WO2017205687A1 - Contact dispensing of cells into multi-well devices - Google Patents

Abstract

The present disclosure provides methods, devices, assemblies, and systems for contact dispensing of cells into multi-well devices. For example, provided herein are systems and methods for contact dispensing a dispense volume into a plurality of wells of a multi-well device, where the multi-well device has a hydrophobic top surface (e.g., a contact agent greater than 140 degrees, including greater than 165 degrees) and wells which have a relatively hydrophilic well surface (e.g., contact angle of 65-80 degrees). In some embodiments, a dispensing tip has a hanging drop of liquid (e.g., containing a cell) that is touched off onto the hydrophobic top surface of the multi-well device such that is repelled by the top surface and collected into, and attracted by, the relatively hydrophilic surface of the wells.

Description

CONTACT DISPENSING OF CELLS INTO MULTI-WELL DEVICES

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of the United States Provisional Patent Application Serial No. 62/342,496, filed May 27, 2016; the disclosure of which application is herein incorporated by reference.

INTRODUCTION

Geneticists are striving to characterize complex diseases like cancer, autoimmune and neurological disorders, but finding the underlying mechanisms driving these diseases has been elusive. Somatic mutations, spontaneous variants that accumulate in cells over a lifetime, are a major factor that drives disease onset and reoccurrence. As cells accumulate new mutations, they form polyclonal cell populations that co-exist with normal cells. Sequencing bulk cell populations can mask the underlying heterogeneity of these unique rare cell types, making it difficult to distinguish them from normal germline mutations. The best way to reveal these differences and visualize the clonal architecture is to sequence individual cells in the population. While single-cell sequencing can help uncover mechanisms of complex disease, traditional approaches are expensive, labor intensive, and require large sample input.

SUMMARY

The present disclosure provides methods, devices, assemblies, and systems for contact dispensing of cells into multi-well devices. For example, provided herein are systems and methods for contact dispensing a dispense volume into a plurality of wells of a multi-well device, where the multi-well device has a hydrophobic top surface (e.g., a contact agent greater than 140 degrees, including greater than 165 degrees) and wells which have a relatively hydrophilic well surface (e.g., contact angle of 65-80 degrees). In some embodiments, a dispensing tip has a hanging drop of liquid (e.g., containing a cell) that is touched off onto the hydrophobic top surface of the multi-well device such that is repelled by the top surface and collected into, and attracted by, the relatively hydrophilic surface of the wells.

In some embodiments, provided herein are methods comprising: dispensing a hanging drop of liquid from a dispense tip onto a hydrophobic top surface of a multi-well device, wherein the multi-well device comprises a plurality of hydrophilic wells formed in the hydrophobic top surface, wherein the dispensing comprises contacting the hanging drop with the hydrophobic surface while the hanging drop is still in contact with the dispense tip, and wherein the dispensing causes the hanging drop to separate from the dispense tip and move along the hydrophobic surface and into one of the hydrophilic wells.

In some embodiments, provided herein are systems comprising: a) a multi-well device, wherein the multi-well devices comprises a hydrophobic top surface with a plurality of relatively hydrophilic wells formed therein; and b) a fluid movement component comprising at least one fluidic channel, wherein the fluidic channel comprises a dispense tip configured to form a hanging drop of liquid when liquid is in the fluidic channel.

In some embodiments, the hydrophobic top surface of the multi-well device has a water contact angle greater than about 135 degrees (e.g., greater than 135 ... 160 ... 165 ... or 175 degrees; such as 135-185 degrees or 140-175). In some embodiments, the hydrophilic surface of the wells has a water contact angle of about 60-85 degrees (e.g., 60 ... 70 ... 80 ... or 85 degrees). In some embodiments, the wells are composed of, or coated with, a hydrophilic material such as, for example, an ACULON hydrophilic coating; PI 00, HI 00, SI 00 & XI 00 hydrophilic coatings from JONSMAN INNOVATION; a hydrophilic polymer disclosed in U.S. Pat. 6,866,936 (herein incorporated by reference in its entirety), such as a hydrophilic polymer selected from poly(N-vinyl lactams), poly(ethylene oxide), poly(propylene oxide),

polyacrylamides, cellulosics, polyacrylic acids, polyvinyl alcohols, and polyvinyl ethers; a hydrophilic polymer disclosed in U.S. Pat. 6,238,799 (herein incorporated by reference in its entirety); or a poly dopamine or similar material (see, Kang and Choi, Bull. Korean, Soc, 2013, 34(8):2525-2527, herein incorporated by reference in its entirety).

