be, for example, a relatively rigid structure substantially surrounding the microfluidic circuit material 116.
  • the frame 114 can comprise a metal material.
  • the microfluidic circuit structure need not include a frame 114.
  • the microfluidic circuit structure can consist of (or consist essentially of) the microfluidic circuit material 116.
  • the microfluidic circuit material 116 can be patterned with cavities or the like to define the circuit elements and interconnections of the microfluidic circuit 120, such as chambers, pens and microfluidic channels.
  • the microfluidic circuit material 116 can comprise a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or the like), which can be gas permeable.
  • a flexible polymer e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or the like
  • Other examples of materials that can form the microfluidic circuit material 116 include molded glass, an etchable material such as silicone (e.g., photo-patternable silicone or “PPS”), photo-resist (e.g., SU8), or the like.
  • microfluidic circuit material 116 can be rigid and/or substantially impermeable to gas.
  • microfluidic circuit material 116 can be disposed on the support structure 104 and inside the frame 114.
  • the microfluidic circuit 120 can include a flow region in which one or more chambers can be disposed and/or fluidically connected thereto.
  • a chamber can have one or more openings fluidically connecting the chamber with one or more flow regions.
  • a flow region comprises or corresponds to a microfluidic channel 122.
  • suitable microfluidic devices can include a plurality (e.g., 2 or 3) of such microfluidic circuits.
  • the microfluidic device 100 can be configured to be a nanofluidic device.
  • the microfluidic circuit 120 may include a plurality of microfluidic sequestration pens 124, 126, 128, and 130, where each sequestration pens may have one or more openings.
  • a sequestration pen may have only a single opening in fluidic communication with the flow path 106.
  • a sequestration pen may have more than one opening in fluidic communication with the flow path 106, e.g., n number of openings, but with n-1 openings that are valved, such that all but one opening is Attorney Docket No.01149-0025-00PCT closable. When all the valved openings are closed, the sequestration pen limits exchange of materials from the flow region into the sequestration pen to occur only by diffusion.
  • the sequestration pens comprise various features and structures (e.g., isolation regions) that have been optimized for retaining micro-objects within the sequestration pen (and therefore within a microfluidic device such as microfluidic device 100) even when a medium 180 is flowing through the flow path 106.
  • the cover 110 can be an integral part of the frame 114 and/or the microfluidic circuit material 116.
  • the cover 110 can be a structurally distinct element, as illustrated in FIG. 1A.
  • the cover 110 can comprise the same or different materials than the frame 114 and/or the microfluidic circuit material 116.
  • the cover 110 can be an integral part of the microfluidic circuit material 116.
  • the support structure 104 can be a separate structure from the frame 114 or microfluidic circuit material 116 as illustrated, or an integral part of the frame 114 or microfluidic circuit material 116.
  • the frame 114 and microfluidic circuit material 116 can be separate structures as shown in FIG. 1A or integral portions of the same structure.
  • the microfluidic device can retain a three-layer structure that includes a base layer and a cover layer that sandwich a middle layer in which the microfluidic circuit 120 is located.
  • the cover 110 can comprise a rigid material.
  • the rigid material may be glass or a material with similar properties.
  • the cover 110 can comprise a deformable material.
  • the deformable material can be a polymer, such as PDMS.
  • the cover 110 can comprise both rigid and deformable materials.
  • one or more portions of cover 110 e.g., one or more portions positioned over sequestration pens 124, 126, 128, 130
  • Microfluidic devices having covers that include both rigid and deformable materials have been described, for example, in U.S. Patent No.10,058,865 (Breinlinger et al.), the contents of which are incorporated herein by reference.
  • the cover 110 can further include one or more electrodes.
  • the one or more electrodes can comprise a conductive oxide, such as indium-tin-oxide (ITO), which may be coated on glass or a similarly insulating material.
  • the one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of electrically conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS).
  • a conductive oxide such as indium-tin-oxide (ITO)
  • ITO indium-tin-oxide
  • the one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of electrically conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS).
  • a polymer e.g., PDMS
  • the cover 110 and/or the support structure 104 can be transparent to light.
  • the cover 110 may also include at least one material that is gas permeable (e.g., PDMS or PPS).
  • the microfluidic circuit 120 is illustrated as comprising a microfluidic channel 122 and sequestration pens 124, 126, 128, 130. Each pen comprises an opening to channel 122, but otherwise is enclosed such that the pens can substantially isolate micro-objects inside the pen from fluidic medium 180 and/or micro- objects in the flow path 106 of channel 122 or in other pens.
  • the walls of the sequestration pen extend from the inner surface 109 of the base to the inside surface of the cover 110 to provide enclosure.
  • the opening of the sequestration pen to the microfluidic channel 122 is oriented at an angle to the flow 106 of fluidic medium 180 such that flow 106 is not directed into the pens.
  • the vector of bulk fluid flow in channel 122 may be tangential or parallel to the plane of the opening of the sequestration pen, and is not directed into the opening of the pen.
  • pens 124, 126, 128, 130 are configured to physically isolate one or more micro-objects within the microfluidic circuit 120.
  • Sequestration pens in accordance with the present disclosure can comprise various shapes, surfaces and features that are optimized for use with DEP, OET, OEW, fluid flow, magnetic forces, centripetal, and/or gravitational forces, as will be discussed and shown in detail below.
  • the microfluidic circuit 120 may comprise any number of microfluidic sequestration pens. Although five sequestration pens are shown, microfluidic circuit 120 may have fewer or more sequestration pens. As shown, microfluidic sequestration pens 124, 126, 128, and 130 of microfluidic circuit 120 each comprise differing features and shapes which may provide one or more benefits useful for maintaining, isolating, assaying or culturing biological micro-objects.
  • the microfluidic circuit 120 comprises a plurality of identical microfluidic sequestration pens. [0133] In the embodiment illustrated in FIG.1A, a single flow path 106 containing a single channel 122 is shown. However, other embodiments may contain multiple channels 122 within a single flow path 106, as shown in FIG. 1B.
  • the microfluidic circuit 120 further comprises an inlet valve or port 107 in fluid communication with the flow path 106, whereby fluidic medium 180 can access the flow path 106 (and channel 122). In some instances, the flow path 106 comprises a substantially straight path.
  • the flow path 106 is arranged in a non-linear or winding manner, such as a zigzag pattern, whereby the flow path 106 travels across the microfluidic device 100 two or more times, e.g., in alternating directions.
  • the flow in the flow path 106 may proceed from inlet to outlet or may be reversed and proceed from outlet to inlet.
  • Attorney Docket No.01149-0025-00PCT [0134]
  • FIG.1B One example of a multi-channel device, microfluidic device 175, is shown in FIG.1B, which may be like microfluidic device 100 in other respects.
  • Microfluidic device 175 and its constituent circuit elements may have any of the dimensions discussed herein.
  • the microfluidic circuit illustrated in FIG.1B has two inlet/outlet ports 107 and a flow path 106 containing four distinct channels 122.
  • the number of channels into which the microfluidic circuit is sub-divided may be chosen to reduce fluidic resistance.
  • the microfluidic circuit may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more channels to provide a selected range of fluidic resistance.
  • Microfluidic device 175 further comprises a plurality of sequestration pens opening off of each channel 122, where each of the sequestration pens is similar to sequestration pen 128 of FIG.
  • microfluidic device 175 may have any of the dimensions or functions of any sequestration pen as described herein.
  • the sequestration pens of microfluidic device 175 can have different shapes, such as any of the shapes of sequestration pens 124, 126, or 130 of FIG.1A or as described anywhere else herein.
  • microfluidic device 175 can include sequestration pens having a mixture of different shapes.
  • a plurality of sequestration pens is configured (e.g., relative to a channel 122) such that the sequestration pens can be loaded with target micro-objects in parallel.
  • microfluidic circuit 120 further may include one or more optional micro-object traps 132.
  • the optional traps 132 may be formed in a wall forming the boundary of a channel 122, and may be positioned opposite an opening of one or more of the microfluidic sequestration pens 124, 126, 128, 130.
  • the optional traps 132 may be configured to receive or capture a single micro-object from the flow path 106, or may be configured to receive or capture a plurality of micro-objects from the flow path 106.
  • the optional traps 132 comprise a volume approximately equal to the volume of a single target micro-object.
  • the trap 132 comprises a side passage 134 that is smaller than the target micro-object in order to facilitate flow through the trap 132. [0136] Sequestration pens.
  • the microfluidic devices described herein may include one or more sequestration pens, where each sequestration pen is suitable for holding one or more micro-objects (e.g., biological cells, or groups of cells that are associated together).
  • the sequestration pens may be disposed within and open to a flow region, which in some embodiments is a microfluidic channel.
  • Each of the sequestration pens can have one or more openings for fluidic communication to one or more microfluidic channels.
  • a sequestration pen may have only one opening to a microfluidic channel.
  • FIG. 2A-2C show sequestration pens 224, 226, and 228 of a microfluidic device 200, which may be like sequestration pen 128 of FIG.1A.
  • Each sequestration pen 224, 226, and 228 can comprise an isolation region 240 and a connection region 236 fluidically connecting the isolation region 240 to a flow region, which may, in some embodiments include a microfluidic channel, such as channel 122.
  • the connection region 236 can comprise a proximal opening 234 to the flow region (e.g., microfluidic channel 122) and a distal opening 238 to the isolation region 240.
  • connection region 236 can be configured so that the maximum penetration depth of a flow of a fluidic medium (not shown) flowing in the microfluidic channel 122 past the sequestration pen 224, 226, and 228 does not extend into the isolation region 240, as discussed below for FIG.2C. In some embodiments, streamlines from the flow in the microfluidic channel do not enter the isolation region. Thus, due to the connection region 236, a micro-object (not shown) or other material (not shown) disposed in the isolation region 240 of a sequestration pen 224, 226, and 228 can be isolated from, and not substantially affected by, a flow of fluidic medium 180 in the microfluidic channel 122.
  • the sequestration pens 224, 226, and 228 of FIGS.2A-2C each have a single opening which opens directly to the microfluidic channel 122.
  • the opening of the sequestration pen may open laterally from the microfluidic channel 122, as shown in FIG.2A, which depicts a vertical cross-section of microfluidic device 200.
  • FIG.2B shows a horizontal cross-section of microfluidic device 200.
  • An electrode activation substrate 206 can underlie both the microfluidic channel 122 and the sequestration pens 224, 226, and 228.
  • the upper surface of the electrode activation substrate 206 within an enclosure of a sequestration pen, forming the floor of the sequestration pen, can be disposed at the same level or substantially the same level of the upper surface the of electrode activation substrate 206 within the microfluidic channel 122 (or flow region if a channel is not present), forming the floor of the flow channel (or flow region, respectively) of the microfluidic device.
  • the electrode activation substrate 206 may be featureless or may have an irregular or patterned surface that varies from its highest elevation to its lowest depression by less than about 3 micrometers (microns), 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns or less.
  • the variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 10%, 7%, 5%, 3%, 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the height of the walls of the sequestration pen.
  • the variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3%, 0.2%, or 0.1% of the height of the Attorney Docket No.01149-0025-00PCT substrate. While described in detail for the microfluidic device 200, this may also apply to any of the microfluidic devices described herein. [0139]
  • the microfluidic channel 122 and connection region 236 can be examples of swept regions, and the isolation regions 240 of the sequestration pens 224, 226, and 228 can be examples of unswept regions.
  • Sequestration pens like 224, 226, 228 have isolation regions wherein each isolation region has only one opening, which opens to the connection region of the sequestration pen. Fluidic media exchange in and out of the isolation region so configured can be limited to occurring substantially only by diffusion.
  • the microfluidic channel 122 and sequestration pens 224, 226, and 228 can be configured to contain one or more fluidic media 180.
  • ports 222 are connected to the microfluidic channel 122 and allow the fluidic medium 180 to be introduced into or removed from the microfluidic device 200. Prior to introduction of the fluidic medium 180, the microfluidic device may be primed with a gas such as carbon dioxide gas.
  • FIG.2C illustrates a detailed view of an example of a sequestration pen 224, which may contain one or more micro-objects 246, according to some embodiments.
  • the flow 242 of fluidic medium 180 in the microfluidic channel 122 past the proximal opening 234 of the connection region 236 of sequestration pen 224 can cause a secondary flow 244 of the fluidic medium 180 into and out of the sequestration pen 224.
  • the length Lcon of the connection region 236 of the sequestration pen 224 i.e., from the proximal opening 234 to the distal opening 238) should be greater than the penetration depth Dp of the secondary flow 244 into the connection region 236.
  • the penetration depth D p depends upon a number of factors, including the shape of the microfluidic channel 122, which may be defined by a width Wcon of the connection region 236 at the proximal opening 234; a width Wch of the microfluidic channel 122 at the proximal opening 234; a height H ch of the channel 122 at the proximal opening 234; and the width of the distal opening 238 of the connection region 236.
  • the width Wcon of the connection region 236 at the proximal opening 234 and the height Hch of the channel 122 at the proximal opening 234 tend to be the most significant.
  • the penetration depth D p can be influenced by the velocity of the fluidic medium Attorney Docket No.01149-0025-00PCT 180 in the channel 122 and the viscosity of fluidic medium 180.
  • these factors i.e., velocity and viscosity
  • these factors can vary widely without dramatic changes in penetration depth Dp.
  • the penetration depth Dp of the secondary flow 244 ranges from less than 1.0 times W con (i.e., less than 50 microns) at a flow rate of 0.1 microliters/sec to about 2.0 times Wcon (i.e., about 100 microns) at a flow rate of 20 microliters/sec, which represents an increase in Dp of only about 2.5-fold over a 200-fold increase in the velocity of the fluidic medium 180.
  • the walls of the microfluidic channel 122 and sequestration pen 224, 226, or 228 can be oriented as follows with respect to the vector of the flow 242 of fluidic medium 180 in the microfluidic channel 122: the microfluidic channel width W ch (or cross-sectional area of the microfluidic channel 122) can be substantially perpendicular to the flow 242 of medium 180; the width Wcon (or cross-sectional area) of the connection region 236 at opening 234 can be substantially parallel to the flow 242 of medium 180 in the microfluidic channel 122; and/or the length L con of the connection region can be substantially perpendicular to the flow 242 of medium 180 in the microfluidic channel 122.
  • the configurations of the microfluidic channel 122 and the opening 234 may be fixed, whereas the rate of flow 242 of fluidic medium 180 in the microfluidic channel 122 may be variable. Accordingly, for each sequestration pen 224, a maximal velocity V max for the flow 242 of fluidic medium 180 in channel 122 may be identified that ensures that the penetration depth Dp of the secondary flow 244 does not exceed the length Lcon of the connection region 236.
  • the resulting secondary flow 244 can be wholly contained within the connection region 236 and does not enter the isolation region 240.
  • the flow 242 of fluidic medium 180 in the microfluidic channel 122 (swept region) is prevented from drawing micro-objects 246 out of the isolation region 240, which is an unswept region of the microfluidic circuit, resulting in the micro-objects 246 being retained within the isolation region 240.
  • microfluidic circuit element dimensions and further selection of the operating parameters can prevent contamination of the isolation region 240 of sequestration pen 224 by materials from the microfluidic channel 122 or another sequestration Attorney Docket No.01149-0025-00PCT pen 226 or 228. It should be noted, however, that for many microfluidic chip configurations, there is no need to worry about Vmax per se, because the chip will break from the pressure associated with flowing fluidic medium 180 at high velocity through the chip before Vmax can be achieved.
  • Components (not shown) in the first fluidic medium 180 in the microfluidic channel 122 can mix with the second fluidic medium 248 in the isolation region 240 substantially only by diffusion of components of the first medium 180 from the microfluidic channel 122 through the connection region 236 and into the second fluidic medium 248 in the isolation region 240.
  • components (not shown) of the second medium 248 in the isolation region 240 can mix with the first medium 180 in the microfluidic channel 122 substantially only by diffusion of components of the second medium 248 from the isolation region 240 through the connection region 236 and into the first medium 180 in the microfluidic channel 122.
  • the extent of fluidic medium exchange between the isolation region of a sequestration pen and the flow region by diffusion is greater than about 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% of fluidic exchange.
  • the first medium 180 can be the same medium or a different medium than the second medium 248.
  • the first medium 180 and the second medium 248 can start out being the same, then become different (e.g., through conditioning of the second medium 248 by one or more cells in the isolation region 240, or by changing the medium 180 flowing through the microfluidic channel 122).
  • the width Wcon of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238.
  • the width Wcon of the connection region 236 at the distal opening 238 can be any of the values identified herein for the width Wcon of the connection region 236 at the proximal opening 234.
  • the width of the isolation region 240 at the distal opening 238 can be substantially the same as the width W con of the connection region 236 at the proximal opening 234.
  • the width W con of the connection region 236 at the distal opening 238 can be different (e.g., larger or smaller) than the width Wcon of the connection region 236 at the proximal opening 234.
  • the width W con of the connection region 236 may be narrowed or widened between the proximal opening 234 and distal opening 238.
  • the connection region 236 may be narrowed or widened between the proximal opening and the distal opening, using a variety of different geometries (e.g., chamfering the connection region, beveling the connection region).
  • any part or subpart of the Attorney Docket No.01149-0025-00PCT connection region 236 may be narrowed or widened (e.g., a portion of the connection region adjacent to the proximal opening 234).
  • microfluidic device 300 depicts another exemplary embodiment of a microfluidic device 300 containing microfluidic circuit structure 308, which includes a channel 322 and sequestration pen 324, which has features and properties like any of the sequestration pens described herein for microfluidic devices 100, 175, 200, 400, 520 and any other microfluidic devices described herein.
  • the exemplary microfluidic devices of FIG. 3 include a microfluidic channel 322, having a width Wch, as described herein, and containing a flow 310 of first fluidic medium 302 and one or more sequestration pens 324 (only one illustrated in FIG.3).
  • the sequestration pens 324 each have a length L s , a connection region 336, and an isolation region 340, where the isolation region 340 contains a second fluidic medium 304.
  • the connection region 336 has a proximal opening 334, having a width Wcon1, which opens to the microfluidic channel 322, and a distal opening 338, having a width W con2 , which opens to the isolation region 340.
  • the width Wcon1 may or may not be the same as Wcon2, as described herein.
  • the walls of each sequestration pen 324 may be formed of microfluidic circuit material 316, which may further form the connection region walls 330.
  • a connection region wall 330 can correspond to a structure that is laterally positioned with respect to the proximal opening 334 and at least partially extends into the enclosed portion of the sequestration pen 324.
  • the length L con of the connection region 336 is at least partially defined by length L wall of the connection region wall 330.
  • the connection region wall 330 may have a length L wall , selected to be more than the penetration depth Dp of the secondary flow 344. Thus, the secondary flow 344 can be wholly contained within the connection region without extending into the isolation region 340.
  • the connection region wall 330 may define a hook region 352, which is a sub- region of the isolation region 340 of the sequestration pen 324.
  • connection region wall 330 extends into the inner cavity of the sequestration pen, the connection region wall 330 can act as a physical barrier to shield hook region 352 from secondary flow 344, with selection of the length of Lwall, contributing to the extent of the hook region. In some embodiments, the longer the length L wall of the connection region wall 330, the more sheltered the hook region 352.
  • the isolation region may have a shape and size of any type, and may be selected to regulate diffusion of nutrients, reagents, and/or media into the sequestration pen to reach to a far wall of the Attorney Docket No.01149-0025-00PCT sequestration pen, e.g., opposite the proximal opening of the connection region to the flow region (or microfluidic channel).
  • the size and shape of the isolation region may further be selected to regulate diffusion of waste products and/or secreted products of a biological micro- object out from the isolation region to the flow region via the proximal opening of the connection region of the sequestration pen.
  • the shape of the isolation region is not critical to the ability of the sequestration pen to isolate micro-objects from direct flow in the flow region.
  • the isolation region may have more than one opening fluidically connecting the isolation region with the flow region of the microfluidic device.
  • n- 1 openings can be valved. When the n-1 valved openings are closed, the isolation region has only one effective opening, and exchange of materials into/out of the isolation region occurs only by diffusion.
  • microfluidic devices having pens in which biological micro- objects can be placed, cultured, and/or monitored have been described, for example, in U.S. Patent No.9,857,333 (Chapman, et al.), U.S. Patent No. 10,010,882 (White, et al.), and U.S. Patent No.9,889,445 (Chapman, et al.), each of which is incorporated herein by reference in its entirety.
  • Microfluidic circuit element dimensions are examples of microfluidic circuit element dimensions.
  • Various dimensions and/or features of the sequestration pens and the microfluidic channels to which the sequestration pens open may be selected to limit introduction of contaminants or unwanted micro- objects into the isolation region of a sequestration pen from the flow region/microfluidic channel; limit the exchange of components in the fluidic medium from the channel or from the isolation region to substantially only diffusive exchange; facilitate the transfer of micro-objects into and/or out of the sequestration pens; and/or facilitate growth or expansion of the biological cells.
  • Microfluidic channels and sequestration pens for any of the embodiments described herein, may have any suitable combination of dimensions, may be selected by one of skill from the teachings of this disclosure.
  • a microfluidic channel may have a uniform cross sectional height along its length that is a substantially uniform cross sectional height, and may be any cross sectional height as described herein.
  • the substantially uniform cross sectional height of the channel the upper surface of which is defined by the inner surface of the cover and the lower surface of Attorney Docket No.01149-0025-00PCT which is defined by the inner surface of the base, may be substantially the same as the cross sectional height at any other point along the channel, e.g., having a cross sectional height that is no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% or less, different from the cross-sectional height of any other location within the channel.
  • the chamber(s), e.g., sequestration pen(s), of the microfluidic devices described herein, may be disposed substantially in a coplanar orientation relative to the microfluidic channel into which the chamber(s) open. That is, the enclosed volume of the chamber(s) is formed by an upper surface that is defined by the inner surface of the cover, a lower surface defined by the inner surface of the base, and walls defined by the microfluidic circuit material. Therefore, the lower surface of the chamber(s) may be coplanar to the lower surface of the microfluidic channel, e.g., substantially coplanar.
  • the upper surface of the chamber may be coplanar to the upper surface of the microfluidic channel, e.g., substantially coplanar.
  • the chamber(s) may have a cross-sectional height, which may have any values as described herein, that is the same as the channel, e.g., substantially the same, and the chamber(s) and microfluidic channel(s) within the microfluidic device may have a substantially uniform cross sectional height throughout the flow region of the microfluidic device, and may be substantially coplanar throughout the microfluidic device.
  • Coplanarity of the lower surfaces of the chamber(s) and the microfluidic channel(s) can offer distinct advantage with repositioning micro-objects within the microfluidic device using DEP or magnetic force.
  • the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) that is at least as large as the largest dimension of a micro-object (e.g., a biological cell, which may be a plant cell, such as a plant protoplast) for which the sequestration pen is intended.
  • a micro-object e.g., a biological cell, which may be a plant cell, such as a plant protoplast
  • the proximal opening has a width (e.g., Wcon or Wcon1) of about 20 microns, about 40 microns, about 50 microns, about 60 microns, about 75 microns, about 100 microns, about 150 microns, about 200 microns, or about 300 microns.
  • Wcon or Wcon1 a width of about 20 microns, about 40 microns, about 50 microns, about 60 microns, about 75 microns, about 100 microns, about 150 microns, about 200 microns, or about 300 microns.
  • the width (e.g., W con or W con1 ) of a proximal opening can be selected to be a value between any of the values listed above (e.g., about 20- 200 microns, about 20-150 microns, about 20-100 microns, about 20-75 microns, about 20-60 microns, about 50-300 microns, about 50-200 microns, about 50-150 microns, about 50-100 Attorney Docket No.01149-0025-00PCT microns, about 50-75 microns, about 75-150 microns, about 75-100 microns, about 100-300 microns, about 100-200 microns, or about 200-300 microns).
  • connection region of the sequestration pen may have a length (e.g., L con ) from the proximal opening to the distal opening to the isolation region of the sequestration pen that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25.
  • L con a length from the proximal opening to the distal opening to the isolation region of the sequestration pen that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25.
  • the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), and the connection region may have a length Lcon that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening.
  • the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), and the connection region may have a length L con that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening.
  • the microfluidic channel of a microfluidic device to which a sequestration pen opens may have specified size (e.g., width or height).
  • the height (e.g., H ch ) of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be within any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns.
  • the height (e.g., Hch) of the microfluidic channel (e.g., 122) can be selected to be between any of the values listed above.
  • the height (e.g., H ch ) of the microfluidic channel 122 can be selected to be any of these heights in regions of the microfluidic channel other than at a proximal opening of a sequestration pen.
  • the width (e.g., W ch ) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen can be within any of the following ranges: about 20- 500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30- 150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, Attorney Docket No.01149-0025-00PCT 40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns,
  • the width (e.g., Wch) of the microfluidic channel can be a value selected to be between any of the values listed above.
  • the width (e.g., W ch ) of the microfluidic channel can be selected to be in any of these widths in regions of the microfluidic channel other than at a proximal opening of a sequestration pen.
  • the width Wch of the microfluidic channel at the proximal opening to the connection region of the sequestration pen e.g., taken transverse to the direction of bulk flow of fluid through the channel
  • a cross-sectional area of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be about 500-50,000 square microns, 500-40,000 square microns, 500-30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1,000- 15,000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000- 5,000 square microns, 2,000-20,000 square microns, 2,000-15,000 square microns, 2,000- 10,000 square microns, 2,000-7,500 square microns, 2,000-6,000 square microns, 3,000- 20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000- 7,500 square microns, or 3,000 to 6,000 square microns,
  • the cross-sectional area of the microfluidic channel at the proximal opening can be selected to be between any of the values listed above.
  • the cross-sectional area of the microfluidic channel at regions of the microfluidic channel other than at the proximal opening can also be selected to be between any of the values listed above.
  • the cross-sectional area is selected to be a substantially uniform value for the entire length of the microfluidic channel.
  • the microfluidic chip is configured such that the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., W con or W con1 ) from about 20 microns to about 200 microns (e.g., about 50 Attorney Docket No.01149-0025-00PCT microns to about 150 microns), the connection region may have a length L con (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., Hch) at the proximal opening of about 30 microns to about 60 microns.
  • W con or W con1 e.g., W con or W con1
  • the connection region may have a length L con (e.g., 236 or 336) that is at least 1.0 times (e.g.,
  • the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), the connection region may have a length L con (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., Hch) at the proximal opening of about 30 microns to about 60 microns.
  • Wcon or Wcon1 width from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns)
  • the connection region may have a length L con (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least
  • the width (e.g., W con or W con1 ) of the proximal opening (e.g., 234 or 274), the length (e.g., L con ) of the connection region, and/or the width (e.g., Wch) of the microfluidic channel (e.g., 122 or 322) can be a value selected to be between any of the values listed above.
  • the width (Wcon or Wcon1) of the proximal opening of the connection region of a sequestration pen is less than the width (W ch ) of the microfluidic channel.
  • the width (Wcon or Wcon1) of the proximal opening is about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, or 30% of the width (W ch ) of the microfluidic channel. That is, the width (W ch ) of the microfluidic channel may be at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times or at least 10.0 times the width (Wcon or Wcon1) of the proximal opening of the connection region of the sequestration pen.
  • the size W C (e.g., cross-sectional width W ch , diameter, area, or the like) of the channel 122, 322, 618, 718 can be about one and a quarter (1.25), about one and a half (1.5), about two, about two and a half (2.5), about three (3), or more times the size W O (e.g., cross-sectional width W con , diameter, area, or the like) of a chamber opening, e.g., sequestration pen opening 234, 334, and the like.
  • a chamber opening e.g., sequestration pen opening 234, 334, and the like.
  • a selected chamber e.g., like sequestration pens 224, 226 of FIG. 2B
  • the rate of diffusion of a molecule is dependent on a number of factors, including (without limitation) temperature, viscosity of the medium, and the coefficient of diffusion D 0 of the molecule.
  • the D0 for an IgG antibody in aqueous solution at about 20°C is about 4.4x10 -7 cm 2 /sec, while the kinematic viscosity of cell culture medium is about 9x10 -4 m 2 /sec.
  • an antibody in cell culture medium at about 20°C can have a rate of diffusion of about Attorney Docket No.01149-0025-00PCT 0.5 microns/sec.
  • a time period for diffusion from a biological micro-object located within a sequestration pen such as 224, 226, 228, 324 into the channel 122, 322, 618, 718 can be about 10 minutes or less (e.g., about 9, 8, 7, 6, 5 minutes, or less).
  • the time period for diffusion can be manipulated by changing parameters that influence the rate of diffusion.
  • the temperature of the media can be increased (e.g., to a physiological temperature such as about 37°C) or decreased (e.g., to about 15°C, 10°C, or 4°C) thereby increasing or decreasing the rate of diffusion, respectively.
  • the concentrations of solutes in the medium can be increased or decreased as discussed herein to isolate a selected pen from solutes from other upstream pens.
  • the width (e.g., W ch ) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 50 to 500 microns, about 50 to 300 microns, about 50 to 200 microns, about 70 to 500 microns, about to 70-300 microns, about 70 to 250 microns, about 70 to 200 microns, about 70 to 150 microns, about 70 to 100 microns, about 80 to 500 microns, about 80 to 300 microns, about 80 to 250 microns, about 80 to 200 microns, about 80 to 150 microns, about 90 to 500 microns, about 90 to 300 microns, about 90 to 250 microns, about 90 to 200 microns, about 90 to 150 microns, about 100 to 500 microns, about 100 to 300 microns, about 100 to 250 microns, about 100 to 200 microns, or about 100 to 150 microns.
  • the width W ch of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 70 to 250 microns, about 80 to 200 microns, or about 90 to 150 microns.
  • the width W con of the opening of the chamber (e.g., sequestration pen) may be about 20 to 100 microns; about 30 to 90 microns; or about 20 to 60 microns.
  • Wch is about 70-250 microns and Wcon is about 20 to 100 microns; Wch is about 80 to 200 microns and W con is about 30 to 90 microns; W ch is about 90 to 150 microns, and W con is about 20 to 60 microns; or any combination of the widths of Wch and Wcon thereof.
  • the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen has a width (e.g., W con or W con1 ) that is 2.0 times or less (e.g., 2.0, 1.9, 1.8, 1.5, 1.3, 1.0, 0.8, 0.5, or 0.1 times) the height (e.g., H ch ) of the flow region/ microfluidic channel at the proximal opening, or has a value that lies within a range defined by any two of the foregoing values.
  • the width W con1 of a proximal opening (e.g., 234 or 334) of a connection region of a sequestration pen may be the same as a width Wcon2 of the distal opening (e.g., 238 or 338) to the isolation region thereof.
  • the width W con1 of the proximal opening may be different than a width W con2 of the distal opening, and Attorney Docket No.01149-0025-00PCT W con1 and/or W con2 may be selected from any of the values described for W con or W con1 .
  • the walls (including a connection region wall) that define the proximal opening and distal opening may be substantially parallel with respect to each other.
  • the length (e.g., Lcon) of the connection region can be about 1-600 microns, 5- 550 microns, 10-500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, about 100-150 microns, about 20-300 microns, about 20 -250 microns, about 20-200 microns, about 20-150 microns, about 20-100 microns, about 30-250 microns, about 30-200 microns, about 30- 150 microns, about 30-100 microns, about 30-80 microns, about 30-50 microns, about 45-250 microns, about 45-200 microns, about 45-100 microns, about 45- 80 microns, about 45-60 microns, about 60-200 microns, about 60
  • connection region wall of a sequestration pen may have a length (e.g., Lwall) that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, or at least 3.5 times the width (e.g., W con or W con1 ) of the proximal opening of the connection region of the sequestration pen.
  • width e.g., W con or W con1
  • connection region wall may have a length Lwall of about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-80 microns, or about 20-50 microns.
  • a sequestration pen may have a length Ls of about 40-600 microns, about 40- 500 microns, about 40-400 microns, about 40-300 microns, about 40-200 microns, about 40- 100 microns or about 40-80 microns.
  • a sequestration pen may have a length Ls selected to be between any of the values listed above.
  • a sequestration pen may have a specified height (e.g., H s ).
  • a sequestration pen has a height H s of about 20 microns to about 200 microns (e.g., about 20 microns to about 150 microns, about 20 microns to about 100 microns, about 20 microns to about 60 microns, about 30 microns to about 150 microns, about 30 microns to about 100 microns, about 30 microns to about 60 microns, about 40 Attorney Docket No.01149-0025-00PCT microns to about 150 microns, about 40 microns to about 100 microns, or about 40 microns to about 60 microns).
  • a sequestration pen can have a height Hs selected to be between any of the values listed above.
  • the height H con of a connection region at a proximal opening of a sequestration pen can be a height within any of the following heights: 20-100 microns, 20-90 microns, 20- 80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns.
  • the height Hcon of the connection region can be selected to be between any of the values listed above.
  • the height H con of the connection region is selected to be the same as the height H ch of the microfluidic channel at the proximal opening of the connection region.
  • the height Hs of the sequestration pen is typically selected to be the same as the height Hcon of a connection region and/or the height Hch of the microfluidic channel.
  • H s , H con , and H ch may be selected to be the same value of any of the values listed above for a selected microfluidic device.
  • the isolation region can be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. In other embodiments, the isolation region may contain more than 10, more than 50 or more than 100 micro-objects. Accordingly, the volume of an isolation region can be, for example, at least 1x10 4 , 1x10 5 , 5x10 5 , 8x10 5 , 1x10 6 , 2x10 6 , 4x10 6 , 6x10 6 , 1x10 7 , 3x10 7 , 5x10 7 1x10 8 , 5x10 8 , or 8x10 8 cubic microns, or more.
  • the isolation region can be configured to contain numbers of micro-objects and volumes selected to be between any of the values listed above (e.g., a volume between 1x10 5 cubic microns and 5x10 5 cubic microns, between 5x10 5 cubic microns and 1x10 6 cubic microns, between 1x10 6 cubic microns and 2x10 6 cubic microns, or between 2x10 6 cubic microns and 1x10 7 cubic microns).
  • a sequestration pen of a microfluidic device may have a specified volume.
  • the specified volume of the sequestration pen may be selected such that a single cell or a small number of cells (e.g., 2-10 or 2-5) can rapidly condition the medium and thereby attain favorable (or optimal) growth conditions.
  • the sequestration pen has a volume of about 5x10 5 , 6x10 5 , 8x10 5 , 1x10 6 , 2x10 6 , 4x10 6 , 8x10 6 , 1x10 7 , 3x10 7 , 5x10 7 , or about 8x10 7 cubic microns, or more.
  • the sequestration pen has a volume of about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2 nanoliters to about 10 nanoliters.
  • Attorney Docket No.01149-0025-00PCT The foregoing are examples only, and a sequestration pen can have a volume selected to be any value that is between any of the values listed above.
  • the flow of fluidic medium within the microfluidic channel e.g., 122 or 322
  • the maximum velocity (e.g., Vmax) may be set at around 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, or 25 microliters/sec.
  • Vmax maximum velocity
  • the foregoing are examples only, and the flow of fluidic medium within the microfluidic channel can have a maximum velocity (e.g., Vmax) selected to be a value between any of the values listed above.
  • the flow of fluidic medium within the microfluidic channel typically may be flowed at a rate less than the V max .
  • a fluidic medium may be flowed at about 0.1 microliters/sec to about 20 microliters/sec; about 0.1 microliters/sec to about 15 microliters/sec; about 0.1 microliters/sec to about 12 microliters/sec, about 0.1 microliters/sec to about 10 microliters/sec; about 0.1 microliter/sec to about 7 microliters/sec without exceeding the Vmax.
  • a flow rate of a fluidic medium may be about 0.1 microliters/sec; about 0.5 microliters/sec; about 1.0 microliters/sec; about 2.0 microliters/sec; about 3.0 microliters/sec; about 4.0 microliters/sec; about 5.0 microliters/sec; about 6.0 microliters/sec; about 7.0 microliters/sec; about 8.0 microliters/sec; about 9.0 microliters/sec; about 10.0 microliters/sec; about 11.0 microliters/sec; or any range defined by two of the foregoing values, e.g., 1-5 microliters/sec or 5-10 microliters/sec.
  • the flow rate of a fluidic medium in the microfluidic channel may be equal to or less than about 12 microliters/sec; about 10 microliters/sec; about 8 microliters/sec, or about 6 microliters/sec.
  • the microfluidic device has sequestration pens configured as in any of the embodiments discussed herein where the microfluidic device has about 5 to about 10 sequestration pens, about 10 to about 50 sequestration pens, about 25 to about 200 sequestration pens, about 100 to about 500 sequestration pens, about 200 to about 1000 sequestration pens, about 500 to about 1500 sequestration pens, about 1000 to about 2500 sequestration pens, about 2000 to about 5000 sequestration pens, about 3500 to about 7000 sequestration pens, about 5000 to about 10,000 sequestration pens, about 7,500 to about 15,000 sequestration pens, about 12,500 to about 20,000 sequestration pens, about 15,000 to about 25,000 sequestration pens, about 20,000 to about
  • At least one inner surface of the microfluidic device includes a coating material that provides a layer of organic and/or hydrophilic molecules suitable for maintenance, expansion and/or movement of biological micro-object(s) (i.e., the biological micro-object exhibits increased viability, greater expansion and/or greater portability within the microfluidic device).
  • the conditioned surface may reduce surface fouling, participate in providing a layer of hydration, and/or otherwise shield the biological micro-objects from contact with the non-organic materials of the microfluidic device interior.
  • substantially all the inner surfaces of the microfluidic device include the coating material.
  • the coated inner surface(s) may include the surface of a flow region (e.g., channel), chamber, or sequestration pen, or a combination thereof.
  • each of a plurality of sequestration pens has at least one inner surface coated with coating materials.
  • each of a plurality of flow regions or channels has at least one inner surface coated with coating materials.
  • each of a plurality of sequestration pens and each of a plurality of channels is coated with coating materials.
  • the coating may be applied before or after introduction of biological micro-object(s), or may be introduced concurrently with the biological micro- object(s).
  • the biological micro-object(s) may be imported into the microfluidic device in a fluidic medium that includes one or more coating agents.
  • the inner surface(s) of the microfluidic device may be treated or “primed” with a coating solution comprising a coating agent prior to introduction of the biological micro-object(s) into the microfluidic device.
  • a coating solution comprising a coating agent prior to introduction of the biological micro-object(s) into the microfluidic device.
  • Any convenient coating agent/coating solution can be used, including but not limited to: serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof.
  • BSA bovine serum albumin
  • the at least one inner surface may include a coating material that comprises a polymer.
  • the polymer may be non-covalently bound (e.g., it may be non-specifically adhered) to the at least one surface.
  • the polymer may have a variety of structural motifs, such as found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein.
  • a wide variety of alkylene ether containing Attorney Docket No.01149-0025-00PCT polymers may be suitable for use in the microfluidic devices described herein, including but not limited to Pluronic® polymers such as Pluronic® L44, L64, P85, and F127 (including F127NF).
  • the at least one inner surface includes covalently linked molecules that provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) within the microfluidic device, providing a conditioned surface for such cells.
  • the covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device, as described below.
  • the linking group is also covalently linked to a surface modifying moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/ expansion/ movement of biological micro- object(s).
  • the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) may include alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono- or polysaccharides (which may include but is not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyalcohols, including but not limited to polyvinyl alcohol; alkylene ethers, including but not limited to polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinyl phosphonic acid); amino groups (including derivatives thereof, such as, but not limited to alkylated amines, hydroxyalkylated amino group, guanidinium, and heterocylic groups containing an unaromatized nitrogen ring atom, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids including but not limited to prop
  • the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device may include non-polymeric moieties such as an alkyl moiety, amino acid moiety, alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety.
  • the covalently linked moiety may include polymeric moieties, which may include any of these moieties.
  • a microfluidic device may have a hydrophobic layer upon the inner surface of the base which includes a covalently linked alkyl moiety.
  • the covalently linked alkyl moiety may comprise carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be an unbranched alkyl moiety.
  • the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group can be fluorinated or perfluorinated).
  • the alkyl group may include a first segment, which may include a perfluoroalkyl group, joined to a second segment, which may include a non-substituted alkyl group, where the first and second segments may be joined directly or indirectly (e.g., by means of an ether linkage).
  • the first segment of the alkyl group may be located distal to the linking group, and the second segment of the alkyl group may be located proximal to the linking group.
  • the covalently linked moiety may include at least one amino acid, which may include more than one type of amino acid.
  • the covalently linked moiety may include a peptide or a protein.
  • the covalently linked moiety may include an amino acid which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof.
  • the covalently linked moiety may further include a streptavidin or biotin moiety.
  • a modified biological moiety such as, for example, a biotinylated protein or peptide may be introduced to the inner surface of a microfluidic device bearing covalently linked streptavidin, and couple via the covalently linked streptavidin to the surface, thereby providing a modified surface presenting the protein or peptide.
  • the covalently linked moiety may include at least one alkylene oxide moiety and may include any alkylene oxide polymer as described above.
  • One useful class of alkylene ether containing polymers is polyethylene glycol (PEG Mw ⁇ 100,000Da) or alternatively polyethylene oxide (PEO, Mw>100,000).
  • PEG polyethylene glycol
  • PEO polyethylene oxide
  • a PEG may have an M w of about 1000Da, 5000Da, 10,000Da or 20,000Da.
  • the PEG polymer may further be substituted with a hydrophilic or charged moiety, such as but not limited to an alcohol functionality or a carboxylic acid moiety.
  • the covalently linked moiety may include one or more saccharides.
  • the covalently linked saccharides may be mono-, di-, or polysaccharides.
  • the covalently linked saccharides may be modified to introduce a reactive pairing moiety which permits coupling or elaboration for attachment to the surface.
  • One exemplary covalently linked moiety may include Attorney Docket No.01149-0025-00PCT a dextran polysaccharide, which may be coupled indirectly to a surface via an unbranched linker.
  • the coating material providing a conditioned surface may comprise only one kind of covalently linked moiety or may include more than one different kind of covalently linked moiety.
  • a polyethylene glycol conditioned surface may have covalently linked alkylene oxide moieties having a specified number of alkylene oxide units which are all the same, e.g., having the same linking group and covalent attachment to the surface, the same overall length, and the same number of alkylene oxide units.
  • the coating material may have more than one kind of covalently linked moiety attached to the surface.
  • the coating material may include the molecules having covalently linked alkylene oxide moieties having a first specified number of alkylene oxide units and may further include a further set of molecules having bulky moieties such as a protein or peptide connected to a covalently attached alkylene oxide linking moiety having a greater number of alkylene oxide units.
  • the conditioned surface having a mixture of first molecules having a chemical structure having a first specified number of alkylene oxide units and second molecules including peptide or protein moieties, which may be coupled via a biotin/streptavidin binding pair to the covalently attached alkylene linking moiety may have a ratio of first molecules: second molecules of about 99:1; about 90:10; about 75:25; about 50:50; about 30:70; about 20:80; about 10:90; or any ratio selected to be between these values.
  • the first set of molecules having different, less sterically demanding termini and fewer backbone atoms can help to functionalize the entire substrate surface and thereby prevent undesired adhesion or contact with the silicon/silicon oxide, hafnium oxide or alumina making up the substrate itself.
  • the selection of the ratio of mixture of first molecules to second molecules may also modulate the surface modification introduced by the second molecules bearing peptide or protein moieties.
  • Conditioned surface properties Various factors can alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid phase deposition, spin coating, flooding, and electrostatic coating).
  • the conditioned surface may have a thickness of about 1nm to about 10nm.
  • the covalently linked moieties of the conditioned surface may form a monolayer when covalently linked to the surface of the microfluidic device (which may include an electrode activation substrate having dielectrophoresis (DEP) or electrowetting (EW) electrodes) and may have a thickness of less Attorney Docket No.01149-0025-00PCT than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). These values are in contrast to that of a surface prepared by spin coating, for example, which may typically have a thickness of about 30nm.
  • DEP dielectrophoresis
  • EW electrowetting
  • the conditioned surface does not require a perfectly formed monolayer to be suitably functional for operation within a DEP-configured microfluidic device.
  • the conditioned surface formed by the covalently linked moieties may have a thickness of about 10 nm to about 50 nm.
  • the covalently linked coating material may be formed by reaction of a molecule which already contains the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device, and may have a structure of Formula I, as shown below.
  • the covalently linked coating material may be formed in a two-part sequence, having a structure of Formula II, by coupling the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance and/or expansion of biological micro-object(s) to a surface modifying ligand that itself has been covalently linked to the surface.
  • the surface may be formed in a two- part or three-part sequence, including a streptavidin/biotin binding pair, to introduce a protein, peptide, or mixed modified surface. or Formula I Formula II [0189]
  • the coating material may be linked covalently to oxides of the surface of a DEP-configured or EW- configured substrate.
  • the coating material may be attached to the oxides via a linking group (“LG”), which may be a siloxy or phosphonate ester group formed from the reaction of a siloxane or phosphonic acid group with the oxides.
  • LG linking group
  • the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device can be any of the moieties described herein.
  • the linking group LG may be directly or indirectly connected to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device.
  • linker L When the Attorney Docket No.01149-0025-00PCT linking group LG is directly connected to the moiety, optional linker (“L”) is not present and n is 0. When the linking group LG is indirectly connected to the moiety, linker L is present and n is 1.
  • the linker L may have a linear portion where a backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and/or phosphorus atoms, subject to chemical bonding limitations as is known in the art. It may be interrupted with any combination of one or more moieties, which may be chosen from ether, amino, carbonyl, amido, and/or phosphonate groups, arylene, heteroarylene, or heterocyclic groups.
  • the coupling group CG represents the resultant group from reaction of a reactive moiety Rx and a reactive pairing moiety R px (i.e., a moiety configured to react with the reactive moiety R x ).
  • CG may be a carboxamidyl group, a triazolylene group, substituted triazolylene group, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or alkenyl group, or any other suitable group that may be formed upon reaction of a reactive moiety with its respective reactive pairing moiety.
  • CG may further represent a streptavidin/biotin binding pair.
  • control and monitoring equipment of the system can comprise a motive module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of a microfluidic device.
  • the motive technology(ies) may include, for example, dielectrophoresis (DEP), electrowetting (EW), and/or other motive technologies.
  • DEP dielectrophoresis
  • EW electrowetting
  • the microfluidic device can have a variety of motive configurations, depending upon the type of object being moved and other considerations.
  • the support structure 104 and/or cover 110 of the microfluidic device 100 can comprise DEP electrode activation substrates for selectively inducing motive forces on micro-objects in the fluidic medium 180 in the microfluidic circuit 120 and thereby select, capture, and/or move individual micro-objects or groups of micro-objects.
  • motive forces are applied across the fluidic medium 180 (e.g., in the flow path and/or in the sequestration pens) via one or more electrodes Attorney Docket No.01149-0025-00PCT (not shown) to manipulate, transport, separate and sort micro-objects located therein.
  • motive forces are applied to one or more portions of microfluidic circuit 120 in order to transfer a single micro-object from the flow path 106 into a desired microfluidic sequestration pen.
  • motive forces are used to prevent a micro-object within a sequestration pen from being displaced therefrom.
  • motive forces are used to selectively remove a micro-object from a sequestration pen that was previously collected in accordance with the embodiments of the current disclosure.
  • the microfluidic device is configured as an optically-actuated electrokinetic device, such as in optoelectronic tweezer (OET) and/or optoelectrowetting (OEW) configured device.
  • OET optoelectronic tweezer
  • OEW optoelectrowetting
  • suitable OET configured devices e.g., containing optically actuated dielectrophoresis electrode activation substrates
  • suitable OET configured devices can include those illustrated in U.S. Patent No. RE 44,711 (Wu, et al.) (originally issued as U.S. Patent No. 7,612,355), U.S. Patent No. 7,956,339 (Ohta, et al.), U.S. Patent No. 9,908,115 (Hobbs et al.), and U.S. Patent No.9,403,172 (Short et al), each of which is incorporated herein by reference in its entirety.
  • suitable OEW configured devices can include those illustrated in U.S. Patent No.
  • optically-actuated electrokinetic devices that include combined OET/OEW configured devices can include those illustrated in U.S. Patent Application Publication No. 2015/0306598 (Khandros, et al.), U.S. Patent Application Publication No 2015/0306599 (Khandros, et al.), and U.S. Patent Application Publication No.2017/0173580 (Lowe, et al.), each of which is incorporated herein by reference in its entirety.
  • FIGS. 1-5B may illustrate portions of microfluidic devices while not depicting other portions. Further, FIGS. 1-5B may be part of, and implemented as, one or more microfluidic systems.
  • FIGS. 4A and 4B show a side cross- sectional view and a top cross-sectional view, respectively, of a portion of an enclosure 102 of the microfluidic device 400 having a region/chamber 402, which may be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, a sequestration pen (which may be like any sequestration pen described herein), a flow region, or a flow channel.
  • microfluidic device 400 may be similar to microfluidic devices 100, 175, 200, 300, 520 or any other microfluidic device as described herein.
  • the microfluidic device 400 may include other fluidic circuit elements and may be part of a system including control Attorney Docket No.01149-0025-00PCT and monitoring equipment 152, described above, having one or more of the media module 160, motive module 162, imaging module 164, optional tilting module 166, and other modules 168.
  • Microfluidic devices 175, 200, 300, 520 and any other microfluidic devices described herein may similarly have any of the features described in detail for FIGS.1A-1B and 4A-4B. [0195] As shown in the example of FIG.
  • the microfluidic device 400 includes a support structure 104 having a bottom electrode 404 and an electrode activation substrate 406 overlying the bottom electrode 404, and a cover 110 having a top electrode 410, with the top electrode 410 spaced apart from the bottom electrode 404.
  • the top electrode 410 and the electrode activation substrate 406 define opposing surfaces of the region/chamber 402.
  • a fluidic medium 180 contained in the region/chamber 402 thus provides a resistive connection between the top electrode 410 and the electrode activation substrate 406.
  • a power source 412 configured to be connected to the bottom electrode 404 and the top electrode 410 and create a biasing voltage between the electrodes, as required for the generation of DEP forces in the region/chamber 402, is also shown.
  • the power source 412 can be, for example, an alternating current (AC) power source.
  • AC alternating current
  • the microfluidic device 200 illustrated in FIGS. 4A and 4B can have an optically-actuated DEP electrode activation substrate. Accordingly, changing patterns of light 418 from the light source 416, which may be controlled by the motive module 162, can selectively activate and deactivate changing patterns of DEP electrodes at regions 414 of the inner surface 408 of the electrode activation substrate 406.
  • DEP electrode regions regions 414 of a microfluidic device having a DEP electrode activation substrate are referred to as “DEP electrode regions.”
  • a light pattern 418 directed onto the inner surface 408 of the electrode activation substrate 406 can illuminate select DEP electrode regions 414a (shown in white) in a pattern, such as a square.
  • the non-illuminated DEP electrode regions 414 cross-hatched are hereinafter referred to as “dark” DEP electrode regions 414.
  • the relative electrical impedance through the DEP electrode activation substrate 406 (i.e., from the bottom electrode 404 up to the inner surface 408 of the electrode activation substrate 406 which interfaces with the fluidic medium 180 in the flow region 106) is greater than the relative electrical impedance through the fluidic medium 180 in the region/chamber 402 (i.e., from the inner surface 408 of the electrode activation substrate 406 to the top electrode 410 of the cover 110) at each dark DEP electrode region 414.
  • An illuminated DEP electrode region 414a exhibits a reduced relative impedance through the electrode activation substrate 406 that is less than the relative impedance through the fluidic medium 180 in the region/chamber 402 at each illuminated DEP electrode region 414a.
  • Whether the DEP forces attract or repel nearby micro- objects can depend on such parameters as the frequency of the power source 412 and the dielectric properties of the fluidic medium 180 and/or micro-objects (not shown).
  • negative DEP forces may be produced. Negative DEP forces may repel the micro-objects away from the location of the induced non-uniform electrical field.
  • a microfluidic device incorporating DEP technology may generate negative DEP forces.
  • the electrode activation substrate 406 can comprise or consist of a photoconductive material.
  • the inner surface 408 of the electrode activation substrate 406 can be featureless.
  • the electrode activation substrate 406 can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H).
  • the a-Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100 * the number of hydrogen atoms / the total number of hydrogen and silicon atoms).
  • the layer of a-Si:H can have a thickness of about 500 nm to about 2.0 ⁇ m.
  • the DEP electrode regions 414 can be created anywhere and in any pattern on the inner surface 408 of the electrode activation substrate 406, in accordance with the light pattern 418. The number and pattern of the DEP electrode regions 214 thus need not be fixed, but can correspond to the light pattern 418. Examples of microfluidic devices having a DEP configuration comprising a photoconductive layer such as discussed above have been described, for example, in U.S.
  • the electrode activation substrate 406 can comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers that form semiconductor integrated circuits, such as is known in semiconductor fields.
  • the electrode activation substrate 406 can comprise a plurality of phototransistors, including, for example, lateral bipolar phototransistors, with each phototransistor corresponding to a DEP electrode region 414.
  • the electrode activation substrate 406 can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, with each such electrode corresponding to a DEP electrode region 414.
  • the electrode activation substrate 406 can include a pattern of such phototransistors or phototransistor-controlled electrodes.
  • the pattern for example, can be an array of substantially square phototransistors or phototransistor- controlled electrodes arranged in rows and columns.
  • the pattern can be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes that form a hexagonal lattice.
  • electric circuit elements can form electrical connections between the DEP electrode regions 414 at the inner surface 408 of the electrode activation substrate 406 and the bottom electrode 404, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 418, as described above.
  • electrical connections i.e., phototransistors or electrodes
  • Examples of microfluidic devices having electrode activation substrates that comprise phototransistors have been described, for example, in U.S. Patent No.7,956,339 (Ohta et al.) and U.S. Patent No.9,908,115 (Hobbs et al.), the entire contents of each of which are incorporated herein by reference.
  • the top electrode 410 is part of a first wall (or cover 110) of the enclosure 402
  • the electrode activation substrate 406 and bottom electrode 404 are part of a second wall (or support structure 104) of the enclosure 102.
  • the region/chamber 402 can be between the first wall and the second wall.
  • the electrode 410 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 406 and/or the electrode Attorney Docket No.01149-0025-00PCT 410 are part of the first wall (or cover 110).
  • the light source 416 can alternatively be used to illuminate the enclosure 102 from below.
  • a micro-object in the fluidic medium 180 in the region/chamber 402 by projecting a light pattern 418 into the microfluidic device 400 to activate a first set of one or more DEP electrodes at DEP electrode regions 414a of the inner surface 408 of the electrode activation substrate 406 in a pattern (e.g., square pattern 420) that surrounds and captures the micro-object.
  • the motive module 162 can then move the in situ-generated captured micro-object by moving the light pattern 418 relative to the microfluidic device 400 to activate a second set of one or more DEP electrodes at DEP electrode regions 414.
  • the microfluidic device 400 can be moved relative to the light pattern 418.
  • the microfluidic device 400 may be a DEP configured device that does not rely upon light activation of DEP electrodes at the inner surface 408 of the electrode activation substrate 406.
  • the electrode activation substrate 406 can comprise selectively addressable and energizable electrodes positioned opposite to a surface including at least one electrode (e.g., cover 110).
  • Switches e.g., transistor switches in a semiconductor substrate
  • the DEP force can attract or repel a nearby micro-object.
  • a set of DEP electrodes e.g., at a set of DEP electrodes regions 414 that forms a square pattern 420
  • one or more micro-objects in region/chamber 402 can be selected and moved within the region/chamber 402.
  • the motive module 162 in FIG.1A can control such switches and thus activate and deactivate individual ones of the DEP electrodes to select, and move particular micro-objects (not shown) around the region/chamber 402.
  • Microfluidic devices having a DEP electrode activation substrate that includes selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Patent No. 6,294,063 (Becker, et al.) and U.S. Patent No.6,942,776 (Medoro), each of which is incorporated herein by reference in its entirety.
  • a power source 412 can be used to provide a potential (e.g., an AC voltage potential) that powers the electrical circuits of the microfluidic device 400.
  • the power source 412 can be the same as, or a component of, the power source 192 referenced in Fig. 1A.
  • Power source 412 can be configured to provide an AC voltage and/or current to the top electrode 410 and the bottom electrode 404.
  • the power source 412 can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate net DEP forces (or electrowetting forces) strong enough to select and move individual micro-objects (not shown) in the region/chamber 402, as discussed above, and/or to change the wetting properties of the inner surface 408 of the support structure 104 in the region/chamber 202, as also discussed above.
  • Such frequency ranges and average or peak power ranges are known in the art. See, e.g., U.S. Patent No.6,958,132 (Chiou, et al.), US Patent No. RE44,711 (Wu, et al.) (originally issued as US Patent No.
  • Localized fluidic flow which may be operated within the microfluidic channel, within a sequestration pen, or within another kind of chamber (e.g., a reservoir) can also be used to move selected micro-objects. Localized fluidic flow can be used to move selected micro-objects out of the flow region into a non-flow region such as a sequestration pen or the reverse, from a non-flow region into a flow region.
  • the localized flow can be actuated by deforming a deformable wall of the microfluidic device, as described in U.S. Patent No.10,058,865 (Breinlinger, et al.), which is incorporated herein by reference in its entirety.
  • Gravity may be used to move micro-objects within the microfluidic channel, into a sequestration pen, and/or out of a sequestration pen or other chamber, as described in U.S. Patent No. 9,744,533 (Breinlinger, et al.), which is incorporated herein by reference in its entirety.
  • Use of gravity e.g., by tilting the microfluidic device and/or the support to which the microfluidic device is attached
  • Magnetic forces may be employed Attorney Docket No.01149-0025-00PCT to move micro-objects including paramagnetic materials, which can include magnetic micro- objects attached to or associated with a biological micro-object.
  • centripetal forces may be used to move micro-objects within the microfluidic channel, as well as into or out of sequestration pens or other chambers in the microfluidic device.
  • laser-generated dislodging forces may be used to export micro-objects or assist in exporting micro-objects from a sequestration pen or any other chamber in the microfluidic device, as described in International Patent Publication No.
  • DEP forces are combined with other forces, such as fluidic flow (e.g., bulk fluidic flow in a channel or localized fluidic flow actuated by deformation of a deformable surface of the microfluidic device, laser generated dislodging forces, and/or gravitational force), so as to manipulate, transport, separate and sort micro- objects and/or droplets within the microfluidic circuit 120.
  • fluidic flow e.g., bulk fluidic flow in a channel or localized fluidic flow actuated by deformation of a deformable surface of the microfluidic device, laser generated dislodging forces, and/or gravitational force
  • the DEP forces can be applied prior to the other forces. In other embodiments, the DEP forces can be applied after the other forces.
  • the DEP forces can be applied in an alternating manner with the other forces.
  • repositioning of micro-objects may not generally rely upon gravity or hydrodynamic forces to position or trap micro-objects at a selected position.
  • Gravity may be chosen as one form of repositioning force, but the ability to reposition of micro-objects within the microfluidic device does not rely solely upon the use of gravity.
  • fluid flow in the microfluidic channels may be used to introduce micro-objects into the microfluidic channels (e.g., flow region), such regional flow is not relied upon to pen or unpen micro-objects, while localized flow (e.g., force derived from actuating a deformable surface) may, in some embodiments, be selected from amongst the other types of repositioning forces described herein to pen or unpen micro-objects or to export them from the microfluidic device.
  • the electrical power source 192 can, for example, comprise one or more alternating current (AC) and/or direct current (DC) voltage or current sources.
  • System 150 can further include a media source 178.
  • the media source 178 e.g., a container, reservoir, or the like
  • the media source 178 can comprise multiple sections or containers, each for holding a different fluidic medium 180.
  • the media source 178 can be a device that is outside of and separate from the microfluidic device 100, as illustrated in FIG. 1A.
  • the media source 178 can be located in whole or in part inside the enclosure 102 of the microfluidic device 100.
  • the media source 178 can comprise reservoirs that are part of the microfluidic device 100.
  • FIG.1A also illustrates simplified block diagram depictions of examples of control and monitoring equipment 152 that constitute part of system 150 and can be utilized in conjunction with a microfluidic device 100.
  • control and monitoring equipment 152 can include a master controller 154 comprising a media module 160 for controlling the media source 178, a motive module 162 for controlling movement and/or selection of micro-objects (not shown) and/or medium (e.g., droplets of medium) in the microfluidic circuit 120, an imaging module 164 for controlling an imaging device (e.g., a camera, microscope, light source or any combination thereof) for capturing images (e.g., digital images), and an optional tilting module 166 for controlling the tilting of the microfluidic device 100.
  • an imaging module 164 for controlling an imaging device (e.g., a camera, microscope, light source or any combination thereof) for capturing images (e.g., digital images)
  • an optional tilting module 166 for controlling the tilting of the microfluidic device 100.
  • the control equipment 152 can also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the monitoring equipment 152 can further include a display device 170 and an input/output device 172.
  • the master controller 154 can comprise a control module 156 and a digital memory 158.
  • the control module 156 can comprise, for example, a digital processor configured to operate in accordance with machine executable instructions (e.g., software, firmware, source code, or the like) stored as non-transitory data or signals in the memory 158. Alternatively, or in addition, the control module 156 can comprise hardwired digital circuitry and/or analog circuitry.
  • the media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 can be similarly configured.
  • functions, processes acts, actions, or steps of a process discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus can be performed by any one or more of the master controller 154, media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 configured Attorney Docket No.01149-0025-00PCT as discussed above.
  • the media module 160 controls the media source 178.
  • the media module 160 can control the media source 178 to input a selected fluidic medium 180 into the enclosure 102 (e.g., through an inlet port 107).
  • the media module 160 can also control removal of media from the enclosure 102 (e.g., through an outlet port (not shown)).
  • One or more media can thus be selectively input into and removed from the microfluidic circuit 120.
  • the media module 160 can also control the flow of fluidic medium 180 in the flow path 106 inside the microfluidic circuit 120.
  • the media module 160 may also provide conditioning gaseous conditions to the media source 178, for example, providing an environment containing 5% CO2 (or higher).
  • the media module 160 may also control the temperature of an enclosure of the media source, for example, to provide feeder cells in the media source with proper temperature control.
  • Motive module The motive module 162 can be configured to control selection and movement of micro-objects (not shown) in the microfluidic circuit 120.
  • the enclosure 102 of the microfluidic device 100 can comprise one or more electrokinetic mechanisms including a dielectrophoresis (DEP) electrode activation substrate, optoelectronic tweezers (OET) electrode activation substrate, electrowetting (EW) electrode activation substrate, and/or an opto-electrowetting (OEW) electrode activation substrate, where the motive module 162 can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects and/or droplets in the flow path 106 and/or within sequestration pens 124, 126, 128, and 130.
  • DEP dielectrophoresis
  • OET optoelectronic tweezers
  • EW electrowetting
  • OEW opto-electrowetting
  • the electrokinetic mechanism(s) may be any suitable single or combined mechanism as described within the paragraphs describing motive technologies for use within the microfluidic device.
  • a DEP configured device may include one or more electrodes that apply a non-uniform electric field in the microfluidic circuit 120 sufficient to exert a dielectrophoretic force on micro-objects in the microfluidic circuit 120.
  • An OET configured device may include photo-activatable electrodes to provide selective control of movement of micro-objects in the microfluidic circuit 120 via light-induced dielectrophoresis.
  • the imaging module 164 can control the imaging device. For example, the imaging module 164 can receive and process image data from the imaging device.
  • Image data from the imaging device can comprise any type of information captured by the imaging Attorney Docket No.01149-0025-00PCT device (e.g., the presence or absence of micro-objects, droplets of medium, accumulation of label, such as fluorescent label, etc.).
  • the imaging module 164 can further calculate the position of objects (e.g., micro-objects, droplets of medium) and/or the rate of motion of such objects within the microfluidic device 100.
  • the imaging device (part of imaging module 164, discussed below) can comprise a device, such as a digital camera, for capturing images inside microfluidic circuit 120.
  • the imaging device further comprises a detector having a fast frame rate and/or high sensitivity (e.g., for low light applications).
  • the imaging device can also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein).
  • the emitted light beams may be in the visible spectrum and may, e.g., include fluorescent emissions.
  • the reflected light beams may include reflected emissions originating from an LED or a wide spectrum lamp, such as a mercury lamp (e.g., a high-pressure mercury lamp) or a Xenon arc lamp.
  • the imaging device may further include a microscope (or an optical train), which may or may not include an eyepiece.
  • System 150 may further comprise a support structure 190 configured to support and/or hold the enclosure 102 comprising the microfluidic circuit 120.
  • the optional tilting module 166 can be configured to activate the support structure 190 to rotate the microfluidic device 100 about one or more axes of rotation.
  • the optional tilting module 166 can be configured to support and/or hold the microfluidic device 100 in a level orientation (i.e., at 0° relative to x- and y-axes), a vertical orientation (i.e., at 90° relative to the x-axis and/or the y-axis), or any orientation therebetween.
  • support structure 190 can optionally be used to tilt the microfluidic device 100 (e.g., as controlled by optional tilting module 166) to 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relative to the x-axis or any degree therebetween.
  • the support structure 190 can hold the microfluidic device 100 at a fixed angle of 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, or 10° Attorney Docket No.01149-0025-00PCT relative to the x-axis (horizontal), so long as DEP is an effective force to move micro-objects out of the sequestration pens into the microfluidic channel.
  • the microfluidic device 100 may be disposed in an orientation such that the inner surface of the base of the flow path 106 is positioned at an angle above or below the inner surface of the base of the one or more sequestration pens opening laterally to the flow path.
  • the term “above” as used herein denotes that the flow path 106 is positioned higher than the one or more sequestration pens on a vertical axis defined by the force of gravity (i.e., an object in a sequestration pen above a flow path 106 would have a higher gravitational potential energy than an object in the flow path), and inversely, for positioning of the flow path 106 below one or more sequestration pens.
  • the support structure 190 may be held at a fixed angle of less than about 5°, about 4°, about 3° or less than about 2 ° relative to the x-axis (horizontal), thereby placing the sequestration pens at a lower potential energy relative to the flow path.
  • the device when long term culturing (e.g., for more than about 2, 3, 4, 5, 6, 7 or more days) is performed within the microfluidic device, the device may be supported on a culturing support and may be tilted at a greater angle of about 10°, 15°, 20°, 25°, 30°, or any angle therebetween to retain biological micro-objects within the sequestration pens during the long-term culturing period.
  • the microfluidic device containing the cultured biological micro-objects may be returned to the support 190 within system 150, where the angle of tilting is decreased to values as described above, affording the use of DEP to move the biological micro-objects out of the sequestration pens.
  • the system 150 can include a structure (also referred to as a “nest”) 500 configured to hold a microfluidic device 520, which may be like microfluidic device 100, 200, or any other microfluidic device described herein.
  • a structure also referred to as a “nest” 500 configured to hold a microfluidic device 520, which may be like microfluidic device 100, 200, or any other microfluidic device described herein.
  • the nest 500 can include a socket 502 capable of interfacing with the microfluidic device 520 (e.g., an optically actuated electrokinetic device 100, 200, etc.) and providing electrical connections from power source 192 to microfluidic device 520.
  • the nest 500 can further include an integrated electrical signal generation subsystem 504.
  • the electrical signal generation Attorney Docket No.01149-0025-00PCT subsystem 504 can be configured to supply a biasing voltage to socket 502 such that the biasing voltage is applied across a pair of electrodes in the microfluidic device 520 when it is being held by socket 502.
  • the electrical signal generation subsystem 504 can be part of power source 192.
  • the nest 500 can include a printed circuit board assembly (PCBA) 522.
  • the electrical signal generation subsystem 504 can be mounted on and electrically integrated into the PCBA 522.
  • the exemplary support includes socket 502 mounted on PCBA 522, as well.
  • the nest 500 can comprise an electrical signal generation subsystem 504 configured to measure the amplified voltage at the microfluidic device 520 and then adjust its own output voltage as needed such that the measured voltage at the microfluidic device 520 is the desired value.
  • the waveform amplification circuit can have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, resulting in a signal of up to 13 Vpp at the microfluidic device 520.
  • the nest 500 further comprises a controller 508, such as a microprocessor used to sense and/or control the electrical signal generation subsystem 504.
  • the controller 508 may be used to perform functions and analysis or may communicate with an external master controller 154 (shown in FIG. 1A) to perform functions and analysis. In the embodiment illustrated in FIG. 5A the controller 508 communicates with the master controller 154 (of FIG.1A) through an interface (e.g., a plug or connector).
  • the support structure 500 e.g., nest
  • the thermal control subsystem 506 can be configured to regulate the temperature of microfluidic device 520 held by the support structure 500.
  • the thermal control subsystem 506 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown).
  • the support structure 500 comprises an inlet 516 and an outlet 518 to receive cooled fluid from an external reservoir (not shown) of the cooling unit, introduce the cooled fluid into the fluidic Attorney Docket No.01149-0025-00PCT path 514 and through the cooling block, and then return the cooled fluid to the external reservoir.
  • the Peltier thermoelectric device, the cooling unit, and/or the fluidic path 514 can be mounted on a casing 512 of the support structure 500.
  • the thermal control subsystem 506 is configured to regulate the temperature of the Peltier thermoelectric device so as to achieve a target temperature for the microfluidic device 520. Temperature regulation of the Peltier thermoelectric device can be achieved, for example, by a thermoelectric power supply, such as a PololuTM thermoelectric power supply (Pololu Robotics and Electronics Corp.).
  • the thermal control subsystem 506 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit.
  • the nest 500 can include a serial port 524 which allows the microprocessor of the controller 508 to communicate with an external master controller 154 via the interface.
  • the microprocessor of the controller 508 can communicate (e.g., via a Plink tool (not shown)) with the electrical signal generation subsystem 504 and thermal control subsystem 506.
  • the electrical signal generation subsystem 504 and the thermal control subsystem 506 can communicate with the external master controller 154.
  • the master controller 154 can, among other things, assist the electrical signal generation subsystem 504 by performing scaling calculations for output voltage adjustments.
  • GUI Graphical User Interface
  • a Graphical User Interface (GUI) (not shown) provided via a display device 170 coupled to the external master controller 154, can be configured to plot temperature and waveform data obtained from the thermal control subsystem 506 and the electrical signal generation subsystem 504, respectively.
  • FIG. 5B is a schematic of an optical sub-system 550 having an optical apparatus 510 for imaging and manipulating micro-objects in a microfluidic device 520, which can be any microfluidic device described herein.
  • the optical apparatus 510 can be configured to perform imaging, analysis and manipulation of one or more micro-objects within the enclosure of the microfluidic device 520.
  • the optical apparatus 510 may have a first light source 552, a second light source 554, and a third light source 556.
  • the first light source 552 can transmit light to a structured light modulator 560, which can include a digital mirror device (DMD) or a microshutter array system (MSA), either of which can be configured to receive light from the first light source 552 and selectively transmit a subset of the received light into the optical Attorney Docket No.01149-0025-00PCT apparatus 510.
  • the structured light modulator 560 can include a device that produces its own light (and thus dispenses with the need for a light source 552), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD).
  • OLED organic light emitting diode display
  • LCOS liquid crystal on silicon
  • FLCOS ferroelectric liquid crystal on silicon device
  • LCD transmissive liquid crystal display
  • the structured light modulator 560 can be, for example, a projector. Thus, the structured light modulator 560 can be capable of emitting both structured and unstructured light. In certain embodiments, an imaging module and/or motive module of the system can control the structured light modulator 560. [0229] In embodiments when the structured light modulator 560 includes a mirror, the modulator can have a plurality of mirrors. Each mirror of the plurality of mirrors can have a size of about 5 microns x 5 microns to about 10 microns x10 microns, or any values therebetween.
  • the structured light modulator 560 can include an array of mirrors (or pixels) that is 2000 x 1000, 2580 x 1600, 3000 x 2000, or any values therebetween.
  • the structured light modulator 560 can transmit the selected subset of light to a first dichroic beam splitter 558, which can reflect this light to a first tube lens 562.
  • the first tube lens 562 can have a large clear aperture, for example, a diameter larger than about 40 mm to about 50 mm, or more, providing a large field of view.
  • the first tube lens 562 can have an aperture that is large enough to capture all (or substantially all) of the light beams emanating from the structured light modulator 560.
  • the structured light 515 having a wavelength of about 400 nm to about 710 nm may alternatively or in addition, provide fluorescent excitation illumination to the microfluidic device.
  • the second light source 554 may provide unstructured brightfield illumination.
  • the brightfield illumination light 525 may have any suitable wavelength, and in some embodiments, may have a wavelength of about 400 nm to about 760 nm.
  • the second light source 554 can transmit light to a second dichroic beam splitter 564 (which also may receive illumination light 535 from the third light source 556), and the second light, brightfield illumination light 525, may be transmitted therefrom to the first dichroic beam splitter 558.