In some embodiments, the dispense tip is composed of a material that is less hydrophilic than the relatively hydrophilic wells. For example, the dispense tip, or the hydrophobic top surface, could be composed of hydrophobic materials, such as polyimide, polyester,

polyethylene, polyurethane, TEFLON PTFE, fluorosilane, NEVERWET coatings (from Rust- Oleum), coatings from HYDROBEAD, NANOMYTE coatings (from NEI corp.), ACULONs hydrophobic and superhydrophobic surface treatments, or combinations of any two, any three, any four, any five, or any six or more of such polymers and coatings).

In further embodiments, the hanging drop of liquid comprises a dispense volume of a cell suspension, wherein the cell suspension comprises cells present in the cell suspension at a concentration such that, on average, X cell(s) is/are present in the dispense volume (e.g., so a Poisson type dispensing is achieved or approximated). In some embodiments, X is between 1 and 20 cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ... 15 ... or 20 cells). In some embodiments, X is one. In further embodiments, the method further comprises: dispensing a first additional volume which is equal to the dispense volume, but is free of cells, into at least some of the wells determined to have X cells or more than X cells.

In other embodiments, the methods further comprise, prior to the dispensing, contacting the dispense tip with a cell suspension such that the hanging drop of liquid is formed on the dispense tip. In other embodiments, the dispensing is performed by a robotic liquid dispensing system, wherein the robotic liquid dispensing system comprises a fluid movement component, and wherein the fluid movement component comprises at least one fluidic channel that terminates in the dispense tip.

In some embodiments, the liquid dispensing system comprises: i) a plurality of fluidic dispensing channels, ii) a source container containing the cell suspension; and iii) a robotic movement component attached to the fluidic dispensing channels, wherein the robotic movement component is moveable between the source container and the multi-well device. In other embodiments, the liquid dispensing system is automated and is configured to receive instructions from the computer (e.g., from image analysis software).

In some embodiments, the cells are circulating cancer cells. In other embodiments, the cells are stem cells. In further embodiments, the cells are cancer stem cells (e.g., breast CSCs, ovarian CSCs, colon CSCs, prostate CSCs, pancreatic CSCs, etc.). In further embodiments, the methods further comprise: conducting a biological reaction in at least one of the wells determined to have a single cell. In other embodiments, the methods further comprise:

conducting a biological reaction in at least 50% (e.g., at least 50% ... 70% ... 90% ... or 100%) of the wells determined to have one cell. In some embodiments, the biological reaction comprises a sequencing reaction, and/or a PCR reaction, and/or a cell lysis reaction). In some embodiments, the sequencing reaction employs nucleic acid barcode sequences.

In some embodiments, the multi-well device comprises at least 50 wells (e.g., 50 ... 100 ... 150 ... 400 ... 689 ... 900 ... or more). In additional embodiments, the multi-well device comprises at least 1000 wells (e.g., 1000 ... 1500 ... 2500 ... 5000 ... 5184 ... 10,000 .... 20,000 ... or more). In other embodiments, the multi-well device comprises a multi-well chip. In other embodiments, the dispensing volume is between 25 and 500 nl, or between 50 nl and 1 μΐ.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a multi-well device (30) that has a hydrophobic top surface (37) with a plurality of hydrophilic wells (35) therein. Figure 1 shows a liquid dispensing system with a fluid movement component (10) which has a plurality of fluidic channels (40), each with a dispense tip (42), shown with an associated hanging drop of liquid (45).

Figure 2 shows an exemplary robotic liquid dispensing system (70) enclosed in a hood.

Figure 3 shows an exemplary robotic liquid dispensing system (70) with the hood removed.

Figure 4 shows a close up view of an exemplary robotic liquid dispensing system, including: a fluid movement component (10) which contains a plurality of fluidic channels (40); a source container (20) shown with 384 individual sample source compartments and a first securing component (50) for holding the source container (20) in place; and a multi-well testing device (30), which may be WAFERGEN's 5184-nanowell chip, which is secured in place by a second securing component (60).

DETAILED DESCRIPTION

The present disclosure provides methods, devices, assemblies, and systems for contact dispensing of cells into multi-well devices. For example, provided herein are systems and methods for contact dispensing a dispense volume into a plurality of wells of a multi-well device, where the multi-well device has a hydrophobic top surface (e.g., a contact agent greater than 140 degrees, including greater thanl65 degrees) and wells which have a relatively hydrophilic well surface (e.g., contact angle of 65-80 degrees). In some embodiments, a dispensing tip has a hanging drop of liquid (e.g., containing a cell) that is touched off onto the hydrophobic top surface of the multi-well device such that it is repelled by the top surface and collected into, and attracted by, the relatively hydrophilic surface of the wells.