  • the second light, brightfield illumination light 525 may then be transmitted from the first dichroic beam splitter 558 to the first tube lens 562.
  • the third light source 556 can transmit light through a matched pair relay lens (not shown) to a mirror 566.
  • the third illumination light 535 may therefrom be reflected to the second dichroic beam splitter 5338 and be transmitted therefrom to the first Attorney Docket No.01149-0025-00PCT beam splitter 5338, and onward to the first tube lens 5381.
  • the third illumination light 535 may be a laser and may have any suitable wavelength. In some embodiments, the laser illumination 535 may have a wavelength of about 350 nm to about 900 nm.
  • the laser illumination 535 may be configured to heat portions of one or more sequestration pens within the microfluidic device.
  • the laser illumination 535 may be configured to heat fluidic medium, a micro-object, a wall or a portion of a wall of a sequestration pen, a metal target disposed within a microfluidic channel or sequestration pen of the microfluidic channel, or a photoreversible physical barrier within the microfluidic device, and described in more detail in U. S. Application Publication Nos. 2017/0165667 (Beaumont, et al.) and 2018/0298318 (Kurz, et al.), each of which disclosure is herein incorporated by reference in its entirety.
  • the laser illumination 535 may be configured to initiate photocleavage of surface modifying moieties of a modified surface of the microfluidic device or photocleavage of moieties providing adherent functionalities for micro-objects within a sequestration pen within the microfluidic device. Further details of photocleavage using a laser may be found in International Application Publication No. WO2017/205830 (Lowe, Jr. et al.), which disclosure is herein incorporated by reference in its entirety. [0234] The light from the first, second, and third light sources (552, 554, 556) passes through the first tube lens 562 and is transmitted to a third dichroic beam splitter 568 and filter changer 572.
  • the third dichroic beam splitter 568 can reflect a portion of the light and transmit the light through one or more filters in the filter changer 572 and to the objective 570, which may be an objective changer with a plurality of different objectives that can be switched on demand. Some of the light (515, 525, and/or 535) may pass through the third dichroic beam splitter 568 and be terminated or absorbed by a beam block (not shown). The light reflected from the third dichroic beam splitter 568 passes through the objective 570 to illuminate the sample plane 574, which can be a portion of a microfluidic device 520 such as the sequestration pens described herein.
  • the nest 500 can be integrated with the optical apparatus 510 and be a part of the apparatus 510.
  • the nest 500 can provide electrical connection to the enclosure and be further configured to provide fluidic connections to the enclosure. Users may load the microfluidic apparatus 520 into the nest 500.
  • the nest 500 can be a separate component independent of the optical apparatus 510.
  • Light can be reflected off and/or emitted from the sample plane 574 to pass back through the objective 570, through the filter changer 572, and through the third dichroic beam splitter 568 to a second tube lens 576.
  • the light can pass through the second Attorney Docket No.01149-0025-00PCT tube lens 576 (or imaging tube lens 576) and be reflected from a mirror 578 to an imaging sensor 580.
  • Stray light baffles (not shown) can be placed between the first tube lens 562 and the third dichroic beam splitter 568, between the third dichroic beam splitter 568 and the second tube lens 576, and between the second tube lens 576 and the imaging sensor 580.
  • the optical apparatus can comprise the objective lens 570 that is specifically designed and configured for viewing and manipulating of micro-objects in the microfluidic device 520.
  • conventional microscope objective lenses are designed to view micro-objects on a slide or through 5mm of aqueous fluid, while micro- objects in the microfluidic device 520 are inside the plurality of sequestration pens within the viewing plane 574 which have a depth of 20, 30, 40, 50, 6070, 80 microns or any values therebetween.
  • a transparent cover 520a for example, glass or ITO cover with a thickness of about 750 microns, can be placed on top of the plurality of sequestration pens, which are disposed above a microfluidic substrate 520c.
  • the images of the micro- objects obtained by using the conventional microscope objective lenses may have large aberrations such as spherical and chromatic aberrations, which can degrade the quality of the images.
  • the objective lens 570 of the optical apparatus 510 can be configured to correct the spherical and chromatic aberrations in the optical apparatus 1350.
  • the objective lens 570 can have one or more magnification levels available such as, 4X, 10X, 20X. [0238] Modes of illumination.
  • the structured light modulator 560 can be configured to modulate light beams received from the first light source 552 and transmits a plurality of illumination light beams 515, which are structured light beams, into the enclosure of the microfluidic device, e.g., the region containing the sequestration pens.
  • the structured light beams can comprise the plurality of illumination light beams.
  • the plurality of illumination light beams can be selectively activated to generate a plurality of illuminations patterns.
  • the structured light modulator 560 can be configured to generate an illumination pattern, similarly as described for FIGS.4A-4B, which can be moved and adjusted.
  • the optical apparatus 560 can further comprise a control unit (not shown) which is configured to adjust the illumination pattern to selectively activate the one or more of the plurality of DEP electrodes of a substrate 520c and generate DEP forces to move the one or more micro-objects inside the plurality of sequestration pens within the microfluidic device 520.
  • the plurality of illuminations patterns can be adjusted over time in a controlled manner to manipulate the micro-objects in the microfluidic device 520.
  • Each of the plurality of illumination patterns can be shifted to shift the location of the DEP force generated Attorney Docket No.01149-0025-00PCT and to move the structured light for one position to another in order to move the micro-objects within the enclosure of the microfluidic apparatus 520.
  • the optical apparatus 510 may be configured such that each of the plurality of sequestration pens in the sample plane 574 within the field of view is simultaneously in focus at the image sensor 580 and at the structured light modulator 560.
  • the structured light modulator 560 can be disposed at a conjugate plane of the image sensor 580.
  • the optical apparatus 510 can have a confocal configuration or confocal property.
  • the optical apparatus 510 can be further configured such that only each interior area of the flow region and/or each of the plurality of sequestration pens in the sample plane 574 within the field of view is imaged onto the image sensor 580 in order to reduce overall noise to thereby increase the contrast and resolution of the image.
  • the first tube lens 562 can be configured to generate collimated light beams and transmit the collimated light beams to the objective lens 570.
  • the objective 570 can receive the collimated light beams from the first tube lens 562 and focus the collimated light beams into each interior area of the flow region and each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510.
  • the first tube lens 562 can be configured to generate a plurality of collimated light beams and transmit the plurality of collimated light beams to the objective lens 570.
  • the objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and converge the plurality of collimated light beams into each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510.
  • the optical apparatus 510 can be configured to illuminate the at least a portion of sequestration pens with a plurality of illumination spots.
  • the objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and project the plurality of illumination spots, which may form an illumination pattern, into each of the plurality of sequestration pens in the sample plane 574 within the field of view.
  • each of the plurality of illumination spots can have a size of about 5 microns X 5 microns; 10 microns X 10 microns; 10 microns X 30 microns, 30 microns X 60 microns, 40 microns X 40 microns, 40 microns X 60 microns, 60 microns X 120 microns, 80 microns X 100 microns, 100 microns X 140 microns and any values there between.
  • the illumination spots may individually have a shape that is circular, square, or rectangular.
  • the illumination spots may be grouped within a plurality of illumination spots (e.g., an illumination pattern) to form a larger polygonal shape such as a rectangle, square, or wedge shape.
  • the Attorney Docket No.01149-0025-00PCT illumination pattern may enclose (e.g., surround) an unilluminated space that may be square, rectangular or polygonal.
  • each of the plurality of illumination spots can have an area of about 150 to about 3000, about 4000 to about 10000, or 5000 to about 15000 square microns.
  • An illumination pattern may have an area of about 1000 to about 8000, about 4000 to about 10000, 7000 to about 20000, 8000 to about 22000, 10000 to about 25000 square microns and any values there between.
  • the optical system 510 may be used to determine how to reposition micro-objects and into and out of the sequestration pens of the microfluidic device, as well as to count the number of micro-objects present within the microfluidic circuit of the device. Further details of repositioning and counting micro-objects are found in U. S. Application Publication No.2016/0160259 (Du); U. S. Patent No.9,996,920 (Du et al.); and International Application Publication No. WO2017/102748 (Kim, et al.). The optical system 510 may also be employed in assay methods to determine concentrations of reagents/assay products, and further details are found in U. S.
  • Patent Nos.8,921,055 Chapman
  • 10,010,882 White et al.
  • 9,889,445 Chapman et al.
  • International Application Publication No. WO2017/181135 Lionberger, et al.
  • International Application Serial No. PCT/US2018/055918 Lionberger, et al.
  • Further details of the features of optical apparatuses suitable for use within a system for observing and manipulating micro-objects within a microfluidic device, as described herein, may be found in WO2018/102747 (Lundquist, et al), the disclosure of which is herein incorporated by reference in its entirety.
  • Additional system components for maintenance of viability of cells within the sequestration pens of the microfluidic device In order to promote growth and/or expansion of cell populations, environmental conditions conducive to maintaining functional cells may be provided by additional components of the system. For example, such additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from cells.
  • additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from cells.
  • the method may further include disposing one or more biological cells within the one or more sequestration pens of the microfluidic device. In some embodiments, each one of the one or more biological cells may be disposed in a different one of the one or more sequestration pens.
  • the one or more biological cells may be disposed within the isolation regions of the one or more sequestration pens of the microfluidic device. In some embodiments of the method, at least one of the one or more biological cells may be disposed within a sequestration pen having one of the one or more capture objects disposed Attorney Docket No.01149-0025-00PCT therein. In some embodiments, the one or more biological cells may be a plurality of biological cells from a clonal population. In various embodiments of the method, disposing the one or more biological cells may be performed before disposing the one or more capture objects. [0245] In various embodiments, the capture object may be any capture object as described herein.
  • the capture object may include a magnetic component (e.g., a magnetic bead). Alternatively, the capture object can be non-magnetic.
  • a single biological cell is disposed in a sequestration pen. In some embodiments, a plurality of biological cells, for example, 2 or more, 2 to 10, 3 to 8, 4 to 6, or the like, are disposed within said sequestration pen. [0247] In various embodiments, disposing the biological cell may further include marking the biological cell (e.g., with a marker for nucleic acids, such as Dapi or Hoechst stain).
  • disposing said biological cell within said sequestration pen is performed before disposing said capture object within said sequestration pen. In some embodiments, disposing said capture object within said sequestration pen is performed before disposing said biological cell within said sequestration pen.
  • said enclosure of said microfluidic device comprises at least one coated surface. In some embodiments, the coated surface comprises a covalently linked surface. In some embodiments, the coated surface comprises a hydrophilic or a negatively charged coated surface. The coated surface can be coated with Tris and/or a polymer, such as a PEG-PPG block co-polymer. In yet other embodiments, the enclosure of the microfluidic device may include at least one conditioned surface.
  • the at least one conditioned surface may include a covalently bound hydrophilic moiety or a negatively charged moiety.
  • a covalently bound hydrophilic moiety or negatively charged moiety can be a hydrophilic or negatively charged polymer.
  • said enclosure of the microfluidic device further comprises a dielectrophoretic (DEP®) configuration, and wherein disposing said biological cell and/or disposing said capture object is performed by applying a dielectrophoretic (DEP®) force on or proximal to said biological cell and/or said capture object.
  • said microfluidic device further comprises a plurality of sequestration pens.
  • the method further comprises disposing a plurality of said biological cells within said plurality of sequestration pens.
  • a plurality of said biological cells disposed within said plurality of sequestration pens may have substantially only one biological cell disposed within Attorney Docket No.01149-0025-00PCT sequestration pens of said plurality.
  • each sequestration pen of the plurality having a biological cell disposed therein will generally contain a single biological cell. For example, less than 10%, 7%, 5%, 3% or 1% of sequestration pens occupied by a cell may contain more than one biological cell.
  • the plurality of biological cells may be a clonal population of biological cells.
  • a plurality of said capture objects disposed within said plurality of sequestration pens may have substantially only one capture object disposed within sequestration pens of said plurality.
  • each sequestration pen of the plurality having a capture object disposed therein will generally contain a single capture object. For example, less than 10%, 7%, 5%, 3% or 1% of sequestration pens occupied by a capture object may contain more than one capture object.
  • a plurality of said biological cells and a plurality of capture objects disposed within said plurality of sequestration pens may have substantially only one biological cell and substantially only one capture object disposed within sequestration pens of said plurality.
  • each sequestration pen of the plurality having a biological cell and a capture object disposed therein will generally contain a single biological cell and a single capture object.
  • less than 10%, 7%, 5%, 3% or 1% of sequestration pens occupied by a cell and a capture object may contain more than one biological cell or more than one capture object.
  • the plurality of biological cells may be a clonal population of biological cells.
  • the kit further includes a microfluidic device as described herein.
  • the microfluidic device can comprise an enclosure, where the enclosure includes a flow region and a plurality of sequestration pens opening off of the flow region.
  • compositions of the present disclosure enable activation of memory B cell samples (e.g., mammalian memory B cells, including human memory B cells) from fresh whole blood or frozen PBMC donor samples (FIG.6B, panels A and B).
  • memory B cell samples e.g., mammalian memory B cells, including human memory B cells
  • FIG.6C Cell preparation and assay reagents: As shown in FIG.6C, the methods can begin by either isolating peripheral blood mononuclear cells (PBMCs) from fresh whole blood, or by thawing frozen PBMCs. Memory B cells are then isolated from PBMCs via off the shelf magnetic-activated cell sorting (MACS) kits and activated for 5 days in culture using the presently disclosed compositions for memory B cell activation (FIG.6C).
  • PBMCs peripheral blood mononuclear cells
  • MCS magnetic-activated cell sorting
  • FIG.7A In 35 PBMC samples harvested from 5 vaccinated and/or convalescent human donors the average frequency of memory B cells was 0.56% (range 0.15% - 1.5%, FIG.7A).
  • FIG.11 after 5 days of culturing in activation medium, the pre- and post-activation expression levels of CD20 and CD138 show the differentiation of memory B cells into antibody -secreting plasma cells. At this point, the activated B cells are ready for screening on the Beacon ® Optofluidic system (Bruker Cellular Analysis, Inc.).
  • PBMCs peripheral blood mononuclear cells
  • STEMCELL density gradient centrifugation
  • AllCells pre-isolated format
  • Class-switched memory B cells were enriched from PBMCs using a magnetic-activated cell sorting (MACS) kit (130-093-617, Miltenyi), with expected frequencies ranging from 0.1-1.0% of the original starting population (FIG.10A).
  • Enriched memory B cells were re-suspended in human memory activation medium at a density of 2.5 E4 cells/mL.
  • the activation medium negates the requirement for feeder cell co-culture and comprises: RPMI 1640, 20% FBS, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 ⁇ M 2-mercaptoethanol, 100 ng/mL IL-2, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, 50 ng/mL BAFF, and 2 ⁇ g/mL CpG ODN 2006. These cells were seeded in 96-well, V-bottom plates with 5000 cells (200 ⁇ L of the above suspension) dispensed per well.
  • FIG.14 shows a comparison of B-cell proliferation after 5 days of culture using the activation medium described compared to two different commercial B-cell expansion medias (composition not provided by manufacturer).
  • the disclosed workflows do not require antigen-positive sorting of memory B cells, thus enabling screening of difficult cell-based targets that cannot be recombinantly expressed as soluble molecules.
  • standard enrichment strategies pre-activation and/or post-activation can be adapted to increase the likelihood of finding rare antibodies (dotted lines, FIG.6C).
  • Example 2 Screening of Activated B Cells
  • System and Microfluidic device The Beacon® system and microfluidic device used in Example 1 were manufactured by Bruker Cellular Analysis, Inc.. The system included at least a flow controller, temperature controller, fluidic medium conditioning and pump component, light source for light activated DEP configurations, mounting stage for the microfluidic device, and a camera.
  • the microfluidic device was an OptoSelect® chip configured with OptoElectroPositioning (OEP®) technology.
  • the microfluidic device included a microfluidic channel and a plurality of NanoPen® chambers fluidically connected thereto.
  • the Beacon® system is used to automatically isolate tens of thousands of single activated B cells into NanoPen® chambers on OptoSelect® 11k or 20k chips (Bruker Cellular Analysis, Inc.) in under 1 hour per chip (FIG. 6B, panel C).
  • Antigen-binding and cross- reactivity assays are used to screen and select activated B cells secreting antigen-specific antibodies (FIG.6B, panel D).
  • Single antigen-specific B cells can then be recovered into 96- well plates (FIG.6B, panel E) followed by off-chip cDNA synthesis and amplification using the Opto® B Discovery cDNA Synthesis® Kit (Bruker Cellular Analysis, Inc.; FIG.6B, panel F).
  • Paired antibody heavy/light chain sequences are amplified from the cDNA of recovered cells using the Opto® B Discovery Sanger Prep Kit, Human (Bruker Cellular Analysis, Inc.), which includes a primer for conventional Sanger sequencing (FIG.6B, panels G and H).
  • FIG.6B panels G and H.
  • activated human B cells are loaded onto OptoSelect® 11k or OptoSelect® 20k chips and isolated as single B cells into NanoPen® chambers. Over 7,500 or 12,500 single B cells can be loaded and screened per OptoSelect® 11k chip or OptoSelect® 20k chip, respectively.
  • Bead-based assays for IgG antibody secretion can be used to assess successful activation of memory B cells. From 16 workflows performed on samples from vaccinated and/or convalescent donor samples against SARS CoV-2, 39% of activated single B cells secreted IgG antibodies on average (FIG.10, two left-most panels). Bead-based assays can also be used to discover antigen-specific antibodies.
  • Sequence Recovery The workflow can be configured for recovery of paired heavy/light chain sequences for antigen-specific antibodies via the single cell approach (FIGS. 12A-12B). Using the single cell approach, all molecular biology steps – lysis, reverse transcription, cDNA synthesis and amplification – are performed after recovery of single cells into 96-well plates (FIG. 12B). This is a preferred method for high recovery rates of paired heavy/light chain sequences, especially when there are fewer than 192 hits.
  • the amplified cDNA from the single cell approach can be performed with the aid of the Opto® B Discovery Sanger Prep Kit (Human).
  • the PCR products can then be sequenced using traditional Sanger Sequencing. On average, 75% of pens targeted for export yielded paired heavy/light chain sequences with unambiguous nucleotide bases across the entire V(D)J regions (FIG. 13, Strict). These same samples yielded sequences that had no ambiguous bases across the CDR regions 78% of the time, on average (FIG.13, Moderate), and no ambiguous bases across the CDR3 region 80% of the time, on average (FIG. 13, Relaxed).
  • the first assay was performed immediately after the penning operation was complete, revealing that ca.30% of the activated B cells were secreting IgG molecules across all OptoSelect chips. Thereafter, the chips were perfused with separate culture media formulations in which the concentration of phorbol myristate acetate (PMA) was 100 ng/mL (FIG.16A, 16B) or varied from 0-100 ng/mL (FIG.17).
  • PMA phorbol myristate acetate
  • each media formulation was: RPMI 1640, 10% FBS, 2% B-27 supplement, 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 ⁇ M 2- mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF.
  • a second IgG secretion assay was performed after 24 h of media perfusion. As shown in FIG. 15A, the number of antibody hits, antibody hit rate (%), and hit retention all increased with the addition of PMA relative to the control.
  • Example 4 PMA Addition- Load and Culture Media Comparisons (on- chip) [0274] Human antibody-secreting B cells generated from memory B cells were penned into OptoSelect 11k chips using the opto-electropositioning (OEP) feature of the Beacon system. Isolation of each cell into an individual pen within a microfluidic environment facilitated the screening of antibody secretion on a single-cell basis using the Capture Assay imaging operation. Load media was formulated with and without 100 ng/mL phorbol myristate acetate (PMA) so that any benefits conferred by the molecule during the penning operation could be investigated (see FIG. 17).
  • PMA phorbol myristate acetate
  • each Load Media formulation was: RPMI 1640, 10% FBS, 10% B-27 supplement, 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 ⁇ M 2-mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF. After the penning operation was complete, the chips were perfused with separate Culture Media formulations that were prepared with and without 100 ng/mL PMA (see FIG. 17).
  • each Culture Media formulation was: RPMI 1640, 10% FBS, 2% B-27 supplement, 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 ⁇ M 2-mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF.
  • An initial antibody secretion assay was performed using these Culture Media formulations, with subsequent assays performed after 2 and 4 h of on-chip culture to assess the longevity effects of PMA in the Load and Culture Media.
  • the chips were perfused with separate culture media formulations in which the presence of two phorbol esters, phorbol myristate acetate (PMA, 100 nM) and phorbol 12,13-dibutyrate (PDBu, 100 nM), were investigated.
  • PMA phorbol myristate acetate
  • PDBu phorbol 12,13-dibutyrate
  • PMA phorbol myristate acetate
  • PDBu phorbol 12,13-dibutyrate
  • each media formulation was: RPMI 1640, 10% FBS, B-27 supplement (10% during OEP, 2% during culture), 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 ⁇ M 2-mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF.
  • the first assay was performed immediately after the penning operation was complete, revealing that ca.36% of the activated B cells were secreting IgG molecules across all OptoSelect chips.

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Abstract

Disclosed herein are compositions for culturing B cell lymphocytes. The compositions can be used to activate B cell lymphocytes, including memory B cell lymphocytes, which may be human B cell lymphocytes. Such activation can involve stimulation of cell proliferation and/or induction of maintained antibody expression. Also disclosed are methods of using the disclosed compositions to culture B cell lymphocytes. Such methods can be performed in a well plate, a microfluidic chip, or a combination thereof. Moreover, the methods can be performed as part of a workflow to screen the B cell lymphocytes for production of antibodies, including antibodies that bind to an antigen of interest.

Description

Attorney Docket No.01149-0025-00PCT METHODS FOR CULTURING AND ASSAYING B CELL LYMPHOCYTES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims the benefit of priority of US Provisional Application No.63/540,358, filed September 25, 2023, which is incorporated by reference herein in its entirety for any purpose. INTRODUCTION AND SUMMARY [0002] This application relates to methods of assaying a biological cell, particularly a B cell lymphocyte, such as a mammalian B cell lymphocyte or a human B cell lymphocyte. [0003] Over the past three decades, antibody therapies have been developed for a host of different diseases, ranging from autoimmune disorders to infectious diseases and cancer. The time it takes to screen antibody producing cells for lead candidates is often quite significant, adding to the drug development timeline. For example, after immunizing an animal and harvesting the antibody-producing B lymphocytes (or B cells) from the spleen, bone marrow, or lymph nodes, it can take at least 12 weeks to produce a hybridoma and screen through all of the potential hits. Recent developments in microfluidic chip-based screening systems have allowed direct interrogation of B cells and more rapid selection of lead candidates. Using such approaches, several tens of thousands of B cells can be cloned in parallel in chambers of the microfluidic device, and multiple assays can be performed for thorough characterization of promising lead candidates. In addition, automated cell lysis and reverse transcription can be performed on chip to generate stable cDNA molecules, which can be subsequently recovered for paired heavy/light chain amplification and sequencing. [0004] Human antibodies have desirable therapeutic characteristics, including inherently lower immunogenicity without a need for humanization, since the antibodies and targets are already physiologically relevant. Accessing the human repertoire for antibody discovery can thus accelerate the timeline toward safe and efficacious therapeutics, especially in the context of an urgent emerging disease response and controlling endemic diseases. Beyond antibody therapeutics, directly screening human B cells opens opportunities to study antibody diversity, prevalence, and population kinetics, giving insights into disease progression and humoral immunity that are especially important for vaccine and auto-immunity research. [0005] Single B cell screening approaches provide attractive strategies for discovery of monoclonal antibodies, including human monoclonal antibodies, as compared with hybridoma and phage display owing to the low fusion efficiency and loss of cognate heavy/light chain pairing in the latter two approaches, respectively. A central challenge to single B cell screening Attorney Docket No.01149-0025-00PCT in humans and most other animals is the lack of robust B cell media to maintain durable B cell survival and antibody secretion for the duration of the assay and down selection processes. While certain B cell screening approaches can use plasma B cells, other approaches are typically performed using memory B cells that must be activated to secrete antibodies for screening purposes. There remains a need for improved media for activating memory B cells, including human memory B cells, to secrete antibodies for long enough to allow single-cell screening and sequence recovery. [0006] Disclosed herein are compositions for culturing B cells, including memory B cells, which may be human B cells (e.g., human memory B cells). By culturing B cells in the disclosed compositions, the B cells can be activated, resulting in proliferation and/or immunoglobulin (e.g., antibody) secretion, and/or maintained in a state of sustained immunoglobulin (e.g., antibody) expression/secretion. Also disclosed herein are methods of culturing, activating, and/or screening B cells that involve contacting the B cells with one or more compositions disclosed herein. 1) A composition for culturing B cell lymphocytes, the composition comprising: a protein kinase C (PKC) agonist (e.g., a PKC-specific agonist), wherein the composition lacks feeder cells. 2) The composition of embodiment 1, wherein the PKC agonist is selected from phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PBD), 4-alpha-phorbol 12- myristate 13-acetate (4-alpha-PMA), dPPA, 4-alpha-phorbol (isophorbol), and phorbol 12- myristate 13-acetate 4-O-methyl ether (MPMA). 3) The composition of embodiment 1 or 2, further comprising: IL-6 and/or IL-4. 4) The composition of any one of embodiments 1 to 3, wherein the composition comprises IL-6 at a concentration of at least about 1.0 ng/mL (e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL). 5) The composition of any one of embodiments 1 to 3, wherein the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL (e.g., from about 5.0 ng/mL to about 25 ng/mL, about 10 ng/mL to about 50 ng/mL, about 15 ng/mL to about 75 ng/mL, about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints). 6) The composition of any one of embodiments 3 to 5, wherein the IL-6 is human IL-6. 7) The composition of any one of embodiments 1 to 6, wherein the composition comprises IL-4 at a concentration of at least about 0.5 ng/mL (e.g., at least about 1.0 ng/mL, Attorney Docket No.01149-0025-00PCT at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL). 8) The composition of any one of embodiments 1 to 6, wherein the composition comprises IL-4 at a concentration from about 0.5 ng/mL to about 10 ng/mL (e.g., from about 1.0 ng/mL to about 2.5 ng/mL, about 1.5 ng/mL to about 5.0 ng/mL, about 2.0 ng/mL to about 7.5 ng/mL, about 2.5 ng/mL to about 10 ng/mL, or any range defined by two of the foregoing endpoints). 9) The composition of any one of embodiments 3, 7 and 8, wherein the IL-4 is human IL-4. 10) The composition of any one of embodiments 1 to 9, further comprising: IL-21 and/or BAFF. 11) The composition of any one of embodiments 1 to 10, wherein the composition comprises IL-21 at a concentration of at least about 1.0 ng/mL (e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL). 12) The composition of any one of embodiments 1 to 10, wherein the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL (e.g., from about 2.0 ng/mL to about 20 ng/mL, about 5.0 ng/mL to about 40 ng/mL, about 10 ng/mL to about 60 ng/mL, about 15 ng/mL to about 80 ng/mL, about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints). 13) The composition of any one of embodiments 10 to 12, wherein the IL-21 is human IL- 21. 14) The composition of any one of embodiments 1 to 13, wherein the composition comprises BAFF at a concentration of at least about 10 ng/mL (e.g., at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL). 15) The composition of any one of embodiments 1 to 13, wherein the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL (e.g., from about 20 ng/mL to about 50 ng/mL, about 30 ng/mL to about 60 ng/mL, about 40 ng/mL to about 70 ng/mL, about 50 ng/mL to about 80 ng/mL, about 60 ng/mL to about 90 ng/mL, about 70 ng/mL to about 100 ng/mL, or any range defined by two of the forgoing endpoints). 16) The composition of any one of embodiments 10, 14 and 15, wherein the BAFF is human BAFF. Attorney Docket No.01149-0025-00PCT 17) The composition of any one of embodiments 1 to 16, wherein the composition comprises IL-2 at a concentration of less than about 1.0 ng/mL (e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks IL-2). 18) The composition of any one of embodiments 1 to 17, wherein the composition comprises TNF-alpha at a concentration of less than about 1.0 ng/mL (e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks TNF-alpha). 19) The composition of any one of embodiments 1 to 18, wherein the composition comprises CpG oligodeoxynucleotides (ODNs) at a concentration of less than about 250 nanomolar (nM) (e.g., less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than about 1 nM, or substantially lacks CpG ODNs). 20) The composition of any one of embodiments 1 to 19, wherein the composition comprises a PI3K agonist (e.g., a PI3K-specific agonist) at a concentration of less than about 1.0 ng/mL (e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks PI3K agonist). 21) The composition of embodiment 20, wherein the PI3K agonist comprises naringenin, naringin, butin, eriodictyol, homoeriodictyol, hesperetin, hesperidin, and/or isosakuranetin. 22) The composition of any one of embodiments 1 to 21, wherein the composition comprises a STAT3 agonist (e.g., a STAT3-specific agonist) at a concentration of less than about 1.0 ng/mL (e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks STAT3 agonist). 23) The composition of embodiment 22, wherein the STAT3 agonist comprises leukemia inhibitory factor (LIF) (e.g., human LIF (hLIF)), as well as fragments and variants that retain STAT3 agonist activity. 24) The composition of any one of embodiments 1 to 23, wherein the composition comprises a CD40-clustering agent at a concentration of less than about 1.0 ng/mL (e.g., less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks CD40-clustering agent). Attorney Docket No.01149-0025-00PCT 25) The composition of embodiment 24, wherein the cD40-clustering agent comprises CD40 ligand (i.e., CD40L), active fragments and variants thereof, and/or anti-CD40 agonist antibodies. 26) The composition of any one of embodiments 1 to 25, wherein the PKC agonist is a phorbol compound. 27) The composition of any one of embodiments 1 to 25, wherein the PKC agonist is a phorbol ester. 28) The composition of any one of embodiments 1 to 25, wherein the PKC agonist is phorbol 12-myristate 13-acetate (PMA). 29) The composition of any one of embodiments 1 to 25, wherein the PKC agonist is phorbol 12,13-dibutyrate (PBD). 30) The composition of any one of embodiments 1 to 29, wherein the composition comprises the PKC agonist at a concentration of at least about 10 ng/mL (e.g., at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, at least about 100 ng/mL, at least about 150 ng/mL, at least about 200 ng/mL, at least about 250 ng/mL, at least about 500 ng/mL, or at least about 750 ng/mL). 31) The composition of any one of embodiments 1 to 29, wherein the composition comprises the PKC agonist at a concentration from about 10 ng/mL to about 1000 ng/mL (e.g., from about 25 ng/mL to about 200 ng/mL, from about 50 ng/mL to about 250 ng/mL, from about 75 ng/mL to about 300 ng/mL, from about 100 ng/mL to about 400 ng/mL, from about 150 ng/mL to about 500 ng/mL, from about 200 ng/mL to 600 ng/mL, from about 250 ng/mL to about 700 ng/mL, from about 300 ng/mL to about 800 ng/mL, from about 400 ng/mL to about 900 ng/mL, from about 500 ng/mL to about 1000 ng/mL, or any range defined by two of the foregoing endpoints). 32) The composition of any one of embodiments 1 to 31, further comprising: serum (e.g., any serum suitable for culturing B cell lymphocytes). 33) The composition of embodiment 32, wherein the serum is present at an amount of about 1% to about 20% (v/v) (e.g., about 2% to about 15% (v/v), or about 5% to about 10% (v/v)). 34) The composition of embodiment 32 or 33, wherein the serum is fetal bovine serum. 35) The composition of any one of embodiments 1 to 34, wherein the composition is a liquid medium. 36) A composition for culturing B cell lymphocytes, the composition comprising: a Toll-like receptor 9 (TLR9) agonist; Attorney Docket No.01149-0025-00PCT IL-6; and IL-4, wherein the composition lacks feeder cells. 37) The composition of embodiment 36, wherein the composition comprises IL-6 at a concentration of at least about 1.0 ng/mL (e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL). 38) The composition of embodiment 36, wherein the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL (e.g., from about 5.0 ng/mL to about 25 ng/mL, about 10 ng/mL to about 50 ng/mL, about 15 ng/mL to about 75 ng/mL, about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints). 39) The composition of any one of embodiments 36 to 38, wherein the IL-6 is human IL- 6. 40) The composition of any one of embodiments 36 to 39, wherein the composition comprises IL-4 at a concentration of at least about 0.5 ng/mL (e.g., at least about 1.0 ng/mL, at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL). 41) The composition of any one of embodiments 36 to 39, wherein the composition comprises IL-4 at a concentration from about 0.5 ng/mL to about 10 ng/mL (e.g., from about 1.0 ng/mL to about 2.5 ng/mL, about 1.5 ng/mL to about 5.0 ng/mL, about 2.0 ng/mL to about 7.5 ng/mL, about 2.5 ng/mL to about 10 ng/mL, or any range defined by two of the foregoing endpoints). 42) The composition of any one of embodiments 36 to 41, wherein the IL-4 is human IL- 4. 43) The composition of any one of embodiments 36 to 42, further comprising: IL-2, IL-21 and/or BAFF. 44) The composition of any one of embodiments 36 to 43, wherein the composition comprises IL-2 at a concentration of at least about 1.0 ng/mL (e.g., at least about 10 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, or at least about 100 ng/mL). 45) The composition of any one of embodiments 36 to 43, wherein the composition comprises IL-2 at a concentration from about 1.0 ng/mL to about 100 ng/mL (e.g., from about 10 ng/mL to about 50 ng/mL, about 25 ng/mL to about 75 ng/mL, about 50 ng/mL to about 100 ng/mL, about 5 ng/mL to about 25 ng/mL, about 25 ng/mL to about 50 ng/mL, Attorney Docket No.01149-0025-00PCT about 50 ng/mL to about 75 ng/mL, about 75 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints). 46) The composition of any one of embodiments 43 to 45, wherein the IL-2 is human IL- 2. 47) The composition of any one of embodiments 36 to 46, wherein the composition comprises IL-21 at a concentration of at least about 1.0 ng/mL (e.g., at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL). 48) The composition of any one of embodiments 36 to 46, wherein the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL (e.g., from about 2.0 ng/mL to about 20 ng/mL, about 5.0 ng/mL to about 40 ng/mL, about 10 ng/mL to about 60 ng/mL, about 15 ng/mL to about 80 ng/mL, about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints). 49) The composition of any one of embodiments 43, 47 and 48, wherein the IL-21 is human IL-21. 50) The composition of any one of embodiments 36 to 49, wherein the composition comprises BAFF at a concentration of at least about 10 ng/mL (e.g., at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL). 51) The composition of any one of embodiments 36 to 49, wherein the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL (e.g., from about 20 ng/mL to about 50 ng/mL, about 30 ng/mL to about 60 ng/mL, about 40 ng/mL to about 70 ng/mL, about 50 ng/mL to about 80 ng/mL, about 60 ng/mL to about 90 ng/mL, about 70 ng/mL to about 100 ng/mL, or any range defined by two of the forgoing endpoints). 52) The composition of any one of embodiments 43, 50 and 51, wherein the BAFF is human BAFF. 53) The composition of any one of embodiments 36 to 52, wherein the composition comprises a protein kinase C (PKC) agonist (e.g., a PKC-specific agonist) at a concentration of less than about 10 ng/mL (e.g., less than about 5.0 ng/mL, less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, or substantially lacks PKC agonist). 54) The composition of any one of embodiments 36 to 53, wherein the TLR9 agonist is a CpG oligodeoxynucleotide (ODN). Attorney Docket No.01149-0025-00PCT 55) The composition of embodiment 54, wherein the CpG ODN is a B-class CpG ODN and/or has a linear structure. 56) The composition of embodiment 54 or 55, wherein the composition comprises CpG ODN at a concentration of at least about 0.25 micromolar (µM) (e.g., at least about 0.5 µM, at least about 1.0 µM, at least about 1.5 µM, at least about 2.0 µM, or at least about 2.5 µM). 57) The composition of embodiment 54 or 55, wherein the composition comprises CpG ODN at a concentration of from about 0.25 µM to about 5.0 µM (e.g., from about 0.25 µM to about 4.0 µM, from about 0.5 µM to about 3.0 µM, from about 1.0 µM to about 2.0 µM, from about 0.25 µM to about 1.0 µM, from about 0.5 µM to about 1.5 µM, from about 1.0 µM to about 2.5 µM, from about 1.5 µM to about 3.0 µM, from about 2.0 µM to about 4.0 µM, from about 2.5 µM to about 5.0 µM, or any range defined by two of the foregoing endpoints). 58) The composition of any one of embodiments 36 to 57, further comprising: serum (e.g., any serum suitable for culturing B cell lymphocytes). 59) The composition of embodiment 58, wherein the serum is present at an amount of about 1% to about 20% (v/v) (e.g., about 2% to about 15% (v/v), or about 5% to about 10% v/v) 60) The composition of embodiment 58 or 59, wherein the serum is fetal bovine serum. 61) The composition of any one of embodiments 36 to 60, wherein the composition is a liquid medium. 62) A method of culturing a B cell lymphocyte, the method comprising: incubating the B cell lymphocyte in a composition of any one of embodiments 1 to 35, wherein the incubating maintains the expression of antibodies by the B cell lymphocyte, optionally wherein a population of B cell lymphocytes incubated in the composition exhibit at least a 60% (e.g., at least 65%, at least 70%, at least 75%, or at least 80%) maintenance of antibody expression after 24 hours of culture. 63) The method of embodiment 62, wherein the B cell lymphocyte is a mammalian B cell. 64) The method of embodiment 62, wherein the B cell lymphocyte is a human B cell. 65) The method of any one of embodiments 62 to 64, wherein the B cell lymphocyte is a memory B cell or a derivative thereof (e.g., an activated memory B cell). 66) The method of any one of embodiments 62 to 65, wherein the incubating is performed for at least 12 hours (e.g., at least 15 hours, at least 18 hours, at least 21 hours, at least 24 hours, at least 27 hours, at least 30 hours, at least 33 hours, or at least 36 hours). 67) The method of embodiment 66, wherein the incubating is performed continuously. Attorney Docket No.01149-0025-00PCT 68) The method of any one of embodiments 62 to 67, wherein the incubating is performed in a microfluidic chip (e.g., a chamber of a microfluidic chip, where the microfluidic chip can be any microfluidic chip described herein). 69) The method of any one of embodiments 62 to 68, wherein the incubating is performed in 10 nanoliters or less of the composition (e.g., 5 nanoliters or less, 2 nanoliters or less, or 1 nanoliter or less). 70) A method of culturing a B cell lymphocyte, the method comprising: incubating the B cell lymphocyte in a first composition, wherein the first composition is a composition of embodiment 61; and incubating the B cell lymphocyte in a second composition, wherein the second composition is a composition of embodiment 35; wherein the first composition stimulates activation and/or expression of antibody by the B cell lymphocyte, and wherein the second composition maintains continued expression of antibody by the B cell lymphocyte. 71) The method of embodiment 70, wherein the B cell lymphocyte is a mammalian B cell. 72) The method of embodiment 70, wherein the B cell lymphocyte is a human B cell. 73) The method of any one of embodiments 70 to 72, wherein the B cell lymphocyte is a memory B cell. 74) The method of any one of embodiments 70 to 73, wherein the incubation in the first composition is performed in a well of a well plate. 75) The method of any one of embodiments 70 to 74, wherein the incubation in the first composition is performed in about 200 microliters or less of the first composition (e.g., 150 microliters or less, 120 microliters or less, 100 microliters or less, 80 microliters or less, 60 microliters or less, or 50 microliters or less), optionally wherein at least 1.0 E4 cells/mL, at least 2.0 E4 cells/mL, at least 3.0 E4 cells/mL, at least 4.0 E4 cells/mL, at least 5.0 E4 cells/ mL, at least 6.0 E4 cells/ mL, at least 7.0 E4 cells/ mL, at least 8.0 E4 cells/ mL, at least 9.0 E4 cells/ mL, or at least 1.0 E6 cells/mL. 76) The method of any one of embodiments 70 to 75, wherein the incubation in the first composition is performed for at least 24 hours (e.g., at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, or at least 120 hours). 77) The method of any one of embodiments 70 to 76, wherein the incubation in the second composition is performed in a microfluidic chip (e.g., a chamber of a microfluidic chip, where the microfluidic chip can be any microfluidic chip described herein). Attorney Docket No.01149-0025-00PCT 78) The method of any one of embodiments 70 to 77, wherein wherein incubating with the second composition comprises perfusing the second composition through the microfluidic chip, and/or wherein the incubation in the second composition is performed in about 20 microliters or less of the second composition (e.g., about 15 microliters or less, about 12 microliters or less, about 10 microliters or less, about 8 microliters or less, about 6 microliters or less, or about 4 microliters or less). 79) The method of any one of embodiments 70 to 77, wherein the incubation in the second composition is performed in a well of a well plate. 80) The method of any one of embodiments 70 to 77 and 79, wherein the incubation in the second composition is performed in about 200 microliters or less of the second composition (e.g., 150 microliters or less, 120 microliters or less, 100 microliters or less, 80 microliters or less, 60 microliters or less, or 50 microliters or less). 81) The method of any one of embodiments 70 to 80, wherein the incubation in the second composition is performed for at least 12 hours (e.g., at least 15 hours, at least 18 hours, at least 21 hours, at least 24 hours, at least 27 hours, at least 30 hours, at least 33 hours, or at least 36 hours). 82) A method of inducing proliferation of a B cell lymphocyte, the method comprising: incubating the B cell lymphocyte in a composition of any one of embodiments 36 to 61, wherein the incubating is performed for a time sufficient to induce proliferation of the B cell lymphocyte. 83) A method of maintaining immunoglobulin (e.g., antibody) expression by a B cell lymphocyte, the method comprising: incubating the B cell lymphocyte in a composition of any one of embodiments 1 to 35, wherein the incubating is performed for a time sufficient to induce and/or maintain immunoglobulin expression by the B cell lymphocyte. 84) The method of embodiment 83, wherein the immunoglobulin is IgG. 85) The method of embodiment 83 or 84, wherein immunoglobulin expression is maintained for at least 4 hours (e.g., at least 8 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 21 hours, at least 24 hours, or more). 86) The method of any one of embodiments 82 to 86, wherein the B cell lymphocyte is a mammalian B cell. 87) The method of any one of embodiments 82 to 86, wherein the B cell lymphocyte is a human B cell. Attorney Docket No.01149-0025-00PCT 88) A method for screening a B cell lymphocyte for expression of an antibody that specifically binds to an antigen of interest, the method comprising: culturing the B cell lymphocyte according to the method of any one of embodiments 62 to 69; providing the antigen of interest; and detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte. 89) The method of embodiment 88, wherein detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte is performed using a sandwich assay (e.g., an ELISA-type sandwich assay). 90) The method of embodiment 88 or 89, wherein detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte comprises using a solid support to capture the immunoglobulin expressed by the B cell lymphocyte. 91) The method of embodiment 90, wherein the solid support is a bead (or a hydrogel structure). 92) The method of any one of embodiments 88 to 91, wherein the antigen of interest comprises a fluorescent label. 93) The method of embodiment 88 or 89, wherein detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte comprises using a cell that expresses the antigen of interest to capture the immunoglobulin expressed by the B cell lymphocyte. 94) The method of any one of embodiments 88 to 93, wherein detecting the interaction between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte is performed within a well of a well plate. 95) The method of any one of embodiments 88 to 93, wherein detecting the interaction between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte is performed within a chamber of a microfluidic chip (e.g., any of the microfluidic chips described herein). 96) The method of embodiment 95, wherein the chamber is a sequestration pen (e.g., as described herein). 97) The method of any one of embodiments 88 to 93, wherein detecting the interaction between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte is performed within a channel of a microfluidic chip (e.g., any of the microfluidic chips described herein). Attorney Docket No.01149-0025-00PCT 98) The method of any one of embodiments 88 to 97, wherein prior to culturing the B cell lymphocyte according to the method of any one of embodiments 62 to 69, the method comprises incubating the B cell lymphocyte in a composition of any one of embodiments 36 to 61, wherein the incubating is performed for a time sufficient to induce proliferation and/or activation of the B cell lymphocyte. [0007] These and other features and advantages of the disclosed compositions and methods will be set forth or will become more fully apparent in the description that follows and in the appended claims. The features and advantages may be realized and obtained by means of the objects and combinations particularly pointed out in the appended examples, partial listing of embodiments, and claims. Furthermore, the features and advantages of the described compositions and methods may be learned by the practice or will be obvious from the description, as set forth hereinafter. [0008] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS [0009] FIG.1A illustrates a microfluidic device and a system with associated control equipment according to some embodiments of the disclosure. [0010] FIG.1B illustrates a microfluidic device with sequestration pens according to an embodiment of the disclosure. [0011] FIGS. 2A to 2B illustrate a microfluidic device having sequestration pens according to some embodiments of the disclosure. [0012] FIG. 2C illustrates a sequestration pen of a microfluidic device according to some embodiments of the disclosure. [0013] FIG.3 illustrates a sequestration pen of a microfluidic device according to some embodiments of the disclosure. [0014] FIGS. 4A to 4B illustrate electrokinetic features of a microfluidic device according to some embodiments of the disclosure. [0015] FIG.5A illustrates a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure. [0016] FIG. 5B illustrates an imaging device according to some embodiments of the disclosure. Attorney Docket No.01149-0025-00PCT [0017] FIGS.6A to 6B illustrate workflows for antibody discovery according to some embodiments of the disclosure. [0018] FIG.6C illustrates a workflow for activating a memory B cell according to some embodiments of the disclosure. [0019] FIGS. 7A-7C summarize data regarding the frequency of memory B cells in human PBMCs (7A) and the extent of proliferation observed upon contacting the memory B cells with an activating composition (7B), and the viability of memory B cells following such activation (7C), as further described in the Example 1, according to embodiments of the present disclosure. [0020] FIG.8 summarizes data regarding the pre- and post-activation expression levels of CD20 and CD138, showing differentiation of memory B cells into antibody-secreting plasma cells, according to embodiments of the present disclosure. [0021] FIG.9 summarizes data regarding the loading of single, activated human B cells into chambers of OptoSelect® 11k and 20k microfluidic chips, according to embodiments of the present disclosure. [0022] FIG.10 summarizes data regarding the number of human B cells secreting IgG antibodies (two left-most columns) and SARS-CoV-2-specific antibodies (two right-most columns), activated according to embodiments of the present disclosure. [0023] FIG. 11 summarizes data regarding the percentage of activated B cells that maintained immunoglobulin (i.e., antibody) secretion after being cultured in a maintaining composition according to embodiments of the present disclosure. [0024] FIGS.12A to 12B illustrate approaches for amplification of antibody heavy and light chain sequences according to embodiments of the present disclosure. [0025] FIG. 13 summarizes data regarding the recovery of antigen-specific antibody sequences from single B cells according to embodiments of the present disclosure. [0026] FIG.14 summarizes data showing a comparison of B-cell proliferation after 5 days of culture using the activation medium described compared to two different commercial B-cell expansion medias. [0027] FIGS. 15A and 15B summarize data showing a comparison of the addition of PMA to the activation media (18A) and the addition of PMA and/or TNF-α to the activation media (18B). [0028] FIG.16 summarizes data showing dose-dependency of PMA on the retention of IgG secretion over time by the activated B cells. Attorney Docket No.01149-0025-00PCT [0029] FIG. 17 summarizes data showing a comparison of PMA addition to the load and culture media of activated B cells. [0030] FIG.18 summarizes data showing a comparison of PKC agonists on activated B cells. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0031] This specification describes exemplary embodiments and applications of the disclosure. The disclosure, however, is not limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Moreover, the figures may show simplified or partial views, and the dimensions of elements in the figures may be exaggerated or otherwise not in proportion. In addition, as the terms “on,” “attached to,” “connected to,” “coupled to,” or similar words are used herein, one element (e.g., a material, a layer, a substrate, etc.) can be “on,” “attached to,” “connected to,” or “coupled to” another element regardless of whether the one element is directly on, attached to, connected to, or coupled to the other element or there are one or more intervening elements between the one element and the other element. Also, unless the context dictates otherwise, directions (e.g., above, below, top, bottom, side, up, down, under, over, upper, lower, horizontal, vertical, “x,” “y,” “z,” etc.), if provided, are relative and provided solely by way of example and for ease of illustration and discussion and not by way of limitation. In addition, where reference is made to a list of elements (e.g., elements a, b, c), such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or a combination of all of the listed elements. Section divisions in the specification are for ease of review only and do not limit any combination of elements discussed. [0032] Where dimensions of microfluidic features are described as having a width or an area, the dimension typically is described relative to an x-axial and/or y-axial dimension, both of which lie within a plane that is parallel to the substrate and/or cover of the microfluidic device. The height of a microfluidic feature may be described relative to a z-axial direction, which is perpendicular to a plane that is parallel to the substrate and/or cover of the microfluidic device. In some instances, a cross sectional area of a microfluidic feature, such as a channel or a passageway, may be in reference to a x-axial/z-axial, a y-axial/z-axial, or an x-axial/y-axial area. Attorney Docket No.01149-0025-00PCT I. Definitions [0033] Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention. [0034] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps. [0035] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal Attorney Docket No.01149-0025-00PCT to, and equal to 10 and 15 are considered disclosed as well as from 10 to 15. It is also understood that each unit between two particular units are also disclosed as well as the endpoints. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. [0036] As used herein, “substantially” means sufficient to work for the intended purpose. The term "substantially" thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, “substantially” means within ten percent. [0037] The term “ones” means more than one. As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. [0038] As used herein: µm means micrometer, µm3 means cubic micrometer, pL means picoliter, nL means nanoliter, and μL (or uL) means microliter. [0039] As used herein, “air” refers to the composition of gases predominating in the atmosphere of the earth. The four most plentiful gases are nitrogen (typically present at a concentration of about 78% by volume, e.g., in a range from about 70-80%), oxygen (typically present at about 20.95% by volume at sea level, e.g. in a range from about 10% to about 25%), argon (typically present at about 1.0% by volume, e.g. in a range from about 0.1% to about 3%), and carbon dioxide (typically present at about 0.04%, e.g., in a range from about 0.01% to about 0.07%). Air may have other trace gases such as methane, nitrous oxide or ozone, trace pollutants and organic materials such as pollen, diesel particulates and the like. Air may include water vapor (typically present at about 0.25%, or may be present in a range from about 10ppm to about 5% by volume). Air may be provided for use in culturing experiments as a filtered, controlled composition and may be conditioned as described herein. [0040] As used herein, the term “disposed” encompasses within its meaning “located.” [0041] As used herein, a “microfluidic device” or “microfluidic apparatus” is a device that includes one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprised of fluidically interconnected circuit elements, including but not limited to region(s), flow path(s), channel(s), chamber(s), and/or pen(s), and at least one port configured to allow the fluid (and, optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, a microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel, and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 µL. In certain Attorney Docket No.01149-0025-00PCT embodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 µL. The microfluidic circuit may be configured to have a first end fluidically connected with a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected with a second port (e.g., an outlet) in the microfluidic device. [0042] As used herein, a “nanofluidic device” or “nanofluidic apparatus” is a type of microfluidic device having a microfluidic circuit that contains at least one circuit element configured to hold a volume of fluid of less than about 1 µL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. A nanofluidic device may comprise a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one circuit elements is configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one circuit elements are configured to hold a volume of fluid of about 20 nL to 200nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL. [0043] A microfluidic device or a nanofluidic device may be referred to herein as a “microfluidic chip” or a “chip”; or “nanofluidic chip” or “chip”. [0044] A “microfluidic channel” or “flow channel” as used herein refers to flow region of a microfluidic device having a length that is significantly longer than both the horizontal and vertical dimensions. The length of the channel is generally defined by the flow path of the channel. In the case of a straight channel, the length would be the “longitudinal axis” of the channel. The “horizontal dimension” or “width” of the channel is the horizontal dimension as observed in a transverse section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangential to the flow path of the channel at the plane of the transverse section). The “vertical dimension” or “height” of the channel is the vertical dimension as observed in a transverse section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangential to the flow path of the channel at the plane of the transverse section). Attorney Docket No.01149-0025-00PCT [0045] For example, the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of a flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween. In some embodiments, the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns, (e.g., from about 40 to about 150 microns). It is noted that a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element. For example, a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof. In addition, a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein. The flow channel may include valves, and the valves may be of any type known in the art of microfluidics. Examples of microfluidic channels that include valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, each of which is herein incorporated by reference in its entirety. [0046] For example, the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of a flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween. In some embodiments, the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns, (e.g., from about 40 to about 150 microns). It is noted that a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element. For example, a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof. In addition, a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein. The flow channel may include valves, and the valves may be of any type known in the art of microfluidics. Examples of microfluidic channels that include valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, each of which is herein incorporated by reference in its entirety. Attorney Docket No.01149-0025-00PCT [0047] The direction of fluid flow through the flow region (e.g., channel), or other circuit element (e.g., a chamber), dictates an “upstream” and a “downstream” orientation of the flow region or circuit element. Accordingly, an inlet will be located at an upstream position, and an outlet will be generally located at a downstream position. It will be appreciated by a person of skill in the art, that the designation of an “inlet” or an “outlet” may be changed by reversing the flow within the device or by opening one or more alternative aperture(s). [0048] As used herein, the term “transparent” refers to a material which allows visible light to pass through without substantially altering the light as is passes through. [0049] As used herein, “brightfield” illumination and/or image refers to white light illumination of the microfluidic field of view from a broad-spectrum light source, where contrast is formed by absorbance of light by objects in the field of view. [0050] As used herein, “structured light” is projected light that is modulated to provide one or more illumination effects. A first illumination effect may be projected light illuminating a portion of a surface of a device without illuminating (or at least minimizing illumination of) an adjacent portion of the surface, e.g., a projected light pattern, as described more fully below, used to activate DEP forces within a DEP substrate. When using structured light patterns to activate DEP forces, the intensity, e.g., variation in duty cycle of a structured light modulator such as a DMD, may be used to change the optical power applied to the light activated DEP actuators, and thus change DEP force without changing the nominal voltage or frequency. Another illumination effect that may be produced by structured light includes projected light that may be corrected for surface irregularities and for irregularities associated with the light projection itself, e.g., fall-off at the edge of an illuminated field. Structured light is typically generated by a structured light modulator, such as a digital mirror device (DMD), a microshutter array system (MSA), a liquid crystal display (LCD), or the like. Illumination of a small area of the surface, e.g., a selected area of interest, with structured light improves the signal-to-noise-ratio (SNR), as illumination of only the selected area of interest reduces stray/scattered light, thereby lowering the dark level of the image. An important aspect of structured light is that it may be changed quickly over time. A light pattern from the structured light modulator, e.g., DMD, may be used to autofocus on difficult targets such as clean mirrors or surfaces that are far out of focus. Using a clean mirror, a number of self-test features may be replicated such as measurement of modulation transfer function and field curvature/tilt, without requiring a more expensive Shack-Hartmann sensor. In another use of structured light patterns, spatial power distribution may be measured at the sample surface with a simple power meter, in place of a camera. Structured light patterns may also be used as a reference feature Attorney Docket No.01149-0025-00PCT for optical module/system component alignment as well used as a manual readout for manual focus. Another illumination effect made possible by use of structured light patterns is selective curing, e.g., solidification of hydrogels within the microfluidic device. [0051] As used herein, the term “micro-object” refers generally to any microscopic object that may be isolated and/or manipulated in accordance with the present disclosure. Non- limiting examples of micro-objects include: inanimate micro-objects such as microparticles; microbeads (e.g., polystyrene beads, glass beads, amorphous solid substrates, Luminex™ beads, or the like); magnetic beads; microrods; microwires; quantum dots, and the like; biological micro-objects such as cells; biological organelles; vesicles, or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts, and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated micro-beads, liposome-coated magnetic beads, or the like). Beads may include moieties/molecules covalently or non-covalently attached, such as fluorescent labels, proteins (including receptor molecules), carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species capable of use in an assay. In some variations, beads/solid substrates including moieties/molecules may be capture beads, e.g., configured to bind molecules including small molecules, peptides, proteins or nucleic acids present in proximity either selectively or non-selectively. In one non-limiting example, a capture bead may include a nucleic acid sequence configured to bind nucleic acids having a specific nucleic acid sequence or the nucleic acid sequence of the capture bead may be configured to bind a set of nucleic acids having related nucleic acid sequences. Either type of binding may be understood to be selective. Capture beads containing moieties/molecules may bind non-selectively when binding of structurally different but physico-chemically similar molecules is performed, for example, size exclusion beads or zeolites configured to capture molecules of selected size or charge. Lipid nanorafts have been described, for example, in Ritchie et al. (2009) “Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231. [0052] As used herein, the term “cell” is used interchangeably with the term “biological cell.” Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, or the like, prokaryotic cells, bacterial cells, fungal cells, protozoan cells, or the like, cells dissociated from a tissue, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like, immunological cells, such as T cells, B cells, natural killer cells, macrophages, and the like, embryos (e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells, cells from a cell line, cancer cells, Attorney Docket No.01149-0025-00PCT infected cells, transfected and/or transformed cells, reporter cells, and the like. A mammalian cell can be, for example, from a human, a mouse, a rat, a rabbit, a horse, a goat, a sheep, camelid (including llama and alpaca), a cow, a pig, a primate (e.g., monkey), a bird (e.g., chicken), or the like. [0053] As used herein, the term “B Cell” equates to “B Cell Lymphocyte.” [0054] A colony of biological cells is “clonal” if all of the living cells in the colony that are capable of reproducing are daughter cells derived from a single parent cell. In certain embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 10 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 14 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 17 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 20 divisions. The term “clonal cells” refers to cells of the same clonal colony. [0055] As used herein, a “colony” of biological cells refers to 2 or more cells (e.g. about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells). [0056] As used herein, the term “maintaining (a) cell(s)” refers to providing an environment comprising both fluidic and gaseous components and, optionally a surface, that provides the conditions necessary to keep the cells viable and/or expanding and/or expressing immunoglobulin (i.e., antibody). [0057] As used herein, the term “expanding” when referring to cells, refers to increasing in cell number. [0058] As referred to herein, “gas permeable” means that the material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, the gas permeable material or structure is permeable to more than one of oxygen, carbon dioxide and nitrogen and may further be permeable to all three of these gases. [0059] A “component” of a fluidic medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, or the like. [0060] As referred to herein, a composition “substantially lacks” a component means that the composition is completely devoid of the component (e.g. undetectable) or has a Attorney Docket No.01149-0025-00PCT substantially reduced amount of the component, (e.g., less than 1%, 0.5%, or 0.1%), or that the component is not added to the composition. If any one or more of the conditions, undetectable, less than 1%, 0.5%, or 0.1%, or not added to the composition, are met, the composition substantially lacks a component. [0061] As referred to herein, a composition comprises a “non-effective” concentration or amount of a component means that the concentration or amount does not produce an intended or desired effect. [0062] As referred to herein, certain components of the composition, may include one or more of IL-6, IL-4, IL-21, BAFF, IL-2, which include known wild-type sequences and functionally-active fragments and variants thereof (e.g., at least 90%, 95%, 98% or greater identity to wild-type sequence and/or conservative amino acid substitutions). [0063] As referred to herein, an “agonist” includes any component that directly binds and activates a receptor or which indirectly activates a receptor by forming a complex with another component that binds the receptor or by causing the modification of another component that thereupon directly binds and activates the receptor. As used herein, an “agonist” that is a component of a composition means that the agonist is not made by the cells in culture (e.g., a PKC agonist or a TLR9 agonist). [0064] As used herein in reference to a fluidic medium, “diffuse” and “diffusion” refer to thermodynamic movement of a component of the fluidic medium down a concentration gradient. [0065] The phrase “flow of a medium” means bulk movement of a fluidic medium primarily due to any mechanism other than diffusion, and may encompass perfusion. For example, flow of a medium can involve movement of the fluidic medium from one point to another point due to a pressure differential between the points. Such flow can include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of the liquid, or any combination thereof. When one fluidic medium flows into another fluidic medium, turbulence and mixing of the media can result. Flowing can comprise pulling solution through and out of the microfluidic channel (e.g., aspirating) or pushing fluid into and through a microfluidic channel (e.g. perfusing). [0066] The phrase “substantially no flow” refers to a rate of flow of a fluidic medium that, when averaged over time, is less than the rate of diffusion of components of a material (e.g., an analyte of interest) into or within the fluidic medium. The ratio of a rate of flow of a component in a fluidic medium (i.e., advection) divided by the rate of diffusion of such component can be expressed by a dimensionless Peclet number. Thus, a region within a Attorney Docket No.01149-0025-00PCT microfluidic device that experiences substantially no flow in one in which the Peclet number is less than 1. The Peclet number associated with a particular region within the microfluidic device can vary with the component or components of the fluidic medium being considered (e.g., the analyte of interest), as the rate of diffusion of a component or components in a fluidic medium can depend on, for example, temperature, the size, mass, and/or shape of the component(s), and the strength of interactions between the component(s) and the fluidic medium. In certain embodiments, the Peclet number associated with a particular region of the microfluidic device and a component located therein can be 0.95 or less, 0.9 or less, 0.85 or less, 0.8 or less, 0.75 or less, 0.7 or less, 0.65 or less, 0.6 or less, 0.55 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less. [0067] As used herein in reference to different regions within a microfluidic device, the phrase “fluidically connected” means that, when the different regions are substantially filled with fluid, such as fluidic media, the fluid in each of the regions is connected so as to form a single body of fluid. This does not mean that the fluids (or fluidic media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) which are in flux as solutes move down their respective concentration gradients and/or fluids flow through the device. [0068] As used herein, a “flow path” refers to one or more fluidically connected circuit elements (e.g., channel(s), region(s), chamber(s) and the like) that define, and are subject to, the trajectory of a flow of medium. A flow path is thus an example of a swept region of a microfluidic device. Other circuit elements (e.g., unswept regions) may be fluidically connected with the circuit elements that comprise the flow path without being subject to the flow of medium in the flow path. [0069] As used herein, “isolating a micro-object” confines a micro-object to a defined area within the microfluidic device. [0070] The defined area can be, for example, a chamber. As used herein, a “chamber” is a region within a microfluidic device (e.g., a circuit element) that allows one or more micro- object(s) to be isolated from other micro-objects located within the microfluidic device. Examples of chambers include microwells, which may be regions etched out of a substrate (e.g., a planar substrate), as described in U.S. Patent Application Publication Nos. 2013/0130232 (Weibel et al.) and 2013/0204076 (Han et al.), or a region formed in a multi- layer device, such as the microfluidic devices described in WO 2010/040851 (Dimov et al.) or U.S. Patent Application No. 2012/0009671 (Hansen et al.). Other examples of chambers Attorney Docket No.01149-0025-00PCT include valved chambers, such as described in WO 2004/089810 (McBride et al.) and U.S. Patent Application Publication No.2012/0015347 (Singhal et al.). Other examples of chambers include the chambers described in: Somaweera et al. (2013), “Generation of a Chemical Gradient Across an Array of 256 Cell Cultures in a Single Chip”, Analyst., Vol. 138(19), pp 5566-5571; U.S. Patent Application Publication No. 2011/0053151 (Hansen et al.); and U.S. Patent Application Publication No. 2006/0154361 (Wikswo et al.). Still other examples of chambers include the sequestration pens described in detail herein. In certain embodiments, the chamber can be configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, the chamber can be configured to hold a volume of fluid of about 20 nL to 200nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL. [0071] As used herein, “pen” or “penning” specifically refers to disposing micro- objects within a sequestration pen within the microfluidic device. Forces used to pen a micro- object may be any suitable force as described herein such as dielectrophoresis (DEP), e.g., an optically actuated dielectrophoretic force (OEP); gravity; magnetic forces; locally actuated fluid flow; or tilting. In some embodiments, penning a plurality of micro-objects may reposition substantially all the micro-objects. In some other embodiments, a selected number of the plurality of micro-objects may be penned, and the remainder of the plurality may not be penned. In some embodiments, when selected micro-objects are penned, a DEP force, e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects. Typically, micro-objects may be introduced to a flow region, e.g., a microfluidic channel, of the microfluidic device and thereafter introduced into a chamber by penning. [0072] As used herein, “unpen” or “unpenning” refers to repositioning micro-objects from within a sequestration pen to a new location within a flow region, e.g., a microfluidic channel, of the microfluidic device. Forces used to unpen a micro-object may be any suitable force as described herein such as dielectrophoresis, e.g., an optically actuated dielectrophoretic force; gravity; magnetic forces; locally actuated fluid flow; or tilting. In some embodiments, unpenning a plurality of micro-objects may reposition substantially all the micro-objects. In some other embodiments, a selected number of the plurality of micro-objects may be unpenned, and the remainder of the plurality may not be unpenned. In some embodiments, when selected Attorney Docket No.01149-0025-00PCT micro-objects are unpenned, a DEP force, e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects. [0073] As used herein, “export” or “exporting” can include, consist of, or consist essentially of repositioning micro-objects from a location within a microfluidic device, e.g., a flow region, a microfluidic channel, a chamber, etc., to a location outside of the microfluidic device, such as a well plate, a tube, or other receiving vessel. In some embodiments, exporting a micro-object comprises withdrawing (e.g., micro-pipetting) a volume of medium containing the micro-object from within the microfluidic device and depositing the volume of medium in or upon the location outside of the microfluidic device. In some related embodiments, withdrawing the volume of medium is preceded by disassembling the microfluidic device (e.g., removing an upper layer, such as a cover or lid, of the microfluidic device from a lower layer , such as a base or substrate, of the microfluidic device) to facilitate access (e.g., of a micro- pipetted) to the internal regions of the microfluidic device. In other embodiments, exporting a micro-object comprises flowing a volume of fluid containing the micro-object through the flow region (including, e.g., a microfluidic channel) of the microfluidic device, out through an outlet of the microfluidic device, and depositing the volume of medium in or upon the location outside of the microfluidic device. In such embodiments, micro-object(s) within the microfluidic channel may be exported without requiring disassembly (e.g., removal of the cover of the device) or insertion of a tool into an interior region of the microfluidic device to remove micro- objects for further processing. “Export” or “exporting” may further comprise repositioning micro-objects from within a chamber, which may include a sequestration pen, to a new location within a flow region, such as a microfluidic channel, as described above with regard to “unpenning”. A planar orientation of the chamber(s) with respect to the microfluidic channel, such that the chamber(s) opens laterally from the microfluidic channel, as described herein with regard to sequestration pens, permits easy export of micro-objects that have been positioned or repositioned (e.g., unpenned from a chamber) to be disposed within the microfluidic channel. [0074] A microfluidic (or nanofluidic) device can comprise “swept” regions and “unswept” regions. As used herein, a “swept” region is comprised of one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which experiences a flow of medium when fluid is flowing through the microfluidic circuit. The circuit elements of a swept region can include, for example, regions, channels, and all or parts of chambers. As used herein, an “unswept” region is comprised of one or more fluidically interconnected circuit element of a microfluidic circuit, each of which experiences substantially no flux of fluid when fluid is flowing through the microfluidic circuit. An unswept region can be fluidically connected to a Attorney Docket No.01149-0025-00PCT swept region, provided the fluidic connections are structured to enable diffusion but substantially no flow of media between the swept region and the unswept region. The microfluidic device can thus be structured to substantially isolate an unswept region from a flow of medium in a swept region, while enabling substantially only diffusive fluidic communication between the swept region and the unswept region. For example, a flow channel of a micro-fluidic device is an example of a swept region while an isolation region (described in further detail below) of a microfluidic device is an example of an unswept region. [0075] As used herein, a “non-sweeping” rate of fluidic medium flow means a rate of flow sufficient to permit components of a second fluidic medium in an isolation region of the sequestration pen to diffuse into the first fluidic medium in the flow region and/or components of the first fluidic medium to diffuse into the second fluidic medium in the isolation region; and further wherein the first medium does not substantially flow into the isolation region. [0076] As used herein, an “isolation region” refers to a region within a microfluidic device that is configured to hold a micro-object such that the micro-object is not drawn away from the region as a result of fluid flowing through the microfluidic device. Depending upon context, the term “isolation region” can further refer to the structures that define the region, which can include a base/substrate, walls (e.g., made from microfluidic circuit material), and a cover. [0077] As used herein, “antibody” refers to an immunoglobulin (Ig) and includes both polyclonal and monoclonal antibodies; multichain antibodies, such as IgG, IgM, IgA, IgE, and IgD antibodies; single chain antibodies, such as camelid antibodies; mammalian antibodies, including primate antibodies (e.g., human), rodent antibodies (e.g., mouse, rat, guinea pig, hamster, and the like), lagomorph antibodies (e.g., rabbit), ungulate antibodies (e.g., cow, pig, horse, donkey, camel, and the like), and canidae antibodies (e.g., dog); primatized (e.g., humanized) antibodies; chimeric antibodies, such as mouse-human, mouse-primate antibodies, or the like; and may be an intact molecule or a fragment thereof (such as a light chain variable region (VL), heavy chain variable region (VH), scFv, Fv, Fd, Fab, Fab' and F(ab)'2 fragments), or multimers or aggregates of intact molecules and/or fragments; and may occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering. An “antibody fragment,” as used herein, refers to fragments, derived from or related to an antibody, which bind antigen. In some embodiments, antibody fragments may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues. The capability of biological micro-objects (e.g., biological cells) to produce specific biological materials (e.g., proteins, such as antibodies) can be assayed in such a microfluidic device. In a specific Attorney Docket No.01149-0025-00PCT embodiment of an assay, sample material comprising biological micro-objects (e.g., cells) to be assayed for production of an analyte of interest can be loaded into a swept region of the microfluidic device. Ones of the biological micro-objects (e.g., mammalian cells, such as human cells) can be selected for particular characteristics and disposed in unswept regions. The remaining sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Because the selected biological micro-objects are in unswept regions, the selected biological micro-objects are not substantially affected by the flowing out of the remaining sample material or the flowing in of the assay material. The selected biological micro-objects can be allowed to produce the analyte of interest, which can diffuse from the unswept regions into the swept region, where the analyte of interest can react with the assay material to produce localized detectable reactions, each of which can be correlated to a particular unswept region. Any unswept region associated with a detected reaction can be analyzed to determine which, if any, of the biological micro-objects in the unswept region are sufficient producers of the analyte of interest. [0078] An antigen, as referred to herein, is a molecule or portion thereof that can bind with specificity to another molecule, such as an Ag-specific receptor. An antigen may be any portion of a molecule, such as a conformational epitope or a linear molecular fragment, and often can be recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system. An antigen may include a peptide, polysaccharide, or lipid. An antigen may be characterized by its ability to bind to an antibody's variable Fab region. Different antibodies have the potential to discriminate among different epitopes present on the antigen surface, the structure of which may be modulated by the presence of a hapten, which may be a small molecule. [0079] In some embodiments, an antigen is a cancer cell- associated antigen. The cancer cell-associated antigen can be simple or complex; the antigen can be an epitope on a protein, a carbohydrate group or chain, a biological or chemical agent other than a protein or carbohydrate, or any combination thereof; the epitope may be linear or conformational. [0080] The cancer cell-associated antigen can be an antigen that uniquely identifies cancer cells (e.g., one or more particular types of cancer cells) or is upregulated on cancer cells as compared to its expression on normal cells. Typically, the cancer cell-associated antigen is present on the surface of the cancer cell, thus ensuring that it can be recognized by an antibody. The antigen can be associated with any type of cancer cell, including any type of cancer cell that can be found in a tumor known in the art or described herein. In particular, the antigen can be associated with lung cancer, breast cancer, melanoma, and the like. As used herein, the term Attorney Docket No.01149-0025-00PCT “associated with a cancer cells,” when used in reference to an antigen, means that the antigen is produced directly by the cancer cell or results from an interaction between the cancer cell and normal cells. [0081] The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. [0082] As used herein, “B” used to denote a single nucleotide, is a nucleotide selected from G (guanosine), C (cytidine) and T (thymidine) nucleotides but does not include A (adenine). [0083] As used herein, “H” used to denote a single nucleotide, is a nucleotide selected from A, C and T, but does not include G. [0084] As used herein, “D” used to denote a single nucleotide, is a nucleotide selected from A, G, and T, but does not include C. [0085] As used herein, “K” used to denote a single nucleotide, is a nucleotide selected from G and T. [0086] As used herein, “M” used to denote a single nucleotide, is a nucleotide selected from A or C. [0087] As used herein, “N” used to denote a single nucleotide, is a nucleotide selected from A, C, G, and T. [0088] As used herein, “R” used to denote a single nucleotide, is a nucleotide selected from A and G. [0089] As used herein, “S” used to denote a single nucleotide, is a nucleotide selected from G and C. [0090] As used herein, “V” used to denote a single nucleotide, is a nucleotide selected from A, G, and C, and does not include T. [0091] As used herein, “Y” used to denote a single nucleotide, is a nucleotide selected from C and T. II. Compositions for Culturing and/or Maintaining B Cells [0092] In some embodiments, compositions for culturing B cells, including memory B cells, which may be human B cells (e.g., human memory B cells) are provided. By culturing B cells in the disclosed compositions, the B cells can be activated, resulting in proliferation and/or immunoglobulin (e.g., antibody) secretion, and/or maintained in a state of sustained Attorney Docket No.01149-0025-00PCT immunoglobulin (e.g., antibody) expression/secretion. Methods of culturing, activating, and/or screening B cells that involve contacting the B cells with one or more compositions disclosed herein are also provided. [0093] In some embodiments, the composition may be an activation composition that promotes B cell activation and/or expansion. In some embodiments, the composition results in proliferation of B cells, and/or increasing the size of B cells, and/or initiating and/or increasing immunoglobulin expression in B cells, for example by differentiating memory B cells into plasmablasts. In some embodiments, the activation composition can comprise a Toll-like receptor 9 (TLR9) agonist (e.g., a TLR9-specific agonist), IL-6, and IL-4, and lack feeder cells. In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide (ODN), which may be a B-class CpG ODN and/or have a linear structure. In some embodiments, the CpG ODN is present at a concentration of at least about 0.25 micromolar (µM), at least about 0.5 µM, at least about 1.0 µM, at least about 1.5 µM, at least about 2.0 µM, or at least about 2.5 µM, or in some embodiments at least about 5 µM. In some embodiments, the CpG ODN is present at a concentration from about 0.25 µM to about 5.0 µM, from about 0.25 µM to about 4.0 µM, from about 0.5 µM to about 3.0 µM, from about 1.0 µM to about 2.0 µM, from about 0.25 µM to about 1.0 µM, from about 0.5 µM to about 1.5 µM, from about 1.0 µM to about 2.5 µM, from about 1.5 µM to about 3.0 µM, from about 2.0 µM to about 4.0 µM, from about 2.5 µM to about 5.0 µM, or any range defined by two of the foregoing endpoints. In some embodiments, the composition comprises IL-6 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL. In some embodiments, the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 5.0 ng/mL to about 25 ng/mL, from about 10 ng/mL to about 50 ng/mL, from about 15 ng/mL to about 75 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints In some embodiments, the IL-6 is human IL-6. In some embodiments, the composition comprises IL-4 at a concentration of at least about 0.5 ng/mL, at least about 1.0 ng/mL, at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL. In some embodiments, the composition comprises IL- 4 at a concentration from about 0.5 ng/mL to about 10 ng/mL, from about 1.0 ng/mL to about 2.5 ng/mL, from about 1.5 ng/mL to about 5.0 ng/mL, from about 2.0 ng/mL to about 7.5 ng/mL, from about 2.5 ng/mL to about 10 ng/mL, or any range defined by two of the foregoing endpoints. In some embodiments, the IL-4 is human IL-4. Attorney Docket No.01149-0025-00PCT [0094] In certain embodiments, the activation composition further comprises IL-2, IL- 21, BAFF, or any combination thereof. For example, in certain embodiments, the composition comprises IL-2, IL-21, and/or BAFF. The IL-2, IL-21, and BAFF can each, independently, be a human protein. In some embodiments, the composition comprises IL-2. In some embodiments, the composition comprises IL-2 at a concentration of at least about 1.0 ng/mL, at least about 10 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, or at least about 100 ng/mL. In some embodiments, the composition comprises IL-2 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 10 ng/mL to about 50 ng/mL, from about 25 ng/mL to about 75 ng/mL, from about 50 ng/mL to about 100 ng/mL, from about 5 ng/mL to about 25 ng/mL, from about 25 ng/mL to about 50 ng/mL, from about 50 ng/mL to about 75 ng/mL, from about 75 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints. In some embodiments, the IL-2 is human IL-2. In some embodiments, the composition comprises IL-21. In some embodiments, the composition comprises IL-21 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL. In some embodiments, the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 2.0 ng/mL to about 20 ng/mL, from about 5.0 ng/mL to about 40 ng/mL, from about 10 ng/mL to about 60 ng/mL, from about 15 ng/mL to about 80 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints. In some embodiments, the IL-21 is human IL-21. In some embodiments, the composition comprises BAFF. In some embodiments, the composition comprises BAFF at a concentration of at least about 10 ng/mL, at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL. In some embodiments, the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL, from about 20 ng/mL to about 50 ng/mL, from about 30 ng/mL to about 60 ng/mL, from about 40 ng/mL to about 70 ng/mL, from about 50 ng/mL to about 80 ng/mL, from about 60 ng/mL to about 90 ng/mL, from about 70 ng/mL to about 100 ng/mL, or any range defined by two of the forgoing endpoints. In some embodiments, the BAFF is human BAFF. [0095] In certain embodiments, the activation composition comprises a non-effective concentration of a protein kinase C (PKC) agonist (e.g., less than about 10 ng/mL) or substantially lacks PKC agonist. In some embodiments, the composition comprises a PKC agonist at a concentration of less than about 10 ng/mL, less than about 5.0 ng/mL, less than Attorney Docket No.01149-0025-00PCT about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, or substantially lacks PKC agonist. [0096] In certain embodiments, the activation composition can further comprise serum (e.g., any serum suitable for culturing B cell lymphocytes), which can be present at an amount of about 1% to about 20% v/v, about 2% to about 15%, or about 5% to about 10%. The serum can be, for example, fetal bovine serum. In certain embodiments, the composition is a liquid medium. [0097] In some embodiments, the composition may be a maintaining composition for maintaining immunoglobulin expression in the B cells. In some embodiments, the maintaining composition can comprise a protein kinase C (PKC) agonist (e.g., a PKC-specific agonist) and lack feeder cells. In certain embodiments, the PKC agonist is a phorbol compound, which may be a phorbol ester. In some embodiments, the phorbol ester may be phorbol 12-myristate 13- acetate (PMA), phorbol 12,13-dibutyrate (PBD), 4-alpha-phorbol 12-myristate 13-acetate (4- alpha-PMA), dPPA, 4-alpha-phorbol (isophorbol), phorbol 12-myristate 13-acetate 4-O- methyl ether (MPMA), or the like. Examples of non-limiting phorbol esters are shown in Table 1 below. In certain embodiments, the PKC agonist is present at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 25 ng/mL, at least about 50 ng/mL, at least about 75 ng/mL, at least about 100 ng/mL, at least about 150 ng/mL, at least about 200 ng/mL, at least about 250 ng/mL, at least about 500 ng/mL, or at least about 750 ng/mL. In some embodiments, the PKC agonist is present at a concentration from about 10 ng/mL to about 1000 ng/mL, from about 25 ng/mL to about 200 ng/mL, from about 50 ng/mL to about 250 ng/mL, from about 75 ng/mL to about 300 ng/mL, from about 100 ng/mL to about 400 ng/mL, from about 150 ng/mL to about 500 ng/mL, from about 200 ng/mL to about 600 ng/mL, from about 250 ng/mL to about 700 ng/mL, from about 300 ng/mL to about 800 ng/mL, from about 400 ng/mL to about 900 ng/mL, from about 500 ng/mL to about 1000 ng/mL, or any range defined by two of the foregoing endpoints.
Figure imgf000032_0001
Attorney Docket No.01149-0025-00PCT
Figure imgf000033_0001
[0098] In certain embodiments, the maintaining composition further comprises IL-6, IL-4, IL-21, BAFF, or any combination thereof. For example, in certain embodiments, the Attorney Docket No.01149-0025-00PCT composition comprises IL-6 and/or IL-4. The IL-6, IL-4, IL-21, and BAFF can each, independently, be a human protein. In some embodiments, the composition comprises IL-6 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 20 ng/mL, or at least about 40 ng/mL. In some embodiments, the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 5.0 ng/mL to about 25 ng/mL, from about 10 ng/mL to about 50 ng/mL, from about 15 ng/mL to about 75 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints. In some embodiments, the IL-6 is human IL-6. In some embodiments, the composition comprises IL-4 at a concentration of at least about 0.5 ng/mL, at least about 1.0 ng/mL, at least about 1.5 ng/mL, at least about 2.0 ng/mL, at least about 2.5 ng/mL, at least about 3.0 ng/mL, at least about 3.5 ng/mL, or at least about 4 ng/mL. In some embodiments, the composition comprises IL- 4 at a concentration from about 0.5 ng/mL to about 10 ng/mL, from about 1.0 ng/mL to about 2.5 ng/mL, from about 1.5 ng/mL to about 5.0 ng/mL, from about 2.0 ng/mL to about 7.5 ng/mL, from about 2.5 ng/mL to about 10 ng/mL, or any range defined by two of the foregoing endpoints. In some embodiments, the IL- 4 is human IL-4. In some embodiments, the composition comprises IL-21. In some embodiments, the composition comprises IL-21 at a concentration of at least about 1.0 ng/mL, at least about 2.0 ng/mL, at least about 5.0 ng/mL, at least about 10 ng/mL, at least about 15 ng/mL, or at least about 20 ng/mL. In some embodiments, the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL, from about 2.0 ng/mL to about 20 ng/mL, from about 5.0 ng/mL to about 40 ng/mL, from about 10 ng/mL to about 60 ng/mL, from about 15 ng/mL to about 80 ng/mL, from about 20 ng/mL to about 100 ng/mL, or any range defined by two of the foregoing endpoints. In some embodiments, the IL-21 is human IL-21. In some embodiments, the composition comprises BAFF. In some embodiments, the composition comprises BAFF at a concentration of at least about 10 ng/mL, at least about 20 ng/mL, at least about 30 ng/mL, at least about 40 ng/mL, at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, or at least about 90 ng/mL. In some embodiments, the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL, from about 20 ng/mL to about 50 ng/mL, from about 30 ng/mL to about 60 ng/mL, from about 40 ng/mL to about 70 ng/mL, from about 50 ng/mL to about 80 ng/mL, from about 60 ng/mL to about 90 ng/mL, from about 70 ng/mL to about 100 ng/mL, or any range defined by two of the forgoing endpoints. In some embodiments, the BAFF is human BAFF. Attorney Docket No.01149-0025-00PCT [0099] In certain embodiments, the maintaining composition comprises a non-effective concentration of IL-2. In some embodiments, the non-effective concentration of IL-2 comprises a concentration of less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL or substantially lacks IL-2. In certain embodiments, the maintaining composition comprises a non-effective concentration of TNF-alpha. In certain embodiments, the non-effective concentration of TNF-alpha comprises a concentration of less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks TNF-alpha. In certain embodiments, the maintaining composition comprises a non-effective concentration of CpG oligodeoxynucleotides (ODNs). In some embodiments, the non-effective concentration of CpG oligodeoxynucleotides (ODNs) comprises a concentration of less than about 250 nanomolar (nM), less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 10 nM, less than about 1 nM, or substantially lacks CpG ODNs. In some embodiments, the ODNs, may include class B/linear ODNs, and in particular ODN 2006. In certain embodiments, the maintaining composition comprises a non-effective concentration of a PI3K agonist. In some embodiments, the non-effective concentration of a PI3K agonist comprises less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks PI3K agonist. In some of the embodiments, the PI3K agonist may include naringenin, naringin, butin, eriodictyol, homoeriodictyol, hesperetin, hesperidin, and isosakuranetin. In certain embodiments, the maintaining composition comprises a non- effective amount of a STAT3 agonist. In some embodiments, the non-effective amount of a STAT3 agonist comprises a concentration of less than about 1.0 ng/mL, less than about 0.5 ng/mL, less than about 0.1 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks STAT3 agonist. In some embodiments, the STAT3 agonist may include leukemia inhibitory factor (LIF) (e.g., human LIF (hLIF)), as well as fragments and variants that retain STAT3 agonist activity. In some embodiments, LIF the non-effective amount of LIF comprises a concentration of less than about 10 ng/ml, less than about 1.0 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks LIF. In certain embodiments, the maintaining composition comprises a non-effective amount of a CD40-clustering agent. In some embodiments, the non-effective amount of a Attorney Docket No.01149-0025-00PCT CD40-clustering agent comprises a concentration of less than about 1.0 ng/mL, less than about 0.05 ng/mL, less than about 0.01 ng/mL, less than about 0.005 ng/mL, less than about 0.001 ng/mL, or substantially lacks CD40-clustering agent. In some embodiments, the CD40- clustering agent may include CD40 ligand (i.e., CD40L), active fragments and variants thereof, and anti-CD40 agonist antibodies, and in some embodiments, the CD40 clustering agent may be attached to a bead that contacts the cell. [0100] In certain embodiments, the maintaining composition can further comprise serum (e.g., any serum suitable for culturing B cell lymphocytes), which can be present at an amount of about 1% to about 20% v/v, or about 2% to about 15%, or about 5% to about 10%. The serum can be, for example, fetal bovine serum. In certain embodiments, the composition is a liquid medium. [0101] In some embodiments, the B cells may be cultured in the activating composition followed by culture in the maintaining composition in accordance with any of the embodiments described herein. III. Methods for Antibody Discovery [0102] As mentioned above, the time needed in screening cells for lead candidates using macroscale workflows that are typically currently used, significantly adds to the drug development timeline. Thus, it is urgently needed to reduce the time needed for screening cells capable of secreting a desired antibody, to thereby accelerate antibody discovery. FIG. 6A shows a general workflow which is directed to providing acceleration of antibody discovery campaigns. The method includes isolating B cells and importing the cells in a microfluidic device, preferably the microfluidic device as disclosed in the following sections. The cells can be loaded into the channel or chamber of the microfluidic device and cultured individually. In some embodiments, up to 50k single B cells may be loaded. In some embodiments, cells that are determined to be healthy (e.g., viable), substantially healthy, or enriched in a proportion of cells that are healthy, may be introduced preferentially to the chamber(s) of the microfluidic device. [0103] The method may also include conducting binding or functional assays, which can be, but is not limited to bead-based analyses for testing the IgG-antigen specificity of the antibodies secreted in each pen. The method may further include loading nucleic acid capture objects, which may be any nucleic acid capture object at described herein, and performing on- chip lysis, nucleic acid capture and reverse transcription. As explained in more detail in the following sections, barcoded cDNA sequences are generated through these steps by using the Attorney Docket No.01149-0025-00PCT capture objects of the present disclosure. The nucleic acid capture objects additionally are labelled, to permit correlation of the binding/functional assay results with the specific nucleic acid isolated from the cell(s) responsible for the assay results. Detection of the labels may be performed at any point during the workflow to identify the label for each capture object in each chamber. [0104] Subsequently, the barcoded cDNA sequences, which are captured on the capture objects and comprise the BCR sequence (i.e., barcoded BCR beads), may be exported to an off-chip culture plate. In some embodiments, barcoded BCR beads from over 1000 pens can be unloaded to a single 96-well plate and permit multiplexing of subsequent processes. [0105] As explained in more detail in WO2022051570, the capture objects enable the identification of the origin of the barcoded BCR beads on the 96-well plate. Last, subsequent analyses including sequencing and/or selective cloning of BCR sequences, performing bioinformatics visualization or re-expression of BCR sequences may be performed. Further, in some embodiments, secondary screenings can be conducted. In some embodiments, the method of the present disclosure aims at increasing screening throughput to up to 50k single plasma B cells and over 1000 exports of target B cell receptor (BCR) sequences. Overall, this workflow provides for high throughput antibody discovery methods. IV. Methods for Identification of Healthy Cells Prior to Importation into a Chamber. [0106] Identifying healthy cells before importing the cells into a chamber can offer benefits in the methods of the present disclosure. As referred to herein, a healthy cell is a cell demonstrating characteristics of viability, e.g., is a viable cell and has the ability to continue to grow and optionally, produce either biomolecules of interest and/or produce daughter cells having the same capabilities. Disposing into the chambers, e.g., sequestration pens, of the microfluidic device only, substantially only or an increased proportion of healthy cells out of an imported population can increase the likelihood of identifying useful cells/clonal populations thereof. Further, resources used during a biomolecule production development/identification campaign are not expended upon non-viable cells, reducing waste and preserving the use of the pre-defined number of chambers for cells that have some possibility of expressing the biomolecule of interest. [0107] Thus, another aspect of the present disclosure is to identify healthy cells before importing them into the chamber of the microfluidic device. However, identifying healthy cells within a microfluidic device can be difficult, due to the very nature of the small scale of the microfluidic device. Furthermore, for a single cell culture scheme, only a relatively small Attorney Docket No.01149-0025-00PCT number of cells may be imported into the device, and staining of such small number of cells may not be able to generate a fluorescent intensity sufficient for meaningful detection. Additionally, for some biomolecule production methods, it may be desirable to not include any sort of dyes or stains to the cells themselves, depending on the downstream uses of the cells. Therefore, it is useful to develop a method of identifying and importing healthy cells which does not rely upon staining every batch of cells to be imported into sequestration pens. [0108] In some embodiments, a staining method can be combined with a brightfield image observation for the purposes of identifying healthy cells. [0109] In some embodiments, identifying a healthy cell can involve the use of a machine learning algorithm to process image data. In some embodiment, the machine learning algorithm is capable of identifying healthy cells without staining. The machine learning algorithm can include a neural network, such as a convolutional neural network. A convolutional neural network (CNN) generally accomplishes an advanced form of image processing and classification/detection by first looking for low level features such as, for example, edges and curves, and then advancing to more abstract (e.g., unique to the type of images being classified) concepts through a series of convolutional layers. A CNN can do this by passing an image through a series of convolutional, nonlinear, pooling (or downsampling, as will be discussed in more detail below), and fully connected layers, and get an output. The output can be a single class or a probability of classes that best describes the image or detects objects on the image. Some examples of CNNs useful in these methods include have been described, for example, International Application Publication No. WO 2019/232473, entitled “Automated Detection and Characterization of Micro-Object in Microfluidic Devices”, filed on May 31, 2019; and in International Application Publication No. WO2018102748, entitled “Automated Detection and Characterization of Micro-Object in Microfluidic Devices”, filed on December 1, 2017, each of which disclosures are incorporated herein by reference. [0110] In some embodiments, a training data used in establishing the CNN model of the present disclosure may include a fluorescent image having the cells of interest stained, a brightfield image having the cells of interest annotated, or combinations thereof. The dyes suitable in the present disclosure may include but are not limited to calcein, zombie violet stain, annexin, acridine orange, propidium iodide, or combinations thereof. Any suitable stain that discriminates between a healthy cell and a dead/dying and/or non-viable cell, as is known to one of skill in the art may be used. In some embodiments, other dyes that are specific to a marker of interest can also be used, for instance, Alexa Fluor® 647 anti-mouse CD138 (Syndecan-1) Antibody (BioLegend), which is highly specific for terminally differentiated live Attorney Docket No.01149-0025-00PCT plasma cells and stains CD138 presented on the surface. In some embodiments, two or more dyes can be used in staining a sample to provide cross-reference or verification. [0111] In a particular embodiment, a training data includes images of cells stained with a fluorescent dye in combination with images of the cells under brightfield. A healthy cell can be identified, for example, by observing the morphologies of the cells under brightfield. In some embodiments, a healthy cell, e.g., a viable cell, can be characterized as having a clear cell boundary, good contrast, round shape, or combinations thereof. In some embodiment, a healthy cell can be determined by identifying the unhealthy ones. For instance, an unhealthy cell can be characterized as having debris-like appearance, unclear or different contrast, or combinations thereof. In many embodiments, assessment of viability can be made in a relative manner by comparing cells in a sample. For instance, a healthy cell can have a larger diameter while other cells having a smaller diameter are more likely to be unhealthy/dead or merely cell debris. [0112] In the training regime, cells may be first detected under brightfield and then labeled as live/dead based on the fluorescent intensity. In some embodiment, labeling of live/dead cells is based on a cutoff value of the fluorescent intensity, which can be selected in accordance with the user’s likings or needs. [0113] After training is accomplished with the type of cell under investigation, the method of penning healthy cells using the trained machine learning algorithm may be employed to increase the penning efficiency of healthy cells, and decrease the numbers of non-viable cells penned. In some embodiments, the percentage of healthy cells relative to non-viable cells imported into the chambers, e.g., sequestration pens, after identification by the algorithm, may be improved by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. V. A Method of Assaying For A Specific Binding Interaction Between A First Molecule And A Second Molecule [0114] Binding interactions between a first molecule and a second molecule can be measured in a chamber of a microfluidic chip. The chamber can be any of the chambers described or referenced herein, including a microwell or a sequestration pen, and the assay formats can vary widely. For example, the assay can be a “sandwich” assay in which a surface, such as a bead or an internal surface of a wall of the microfluidic device, is configured to capture and/or present the first molecule; binding of the second molecule is detected via a third molecule that is labeled and capable of binding to the complex formed by the second molecule binding to the first molecule, thereby associating the label of the third molecule with the surface Attorney Docket No.01149-0025-00PCT in a detectable manner. In such assays, the second molecule can be produced by a biological cell. The assay surface can be in the chamber (e.g., as described in U.S. Patent Application Publication No. 2015/0165436 and PCT International Publication No. WO 2010/040851) or proximal to the chamber, such as in a channel that the chamber connects with (e.g., as described in U.S. Patent Application Publication No.2015/0151298). Alternatively, the assay can be a diffusion gradient assay in which the second molecule has a label (which can be linked to the second molecule, or can be an intrinsic property of the second molecule, such as auto- fluorescence) and the diffusive properties of the labeled second molecule in the presence of the first molecule can be monitored, e.g., as described in PCT International Publication No. WO 2017/181135. In such assays, the first molecule can be produced by a biological cell. Still other assays can feature a blocking interaction in which a molecule of interest binds to the first molecule and thereby blocks an interaction of the first molecule with the second molecule. In such assays, the molecule of interest can be produced by a biological cell, and the second molecule can comprise a label. As with the sandwich assays, the blocking assays can feature the first molecule bound to a surface. The surface can be located, for example, in the chamber or a region proximal to the chamber, such as a channel. Examples of blocking assays are described below and elsewhere herein, including in the examples and in the claims. [0115] In-channel Binding Assay. In some embodiments, a method of assay for a specific binding interaction between a first molecule and a second molecule is provided. The method can be performed within a microfluidic device having a channel and a chamber, such as a microwell or a sequestration pen, fluidically connecting to the channel. The method can include: introducing each of a plurality of biological cells into a respective one of a plurality of chambers; incubating the biological cells and allowing the biological cell to produce and/or secrete a molecule of interest; introducing a micro-object including a plurality of first molecules into the channel; and monitoring an accumulation of the molecule of interest on the micro-object. [0116] In some embodiments, monitoring an accumulating of the molecule of interest on the micro-object including introducing a third molecule that is labeled and capable of binding to the complex formed by the molecule of interest binding to the first molecule, thereby associating the label of the third molecule with the accumulation of the molecule of interest on the micro-object. Some aspects of an in-channel assay using micro-objects comprising beads having a plurality of first molecules are further described in an International Application filed on October 22, 2014, and published as International Publication WO2015/061497. Attorney Docket No.01149-0025-00PCT [0117] In some embodiments, introducing a micro-object including a plurality of first molecules, e.g., a reporter cell, into the channel including introducing a plurality of the micro- objects and allowing the plurality of the micro-objects fill the channel at a density. In some embodiments, an optimal density is such that nearly the entire channel is filled with the micro- objects. A density that is below optimal might result in a sparse number of micro-objects in the channel and an under-sampling of the secreted molecule of interest, making unambiguous identification of the secreting chamber difficult. On the other hand, an overly concentrated density might lead to higher risk of channel blockages, poor uniformity across the chip, and might lead to the micro-objects getting pushed into the chambers. In some embodiments, the optimal density can vary depending on the size of the micro-objects introduced. In certain embodiments, the micro-objects are biological cells, and the density can be from about 107 to 109, or about 108 to 2x108 cells/mL. In some embodiments, the micro-object including the plurality of first molecules, e.g., reporter cells, may be cells that may be cells that culture in suspension. In other embodiments, adherent cell types may be used as reporter cells when detachment protocols are used. For example, adherent CHO cells may be successfully used when a detachment protocol may include: culturing to confluence prior to importation, e.g. not exceeding confluence; and treating with a detachment reagent such as Accutase (ThermoFisher Scientific, A1110501), TrypLE or the like, e.g. treatment at about 22°C for 10 min with no agitation. The adherent CHO cells were then successfully importable as monodisperse cells and the target cell densities were achieved. Specific detachment protocols may be determined as needed for other cell types. The preparatory culture density may be varied, e.g., less than about 100% confluent, less than about 90% confluent, less than about 80% confluent, less than about 60% confluent, or less than about 50% confluent. The detachment reagent may be varied. The duration of the detachment treatment may be varied, e.g., from about 5 min to about 1h, about 10 min, about 15 min, about 20 min, about 30 min, about 45 min, about 60 min or any value therebetween. In some embodiments, agitation is not employed. In yet other embodiments, the cells may be agitated during the detachment treatment. Temperature may be varied in order to successfully detach the cells, and may be varied from about 15°C to about 36°C, about 10°C to about 40°C, or any temperature therebetween. Filtration through a cell strainer may be useful to remove cell clumps or other large debris, and may be performed before concentrating the cells to target import concentration. The cells may be concentrated by centrifuging at 400 x g for 5 min, and resuspended to the desired concentration. The third molecule, which is labeled and capable of binding to the complex formed by the molecule of Attorney Docket No.01149-0025-00PCT interest binding to the first molecule, e.g., a labelled antibody, may be added to the media when resuspending the cells. [0118] In some embodiments, the first molecule and/or the molecule of interest can be a protein. The protein can be, for example, a cell surface protein or an extracellular protein. The protein can be a modified protein, such as a glycosylated protein, a lipid-anchored protein, or the like. In some embodiments, the molecule of interest can specifically bind to the first molecule. In certain embodiments, the first molecule and molecule of interest can be an antigen-antibody pair. For example, the biological cell can be a B cell producing an antibody of interest (i.e., molecule of interest) and the first molecule presented on the surface of the micro-objects can be an antigen or epitope of the produced antibody. In some embodiments, the third molecule can be a secondary antibody binding to the produced antibodies (i.e., the secreted second molecule), and the detection thereof is associated with the binding of the first molecule and the molecule of interest. [0119] In certain embodiments, the micro-object can be one or more beads or cells that express the first molecule. If a cell, the cell can express the first molecule naturally or can be genetically modified (e.g., stably or transiently transfected) to express the first molecule. If a bead, the bead can be created by conjugating the first molecule on its surface. VI. Methods of Assembling Full Length V(D)J sequences from fragmented NGS (Next Generation Sequencing, massively parallel sequencing) data [0120] Methods for assembly of complete V(D)J sequences from fragmented NGS data originating from a single antibody producing cell (e.g., a B-Cell) may be used, as described, for example, in WO2022051570, published on March 10, 2022. VII. Device and System [0121] Microfluidic device/system feature cross- applicability. It should be appreciated that various features of microfluidic devices, systems, and motive technologies described herein may be combinable or interchangeable. For example, features described herein with reference to the microfluidic device 100, 175, 200, 300, 320, 400, 450, 520 and system attributes as described in FIGS.1A-5B may be combinable or interchangeable. [0122] Microfluidic devices. FIG.1A illustrates an example of a microfluidic device 100. A perspective view of the microfluidic device 100 is shown having a partial cut-away of its cover 110 to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally comprises a microfluidic circuit 120 comprising a flow path 106 through Attorney Docket No.01149-0025-00PCT which a fluidic medium 180 can flow, optionally carrying one or more micro-objects (not shown) into and/or through the microfluidic circuit 120. [0123] As generally illustrated in FIG.1A, the microfluidic circuit 120 is defined by an enclosure 102. Although the enclosure 102 can be physically structured in different configurations, in the example shown in FIG.1A the enclosure 102 is depicted as comprising a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110. The support structure 104, microfluidic circuit structure 108, and cover 110 can be attached to each other. For example, the microfluidic circuit structure 108 can be disposed on an inner surface 109 of the support structure 104, and the cover 110 can be disposed over the microfluidic circuit structure 108. Together with the support structure 104 and cover 110, the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120, forming a three-layer structure. [0124] The support structure 104 can be at the bottom and the cover 110 at the top of the microfluidic circuit 120 as illustrated in FIG.1A. Alternatively, the support structure 104 and the cover 110 can be configured in other orientations. For example, the support structure 104 can be at the top and the cover 110 at the bottom of the microfluidic circuit 120. Regardless, there can be one or more ports 107 each comprising a passage into or out of the enclosure 102. Examples of a passage include a valve, a gate, a pass-through hole, or the like. As illustrated, port 107 is a pass-through hole created by a gap in the microfluidic circuit structure 108. However, the port 107 can be situated in other components of the enclosure 102, such as the cover 110. Only one port 107 is illustrated in FIG.1A but the microfluidic circuit 120 can have two or more ports 107. For example, there can be a first port 107 that functions as an inlet for fluid entering the microfluidic circuit 120, and there can be a second port 107 that functions as an outlet for fluid exiting the microfluidic circuit 120. Whether a port 107 function as an inlet or an outlet can depend upon the direction that fluid flows through flow path 106. [0125] The support structure 104 can comprise one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 can comprise one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates can be electrically connected to a single electrode). The support structure 104 can further comprise a printed circuit board assembly (“PCBA”). For example, the semiconductor substrate(s) can be mounted on a PCBA. [0126] The microfluidic circuit structure 108 can define circuit elements of the microfluidic circuit 120. Such circuit elements can comprise spaces or regions that can be Attorney Docket No.01149-0025-00PCT fluidly interconnected when microfluidic circuit 120 is filled with fluid, such as flow regions (which may include or be one or more flow channels), chambers (which class of circuit elements may also include sub-classes including sequestration pens), traps, and the like. Circuit elements can also include barriers, and the like. In the microfluidic circuit 120 illustrated in FIG. 1A, the microfluidic circuit structure 108 comprises a frame 114 and a microfluidic circuit material 116. The frame 114 can partially or completely enclose the microfluidic circuit material 116. The frame 114 can be, for example, a relatively rigid structure substantially surrounding the microfluidic circuit material 116. For example, the frame 114 can comprise a metal material. However, the microfluidic circuit structure need not include a frame 114. For example, the microfluidic circuit structure can consist of (or consist essentially of) the microfluidic circuit material 116. [0127] The microfluidic circuit material 116 can be patterned with cavities or the like to define the circuit elements and interconnections of the microfluidic circuit 120, such as chambers, pens and microfluidic channels. The microfluidic circuit material 116 can comprise a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or the like), which can be gas permeable. Other examples of materials that can form the microfluidic circuit material 116 include molded glass, an etchable material such as silicone (e.g., photo-patternable silicone or “PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, such materials—and thus the microfluidic circuit material 116—can be rigid and/or substantially impermeable to gas. Regardless, microfluidic circuit material 116 can be disposed on the support structure 104 and inside the frame 114. [0128] The microfluidic circuit 120 can include a flow region in which one or more chambers can be disposed and/or fluidically connected thereto. A chamber can have one or more openings fluidically connecting the chamber with one or more flow regions. In some embodiments, a flow region comprises or corresponds to a microfluidic channel 122. Although a single microfluidic circuit 120 is illustrated in FIG. 1A, suitable microfluidic devices can include a plurality (e.g., 2 or 3) of such microfluidic circuits. In some embodiments, the microfluidic device 100 can be configured to be a nanofluidic device. As illustrated in FIG. 1A, the microfluidic circuit 120 may include a plurality of microfluidic sequestration pens 124, 126, 128, and 130, where each sequestration pens may have one or more openings. In some embodiments of sequestration pens, a sequestration pen may have only a single opening in fluidic communication with the flow path 106. In some other embodiments, a sequestration pen may have more than one opening in fluidic communication with the flow path 106, e.g., n number of openings, but with n-1 openings that are valved, such that all but one opening is Attorney Docket No.01149-0025-00PCT closable. When all the valved openings are closed, the sequestration pen limits exchange of materials from the flow region into the sequestration pen to occur only by diffusion. In some embodiments, the sequestration pens comprise various features and structures (e.g., isolation regions) that have been optimized for retaining micro-objects within the sequestration pen (and therefore within a microfluidic device such as microfluidic device 100) even when a medium 180 is flowing through the flow path 106. [0129] The cover 110 can be an integral part of the frame 114 and/or the microfluidic circuit material 116. Alternatively, the cover 110 can be a structurally distinct element, as illustrated in FIG. 1A. The cover 110 can comprise the same or different materials than the frame 114 and/or the microfluidic circuit material 116. In some embodiments, the cover 110 can be an integral part of the microfluidic circuit material 116. Similarly, the support structure 104 can be a separate structure from the frame 114 or microfluidic circuit material 116 as illustrated, or an integral part of the frame 114 or microfluidic circuit material 116. Likewise, the frame 114 and microfluidic circuit material 116 can be separate structures as shown in FIG. 1A or integral portions of the same structure. Regardless of the various possible integrations, the microfluidic device can retain a three-layer structure that includes a base layer and a cover layer that sandwich a middle layer in which the microfluidic circuit 120 is located. [0130] In some embodiments, the cover 110 can comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover 110 can comprise a deformable material. The deformable material can be a polymer, such as PDMS. In some embodiments, the cover 110 can comprise both rigid and deformable materials. For example, one or more portions of cover 110 (e.g., one or more portions positioned over sequestration pens 124, 126, 128, 130) can comprise a deformable material that interfaces with rigid materials of the cover 110. Microfluidic devices having covers that include both rigid and deformable materials have been described, for example, in U.S. Patent No.10,058,865 (Breinlinger et al.), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 can further include one or more electrodes. The one or more electrodes can comprise a conductive oxide, such as indium-tin-oxide (ITO), which may be coated on glass or a similarly insulating material. Alternatively, the one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of electrically conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices have been described, for example, in U.S. Patent No. 9,227,200 (Chiou et al.), the contents of which are incorporated herein by reference. In some Attorney Docket No.01149-0025-00PCT embodiments, the cover 110 and/or the support structure 104 can be transparent to light. The cover 110 may also include at least one material that is gas permeable (e.g., PDMS or PPS). [0131] In the example shown in FIG.1A, the microfluidic circuit 120 is illustrated as comprising a microfluidic channel 122 and sequestration pens 124, 126, 128, 130. Each pen comprises an opening to channel 122, but otherwise is enclosed such that the pens can substantially isolate micro-objects inside the pen from fluidic medium 180 and/or micro- objects in the flow path 106 of channel 122 or in other pens. The walls of the sequestration pen extend from the inner surface 109 of the base to the inside surface of the cover 110 to provide enclosure. The opening of the sequestration pen to the microfluidic channel 122 is oriented at an angle to the flow 106 of fluidic medium 180 such that flow 106 is not directed into the pens. The vector of bulk fluid flow in channel 122 may be tangential or parallel to the plane of the opening of the sequestration pen, and is not directed into the opening of the pen. In some instances, pens 124, 126, 128, 130 are configured to physically isolate one or more micro-objects within the microfluidic circuit 120. Sequestration pens in accordance with the present disclosure can comprise various shapes, surfaces and features that are optimized for use with DEP, OET, OEW, fluid flow, magnetic forces, centripetal, and/or gravitational forces, as will be discussed and shown in detail below. [0132] The microfluidic circuit 120 may comprise any number of microfluidic sequestration pens. Although five sequestration pens are shown, microfluidic circuit 120 may have fewer or more sequestration pens. As shown, microfluidic sequestration pens 124, 126, 128, and 130 of microfluidic circuit 120 each comprise differing features and shapes which may provide one or more benefits useful for maintaining, isolating, assaying or culturing biological micro-objects. In some embodiments, the microfluidic circuit 120 comprises a plurality of identical microfluidic sequestration pens. [0133] In the embodiment illustrated in FIG.1A, a single flow path 106 containing a single channel 122 is shown. However, other embodiments may contain multiple channels 122 within a single flow path 106, as shown in FIG. 1B. The microfluidic circuit 120 further comprises an inlet valve or port 107 in fluid communication with the flow path 106, whereby fluidic medium 180 can access the flow path 106 (and channel 122). In some instances, the flow path 106 comprises a substantially straight path. In other instances, the flow path 106 is arranged in a non-linear or winding manner, such as a zigzag pattern, whereby the flow path 106 travels across the microfluidic device 100 two or more times, e.g., in alternating directions. The flow in the flow path 106 may proceed from inlet to outlet or may be reversed and proceed from outlet to inlet. Attorney Docket No.01149-0025-00PCT [0134] One example of a multi-channel device, microfluidic device 175, is shown in FIG.1B, which may be like microfluidic device 100 in other respects. Microfluidic device 175 and its constituent circuit elements (e.g., channels 122 and sequestration pens 128) may have any of the dimensions discussed herein. The microfluidic circuit illustrated in FIG.1B has two inlet/outlet ports 107 and a flow path 106 containing four distinct channels 122. The number of channels into which the microfluidic circuit is sub-divided may be chosen to reduce fluidic resistance. For example, the microfluidic circuit may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more channels to provide a selected range of fluidic resistance. Microfluidic device 175 further comprises a plurality of sequestration pens opening off of each channel 122, where each of the sequestration pens is similar to sequestration pen 128 of FIG. 1A, and may have any of the dimensions or functions of any sequestration pen as described herein. However, the sequestration pens of microfluidic device 175 can have different shapes, such as any of the shapes of sequestration pens 124, 126, or 130 of FIG.1A or as described anywhere else herein. Moreover, microfluidic device 175 can include sequestration pens having a mixture of different shapes. In some instances, a plurality of sequestration pens is configured (e.g., relative to a channel 122) such that the sequestration pens can be loaded with target micro-objects in parallel. [0135] Returning to FIG.1A, microfluidic circuit 120 further may include one or more optional micro-object traps 132. The optional traps 132 may be formed in a wall forming the boundary of a channel 122, and may be positioned opposite an opening of one or more of the microfluidic sequestration pens 124, 126, 128, 130. The optional traps 132 may be configured to receive or capture a single micro-object from the flow path 106, or may be configured to receive or capture a plurality of micro-objects from the flow path 106. In some instances, the optional traps 132 comprise a volume approximately equal to the volume of a single target micro-object. In some instances, the trap 132 comprises a side passage 134 that is smaller than the target micro-object in order to facilitate flow through the trap 132. [0136] Sequestration pens. The microfluidic devices described herein may include one or more sequestration pens, where each sequestration pen is suitable for holding one or more micro-objects (e.g., biological cells, or groups of cells that are associated together). The sequestration pens may be disposed within and open to a flow region, which in some embodiments is a microfluidic channel. Each of the sequestration pens can have one or more openings for fluidic communication to one or more microfluidic channels. In some embodiments, a sequestration pen may have only one opening to a microfluidic channel. Attorney Docket No.01149-0025-00PCT [0137] FIGS. 2A-2C show sequestration pens 224, 226, and 228 of a microfluidic device 200, which may be like sequestration pen 128 of FIG.1A. Each sequestration pen 224, 226, and 228 can comprise an isolation region 240 and a connection region 236 fluidically connecting the isolation region 240 to a flow region, which may, in some embodiments include a microfluidic channel, such as channel 122. The connection region 236 can comprise a proximal opening 234 to the flow region (e.g., microfluidic channel 122) and a distal opening 238 to the isolation region 240. The connection region 236 can be configured so that the maximum penetration depth of a flow of a fluidic medium (not shown) flowing in the microfluidic channel 122 past the sequestration pen 224, 226, and 228 does not extend into the isolation region 240, as discussed below for FIG.2C. In some embodiments, streamlines from the flow in the microfluidic channel do not enter the isolation region. Thus, due to the connection region 236, a micro-object (not shown) or other material (not shown) disposed in the isolation region 240 of a sequestration pen 224, 226, and 228 can be isolated from, and not substantially affected by, a flow of fluidic medium 180 in the microfluidic channel 122. [0138] The sequestration pens 224, 226, and 228 of FIGS.2A-2C each have a single opening which opens directly to the microfluidic channel 122. The opening of the sequestration pen may open laterally from the microfluidic channel 122, as shown in FIG.2A, which depicts a vertical cross-section of microfluidic device 200. FIG.2B shows a horizontal cross-section of microfluidic device 200. An electrode activation substrate 206 can underlie both the microfluidic channel 122 and the sequestration pens 224, 226, and 228. The upper surface of the electrode activation substrate 206 within an enclosure of a sequestration pen, forming the floor of the sequestration pen, can be disposed at the same level or substantially the same level of the upper surface the of electrode activation substrate 206 within the microfluidic channel 122 (or flow region if a channel is not present), forming the floor of the flow channel (or flow region, respectively) of the microfluidic device. The electrode activation substrate 206 may be featureless or may have an irregular or patterned surface that varies from its highest elevation to its lowest depression by less than about 3 micrometers (microns), 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns or less. The variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 10%, 7%, 5%, 3%, 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the height of the walls of the sequestration pen. Alternatively, the variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3%, 0.2%, or 0.1% of the height of the Attorney Docket No.01149-0025-00PCT substrate. While described in detail for the microfluidic device 200, this may also apply to any of the microfluidic devices described herein. [0139] The microfluidic channel 122 and connection region 236 can be examples of swept regions, and the isolation regions 240 of the sequestration pens 224, 226, and 228 can be examples of unswept regions. Sequestration pens like 224, 226, 228 have isolation regions wherein each isolation region has only one opening, which opens to the connection region of the sequestration pen. Fluidic media exchange in and out of the isolation region so configured can be limited to occurring substantially only by diffusion. As noted, the microfluidic channel 122 and sequestration pens 224, 226, and 228 can be configured to contain one or more fluidic media 180. In the example shown in FIGS.2A-2B, ports 222 are connected to the microfluidic channel 122 and allow the fluidic medium 180 to be introduced into or removed from the microfluidic device 200. Prior to introduction of the fluidic medium 180, the microfluidic device may be primed with a gas such as carbon dioxide gas. Once the microfluidic device 200 contains the fluidic medium 180, the flow 242 (see FIG.2C) of fluidic medium 180 in the microfluidic channel 122 can be selectively generated and stopped. For example, as shown, the ports 222 can be disposed at different locations (e.g., opposite ends) of the flow region (microfluidic channel 122), and a flow 242 of the fluidic medium can be created from one port 222 functioning as an inlet to another port 222 functioning as an outlet. [0140] FIG. 2C illustrates a detailed view of an example of a sequestration pen 224, which may contain one or more micro-objects 246, according to some embodiments. The flow 242 of fluidic medium 180 in the microfluidic channel 122 past the proximal opening 234 of the connection region 236 of sequestration pen 224 can cause a secondary flow 244 of the fluidic medium 180 into and out of the sequestration pen 224. To sequester the micro-objects 246 in the isolation region 240 of the sequestration pen 224 from the secondary flow 244, the length Lcon of the connection region 236 of the sequestration pen 224 (i.e., from the proximal opening 234 to the distal opening 238) should be greater than the penetration depth Dp of the secondary flow 244 into the connection region 236. The penetration depth Dp depends upon a number of factors, including the shape of the microfluidic channel 122, which may be defined by a width Wcon of the connection region 236 at the proximal opening 234; a width Wch of the microfluidic channel 122 at the proximal opening 234; a height Hch of the channel 122 at the proximal opening 234; and the width of the distal opening 238 of the connection region 236. Of these factors, the width Wcon of the connection region 236 at the proximal opening 234 and the height Hch of the channel 122 at the proximal opening 234 tend to be the most significant. In addition, the penetration depth Dp can be influenced by the velocity of the fluidic medium Attorney Docket No.01149-0025-00PCT 180 in the channel 122 and the viscosity of fluidic medium 180. However, these factors (i.e., velocity and viscosity) can vary widely without dramatic changes in penetration depth Dp. For example, for a microfluidic chip 200 having a width Wcon of the connection region 236 at the proximal opening 234 of about 50 microns, a height Hch of the channel 122 at the proximal opening 122 of about 40 microns, and a width Wch of the microfluidic channel 122 at the proximal opening 122 of about 100 microns to about 150 microns, the penetration depth Dp of the secondary flow 244 ranges from less than 1.0 times Wcon (i.e., less than 50 microns) at a flow rate of 0.1 microliters/sec to about 2.0 times Wcon (i.e., about 100 microns) at a flow rate of 20 microliters/sec, which represents an increase in Dp of only about 2.5-fold over a 200-fold increase in the velocity of the fluidic medium 180. [0141] In some embodiments, the walls of the microfluidic channel 122 and sequestration pen 224, 226, or 228 can be oriented as follows with respect to the vector of the flow 242 of fluidic medium 180 in the microfluidic channel 122: the microfluidic channel width Wch (or cross-sectional area of the microfluidic channel 122) can be substantially perpendicular to the flow 242 of medium 180; the width Wcon (or cross-sectional area) of the connection region 236 at opening 234 can be substantially parallel to the flow 242 of medium 180 in the microfluidic channel 122; and/or the length Lcon of the connection region can be substantially perpendicular to the flow 242 of medium 180 in the microfluidic channel 122. The foregoing are examples only, and the relative position of the microfluidic channel 122 and sequestration pens 224, 226 and 228 can be in other orientations with respect to each other. [0142] In some embodiments, for a given microfluidic device, the configurations of the microfluidic channel 122 and the opening 234 may be fixed, whereas the rate of flow 242 of fluidic medium 180 in the microfluidic channel 122 may be variable. Accordingly, for each sequestration pen 224, a maximal velocity Vmax for the flow 242 of fluidic medium 180 in channel 122 may be identified that ensures that the penetration depth Dp of the secondary flow 244 does not exceed the length Lcon of the connection region 236. When Vmax is not exceeded, the resulting secondary flow 244 can be wholly contained within the connection region 236 and does not enter the isolation region 240. Thus, the flow 242 of fluidic medium 180 in the microfluidic channel 122 (swept region) is prevented from drawing micro-objects 246 out of the isolation region 240, which is an unswept region of the microfluidic circuit, resulting in the micro-objects 246 being retained within the isolation region 240. Accordingly, selection of microfluidic circuit element dimensions and further selection of the operating parameters (e.g., velocity of fluidic medium 180) can prevent contamination of the isolation region 240 of sequestration pen 224 by materials from the microfluidic channel 122 or another sequestration Attorney Docket No.01149-0025-00PCT pen 226 or 228. It should be noted, however, that for many microfluidic chip configurations, there is no need to worry about Vmax per se, because the chip will break from the pressure associated with flowing fluidic medium 180 at high velocity through the chip before Vmax can be achieved. [0143] Components (not shown) in the first fluidic medium 180 in the microfluidic channel 122 can mix with the second fluidic medium 248 in the isolation region 240 substantially only by diffusion of components of the first medium 180 from the microfluidic channel 122 through the connection region 236 and into the second fluidic medium 248 in the isolation region 240. Similarly, components (not shown) of the second medium 248 in the isolation region 240 can mix with the first medium 180 in the microfluidic channel 122 substantially only by diffusion of components of the second medium 248 from the isolation region 240 through the connection region 236 and into the first medium 180 in the microfluidic channel 122. In some embodiments, the extent of fluidic medium exchange between the isolation region of a sequestration pen and the flow region by diffusion is greater than about 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% of fluidic exchange. [0144] In some embodiments, the first medium 180 can be the same medium or a different medium than the second medium 248. In some embodiments, the first medium 180 and the second medium 248 can start out being the same, then become different (e.g., through conditioning of the second medium 248 by one or more cells in the isolation region 240, or by changing the medium 180 flowing through the microfluidic channel 122). [0145] As illustrated in FIG. 2C, the width Wcon of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238. The width Wcon of the connection region 236 at the distal opening 238 can be any of the values identified herein for the width Wcon of the connection region 236 at the proximal opening 234. In some embodiments, the width of the isolation region 240 at the distal opening 238 can be substantially the same as the width Wcon of the connection region 236 at the proximal opening 234. Alternatively, the width Wcon of the connection region 236 at the distal opening 238 can be different (e.g., larger or smaller) than the width Wcon of the connection region 236 at the proximal opening 234. In some embodiments, the width Wcon of the connection region 236 may be narrowed or widened between the proximal opening 234 and distal opening 238. For example, the connection region 236 may be narrowed or widened between the proximal opening and the distal opening, using a variety of different geometries (e.g., chamfering the connection region, beveling the connection region). Further, any part or subpart of the Attorney Docket No.01149-0025-00PCT connection region 236 may be narrowed or widened (e.g., a portion of the connection region adjacent to the proximal opening 234). [0146] FIG. 3 depicts another exemplary embodiment of a microfluidic device 300 containing microfluidic circuit structure 308, which includes a channel 322 and sequestration pen 324, which has features and properties like any of the sequestration pens described herein for microfluidic devices 100, 175, 200, 400, 520 and any other microfluidic devices described herein. [0147] The exemplary microfluidic devices of FIG. 3 include a microfluidic channel 322, having a width Wch, as described herein, and containing a flow 310 of first fluidic medium 302 and one or more sequestration pens 324 (only one illustrated in FIG.3). The sequestration pens 324 each have a length Ls, a connection region 336, and an isolation region 340, where the isolation region 340 contains a second fluidic medium 304. The connection region 336 has a proximal opening 334, having a width Wcon1, which opens to the microfluidic channel 322, and a distal opening 338, having a width Wcon2, which opens to the isolation region 340. The width Wcon1 may or may not be the same as Wcon2, as described herein. The walls of each sequestration pen 324 may be formed of microfluidic circuit material 316, which may further form the connection region walls 330. A connection region wall 330 can correspond to a structure that is laterally positioned with respect to the proximal opening 334 and at least partially extends into the enclosed portion of the sequestration pen 324. In some embodiments, the length Lcon of the connection region 336 is at least partially defined by length Lwall of the connection region wall 330. The connection region wall 330 may have a length Lwall, selected to be more than the penetration depth Dp of the secondary flow 344. Thus, the secondary flow 344 can be wholly contained within the connection region without extending into the isolation region 340. [0148] The connection region wall 330 may define a hook region 352, which is a sub- region of the isolation region 340 of the sequestration pen 324. Since the connection region wall 330 extends into the inner cavity of the sequestration pen, the connection region wall 330 can act as a physical barrier to shield hook region 352 from secondary flow 344, with selection of the length of Lwall, contributing to the extent of the hook region. In some embodiments, the longer the length Lwall of the connection region wall 330, the more sheltered the hook region 352. [0149] In sequestration pens configured like those of FIGS.2A-2C and 3, the isolation region may have a shape and size of any type, and may be selected to regulate diffusion of nutrients, reagents, and/or media into the sequestration pen to reach to a far wall of the Attorney Docket No.01149-0025-00PCT sequestration pen, e.g., opposite the proximal opening of the connection region to the flow region (or microfluidic channel). The size and shape of the isolation region may further be selected to regulate diffusion of waste products and/or secreted products of a biological micro- object out from the isolation region to the flow region via the proximal opening of the connection region of the sequestration pen. In general, the shape of the isolation region is not critical to the ability of the sequestration pen to isolate micro-objects from direct flow in the flow region. [0150] In some other embodiments of sequestration pens, the isolation region may have more than one opening fluidically connecting the isolation region with the flow region of the microfluidic device. However, for an isolation region having a number of n openings fluidically connecting the isolation region to the flow region (or two or more flow regions), n- 1 openings can be valved. When the n-1 valved openings are closed, the isolation region has only one effective opening, and exchange of materials into/out of the isolation region occurs only by diffusion. [0151] Examples of microfluidic devices having pens in which biological micro- objects can be placed, cultured, and/or monitored have been described, for example, in U.S. Patent No.9,857,333 (Chapman, et al.), U.S. Patent No. 10,010,882 (White, et al.), and U.S. Patent No.9,889,445 (Chapman, et al.), each of which is incorporated herein by reference in its entirety. [0152] Microfluidic circuit element dimensions. Various dimensions and/or features of the sequestration pens and the microfluidic channels to which the sequestration pens open, as described herein, may be selected to limit introduction of contaminants or unwanted micro- objects into the isolation region of a sequestration pen from the flow region/microfluidic channel; limit the exchange of components in the fluidic medium from the channel or from the isolation region to substantially only diffusive exchange; facilitate the transfer of micro-objects into and/or out of the sequestration pens; and/or facilitate growth or expansion of the biological cells. Microfluidic channels and sequestration pens, for any of the embodiments described herein, may have any suitable combination of dimensions, may be selected by one of skill from the teachings of this disclosure. [0153] For any of the microfluidic devices described herein, a microfluidic channel may have a uniform cross sectional height along its length that is a substantially uniform cross sectional height, and may be any cross sectional height as described herein. At any point along the microfluidic channel, the substantially uniform cross sectional height of the channel, the upper surface of which is defined by the inner surface of the cover and the lower surface of Attorney Docket No.01149-0025-00PCT which is defined by the inner surface of the base, may be substantially the same as the cross sectional height at any other point along the channel, e.g., having a cross sectional height that is no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% or less, different from the cross-sectional height of any other location within the channel. [0154] Additionally, the chamber(s), e.g., sequestration pen(s), of the microfluidic devices described herein, may be disposed substantially in a coplanar orientation relative to the microfluidic channel into which the chamber(s) open. That is, the enclosed volume of the chamber(s) is formed by an upper surface that is defined by the inner surface of the cover, a lower surface defined by the inner surface of the base, and walls defined by the microfluidic circuit material. Therefore, the lower surface of the chamber(s) may be coplanar to the lower surface of the microfluidic channel, e.g., substantially coplanar. The upper surface of the chamber may be coplanar to the upper surface of the microfluidic channel, e.g., substantially coplanar. Accordingly, the chamber(s) may have a cross-sectional height, which may have any values as described herein, that is the same as the channel, e.g., substantially the same, and the chamber(s) and microfluidic channel(s) within the microfluidic device may have a substantially uniform cross sectional height throughout the flow region of the microfluidic device, and may be substantially coplanar throughout the microfluidic device. [0155] Coplanarity of the lower surfaces of the chamber(s) and the microfluidic channel(s) can offer distinct advantage with repositioning micro-objects within the microfluidic device using DEP or magnetic force. Penning and unpenning of micro-objects, and in particular selective penning/ selective unpenning, can be greatly facilitated when the lower surfaces of the chamber(s) and the microfluidic channel to which the chamber(s) open have a coplanar orientation. [0156] The proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) that is at least as large as the largest dimension of a micro-object (e.g., a biological cell, which may be a plant cell, such as a plant protoplast) for which the sequestration pen is intended. In some embodiments, the proximal opening has a width (e.g., Wcon or Wcon1) of about 20 microns, about 40 microns, about 50 microns, about 60 microns, about 75 microns, about 100 microns, about 150 microns, about 200 microns, or about 300 microns. The foregoing are examples only, and the width (e.g., Wcon or Wcon1) of a proximal opening can be selected to be a value between any of the values listed above (e.g., about 20- 200 microns, about 20-150 microns, about 20-100 microns, about 20-75 microns, about 20-60 microns, about 50-300 microns, about 50-200 microns, about 50-150 microns, about 50-100 Attorney Docket No.01149-0025-00PCT microns, about 50-75 microns, about 75-150 microns, about 75-100 microns, about 100-300 microns, about 100-200 microns, or about 200-300 microns). [0157] In some embodiments, the connection region of the sequestration pen may have a length (e.g., Lcon) from the proximal opening to the distal opening to the isolation region of the sequestration pen that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25. times, at least 2.5 times, at least 2.75 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, or at least 10.0 times the width (e.g., Wcon or Wcon1) of the proximal opening. Thus, for example, the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), and the connection region may have a length Lcon that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening. As another example, the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), and the connection region may have a length Lcon that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening. [0158] The microfluidic channel of a microfluidic device to which a sequestration pen opens may have specified size (e.g., width or height). In some embodiments, the height (e.g., Hch) of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be within any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height (e.g., Hch) of the microfluidic channel (e.g., 122) can be selected to be between any of the values listed above. Moreover, the height (e.g., Hch) of the microfluidic channel 122 can be selected to be any of these heights in regions of the microfluidic channel other than at a proximal opening of a sequestration pen. [0159] The width (e.g., Wch) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen can be within any of the following ranges: about 20- 500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30- 150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, Attorney Docket No.01149-0025-00PCT 40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-300 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 70-100 microns, 80-100 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, 100-120 microns, 200-800 microns, 200-700 microns, or 200-600 microns. The foregoing are examples only, and the width (e.g., Wch) of the microfluidic channel can be a value selected to be between any of the values listed above. Moreover, the width (e.g., Wch) of the microfluidic channel can be selected to be in any of these widths in regions of the microfluidic channel other than at a proximal opening of a sequestration pen. In some embodiments, the width Wch of the microfluidic channel at the proximal opening to the connection region of the sequestration pen (e.g., taken transverse to the direction of bulk flow of fluid through the channel) can be substantially perpendicular to a width (e.g., Wcon or Wcon1) of the proximal opening. [0160] A cross-sectional area of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be about 500-50,000 square microns, 500-40,000 square microns, 500-30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1,000- 15,000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000- 5,000 square microns, 2,000-20,000 square microns, 2,000-15,000 square microns, 2,000- 10,000 square microns, 2,000-7,500 square microns, 2,000-6,000 square microns, 3,000- 20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000- 7,500 square microns, or 3,000 to 6,000 square microns. The foregoing are examples only, and the cross-sectional area of the microfluidic channel at the proximal opening can be selected to be between any of the values listed above. In various embodiments, and the cross-sectional area of the microfluidic channel at regions of the microfluidic channel other than at the proximal opening can also be selected to be between any of the values listed above. In some embodiments, the cross-sectional area is selected to be a substantially uniform value for the entire length of the microfluidic channel. [0161] In some embodiments, the microfluidic chip is configured such that the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) from about 20 microns to about 200 microns (e.g., about 50 Attorney Docket No.01149-0025-00PCT microns to about 150 microns), the connection region may have a length Lcon (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., Hch) at the proximal opening of about 30 microns to about 60 microns. As another example, the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., Wcon or Wcon1) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), the connection region may have a length Lcon (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., Hch) at the proximal opening of about 30 microns to about 60 microns. The foregoing are examples only, and the width (e.g., Wcon or Wcon1) of the proximal opening (e.g., 234 or 274), the length (e.g., Lcon) of the connection region, and/or the width (e.g., Wch) of the microfluidic channel (e.g., 122 or 322), can be a value selected to be between any of the values listed above. Generally, however, the width (Wcon or Wcon1) of the proximal opening of the connection region of a sequestration pen is less than the width (Wch) of the microfluidic channel. In some embodiments, the width (Wcon or Wcon1) of the proximal opening is about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, or 30% of the width (Wch) of the microfluidic channel. That is, the width (Wch) of the microfluidic channel may be at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times or at least 10.0 times the width (Wcon or Wcon1) of the proximal opening of the connection region of the sequestration pen. [0162] In some embodiments, the size WC (e.g., cross-sectional width Wch, diameter, area, or the like) of the channel 122, 322, 618, 718 can be about one and a quarter (1.25), about one and a half (1.5), about two, about two and a half (2.5), about three (3), or more times the size WO (e.g., cross-sectional width Wcon, diameter, area, or the like) of a chamber opening, e.g., sequestration pen opening 234, 334, and the like. This can reduce the extent of secondary flow and the rate of diffusion (or diffusion flux) through the opening 234, 334 for materials diffusing from a selected chamber (e.g., like sequestration pens 224, 226 of FIG. 2B) into channel 122, 322, 618, 718 and subsequently re-entering a downstream or adjacent chamber (e.g., like sequestration pen 228). The rate of diffusion of a molecule (e.g., an analyte of interest, such as an antibody) is dependent on a number of factors, including (without limitation) temperature, viscosity of the medium, and the coefficient of diffusion D0 of the molecule. For example, the D0 for an IgG antibody in aqueous solution at about 20°C is about 4.4x10-7 cm2/sec, while the kinematic viscosity of cell culture medium is about 9x10-4 m2/sec. Thus, an antibody in cell culture medium at about 20°C can have a rate of diffusion of about Attorney Docket No.01149-0025-00PCT 0.5 microns/sec. Accordingly, in some embodiments, a time period for diffusion from a biological micro-object located within a sequestration pen such as 224, 226, 228, 324 into the channel 122, 322, 618, 718 can be about 10 minutes or less (e.g., about 9, 8, 7, 6, 5 minutes, or less). The time period for diffusion can be manipulated by changing parameters that influence the rate of diffusion. For example, the temperature of the media can be increased (e.g., to a physiological temperature such as about 37°C) or decreased (e.g., to about 15°C, 10°C, or 4°C) thereby increasing or decreasing the rate of diffusion, respectively. Alternatively, or in addition, the concentrations of solutes in the medium can be increased or decreased as discussed herein to isolate a selected pen from solutes from other upstream pens. [0163] Accordingly, in some variations, the width (e.g., Wch) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 50 to 500 microns, about 50 to 300 microns, about 50 to 200 microns, about 70 to 500 microns, about to 70-300 microns, about 70 to 250 microns, about 70 to 200 microns, about 70 to 150 microns, about 70 to 100 microns, about 80 to 500 microns, about 80 to 300 microns, about 80 to 250 microns, about 80 to 200 microns, about 80 to 150 microns, about 90 to 500 microns, about 90 to 300 microns, about 90 to 250 microns, about 90 to 200 microns, about 90 to 150 microns, about 100 to 500 microns, about 100 to 300 microns, about 100 to 250 microns, about 100 to 200 microns, or about 100 to 150 microns. In some embodiments, the width Wch of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 70 to 250 microns, about 80 to 200 microns, or about 90 to 150 microns. The width Wcon of the opening of the chamber (e.g., sequestration pen) may be about 20 to 100 microns; about 30 to 90 microns; or about 20 to 60 microns. In some embodiments, Wch is about 70-250 microns and Wcon is about 20 to 100 microns; Wch is about 80 to 200 microns and Wcon is about 30 to 90 microns; Wch is about 90 to 150 microns, and Wcon is about 20 to 60 microns; or any combination of the widths of Wch and Wcon thereof. [0164] In some embodiments, the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen has a width (e.g., Wcon or Wcon1) that is 2.0 times or less (e.g., 2.0, 1.9, 1.8, 1.5, 1.3, 1.0, 0.8, 0.5, or 0.1 times) the height (e.g., Hch) of the flow region/ microfluidic channel at the proximal opening, or has a value that lies within a range defined by any two of the foregoing values. [0165] In some embodiments, the width Wcon1 of a proximal opening (e.g., 234 or 334) of a connection region of a sequestration pen may be the same as a width Wcon2 of the distal opening (e.g., 238 or 338) to the isolation region thereof. In some embodiments, the width Wcon1 of the proximal opening may be different than a width Wcon2 of the distal opening, and Attorney Docket No.01149-0025-00PCT Wcon1 and/or Wcon2 may be selected from any of the values described for Wcon or Wcon1. In some embodiments, the walls (including a connection region wall) that define the proximal opening and distal opening may be substantially parallel with respect to each other. In some embodiments, the walls that define the proximal opening and distal opening may be selected to not be parallel with respect to each other. [0166] The length (e.g., Lcon) of the connection region can be about 1-600 microns, 5- 550 microns, 10-500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, about 100-150 microns, about 20-300 microns, about 20 -250 microns, about 20-200 microns, about 20-150 microns, about 20-100 microns, about 30-250 microns, about 30-200 microns, about 30- 150 microns, about 30-100 microns, about 30-80 microns, about 30-50 microns, about 45-250 microns, about 45-200 microns, about 45-100 microns, about 45- 80 microns, about 45-60 microns, about 60-200 microns, about 60- 150 microns, about 60-100 microns or about 60-80 microns. The foregoing are examples only, and length (e.g., Lcon) of a connection region can be selected to be a value that is between any of the values listed above. [0167] The connection region wall of a sequestration pen may have a length (e.g., Lwall) that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, or at least 3.5 times the width (e.g., Wcon or Wcon1) of the proximal opening of the connection region of the sequestration pen. In some embodiments, the connection region wall may have a length Lwall of about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-80 microns, or about 20-50 microns. The foregoing are examples only, and a connection region wall may have a length Lwall selected to be between any of the values listed above. [0168] A sequestration pen may have a length Ls of about 40-600 microns, about 40- 500 microns, about 40-400 microns, about 40-300 microns, about 40-200 microns, about 40- 100 microns or about 40-80 microns. The foregoing are examples only, and a sequestration pen may have a length Ls selected to be between any of the values listed above. [0169] According to some embodiments, a sequestration pen may have a specified height (e.g., Hs). In some embodiments, a sequestration pen has a height Hs of about 20 microns to about 200 microns (e.g., about 20 microns to about 150 microns, about 20 microns to about 100 microns, about 20 microns to about 60 microns, about 30 microns to about 150 microns, about 30 microns to about 100 microns, about 30 microns to about 60 microns, about 40 Attorney Docket No.01149-0025-00PCT microns to about 150 microns, about 40 microns to about 100 microns, or about 40 microns to about 60 microns). The foregoing are examples only, and a sequestration pen can have a height Hs selected to be between any of the values listed above. [0170] The height Hcon of a connection region at a proximal opening of a sequestration pen can be a height within any of the following heights: 20-100 microns, 20-90 microns, 20- 80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height Hcon of the connection region can be selected to be between any of the values listed above. Typically, the height Hcon of the connection region is selected to be the same as the height Hch of the microfluidic channel at the proximal opening of the connection region. Additionally, the height Hs of the sequestration pen is typically selected to be the same as the height Hcon of a connection region and/or the height Hch of the microfluidic channel. In some embodiments, Hs, Hcon, and Hch may be selected to be the same value of any of the values listed above for a selected microfluidic device. [0171] The isolation region can be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. In other embodiments, the isolation region may contain more than 10, more than 50 or more than 100 micro-objects. Accordingly, the volume of an isolation region can be, for example, at least 1x104, 1x105, 5x105, 8x105, 1x106, 2x106, 4x106, 6x106, 1x107, 3x107, 5x1071x108, 5x108, or 8x108 cubic microns, or more. The foregoing are examples only, and the isolation region can be configured to contain numbers of micro-objects and volumes selected to be between any of the values listed above (e.g., a volume between 1x105 cubic microns and 5x105 cubic microns, between 5x105 cubic microns and 1x106 cubic microns, between 1x106 cubic microns and 2x106 cubic microns, or between 2x106 cubic microns and 1x107 cubic microns). [0172] According to some embodiments, a sequestration pen of a microfluidic device may have a specified volume. The specified volume of the sequestration pen (or the isolation region of the sequestration pen) may be selected such that a single cell or a small number of cells (e.g., 2-10 or 2-5) can rapidly condition the medium and thereby attain favorable (or optimal) growth conditions. In some embodiments, the sequestration pen has a volume of about 5x105, 6x105, 8x105, 1x106, 2x106, 4x106, 8x106, 1x107, 3x107, 5x107, or about 8x107 cubic microns, or more. In some embodiments, the sequestration pen has a volume of about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2 nanoliters to about 10 nanoliters. Attorney Docket No.01149-0025-00PCT The foregoing are examples only, and a sequestration pen can have a volume selected to be any value that is between any of the values listed above. [0173] According to some embodiments, the flow of fluidic medium within the microfluidic channel (e.g., 122 or 322) may have a specified maximum velocity (e.g., Vmax). In some embodiments, the maximum velocity (e.g., Vmax) may be set at around 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, or 25 microliters/sec. The foregoing are examples only, and the flow of fluidic medium within the microfluidic channel can have a maximum velocity (e.g., Vmax) selected to be a value between any of the values listed above. The flow of fluidic medium within the microfluidic channel typically may be flowed at a rate less than the Vmax. While the Vmax may vary depending on the specific size and numbers of channel and sequestration pens opening thereto, a fluidic medium may be flowed at about 0.1 microliters/sec to about 20 microliters/sec; about 0.1 microliters/sec to about 15 microliters/sec; about 0.1 microliters/sec to about 12 microliters/sec, about 0.1 microliters/sec to about 10 microliters/sec; about 0.1 microliter/sec to about 7 microliters/sec without exceeding the Vmax. In some portions of a typical workflow, a flow rate of a fluidic medium may be about 0.1 microliters/sec; about 0.5 microliters/sec; about 1.0 microliters/sec; about 2.0 microliters/sec; about 3.0 microliters/sec; about 4.0 microliters/sec; about 5.0 microliters/sec; about 6.0 microliters/sec; about 7.0 microliters/sec; about 8.0 microliters/sec; about 9.0 microliters/sec; about 10.0 microliters/sec; about 11.0 microliters/sec; or any range defined by two of the foregoing values, e.g., 1-5 microliters/sec or 5-10 microliters/sec. The flow rate of a fluidic medium in the microfluidic channel may be equal to or less than about 12 microliters/sec; about 10 microliters/sec; about 8 microliters/sec, or about 6 microliters/sec. [0174] In various embodiment, the microfluidic device has sequestration pens configured as in any of the embodiments discussed herein where the microfluidic device has about 5 to about 10 sequestration pens, about 10 to about 50 sequestration pens, about 25 to about 200 sequestration pens, about 100 to about 500 sequestration pens, about 200 to about 1000 sequestration pens, about 500 to about 1500 sequestration pens, about 1000 to about 2500 sequestration pens, about 2000 to about 5000 sequestration pens, about 3500 to about 7000 sequestration pens, about 5000 to about 10,000 sequestration pens, about 7,500 to about 15,000 sequestration pens, about 12,500 to about 20,000 sequestration pens, about 15,000 to about 25,000 sequestration pens, about 20,000 to about 30,000 sequestration pens, about 25,000 to about 35,000 sequestration pens, about 30,000 to about 40,000 sequestration pens, about 35,000 to about 45,000 sequestration pens, or about 40,000 to about 50,000 sequestration pens. Attorney Docket No.01149-0025-00PCT The sequestration pens need not all be the same size and may include a variety of configurations (e.g., different widths, different features within the sequestration pen). [0175] Coating solutions and coating agents. In some embodiments, at least one inner surface of the microfluidic device includes a coating material that provides a layer of organic and/or hydrophilic molecules suitable for maintenance, expansion and/or movement of biological micro-object(s) (i.e., the biological micro-object exhibits increased viability, greater expansion and/or greater portability within the microfluidic device). The conditioned surface may reduce surface fouling, participate in providing a layer of hydration, and/or otherwise shield the biological micro-objects from contact with the non-organic materials of the microfluidic device interior. [0176] In some embodiments, substantially all the inner surfaces of the microfluidic device include the coating material. The coated inner surface(s) may include the surface of a flow region (e.g., channel), chamber, or sequestration pen, or a combination thereof. In some embodiments, each of a plurality of sequestration pens has at least one inner surface coated with coating materials. In other embodiments, each of a plurality of flow regions or channels has at least one inner surface coated with coating materials. In some embodiments, at least one inner surface of each of a plurality of sequestration pens and each of a plurality of channels is coated with coating materials. The coating may be applied before or after introduction of biological micro-object(s), or may be introduced concurrently with the biological micro- object(s). In some embodiments, the biological micro-object(s) may be imported into the microfluidic device in a fluidic medium that includes one or more coating agents. In other embodiments, the inner surface(s) of the microfluidic device (e.g., a microfluidic device having an electrode activation substrate such as, but not limited to, a device including dielectrophoresis (DEP) electrodes) may be treated or “primed” with a coating solution comprising a coating agent prior to introduction of the biological micro-object(s) into the microfluidic device. Any convenient coating agent/coating solution can be used, including but not limited to: serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof. [0177] Synthetic polymer-based coating materials. The at least one inner surface may include a coating material that comprises a polymer. The polymer may be non-covalently bound (e.g., it may be non-specifically adhered) to the at least one surface. The polymer may have a variety of structural motifs, such as found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein. A wide variety of alkylene ether containing Attorney Docket No.01149-0025-00PCT polymers may be suitable for use in the microfluidic devices described herein, including but not limited to Pluronic® polymers such as Pluronic® L44, L64, P85, and F127 (including F127NF). Other examples of suitable coating materials are described in US2016/0312165, the contents of which are herein incorporated by reference in their entirety. [0178] Covalently linked coating materials. In some embodiments, the at least one inner surface includes covalently linked molecules that provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) within the microfluidic device, providing a conditioned surface for such cells. The covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device, as described below. The linking group is also covalently linked to a surface modifying moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/ expansion/ movement of biological micro- object(s). [0179] In some embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) may include alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono- or polysaccharides (which may include but is not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyalcohols, including but not limited to polyvinyl alcohol; alkylene ethers, including but not limited to polyethylene glycol; polyelectrolytes ( including but not limited to polyacrylic acid or polyvinyl phosphonic acid); amino groups (including derivatives thereof, such as, but not limited to alkylated amines, hydroxyalkylated amino group, guanidinium, and heterocylic groups containing an unaromatized nitrogen ring atom, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynyl phosphonic acid (which may provide a phosphonate anionic surface); sulfonate anions; carboxybetaines; sulfobetaines; sulfamic acids; or amino acids. [0180] In various embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device may include non-polymeric moieties such as an alkyl moiety, amino acid moiety, alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety. Alternatively, the covalently linked moiety may include polymeric moieties, which may include any of these moieties. Attorney Docket No.01149-0025-00PCT [0181] In some embodiments, a microfluidic device may have a hydrophobic layer upon the inner surface of the base which includes a covalently linked alkyl moiety. The covalently linked alkyl moiety may comprise carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be an unbranched alkyl moiety. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group can be fluorinated or perfluorinated). In some embodiments, the alkyl group may include a first segment, which may include a perfluoroalkyl group, joined to a second segment, which may include a non-substituted alkyl group, where the first and second segments may be joined directly or indirectly (e.g., by means of an ether linkage). The first segment of the alkyl group may be located distal to the linking group, and the second segment of the alkyl group may be located proximal to the linking group. [0182] In other embodiments, the covalently linked moiety may include at least one amino acid, which may include more than one type of amino acid. Thus, the covalently linked moiety may include a peptide or a protein. In some embodiments, the covalently linked moiety may include an amino acid which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof. [0183] In other embodiments, the covalently linked moiety may further include a streptavidin or biotin moiety. In some embodiments, a modified biological moiety such as, for example, a biotinylated protein or peptide may be introduced to the inner surface of a microfluidic device bearing covalently linked streptavidin, and couple via the covalently linked streptavidin to the surface, thereby providing a modified surface presenting the protein or peptide. [0184] In other embodiments, the covalently linked moiety may include at least one alkylene oxide moiety and may include any alkylene oxide polymer as described above. One useful class of alkylene ether containing polymers is polyethylene glycol (PEG Mw <100,000Da) or alternatively polyethylene oxide (PEO, Mw>100,000). In some embodiments, a PEG may have an Mw of about 1000Da, 5000Da, 10,000Da or 20,000Da. In some embodiments, the PEG polymer may further be substituted with a hydrophilic or charged moiety, such as but not limited to an alcohol functionality or a carboxylic acid moiety. [0185] The covalently linked moiety may include one or more saccharides. The covalently linked saccharides may be mono-, di-, or polysaccharides. The covalently linked saccharides may be modified to introduce a reactive pairing moiety which permits coupling or elaboration for attachment to the surface. One exemplary covalently linked moiety may include Attorney Docket No.01149-0025-00PCT a dextran polysaccharide, which may be coupled indirectly to a surface via an unbranched linker. [0186] The coating material providing a conditioned surface may comprise only one kind of covalently linked moiety or may include more than one different kind of covalently linked moiety. For example, a polyethylene glycol conditioned surface may have covalently linked alkylene oxide moieties having a specified number of alkylene oxide units which are all the same, e.g., having the same linking group and covalent attachment to the surface, the same overall length, and the same number of alkylene oxide units. Alternatively, the coating material may have more than one kind of covalently linked moiety attached to the surface. For example, the coating material may include the molecules having covalently linked alkylene oxide moieties having a first specified number of alkylene oxide units and may further include a further set of molecules having bulky moieties such as a protein or peptide connected to a covalently attached alkylene oxide linking moiety having a greater number of alkylene oxide units. The different types of molecules may be varied in any suitable ratio to obtain the surface characteristics desired. For example, the conditioned surface having a mixture of first molecules having a chemical structure having a first specified number of alkylene oxide units and second molecules including peptide or protein moieties, which may be coupled via a biotin/streptavidin binding pair to the covalently attached alkylene linking moiety, may have a ratio of first molecules: second molecules of about 99:1; about 90:10; about 75:25; about 50:50; about 30:70; about 20:80; about 10:90; or any ratio selected to be between these values. In this instance, the first set of molecules having different, less sterically demanding termini and fewer backbone atoms can help to functionalize the entire substrate surface and thereby prevent undesired adhesion or contact with the silicon/silicon oxide, hafnium oxide or alumina making up the substrate itself. The selection of the ratio of mixture of first molecules to second molecules may also modulate the surface modification introduced by the second molecules bearing peptide or protein moieties. [0187] Conditioned surface properties. Various factors can alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid phase deposition, spin coating, flooding, and electrostatic coating). In some embodiments, the conditioned surface may have a thickness of about 1nm to about 10nm. In some embodiments, the covalently linked moieties of the conditioned surface may form a monolayer when covalently linked to the surface of the microfluidic device (which may include an electrode activation substrate having dielectrophoresis (DEP) or electrowetting (EW) electrodes) and may have a thickness of less Attorney Docket No.01149-0025-00PCT than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). These values are in contrast to that of a surface prepared by spin coating, for example, which may typically have a thickness of about 30nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to be suitably functional for operation within a DEP-configured microfluidic device. In other embodiments, the conditioned surface formed by the covalently linked moieties may have a thickness of about 10 nm to about 50 nm. [0188] Unitary or Multi-part conditioned surface. The covalently linked coating material may be formed by reaction of a molecule which already contains the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device, and may have a structure of Formula I, as shown below. Alternatively, the covalently linked coating material may be formed in a two-part sequence, having a structure of Formula II, by coupling the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance and/or expansion of biological micro-object(s) to a surface modifying ligand that itself has been covalently linked to the surface. In some embodiments, the surface may be formed in a two- part or three-part sequence, including a streptavidin/biotin binding pair, to introduce a protein, peptide, or mixed modified surface.
Figure imgf000066_0002
Figure imgf000066_0001
Figure imgf000066_0003
or
Figure imgf000066_0004
Formula I Formula II [0189] The coating material may be linked covalently to oxides of the surface of a DEP-configured or EW- configured substrate. The coating material may be attached to the oxides via a linking group (“LG”), which may be a siloxy or phosphonate ester group formed from the reaction of a siloxane or phosphonic acid group with the oxides. The moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device can be any of the moieties described herein. The linking group LG may be directly or indirectly connected to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device. When the Attorney Docket No.01149-0025-00PCT linking group LG is directly connected to the moiety, optional linker (“L”) is not present and n is 0. When the linking group LG is indirectly connected to the moiety, linker L is present and n is 1. The linker L may have a linear portion where a backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and/or phosphorus atoms, subject to chemical bonding limitations as is known in the art. It may be interrupted with any combination of one or more moieties, which may be chosen from ether, amino, carbonyl, amido, and/or phosphonate groups, arylene, heteroarylene, or heterocyclic groups. In some embodiments, the coupling group CG represents the resultant group from reaction of a reactive moiety Rx and a reactive pairing moiety Rpx (i.e., a moiety configured to react with the reactive moiety Rx). CG may be a carboxamidyl group, a triazolylene group, substituted triazolylene group, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or alkenyl group, or any other suitable group that may be formed upon reaction of a reactive moiety with its respective reactive pairing moiety. In some embodiments, CG may further represent a streptavidin/biotin binding pair. [0190] Further details of suitable coating treatments and modifications, as well as methods of preparation, may be found at U.S. Patent Application Publication No. US2016/0312165 (Lowe, Jr., et al.), U.S. Patent Application Publication No US2017/0173580 (Lowe, Jr., et al), International Patent Application Publication WO2017/205830 (Lowe, Jr., et al.), and International Patent Application Publication WO2019/01880 (Beemiller et al.), each of which disclosures is herein incorporated by reference in its entirety. [0191] Microfluidic device motive technologies. The microfluidic devices described herein can be used with any type of motive technology. As described herein, the control and monitoring equipment of the system can comprise a motive module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of a microfluidic device. The motive technology(ies) may include, for example, dielectrophoresis (DEP), electrowetting (EW), and/or other motive technologies. The microfluidic device can have a variety of motive configurations, depending upon the type of object being moved and other considerations. Returning to FIG. 1A, for example, the support structure 104 and/or cover 110 of the microfluidic device 100 can comprise DEP electrode activation substrates for selectively inducing motive forces on micro-objects in the fluidic medium 180 in the microfluidic circuit 120 and thereby select, capture, and/or move individual micro-objects or groups of micro-objects. [0192] In some embodiments, motive forces are applied across the fluidic medium 180 (e.g., in the flow path and/or in the sequestration pens) via one or more electrodes Attorney Docket No.01149-0025-00PCT (not shown) to manipulate, transport, separate and sort micro-objects located therein. For example, in some embodiments, motive forces are applied to one or more portions of microfluidic circuit 120 in order to transfer a single micro-object from the flow path 106 into a desired microfluidic sequestration pen. In some embodiments, motive forces are used to prevent a micro-object within a sequestration pen from being displaced therefrom. Further, in some embodiments, motive forces are used to selectively remove a micro-object from a sequestration pen that was previously collected in accordance with the embodiments of the current disclosure. [0193] In some embodiments, the microfluidic device is configured as an optically-actuated electrokinetic device, such as in optoelectronic tweezer (OET) and/or optoelectrowetting (OEW) configured device. Examples of suitable OET configured devices (e.g., containing optically actuated dielectrophoresis electrode activation substrates) can include those illustrated in U.S. Patent No. RE 44,711 (Wu, et al.) (originally issued as U.S. Patent No. 7,612,355), U.S. Patent No. 7,956,339 (Ohta, et al.), U.S. Patent No. 9,908,115 (Hobbs et al.), and U.S. Patent No.9,403,172 (Short et al), each of which is incorporated herein by reference in its entirety. Examples of suitable OEW configured devices can include those illustrated in U.S. Patent No. 6,958,132 (Chiou, et al.), and U.S. Patent Application No. 9,533,306 (Chiou, et al.), each of which is incorporated herein by reference in its entirety. Examples of suitable optically-actuated electrokinetic devices that include combined OET/OEW configured devices can include those illustrated in U.S. Patent Application Publication No. 2015/0306598 (Khandros, et al.), U.S. Patent Application Publication No 2015/0306599 (Khandros, et al.), and U.S. Patent Application Publication No.2017/0173580 (Lowe, et al.), each of which is incorporated herein by reference in its entirety. [0194] It should be understood that, for purposes of simplicity, the various examples of FIGS. 1-5B may illustrate portions of microfluidic devices while not depicting other portions. Further, FIGS. 1-5B may be part of, and implemented as, one or more microfluidic systems. In one non-limiting example, FIGS. 4A and 4B show a side cross- sectional view and a top cross-sectional view, respectively, of a portion of an enclosure 102 of the microfluidic device 400 having a region/chamber 402, which may be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, a sequestration pen (which may be like any sequestration pen described herein), a flow region, or a flow channel. For instance, microfluidic device 400 may be similar to microfluidic devices 100, 175, 200, 300, 520 or any other microfluidic device as described herein. Furthermore, the microfluidic device 400 may include other fluidic circuit elements and may be part of a system including control Attorney Docket No.01149-0025-00PCT and monitoring equipment 152, described above, having one or more of the media module 160, motive module 162, imaging module 164, optional tilting module 166, and other modules 168. Microfluidic devices 175, 200, 300, 520 and any other microfluidic devices described herein may similarly have any of the features described in detail for FIGS.1A-1B and 4A-4B. [0195] As shown in the example of FIG. 4A, the microfluidic device 400 includes a support structure 104 having a bottom electrode 404 and an electrode activation substrate 406 overlying the bottom electrode 404, and a cover 110 having a top electrode 410, with the top electrode 410 spaced apart from the bottom electrode 404. The top electrode 410 and the electrode activation substrate 406 define opposing surfaces of the region/chamber 402. A fluidic medium 180 contained in the region/chamber 402 thus provides a resistive connection between the top electrode 410 and the electrode activation substrate 406. A power source 412 configured to be connected to the bottom electrode 404 and the top electrode 410 and create a biasing voltage between the electrodes, as required for the generation of DEP forces in the region/chamber 402, is also shown. The power source 412 can be, for example, an alternating current (AC) power source. [0196] In certain embodiments, the microfluidic device 200 illustrated in FIGS. 4A and 4B can have an optically-actuated DEP electrode activation substrate. Accordingly, changing patterns of light 418 from the light source 416, which may be controlled by the motive module 162, can selectively activate and deactivate changing patterns of DEP electrodes at regions 414 of the inner surface 408 of the electrode activation substrate 406. (Hereinafter the regions 414 of a microfluidic device having a DEP electrode activation substrate are referred to as “DEP electrode regions.”) As illustrated in FIG.4B, a light pattern 418 directed onto the inner surface 408 of the electrode activation substrate 406 can illuminate select DEP electrode regions 414a (shown in white) in a pattern, such as a square. The non-illuminated DEP electrode regions 414 (cross-hatched) are hereinafter referred to as “dark” DEP electrode regions 414. The relative electrical impedance through the DEP electrode activation substrate 406 (i.e., from the bottom electrode 404 up to the inner surface 408 of the electrode activation substrate 406 which interfaces with the fluidic medium 180 in the flow region 106) is greater than the relative electrical impedance through the fluidic medium 180 in the region/chamber 402 (i.e., from the inner surface 408 of the electrode activation substrate 406 to the top electrode 410 of the cover 110) at each dark DEP electrode region 414. An illuminated DEP electrode region 414a, however, exhibits a reduced relative impedance through the electrode activation substrate 406 that is less than the relative impedance through the fluidic medium 180 in the region/chamber 402 at each illuminated DEP electrode region 414a. Attorney Docket No.01149-0025-00PCT [0197] With the power source 412 activated, the foregoing DEP configuration creates an electric field gradient in the fluidic medium 180 between illuminated DEP electrode regions 414a and adjacent dark DEP electrode regions 414, which in turn creates local DEP forces that attract or repel nearby micro-objects (not shown) in the fluidic medium 180. DEP electrodes that attract or repel micro-objects in the fluidic medium 180 can thus be selectively activated and deactivated at many different such DEP electrode regions 414 at the inner surface 408 of the region/chamber 402 by changing light patterns 418 projected from a light source 416 into the microfluidic device 400. Whether the DEP forces attract or repel nearby micro- objects can depend on such parameters as the frequency of the power source 412 and the dielectric properties of the fluidic medium 180 and/or micro-objects (not shown). Depending on the frequency of the power applied to the DEP configuration and selection of fluidic media (e.g., a highly conductive media such as PBS or other media appropriate for maintaining biological cells), negative DEP forces may be produced. Negative DEP forces may repel the micro-objects away from the location of the induced non-uniform electrical field. In some embodiments, a microfluidic device incorporating DEP technology may generate negative DEP forces. [0198] The square pattern 420 of illuminated DEP electrode regions 414a illustrated in FIG.4B is an example only. Any pattern of the DEP electrode regions 414 can be illuminated (and thereby activated) by the pattern of light 418 projected into the microfluidic device 400, and the pattern of illuminated/activated DEP electrode regions 414 can be repeatedly changed by changing or moving the light pattern 418. [0199] In some embodiments, the electrode activation substrate 406 can comprise or consist of a photoconductive material. In such embodiments, the inner surface 408 of the electrode activation substrate 406 can be featureless. For example, the electrode activation substrate 406 can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100 * the number of hydrogen atoms / the total number of hydrogen and silicon atoms). The layer of a-Si:H can have a thickness of about 500 nm to about 2.0 ^m. In such embodiments, the DEP electrode regions 414 can be created anywhere and in any pattern on the inner surface 408 of the electrode activation substrate 406, in accordance with the light pattern 418. The number and pattern of the DEP electrode regions 214 thus need not be fixed, but can correspond to the light pattern 418. Examples of microfluidic devices having a DEP configuration comprising a photoconductive layer such as discussed above have been described, for example, in U.S. Attorney Docket No.01149-0025-00PCT Patent No. RE 44,711 (Wu, et al.) (originally issued as U.S. Patent No. 7,612,355), each of which is incorporated herein by reference in its entirety. [0200] In other embodiments, the electrode activation substrate 406 can comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers that form semiconductor integrated circuits, such as is known in semiconductor fields. For example, the electrode activation substrate 406 can comprise a plurality of phototransistors, including, for example, lateral bipolar phototransistors, with each phototransistor corresponding to a DEP electrode region 414. Alternatively, the electrode activation substrate 406 can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, with each such electrode corresponding to a DEP electrode region 414. The electrode activation substrate 406 can include a pattern of such phototransistors or phototransistor-controlled electrodes. The pattern, for example, can be an array of substantially square phototransistors or phototransistor- controlled electrodes arranged in rows and columns. Alternatively, the pattern can be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes that form a hexagonal lattice. Regardless of the pattern, electric circuit elements can form electrical connections between the DEP electrode regions 414 at the inner surface 408 of the electrode activation substrate 406 and the bottom electrode 404, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 418, as described above. [0201] Examples of microfluidic devices having electrode activation substrates that comprise phototransistors have been described, for example, in U.S. Patent No.7,956,339 (Ohta et al.) and U.S. Patent No.9,908,115 (Hobbs et al.), the entire contents of each of which are incorporated herein by reference. Examples of microfluidic devices having electrode activation substrates that comprise electrodes controlled by phototransistor switches have been described, for example, in U.S. Patent No.9,403,172 (Short et al.), which is incorporated herein by reference in its entirety. [0202] In some embodiments of a DEP configured microfluidic device, the top electrode 410 is part of a first wall (or cover 110) of the enclosure 402, and the electrode activation substrate 406 and bottom electrode 404 are part of a second wall (or support structure 104) of the enclosure 102. The region/chamber 402 can be between the first wall and the second wall. In other embodiments, the electrode 410 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 406 and/or the electrode Attorney Docket No.01149-0025-00PCT 410 are part of the first wall (or cover 110). Moreover, the light source 416 can alternatively be used to illuminate the enclosure 102 from below. [0203] With the microfluidic device 400 of FIGS. 4A-4B having a DEP electrode activation substrate, the motive module 162 of control and monitoring equipment 152, as described for FIG. 1A herein, can select a micro-object (not shown) in the fluidic medium 180 in the region/chamber 402 by projecting a light pattern 418 into the microfluidic device 400 to activate a first set of one or more DEP electrodes at DEP electrode regions 414a of the inner surface 408 of the electrode activation substrate 406 in a pattern (e.g., square pattern 420) that surrounds and captures the micro-object. The motive module 162 can then move the in situ-generated captured micro-object by moving the light pattern 418 relative to the microfluidic device 400 to activate a second set of one or more DEP electrodes at DEP electrode regions 414. Alternatively, the microfluidic device 400 can be moved relative to the light pattern 418. [0204] In other embodiments, the microfluidic device 400 may be a DEP configured device that does not rely upon light activation of DEP electrodes at the inner surface 408 of the electrode activation substrate 406. For example, the electrode activation substrate 406 can comprise selectively addressable and energizable electrodes positioned opposite to a surface including at least one electrode (e.g., cover 110). Switches (e.g., transistor switches in a semiconductor substrate) may be selectively opened and closed to activate or inactivate DEP electrodes at DEP electrode regions 414, thereby creating a net DEP force on a micro-object (not shown) in region/chamber 402 in the vicinity of the activated DEP electrodes. Depending on such characteristics as the frequency of the power source 412 and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber 402, the DEP force can attract or repel a nearby micro-object. By selectively activating and deactivating a set of DEP electrodes (e.g., at a set of DEP electrodes regions 414 that forms a square pattern 420), one or more micro-objects in region/chamber 402 can be selected and moved within the region/chamber 402. The motive module 162 in FIG.1A can control such switches and thus activate and deactivate individual ones of the DEP electrodes to select, and move particular micro-objects (not shown) around the region/chamber 402. Microfluidic devices having a DEP electrode activation substrate that includes selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Patent No. 6,294,063 (Becker, et al.) and U.S. Patent No.6,942,776 (Medoro), each of which is incorporated herein by reference in its entirety. Attorney Docket No.01149-0025-00PCT [0205] Regardless of whether the microfluidic device 400 has a dielectrophoretic electrode activation substrate, an electrowetting electrode activation substrate or a combination of both a dielectrophoretic and an electrowetting activation substrate, a power source 412 can be used to provide a potential (e.g., an AC voltage potential) that powers the electrical circuits of the microfluidic device 400. The power source 412 can be the same as, or a component of, the power source 192 referenced in Fig. 1A. Power source 412 can be configured to provide an AC voltage and/or current to the top electrode 410 and the bottom electrode 404. For an AC voltage, the power source 412 can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate net DEP forces (or electrowetting forces) strong enough to select and move individual micro-objects (not shown) in the region/chamber 402, as discussed above, and/or to change the wetting properties of the inner surface 408 of the support structure 104 in the region/chamber 202, as also discussed above. Such frequency ranges and average or peak power ranges are known in the art. See, e.g., U.S. Patent No.6,958,132 (Chiou, et al.), US Patent No. RE44,711 (Wu, et al.) (originally issued as US Patent No. 7,612,355), and U.S. Patent Application Publication Nos. 2014/0124370 (Short, et al.), 2015/0306598 (Khandros, et al.), 2015/0306599 (Khandros, et al.), and 2017/0173580 (Lowe, Jr. et al.), each of which disclosures are herein incorporated by reference in its entirety. [0206] Other forces may be utilized within the microfluidic devices, alone or in combination, to move selected micro-objects. Bulk fluidic flow within the microfluidic channel may move micro-objects within the flow region. Localized fluidic flow, which may be operated within the microfluidic channel, within a sequestration pen, or within another kind of chamber (e.g., a reservoir) can also be used to move selected micro-objects. Localized fluidic flow can be used to move selected micro-objects out of the flow region into a non-flow region such as a sequestration pen or the reverse, from a non-flow region into a flow region. The localized flow can be actuated by deforming a deformable wall of the microfluidic device, as described in U.S. Patent No.10,058,865 (Breinlinger, et al.), which is incorporated herein by reference in its entirety. [0207] Gravity may be used to move micro-objects within the microfluidic channel, into a sequestration pen, and/or out of a sequestration pen or other chamber, as described in U.S. Patent No. 9,744,533 (Breinlinger, et al.), which is incorporated herein by reference in its entirety. Use of gravity (e.g., by tilting the microfluidic device and/or the support to which the microfluidic device is attached) may be useful for bulk movement of cells into or out of the sequestration pens from/to the flow region. Magnetic forces may be employed Attorney Docket No.01149-0025-00PCT to move micro-objects including paramagnetic materials, which can include magnetic micro- objects attached to or associated with a biological micro-object. Alternatively, or in additional, centripetal forces may be used to move micro-objects within the microfluidic channel, as well as into or out of sequestration pens or other chambers in the microfluidic device. [0208] In another alternative mode of moving micro-objects, laser-generated dislodging forces may be used to export micro-objects or assist in exporting micro-objects from a sequestration pen or any other chamber in the microfluidic device, as described in International Patent Publication No. WO2017/117408 (Kurz, et al.), which is incorporated herein by reference in its entirety. [0209] In some embodiments, DEP forces are combined with other forces, such as fluidic flow (e.g., bulk fluidic flow in a channel or localized fluidic flow actuated by deformation of a deformable surface of the microfluidic device, laser generated dislodging forces, and/or gravitational force), so as to manipulate, transport, separate and sort micro- objects and/or droplets within the microfluidic circuit 120. In some embodiments, the DEP forces can be applied prior to the other forces. In other embodiments, the DEP forces can be applied after the other forces. In still other instances, the DEP forces can be applied in an alternating manner with the other forces. For the microfluidic devices described herein, repositioning of micro-objects may not generally rely upon gravity or hydrodynamic forces to position or trap micro-objects at a selected position. Gravity may be chosen as one form of repositioning force, but the ability to reposition of micro-objects within the microfluidic device does not rely solely upon the use of gravity. While fluid flow in the microfluidic channels may be used to introduce micro-objects into the microfluidic channels (e.g., flow region), such regional flow is not relied upon to pen or unpen micro-objects, while localized flow (e.g., force derived from actuating a deformable surface) may, in some embodiments, be selected from amongst the other types of repositioning forces described herein to pen or unpen micro-objects or to export them from the microfluidic device. [0210] When DEP is used to reposition micro-objects, bulk fluidic flow in a channel is generally stopped prior to applying DEP to micro-objects to reposition the micro- objects within the microfluidic circuit of the device, whether the micro-objects are being repositioned from the channel into a sequestration pen or from a sequestration pen into the channel. Bulk fluidic flow may be resumed thereafter. [0211] System. Returning to FIG. 1A, a system 150 for operating and controlling microfluidic devices is shown, such as for controlling the microfluidic device 100. The electrical power source 192 can provide electric power to the microfluidic device 100, Attorney Docket No.01149-0025-00PCT providing biasing voltages or currents as needed. The electrical power source 192 can, for example, comprise one or more alternating current (AC) and/or direct current (DC) voltage or current sources. [0212] System 150 can further include a media source 178. The media source 178 (e.g., a container, reservoir, or the like) can comprise multiple sections or containers, each for holding a different fluidic medium 180. Thus, the media source 178 can be a device that is outside of and separate from the microfluidic device 100, as illustrated in FIG. 1A. Alternatively, the media source 178 can be located in whole or in part inside the enclosure 102 of the microfluidic device 100. For example, the media source 178 can comprise reservoirs that are part of the microfluidic device 100. [0213] FIG.1A also illustrates simplified block diagram depictions of examples of control and monitoring equipment 152 that constitute part of system 150 and can be utilized in conjunction with a microfluidic device 100. As shown, examples of such control and monitoring equipment 152 can include a master controller 154 comprising a media module 160 for controlling the media source 178, a motive module 162 for controlling movement and/or selection of micro-objects (not shown) and/or medium (e.g., droplets of medium) in the microfluidic circuit 120, an imaging module 164 for controlling an imaging device (e.g., a camera, microscope, light source or any combination thereof) for capturing images (e.g., digital images), and an optional tilting module 166 for controlling the tilting of the microfluidic device 100. The control equipment 152 can also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the monitoring equipment 152 can further include a display device 170 and an input/output device 172. [0214] The master controller 154 can comprise a control module 156 and a digital memory 158. The control module 156 can comprise, for example, a digital processor configured to operate in accordance with machine executable instructions (e.g., software, firmware, source code, or the like) stored as non-transitory data or signals in the memory 158. Alternatively, or in addition, the control module 156 can comprise hardwired digital circuitry and/or analog circuitry. The media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 can be similarly configured. Thus, functions, processes acts, actions, or steps of a process discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus can be performed by any one or more of the master controller 154, media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 configured Attorney Docket No.01149-0025-00PCT as discussed above. Similarly, the master controller 154, media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 may be communicatively coupled to transmit and receive data used in any function, process, act, action or step discussed herein. [0215] The media module 160 controls the media source 178. For example, the media module 160 can control the media source 178 to input a selected fluidic medium 180 into the enclosure 102 (e.g., through an inlet port 107). The media module 160 can also control removal of media from the enclosure 102 (e.g., through an outlet port (not shown)). One or more media can thus be selectively input into and removed from the microfluidic circuit 120. The media module 160 can also control the flow of fluidic medium 180 in the flow path 106 inside the microfluidic circuit 120. The media module 160 may also provide conditioning gaseous conditions to the media source 178, for example, providing an environment containing 5% CO2 (or higher). The media module 160 may also control the temperature of an enclosure of the media source, for example, to provide feeder cells in the media source with proper temperature control. [0216] Motive module. The motive module 162 can be configured to control selection and movement of micro-objects (not shown) in the microfluidic circuit 120. The enclosure 102 of the microfluidic device 100 can comprise one or more electrokinetic mechanisms including a dielectrophoresis (DEP) electrode activation substrate, optoelectronic tweezers (OET) electrode activation substrate, electrowetting (EW) electrode activation substrate, and/or an opto-electrowetting (OEW) electrode activation substrate, where the motive module 162 can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects and/or droplets in the flow path 106 and/or within sequestration pens 124, 126, 128, and 130. The electrokinetic mechanism(s) may be any suitable single or combined mechanism as described within the paragraphs describing motive technologies for use within the microfluidic device. A DEP configured device may include one or more electrodes that apply a non-uniform electric field in the microfluidic circuit 120 sufficient to exert a dielectrophoretic force on micro-objects in the microfluidic circuit 120. An OET configured device may include photo-activatable electrodes to provide selective control of movement of micro-objects in the microfluidic circuit 120 via light-induced dielectrophoresis. [0217] The imaging module 164 can control the imaging device. For example, the imaging module 164 can receive and process image data from the imaging device. Image data from the imaging device can comprise any type of information captured by the imaging Attorney Docket No.01149-0025-00PCT device (e.g., the presence or absence of micro-objects, droplets of medium, accumulation of label, such as fluorescent label, etc.). Using the information captured by the imaging device, the imaging module 164 can further calculate the position of objects (e.g., micro-objects, droplets of medium) and/or the rate of motion of such objects within the microfluidic device 100. [0218] The imaging device (part of imaging module 164, discussed below) can comprise a device, such as a digital camera, for capturing images inside microfluidic circuit 120. In some instances, the imaging device further comprises a detector having a fast frame rate and/or high sensitivity (e.g., for low light applications). The imaging device can also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beams may be in the visible spectrum and may, e.g., include fluorescent emissions. The reflected light beams may include reflected emissions originating from an LED or a wide spectrum lamp, such as a mercury lamp (e.g., a high-pressure mercury lamp) or a Xenon arc lamp. The imaging device may further include a microscope (or an optical train), which may or may not include an eyepiece. [0219] Support Structure. System 150 may further comprise a support structure 190 configured to support and/or hold the enclosure 102 comprising the microfluidic circuit 120. In some embodiments, the optional tilting module 166 can be configured to activate the support structure 190 to rotate the microfluidic device 100 about one or more axes of rotation. The optional tilting module 166 can be configured to support and/or hold the microfluidic device 100 in a level orientation (i.e., at 0° relative to x- and y-axes), a vertical orientation (i.e., at 90° relative to the x-axis and/or the y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) relative to an axis is referred to herein as the “tilt” of the microfluidic device 100 (and the microfluidic circuit 120). For example, support structure 190 can optionally be used to tilt the microfluidic device 100 (e.g., as controlled by optional tilting module 166) to 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relative to the x-axis or any degree therebetween. When the microfluidic device is tilted at angles greater than about 15, tilting may be performed to create bulk movement of micro-objects into/out of sequestration pens from/into the flow region (e.g., microfluidic channel). In some embodiments, the support structure 190 can hold the microfluidic device 100 at a fixed angle of 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, or 10° Attorney Docket No.01149-0025-00PCT relative to the x-axis (horizontal), so long as DEP is an effective force to move micro-objects out of the sequestration pens into the microfluidic channel. Since the surface of the electrode activation substrate is substantially flat, DEP forces may be used even when the far end of the sequestration pen, opposite its opening to the microfluidic channel, is disposed at a position lower in a vertical direction than the microfluidic channel. [0220] In some embodiments where the microfluidic device is tilted or held at a fixed angle relative to horizontal, the microfluidic device 100 may be disposed in an orientation such that the inner surface of the base of the flow path 106 is positioned at an angle above or below the inner surface of the base of the one or more sequestration pens opening laterally to the flow path. The term “above” as used herein denotes that the flow path 106 is positioned higher than the one or more sequestration pens on a vertical axis defined by the force of gravity (i.e., an object in a sequestration pen above a flow path 106 would have a higher gravitational potential energy than an object in the flow path), and inversely, for positioning of the flow path 106 below one or more sequestration pens. In some embodiments, the support structure 190 may be held at a fixed angle of less than about 5°, about 4°, about 3° or less than about 2 ° relative to the x-axis (horizontal), thereby placing the sequestration pens at a lower potential energy relative to the flow path. In some other embodiments, when long term culturing (e.g., for more than about 2, 3, 4, 5, 6, 7 or more days) is performed within the microfluidic device, the device may be supported on a culturing support and may be tilted at a greater angle of about 10°, 15°, 20°, 25°, 30°, or any angle therebetween to retain biological micro-objects within the sequestration pens during the long-term culturing period. At the end of the culturing period, the microfluidic device containing the cultured biological micro-objects may be returned to the support 190 within system 150, where the angle of tilting is decreased to values as described above, affording the use of DEP to move the biological micro-objects out of the sequestration pens. Further examples of the use of gravitational forces induced by tilting are described in U.S. Patent No.9,744,533 (Breinlinger et al.), the contents of which are herein incorporated by reference in its entirety. [0221] Nest. Turning now to FIG. 5A, the system 150 can include a structure (also referred to as a “nest”) 500 configured to hold a microfluidic device 520, which may be like microfluidic device 100, 200, or any other microfluidic device described herein. The nest 500 can include a socket 502 capable of interfacing with the microfluidic device 520 (e.g., an optically actuated electrokinetic device 100, 200, etc.) and providing electrical connections from power source 192 to microfluidic device 520. The nest 500 can further include an integrated electrical signal generation subsystem 504. The electrical signal generation Attorney Docket No.01149-0025-00PCT subsystem 504 can be configured to supply a biasing voltage to socket 502 such that the biasing voltage is applied across a pair of electrodes in the microfluidic device 520 when it is being held by socket 502. Thus, the electrical signal generation subsystem 504 can be part of power source 192. The ability to apply a biasing voltage to microfluidic device 520 does not mean that a biasing voltage will be applied at all times when the microfluidic device 520 is held by the socket 502. Rather, in most cases, the biasing voltage will be applied intermittently, e.g., only as needed to facilitate the generation of electrokinetic forces, such as dielectrophoresis or electro-wetting, in the microfluidic device 520. [0222] As illustrated in FIG.5A, the nest 500 can include a printed circuit board assembly (PCBA) 522. The electrical signal generation subsystem 504 can be mounted on and electrically integrated into the PCBA 522. The exemplary support includes socket 502 mounted on PCBA 522, as well. [0223] In some embodiments, the nest 500 can comprise an electrical signal generation subsystem 504 configured to measure the amplified voltage at the microfluidic device 520 and then adjust its own output voltage as needed such that the measured voltage at the microfluidic device 520 is the desired value. In some embodiments, the waveform amplification circuit can have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, resulting in a signal of up to 13 Vpp at the microfluidic device 520. [0224] In certain embodiments, the nest 500 further comprises a controller 508, such as a microprocessor used to sense and/or control the electrical signal generation subsystem 504. Examples of suitable microprocessors include the Arduino™ microprocessors, such as the Arduino Nano™. The controller 508 may be used to perform functions and analysis or may communicate with an external master controller 154 (shown in FIG. 1A) to perform functions and analysis. In the embodiment illustrated in FIG. 5A the controller 508 communicates with the master controller 154 (of FIG.1A) through an interface (e.g., a plug or connector). [0225] As illustrated in FIG. 5A, the support structure 500 (e.g., nest) can further include a thermal control subsystem 506. The thermal control subsystem 506 can be configured to regulate the temperature of microfluidic device 520 held by the support structure 500. For example, the thermal control subsystem 506 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). In the embodiment illustrated in FIG 5A, the support structure 500 comprises an inlet 516 and an outlet 518 to receive cooled fluid from an external reservoir (not shown) of the cooling unit, introduce the cooled fluid into the fluidic Attorney Docket No.01149-0025-00PCT path 514 and through the cooling block, and then return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and/or the fluidic path 514 can be mounted on a casing 512 of the support structure 500. In some embodiments, the thermal control subsystem 506 is configured to regulate the temperature of the Peltier thermoelectric device so as to achieve a target temperature for the microfluidic device 520. Temperature regulation of the Peltier thermoelectric device can be achieved, for example, by a thermoelectric power supply, such as a Pololu™ thermoelectric power supply (Pololu Robotics and Electronics Corp.). The thermal control subsystem 506 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit. [0226] The nest 500 can include a serial port 524 which allows the microprocessor of the controller 508 to communicate with an external master controller 154 via the interface. In addition, the microprocessor of the controller 508 can communicate (e.g., via a Plink tool (not shown)) with the electrical signal generation subsystem 504 and thermal control subsystem 506. Thus, via the combination of the controller 508, the interface, and the serial port 524, the electrical signal generation subsystem 504 and the thermal control subsystem 506 can communicate with the external master controller 154. In this manner, the master controller 154 can, among other things, assist the electrical signal generation subsystem 504 by performing scaling calculations for output voltage adjustments. A Graphical User Interface (GUI) (not shown) provided via a display device 170 coupled to the external master controller 154, can be configured to plot temperature and waveform data obtained from the thermal control subsystem 506 and the electrical signal generation subsystem 504, respectively. Alternatively, or in addition, the GUI can allow for updates to the controller 508, the thermal control subsystem 506, and the electrical signal generation subsystem 504. [0227] Optical sub-system. FIG. 5B is a schematic of an optical sub-system 550 having an optical apparatus 510 for imaging and manipulating micro-objects in a microfluidic device 520, which can be any microfluidic device described herein. The optical apparatus 510 can be configured to perform imaging, analysis and manipulation of one or more micro-objects within the enclosure of the microfluidic device 520. [0228] The optical apparatus 510 may have a first light source 552, a second light source 554, and a third light source 556. The first light source 552 can transmit light to a structured light modulator 560, which can include a digital mirror device (DMD) or a microshutter array system (MSA), either of which can be configured to receive light from the first light source 552 and selectively transmit a subset of the received light into the optical Attorney Docket No.01149-0025-00PCT apparatus 510. Alternatively, the structured light modulator 560 can include a device that produces its own light (and thus dispenses with the need for a light source 552), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD). The structured light modulator 560 can be, for example, a projector. Thus, the structured light modulator 560 can be capable of emitting both structured and unstructured light. In certain embodiments, an imaging module and/or motive module of the system can control the structured light modulator 560. [0229] In embodiments when the structured light modulator 560 includes a mirror, the modulator can have a plurality of mirrors. Each mirror of the plurality of mirrors can have a size of about 5 microns x 5 microns to about 10 microns x10 microns, or any values therebetween. The structured light modulator 560 can include an array of mirrors (or pixels) that is 2000 x 1000, 2580 x 1600, 3000 x 2000, or any values therebetween. In some embodiments, only a portion of an illumination area of the structured light modulator 560 is used. The structured light modulator 560 can transmit the selected subset of light to a first dichroic beam splitter 558, which can reflect this light to a first tube lens 562. [0230] The first tube lens 562 can have a large clear aperture, for example, a diameter larger than about 40 mm to about 50 mm, or more, providing a large field of view. Thus, the first tube lens 562 can have an aperture that is large enough to capture all (or substantially all) of the light beams emanating from the structured light modulator 560. [0231] The structured light 515 having a wavelength of about 400 nm to about 710 nm, may alternatively or in addition, provide fluorescent excitation illumination to the microfluidic device. [0232] The second light source 554 may provide unstructured brightfield illumination. The brightfield illumination light 525 may have any suitable wavelength, and in some embodiments, may have a wavelength of about 400 nm to about 760 nm. The second light source 554 can transmit light to a second dichroic beam splitter 564 (which also may receive illumination light 535 from the third light source 556), and the second light, brightfield illumination light 525, may be transmitted therefrom to the first dichroic beam splitter 558. The second light, brightfield illumination light 525, may then be transmitted from the first dichroic beam splitter 558 to the first tube lens 562. [0233] The third light source 556 can transmit light through a matched pair relay lens (not shown) to a mirror 566. The third illumination light 535 may therefrom be reflected to the second dichroic beam splitter 5338 and be transmitted therefrom to the first Attorney Docket No.01149-0025-00PCT beam splitter 5338, and onward to the first tube lens 5381. The third illumination light 535 may be a laser and may have any suitable wavelength. In some embodiments, the laser illumination 535 may have a wavelength of about 350 nm to about 900 nm. The laser illumination 535 may be configured to heat portions of one or more sequestration pens within the microfluidic device. The laser illumination 535 may be configured to heat fluidic medium, a micro-object, a wall or a portion of a wall of a sequestration pen, a metal target disposed within a microfluidic channel or sequestration pen of the microfluidic channel, or a photoreversible physical barrier within the microfluidic device, and described in more detail in U. S. Application Publication Nos. 2017/0165667 (Beaumont, et al.) and 2018/0298318 (Kurz, et al.), each of which disclosure is herein incorporated by reference in its entirety. In other embodiments, the laser illumination 535 may be configured to initiate photocleavage of surface modifying moieties of a modified surface of the microfluidic device or photocleavage of moieties providing adherent functionalities for micro-objects within a sequestration pen within the microfluidic device. Further details of photocleavage using a laser may be found in International Application Publication No. WO2017/205830 (Lowe, Jr. et al.), which disclosure is herein incorporated by reference in its entirety. [0234] The light from the first, second, and third light sources (552, 554, 556) passes through the first tube lens 562 and is transmitted to a third dichroic beam splitter 568 and filter changer 572. The third dichroic beam splitter 568 can reflect a portion of the light and transmit the light through one or more filters in the filter changer 572 and to the objective 570, which may be an objective changer with a plurality of different objectives that can be switched on demand. Some of the light (515, 525, and/or 535) may pass through the third dichroic beam splitter 568 and be terminated or absorbed by a beam block (not shown). The light reflected from the third dichroic beam splitter 568 passes through the objective 570 to illuminate the sample plane 574, which can be a portion of a microfluidic device 520 such as the sequestration pens described herein. [0235] The nest 500, as described in FIG.5A, can be integrated with the optical apparatus 510 and be a part of the apparatus 510. The nest 500 can provide electrical connection to the enclosure and be further configured to provide fluidic connections to the enclosure. Users may load the microfluidic apparatus 520 into the nest 500. In some other embodiments, the nest 500 can be a separate component independent of the optical apparatus 510. [0236] Light can be reflected off and/or emitted from the sample plane 574 to pass back through the objective 570, through the filter changer 572, and through the third dichroic beam splitter 568 to a second tube lens 576. The light can pass through the second Attorney Docket No.01149-0025-00PCT tube lens 576 (or imaging tube lens 576) and be reflected from a mirror 578 to an imaging sensor 580. Stray light baffles (not shown) can be placed between the first tube lens 562 and the third dichroic beam splitter 568, between the third dichroic beam splitter 568 and the second tube lens 576, and between the second tube lens 576 and the imaging sensor 580. [0237] Objective. The optical apparatus can comprise the objective lens 570 that is specifically designed and configured for viewing and manipulating of micro-objects in the microfluidic device 520. For example, conventional microscope objective lenses are designed to view micro-objects on a slide or through 5mm of aqueous fluid, while micro- objects in the microfluidic device 520 are inside the plurality of sequestration pens within the viewing plane 574 which have a depth of 20, 30, 40, 50, 6070, 80 microns or any values therebetween. In some embodiments, a transparent cover 520a, for example, glass or ITO cover with a thickness of about 750 microns, can be placed on top of the plurality of sequestration pens, which are disposed above a microfluidic substrate 520c. Thus, the images of the micro- objects obtained by using the conventional microscope objective lenses may have large aberrations such as spherical and chromatic aberrations, which can degrade the quality of the images. The objective lens 570 of the optical apparatus 510 can be configured to correct the spherical and chromatic aberrations in the optical apparatus 1350. The objective lens 570 can have one or more magnification levels available such as, 4X, 10X, 20X. [0238] Modes of illumination. In some embodiments, the structured light modulator 560 can be configured to modulate light beams received from the first light source 552 and transmits a plurality of illumination light beams 515, which are structured light beams, into the enclosure of the microfluidic device, e.g., the region containing the sequestration pens. The structured light beams can comprise the plurality of illumination light beams. The plurality of illumination light beams can be selectively activated to generate a plurality of illuminations patterns. In some embodiments, the structured light modulator 560 can be configured to generate an illumination pattern, similarly as described for FIGS.4A-4B, which can be moved and adjusted. The optical apparatus 560 can further comprise a control unit (not shown) which is configured to adjust the illumination pattern to selectively activate the one or more of the plurality of DEP electrodes of a substrate 520c and generate DEP forces to move the one or more micro-objects inside the plurality of sequestration pens within the microfluidic device 520. For example, the plurality of illuminations patterns can be adjusted over time in a controlled manner to manipulate the micro-objects in the microfluidic device 520. Each of the plurality of illumination patterns can be shifted to shift the location of the DEP force generated Attorney Docket No.01149-0025-00PCT and to move the structured light for one position to another in order to move the micro-objects within the enclosure of the microfluidic apparatus 520. [0239] In some embodiments, the optical apparatus 510 may be configured such that each of the plurality of sequestration pens in the sample plane 574 within the field of view is simultaneously in focus at the image sensor 580 and at the structured light modulator 560. In some embodiments, the structured light modulator 560 can be disposed at a conjugate plane of the image sensor 580. In various embodiments, the optical apparatus 510 can have a confocal configuration or confocal property. The optical apparatus 510 can be further configured such that only each interior area of the flow region and/or each of the plurality of sequestration pens in the sample plane 574 within the field of view is imaged onto the image sensor 580 in order to reduce overall noise to thereby increase the contrast and resolution of the image. [0240] In some embodiments, the first tube lens 562 can be configured to generate collimated light beams and transmit the collimated light beams to the objective lens 570. The objective 570 can receive the collimated light beams from the first tube lens 562 and focus the collimated light beams into each interior area of the flow region and each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510. In some embodiments, the first tube lens 562 can be configured to generate a plurality of collimated light beams and transmit the plurality of collimated light beams to the objective lens 570. The objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and converge the plurality of collimated light beams into each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510. [0241] In some embodiments, the optical apparatus 510 can be configured to illuminate the at least a portion of sequestration pens with a plurality of illumination spots. The objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and project the plurality of illumination spots, which may form an illumination pattern, into each of the plurality of sequestration pens in the sample plane 574 within the field of view. For example, each of the plurality of illumination spots can have a size of about 5 microns X 5 microns; 10 microns X 10 microns; 10 microns X 30 microns, 30 microns X 60 microns, 40 microns X 40 microns, 40 microns X 60 microns, 60 microns X 120 microns, 80 microns X 100 microns, 100 microns X 140 microns and any values there between. The illumination spots may individually have a shape that is circular, square, or rectangular. Alternatively, the illumination spots may be grouped within a plurality of illumination spots (e.g., an illumination pattern) to form a larger polygonal shape such as a rectangle, square, or wedge shape. The Attorney Docket No.01149-0025-00PCT illumination pattern may enclose (e.g., surround) an unilluminated space that may be square, rectangular or polygonal. For example, each of the plurality of illumination spots can have an area of about 150 to about 3000, about 4000 to about 10000, or 5000 to about 15000 square microns. An illumination pattern may have an area of about 1000 to about 8000, about 4000 to about 10000, 7000 to about 20000, 8000 to about 22000, 10000 to about 25000 square microns and any values there between. [0242] The optical system 510 may be used to determine how to reposition micro-objects and into and out of the sequestration pens of the microfluidic device, as well as to count the number of micro-objects present within the microfluidic circuit of the device. Further details of repositioning and counting micro-objects are found in U. S. Application Publication No.2016/0160259 (Du); U. S. Patent No.9,996,920 (Du et al.); and International Application Publication No. WO2017/102748 (Kim, et al.). The optical system 510 may also be employed in assay methods to determine concentrations of reagents/assay products, and further details are found in U. S. Patent Nos.8,921,055 (Chapman), 10,010,882 (White et al.), and 9,889,445 (Chapman et al.); International Application Publication No. WO2017/181135 (Lionberger, et al.); and International Application Serial No. PCT/US2018/055918 (Lionberger, et al.). Further details of the features of optical apparatuses suitable for use within a system for observing and manipulating micro-objects within a microfluidic device, as described herein, may be found in WO2018/102747 (Lundquist, et al), the disclosure of which is herein incorporated by reference in its entirety. [0243] Additional system components for maintenance of viability of cells within the sequestration pens of the microfluidic device. In order to promote growth and/or expansion of cell populations, environmental conditions conducive to maintaining functional cells may be provided by additional components of the system. For example, such additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from cells. A. Disposing biological cells/capture object within chamber [0244] In some embodiments, the method may further include disposing one or more biological cells within the one or more sequestration pens of the microfluidic device. In some embodiments, each one of the one or more biological cells may be disposed in a different one of the one or more sequestration pens. The one or more biological cells may be disposed within the isolation regions of the one or more sequestration pens of the microfluidic device. In some embodiments of the method, at least one of the one or more biological cells may be disposed within a sequestration pen having one of the one or more capture objects disposed Attorney Docket No.01149-0025-00PCT therein. In some embodiments, the one or more biological cells may be a plurality of biological cells from a clonal population. In various embodiments of the method, disposing the one or more biological cells may be performed before disposing the one or more capture objects. [0245] In various embodiments, the capture object may be any capture object as described herein. In some embodiments, the capture object may include a magnetic component (e.g., a magnetic bead). Alternatively, the capture object can be non-magnetic. [0246] In some embodiments, a single biological cell is disposed in a sequestration pen. In some embodiments, a plurality of biological cells, for example, 2 or more, 2 to 10, 3 to 8, 4 to 6, or the like, are disposed within said sequestration pen. [0247] In various embodiments, disposing the biological cell may further include marking the biological cell (e.g., with a marker for nucleic acids, such as Dapi or Hoechst stain). [0248] In some embodiments, disposing said biological cell within said sequestration pen is performed before disposing said capture object within said sequestration pen. In some embodiments, disposing said capture object within said sequestration pen is performed before disposing said biological cell within said sequestration pen. [0249] In some embodiments, said enclosure of said microfluidic device comprises at least one coated surface. In some embodiments, the coated surface comprises a covalently linked surface. In some embodiments, the coated surface comprises a hydrophilic or a negatively charged coated surface. The coated surface can be coated with Tris and/or a polymer, such as a PEG-PPG block co-polymer. In yet other embodiments, the enclosure of the microfluidic device may include at least one conditioned surface. [0250] The at least one conditioned surface may include a covalently bound hydrophilic moiety or a negatively charged moiety. A covalently bound hydrophilic moiety or negatively charged moiety can be a hydrophilic or negatively charged polymer. [0251] In some embodiments, said enclosure of the microfluidic device further comprises a dielectrophoretic (DEP®) configuration, and wherein disposing said biological cell and/or disposing said capture object is performed by applying a dielectrophoretic (DEP®) force on or proximal to said biological cell and/or said capture object. [0252] In some embodiments, said microfluidic device further comprises a plurality of sequestration pens. Optionally, the method further comprises disposing a plurality of said biological cells within said plurality of sequestration pens. [0253] A plurality of said biological cells disposed within said plurality of sequestration pens may have substantially only one biological cell disposed within Attorney Docket No.01149-0025-00PCT sequestration pens of said plurality. Thus, each sequestration pen of the plurality having a biological cell disposed therein will generally contain a single biological cell. For example, less than 10%, 7%, 5%, 3% or 1% of sequestration pens occupied by a cell may contain more than one biological cell. In some embodiments, the plurality of biological cells may be a clonal population of biological cells. [0254] A plurality of said capture objects disposed within said plurality of sequestration pens may have substantially only one capture object disposed within sequestration pens of said plurality. Thus, each sequestration pen of the plurality having a capture object disposed therein will generally contain a single capture object. For example, less than 10%, 7%, 5%, 3% or 1% of sequestration pens occupied by a capture object may contain more than one capture object. [0255] A plurality of said biological cells and a plurality of capture objects disposed within said plurality of sequestration pens may have substantially only one biological cell and substantially only one capture object disposed within sequestration pens of said plurality. Thus, each sequestration pen of the plurality having a biological cell and a capture object disposed therein will generally contain a single biological cell and a single capture object. For example, less than 10%, 7%, 5%, 3% or 1% of sequestration pens occupied by a cell and a capture object may contain more than one biological cell or more than one capture object. In some embodiments, the plurality of biological cells may be a clonal population of biological cells. VIII. Kits [0256] A kit is also provided for use in methods of assaying a biological cell such as any of those disclosed herein. In some embodiments, the kit includes an activation composition and/or a maintaining composition, each as described herein. In some embodiments, the kit further includes a microfluidic device as described herein. For example, in certain embodiments, the microfluidic device can comprise an enclosure, where the enclosure includes a flow region and a plurality of sequestration pens opening off of the flow region. IX. EXAMPLES Example 1: Memory B Cell Isolation and Activation. [0257] The compositions and methods disclosed herein provide an end-to-end workflow that enables robust feeder-free activation of memory B cells, including human memory B cells, followed by on-chip assays and downstream sequence recovery (FIG. 6B). Attorney Docket No.01149-0025-00PCT The compositions of the present disclosure enable activation of memory B cell samples (e.g., mammalian memory B cells, including human memory B cells) from fresh whole blood or frozen PBMC donor samples (FIG.6B, panels A and B). [0258] Cell preparation and assay reagents: As shown in FIG.6C, the methods can begin by either isolating peripheral blood mononuclear cells (PBMCs) from fresh whole blood, or by thawing frozen PBMCs. Memory B cells are then isolated from PBMCs via off the shelf magnetic-activated cell sorting (MACS) kits and activated for 5 days in culture using the presently disclosed compositions for memory B cell activation (FIG.6C). In 35 PBMC samples harvested from 5 vaccinated and/or convalescent human donors the average frequency of memory B cells was 0.56% (range 0.15% - 1.5%, FIG.7A). The activation medium produces activated B cells with robust proliferation (2.6-31.6-fold, n=39 samples; FIG. 7B) and high viability (62-99%, n=48 samples; FIG.7C). As shown in FIG.11, after 5 days of culturing in activation medium, the pre- and post-activation expression levels of CD20 and CD138 show the differentiation of memory B cells into antibody -secreting plasma cells. At this point, the activated B cells are ready for screening on the Beacon ® Optofluidic system (Bruker Cellular Analysis, Inc.). [0259] For the experiment described above, human peripheral blood mononuclear cells (PBMCs) were isolated from fresh leukopak (STEMCELL) using density gradient centrifugation (CL5015, Cedarlane) or obtained in a pre-isolated format (AllCells) for enrichment and screening on the Beacon system (FIG. 6C). Class-switched memory B cells were enriched from PBMCs using a magnetic-activated cell sorting (MACS) kit (130-093-617, Miltenyi), with expected frequencies ranging from 0.1-1.0% of the original starting population (FIG.10A). Enriched memory B cells were re-suspended in human memory activation medium at a density of 2.5 E4 cells/mL. The activation medium negates the requirement for feeder cell co-culture and comprises: RPMI 1640, 20% FBS, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 µM 2-mercaptoethanol, 100 ng/mL IL-2, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, 50 ng/mL BAFF, and 2 µg/mL CpG ODN 2006. These cells were seeded in 96-well, V-bottom plates with 5000 cells (200 µL of the above suspension) dispensed per well. Off-chip activation was conducted over 5 days of culture at 37 ºC and 5% CO2 with no media exchanges. The activation media formulation and culture conditions (e.g., seeding density and timeline) were developed to enable efficient proliferation (FIG.7B), viability (FIG.7C) and IgG secretion after 5 days of culture. Differentiation of these memory B cells into an antibody secreting state is also inferred from the increase in CD138 expression, a surface marker for antibody-secreting plasma B cells, and a decrease in CD20 Attorney Docket No.01149-0025-00PCT expression, a surface marker for memory B cells, after off-chip activation (FIG. 8). Without the activation medium, the cells die overnight and no proliferation or IgG secretion will be observed over five days of off-chip culture. [0260] FIG.14 shows a comparison of B-cell proliferation after 5 days of culture using the activation medium described compared to two different commercial B-cell expansion medias (composition not provided by manufacturer). [0261] The disclosed workflows do not require antigen-positive sorting of memory B cells, thus enabling screening of difficult cell-based targets that cannot be recombinantly expressed as soluble molecules. However, in cases where the antigen-positive memory B cell populations are rare, standard enrichment strategies pre-activation and/or post-activation can be adapted to increase the likelihood of finding rare antibodies (dotted lines, FIG.6C). Example 2: Screening of Activated B Cells [0262] Materials and Methods [0263] System and Microfluidic device: The Beacon® system and microfluidic device used in Example 1 were manufactured by Bruker Cellular Analysis, Inc.. The system included at least a flow controller, temperature controller, fluidic medium conditioning and pump component, light source for light activated DEP configurations, mounting stage for the microfluidic device, and a camera. The microfluidic device was an OptoSelect® chip configured with OptoElectroPositioning (OEP®) technology. The microfluidic device included a microfluidic channel and a plurality of NanoPen® chambers fluidically connected thereto. [0264] The Beacon® system is used to automatically isolate tens of thousands of single activated B cells into NanoPen® chambers on OptoSelect® 11k or 20k chips (Bruker Cellular Analysis, Inc.) in under 1 hour per chip (FIG. 6B, panel C). Antigen-binding and cross- reactivity assays are used to screen and select activated B cells secreting antigen-specific antibodies (FIG.6B, panel D). Single antigen-specific B cells can then be recovered into 96- well plates (FIG.6B, panel E) followed by off-chip cDNA synthesis and amplification using the Opto® B Discovery cDNA Synthesis® Kit (Bruker Cellular Analysis, Inc.; FIG.6B, panel F). Paired antibody heavy/light chain sequences are amplified from the cDNA of recovered cells using the Opto® B Discovery Sanger Prep Kit, Human (Bruker Cellular Analysis, Inc.), which includes a primer for conventional Sanger sequencing (FIG.6B, panels G and H). [0265] Following B cell activation: After 5 days of incubation, activated human B cells are loaded onto OptoSelect® 11k or OptoSelect® 20k chips and isolated as single B cells into NanoPen® chambers. Over 7,500 or 12,500 single B cells can be loaded and screened per OptoSelect® 11k chip or OptoSelect® 20k chip, respectively. Workflows can use up to four Attorney Docket No.01149-0025-00PCT chips at a single time, enabling a total single B cell screening throughput of 7,500 to up to 60,000 cells per workflow (FIG.9). In some samples, the activated B cells are loaded onto the selected chip in cell media composition comprising PMA. In other samples, the activated B cells are loaded onto the selected chip in cell media without PMA in the composition. [0266] On-Chip Assays for Functional Profiling: Following loading, activated B cells can be screened using multiple on-chip assays for antibody specificity and cross- reactivity. Cell Analysis Suite (CAS®) software on the Beacon® system uses machine learning to automatically score assays and identify NanoPen® chambers that contain B cells secreting antigen-specific antibodies. Users can then manually verify assay results using Image Analyzer software. [0267] Bead-based assays for IgG antibody secretion can be used to assess successful activation of memory B cells. From 16 workflows performed on samples from vaccinated and/or convalescent donor samples against SARS CoV-2, 39% of activated single B cells secreted IgG antibodies on average (FIG.10, two left-most panels). Bead-based assays can also be used to discover antigen-specific antibodies. In the same 16 workflows, an average of 0.18% of single B cells were positive for the soluble SARS-CoV-2 antigen (FIG.10, two right-most panels). The culture medium designed for maintenance of antibody expression used in the workflow maintains an average of 86% of IgG secreting B cells 4 hours into the workflow (FIG.11), enabling more assays and longer, more complex assays to be run on precious donor samples. Table 2 illustrates an example working concentration and an example working range of selected reagents in the culture medium for activation/culturing of the B cells (off-chip) and for functionality profiling/maintenance (on chip).
Figure imgf000090_0001
[0268] On-chip assays for specificity against cell membrane antigens can also be performed by replacing beads with cells over-expressing the target antigen. This unlocks the Attorney Docket No.01149-0025-00PCT ability to perform antibody discovery against more challenging membrane antigens that cannot be expressed solubly and screened with other technologies. [0269] Sequence Recovery: The workflow can be configured for recovery of paired heavy/light chain sequences for antigen-specific antibodies via the single cell approach (FIGS. 12A-12B). Using the single cell approach, all molecular biology steps – lysis, reverse transcription, cDNA synthesis and amplification – are performed after recovery of single cells into 96-well plates (FIG. 12B). This is a preferred method for high recovery rates of paired heavy/light chain sequences, especially when there are fewer than 192 hits. The amplified cDNA from the single cell approach can be performed with the aid of the Opto® B Discovery Sanger Prep Kit (Human). [0270] The PCR products can then be sequenced using traditional Sanger Sequencing. On average, 75% of pens targeted for export yielded paired heavy/light chain sequences with unambiguous nucleotide bases across the entire V(D)J regions (FIG. 13, Strict). These same samples yielded sequences that had no ambiguous bases across the CDR regions 78% of the time, on average (FIG.13, Moderate), and no ambiguous bases across the CDR3 region 80% of the time, on average (FIG. 13, Relaxed). Sequences with ambiguous base calls may be recovered with traditional cloning and/or re-sequencing methods. [0271] Example 3: Phorbol Myristate Acetate (PMA) Addition in B Cell Culture (on-chip) [0272] Human antibody-secreting B cells generated from memory B cells were penned into OptoSelect 11k chips using the opto-electropositioning (OEP) feature of the Beacon system. Isolation of each cell into an individual pen within a microfluidic environment facilitated the screening of antibody secretion on a single-cell basis using the Capture Assay imaging operation. The first assay was performed immediately after the penning operation was complete, revealing that ca.30% of the activated B cells were secreting IgG molecules across all OptoSelect chips. Thereafter, the chips were perfused with separate culture media formulations in which the concentration of phorbol myristate acetate (PMA) was 100 ng/mL (FIG.16A, 16B) or varied from 0-100 ng/mL (FIG.17). Common to each media formulation was: RPMI 1640, 10% FBS, 2% B-27 supplement, 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 µM 2- mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF. A second IgG secretion assay was performed after 24 h of media perfusion. As shown in FIG. 15A, the number of antibody hits, antibody hit rate (%), and hit retention all increased with the addition of PMA relative to the control. As shown in FIG.15B, the addition of TNF-α alone Attorney Docket No.01149-0025-00PCT did not show any increase in any of the parameters measured relative to the control and TNF- α with PMA did not show any increase in any of the parameters measured relative to PMA alone. As shown in FIG.16, a dose-dependency of PMA on the retention of IgG secretion over time by the activated B cells occurs for the number of antibody hits, antibody hit rate (%), and hit retention. Increasing the concentration of PMA from 0 to 100 ng/mL increased the percentage of B cells that continued to secrete IgG after this period of on-chip culture as shown in FIG.16. [0273] Example 4: PMA Addition- Load and Culture Media Comparisons (on- chip) [0274] Human antibody-secreting B cells generated from memory B cells were penned into OptoSelect 11k chips using the opto-electropositioning (OEP) feature of the Beacon system. Isolation of each cell into an individual pen within a microfluidic environment facilitated the screening of antibody secretion on a single-cell basis using the Capture Assay imaging operation. Load media was formulated with and without 100 ng/mL phorbol myristate acetate (PMA) so that any benefits conferred by the molecule during the penning operation could be investigated (see FIG. 17). Common to each Load Media formulation was: RPMI 1640, 10% FBS, 10% B-27 supplement, 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 µM 2-mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF. After the penning operation was complete, the chips were perfused with separate Culture Media formulations that were prepared with and without 100 ng/mL PMA (see FIG. 17). Common to each Culture Media formulation was: RPMI 1640, 10% FBS, 2% B-27 supplement, 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 µM 2-mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF. An initial antibody secretion assay was performed using these Culture Media formulations, with subsequent assays performed after 2 and 4 h of on-chip culture to assess the longevity effects of PMA in the Load and Culture Media. As shown in FIG.17, the presence of PMA in both the Load and Culture Media facilitated an improvement in the retention of IgG secretion over time by the activated B cells. [0275] Example 6: Comparison of PKC Agonists on B cells (on-chip) [0276] Human antibody-secreting B cells generated from memory B cells were penned into OptoSelect 11k chips using the opto-electropositioning (OEP) feature of the Beacon system. Isolation of each cell into an individual pen within a microfluidic environment facilitated the screening of antibody secretion on a single-cell basis using the Capture Assay Attorney Docket No.01149-0025-00PCT imaging operation. The chips were perfused with separate culture media formulations in which the presence of two phorbol esters, phorbol myristate acetate (PMA, 100 nM) and phorbol 12,13-dibutyrate (PDBu, 100 nM), were investigated. Compared to PMA, PDBu possesses the same central phorbol ring with matching stereochemistry, though differs in that both the myristate and acetate ester moieties are replaced with butyrate esters. Common to each media formulation was: RPMI 1640, 10% FBS, B-27 supplement (10% during OEP, 2% during culture), 0.1% pluronic F-127, 1x GlutaMAX, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/mL penicillin/streptomycin, 10 µM 2-mercaptoethanol, 2.5 ng/mL IL-4, 10 ng/mL IL-6, 5 ng/mL IL-21, and 50 ng/mL BAFF. The first assay was performed immediately after the penning operation was complete, revealing that ca.36% of the activated B cells were secreting IgG molecules across all OptoSelect chips. Sequential assays were thereafter performed after 4, 24, and 48 h of on-chip culture to assess the longevity effects of both phorbol esters. As shown in FIG. 18, both PMA and PDBu facilitated an improvement in the retention of IgG secretion over time by the activated B cells to the same extent. * * * [0277] In addition to any previously indicated modification, numerous other variations and alternative arrangements may be devised by those skilled in the art without departing from the spirit and scope of this description, and appended claims are intended to cover such modifications and arrangements. Thus, while the information has been described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred aspects, it will be apparent to those of ordinary skill in the art that numerous modifications, including, but not limited to, form, function, manner of operation, and use may be made without departing from the principles and concepts set forth herein. Also, as used herein, the examples and embodiments, in all respects, are meant to be illustrative only and should not be construed to be limiting in any manner. Furthermore, where reference is made herein to a list of elements (e.g., elements a, b, c), such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or a combination of all of the listed elements. Also, as used herein, the terms a, an, and one may each be interchangeable with the terms at least one and one or more. It should also be noted, that while the term step is used herein, that term may be used to simply draw attention to different portions of the described methods and is not meant to delineate a starting point or a stopping point for any portion of the methods, or to be limiting in any other way.

Claims

Attorney Docket No.01149-0025-00PCT WHAT IS CLAIMED: 1. A composition for culturing B cell lymphocytes, the composition comprising: a protein kinase C (PKC) agonist, wherein the composition lacks feeder cells. 2. The composition of claim 1, wherein the PKC agonist is a phorbol compound. 3. The composition of claim 1, wherein the PKC agonist is a phorbol ester. 4. The composition of claim 1, wherein the PKC agonist is selected from phorbol 12- myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PBD), 4-alpha-phorbol 12-myristate 13-acetate (4-alpha-PMA), dPPA, 4-alpha-phorbol (isophorbol), and phorbol 12-myristate 13- acetate 4-O-methyl ether (MPMA). 5. The composition of claim 4, wherein the PKC agonist is phorbol 12-myristate 13- acetate (PMA). 6. The composition of claim 4, wherein the PKC agonist is phorbol 12,13-dibutyrate (PBD). 7. The composition of claim 2, wherein the composition comprises the PKC agonist at a concentration of at least about 10 ng/mL. 8. The composition of claim 2, wherein the composition comprises the PKC agonist at a concentration from about 10 ng/mL to about 1000 ng/mL. 9. The composition of claim 1 further comprising: IL-6 and/or IL-4. 10. The composition of claim 9, wherein the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL and, optionally, wherein the IL-6 is human IL-6. 11. The composition of claim 9, wherein the composition comprises IL-4 at a concentration from about 0.5 ng/mL to about 10 ng/mL and, optionally, wherein the IL-4 is human IL-4. 12. The composition of claim 9 further comprising: IL-21 and/or BAFF. 13. The composition of claim 12, wherein the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL and, optionally, wherein the IL-21 is human IL-21. 14. The composition of claim 12, wherein the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL and, optionally, wherein the BAFF is human BAFF. 15. The composition of claim 1, wherein the composition comprises IL-2 at a concentration of less than about 1.0 ng/mL or substantially lacks IL-2. Attorney Docket No.01149-0025-00PCT 16. The composition of claim 1, wherein the composition comprises TNF-alpha at a concentration of less than about 1.0 ng/mL or substantially lacks TNF-alpha. 17. The composition of claim 1, wherein the composition comprises CpG oligodeoxynucleotides (ODNs) at a concentration of less than about 250 nanomolar (nM) or substantially lacks CpG ODNs. 18. The composition of claim 1, wherein the composition comprises a PI3K agonist at a concentration of less than about 1.0 ng/mL or substantially lacks a PI3K agonist. 19. The composition of claim 1, wherein the composition comprises a STAT3 agonist at a concentration of less than about 1.0 ng/mL or substantially lacks STAT3 agonist. 20. The composition of claim 1, wherein the composition comprises a CD40-clustering agent at a concentration of less than about 1.0 ng/mL or substantially lacks CD40-clustering agent. 21. The composition of claim 1, further comprising: serum at an amount of about 1% to about 20% (v/v). 22. The composition of claim 22, wherein the serum is fetal bovine serum. 23. The composition of any one of claims 1 to 22, wherein the composition is a liquid medium. 24. A composition for culturing B cell lymphocytes, the composition comprising: a Toll-like receptor 9 (TLR9) agonist; IL-6; and IL-4, wherein the composition lacks feeder cells. 25. The composition of claim 24, wherein the composition comprises IL-6 at a concentration from about 1.0 ng/mL to about 100 ng/mL and, optionally, wherein the IL-6 is human IL-6. 26. The composition of claim 24, wherein the composition comprises IL-4 at a concentration from about 0.5 ng/mL to about 10 ng/mL and, optionally, wherein the IL-4 is human IL-4. 27. The composition of claim 24 further comprising: IL-2, IL-21 and/or BAFF. 28. The composition of claim 27, wherein the composition comprises IL-2 at a concentration from about 1.0 ng/mL to about 100 ng/mL and, optionally, wherein the IL-2 is human IL-2. Attorney Docket No.01149-0025-00PCT 29. The composition of claim 27, wherein the composition comprises IL-21 at a concentration from about 1.0 ng/mL to about 100 ng/mL and, optionally, wherein the IL-21 is human IL-21. 30. The composition of claim 27, wherein the composition comprises BAFF at a concentration from about 10 ng/mL to about 100 ng/mL and, optionally, wherein the BAFF is human BAFF. 31. The composition of claim 24, wherein the composition comprises a protein kinase C (PKC) agonist at a concentration of less than about 10 ng/mL or substantially lacks PKC agonist. 32. The composition of claim 24, wherein the TLR9 agonist is a CpG oligodeoxynucleotide (ODN). 33. The composition of claim 32, wherein the CpG ODN is a B-class CpG ODN and/or has a linear structure. 34. The composition of claim 32, wherein the composition comprises CpG ODN at a concentration of at least about 0.25 micromolar (µM). 35. The composition of claim 32, wherein the composition comprises CpG ODN at a concentration of from about 0.25 µM to about 5.0 µM. 36. The composition of claim 24 further comprising serum present at an amount of about 1% to about 20% (v/v). 37. The composition of claim 36, wherein the serum is fetal bovine serum. 38. The composition of any one of claims 24 to 37, wherein the composition is a liquid medium. 39. A method of culturing a B cell lymphocyte, the method comprising: incubating the B cell lymphocyte in a composition of claim 23, wherein the incubating maintains the expression of antibodies by the B cell lymphocyte. 40. The method of claim 39, wherein the B cell lymphocyte is a mammalian B cell. 41. The method of claim 39, wherein the B cell lymphocyte is a human B cell. 42. The method of claim 39, wherein the B cell lymphocyte is a memory B cell or a derivative thereof. 43. The method of claim 39, wherein the incubating is performed for at least 12 hours. 44. The method of claim 39, wherein the incubating is performed in a microfluidic chip. 45. The method of claim 44, wherein the incubating is performed in 10 nanoliters or less of the composition. 46. A method of inducing proliferation of a B cell lymphocyte, the method comprising: Attorney Docket No.01149-0025-00PCT incubating the B cell lymphocyte in a composition of claim 38, wherein the incubating is performed for a time sufficient to induce proliferation of the B cell lymphocyte. 47. A method of culturing a B cell lymphocyte, the method comprising: incubating the B cell lymphocyte in a first composition, wherein the first composition is a composition of claim 24; and incubating the B cell lymphocyte in a second composition, wherein the second composition is a composition of claim 23; wherein the first composition stimulates activation and/or expression of antibody by the B cell lymphocyte, and wherein the second composition maintains continued expression of antibody by the B cell lymphocyte. 48. The method of claim 47, wherein the B cell lymphocyte is a human B cell. 49. The method of claim 47, wherein the B cell lymphocyte is a memory B cell. 50. The method of claim 47, wherein the incubation in the first composition is performed in a well of a well plate and wherein the incubation in the first composition is performed for at least 24 hours. 51. The method of claim 47, wherein the incubation in the second composition is performed in a microfluidic chip and wherein incubating with the second composition comprises perfusing the second composition through the microfluidic chip. 52. The method of claim 47, wherein the incubation in the second composition is performed in a well of a well plate. 53. A method for screening a B cell lymphocyte for expression of an antibody that specifically binds to an antigen of interest, the method comprising: culturing the B cell lymphocyte according to the method of claim 39; providing the antigen of interest; and detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte. 54. A method for screening a B cell lymphocyte for expression of an antibody that specifically binds to an antigen of interest, the method comprising: culturing the B cell lymphocyte according to the method of claim 47; providing the antigen of interest; and detecting binding between the antigen of interest and immunoglobulin expressed by the B cell lymphocyte.
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