A. Contact Dispensing in Multi-Well Devices

Provided herein are methods for contact dispensing into multi-well devices that have hydrophobic top surfaces, having a plurality of hydrophobic wells formed therein. The dispense tip, with associated hanging drop of liquid, is contacted with the hydrophobic top surface of a multi-well device as shown in Figure 1. This causes the drop of liquid to be released from the dispense tip and move along the hydrophobic top source (e.g., repelled by the top surface) until it enters (e.g., attracted by) a hydrophilic well. In some instances, the drop of liquid has a single cell therein such that a well receives a single cell. The dispense tip is designed such that a desired volume of liquid is in the hanging drop of liquid. The cell suspension used as the source of liquid drops in some instances may contain a particular number of cells per volume such that a desired number of cells are likely to be statistically present in a single drop (including e.g., a single cell or multiple cells).

In some embodiments, the dispense tip or tips, are dipped into a cell suspension in order to form the hanging drop of liquid on each dispense tip. The dispense tips are then moved over to a multi-well device (e.g., nano-chip) and the hanging drops are "touched off onto the hydrophobic surface of the multi-well device. In this regard, each hanging drop is "rejected" by the hydrophobic surface of the multi-well device, and is "attracted" by the relatively hydrophilic surface inside each well. The attraction of the hydrophilic wells should be sufficient to pull the drop off (including any cells contained within the drop) and into the well. In some embodiments, the dispense tip is less hydrophilic than the wells of the multi-well device. Dispense tips can be treated during or after their fabrication to meet this parameter.

In some embodiments, cell suspension may be back-filled into the dispense tip or tips. By "back-filled" is meant that the fluid is loaded into the dispensing tips from an end opposite from which the fluid is dispensed, i.e., opposite the dispensing end. In back-filling the dispensing tip the fluid is loaded in the direction of dispensing. For example, a volume of cell suspension is transferred into a reservoir or container connected to one or more channels that extend into the dispense tip or tips, such that cell suspension may be moved through the one or more channels in order to form the hanging drop of liquid on each dispense tip. The dispense tips may then be moved over to a multi-well device and the hanging drops are "touched off, "rejected" "attracted" as described above. Any convenient method of transferring and/or moving the cell suspension, e.g., to or from a reservoir or container, through a channel, through a dispense tip, etc., may be employed including e.g., internal or external pressure sources (including e.g., positive pressure and negative pressure, including aspiration), internal or external mechanical pumps (including e.g., micropumps as employed in microfluidic devices), capillary forces, electrokinetic forces, gravity flow and the like.

In some embodiments, the top surface of the chip is composed of a hydrophobic material or coated with a hydrophobic material. Examples of such materials include, for example, polyimide, polyester, polyethylene, polyurethane, TEFLON PTFE, fluorosilane, NEVERWET coatings (from Rust-Oleum), coatings from HYDROBEAD, NANOMYTE coatings (from NEI corp.), ACULON's hydrophobic and superhydrophobic surface treatments, or combinations of any two, any three, any four, any five, or any six or more of such polymers and coatings). The hydrophobicity of a hydrophobic chip material or coating may vary. Methods of producing a hanging drop of liquid on the end of a dispense tip will vary. In some instances, the dispense tip may be inserted into the liquid and the removed such that, upon removal, the hanging drop is present on the dispense tip. In some instances, hanging drop is held on the dispense tip by the surface tension of the liquid. In some instances, liquid may be drawn into a channel present in the dispense tip. In some instances, the liquid is drawn into a channel of the dispense tip or held on the dispense tip through the action of a fluid movement component that contains or is attached to the dispense tip.

Any convenient protocol may be employed to draw liquid into the dispense tip, including but not limited to e.g., capillary action, negative pressure, etc. For example, in some instances, the dispense tip may include an internal channel dimensioned to draw the liquid (e.g., cell suspension) into the channel by capillary action. In some instances, a fluid movement component that includes a pump (e.g., a syringe pump, a peristaltic pump, a solenoid pump, a pneumatic pump, a piezoelectric pumps, etc.) or an aspirator may be employed to draw liquid into the dispense tip. In some instances, where a dispensing tip is configured with an internal channel, the internal channel may be sized with a sufficient diameter that cells of interest can pass feely into the channel, including e.g., where the internal channel diameter is at least 50 μπι in diameter or more, including e.g., 50 μπι in diameter or more, 75 μπι in diameter or more, 100 μπι in diameter or more, 125 μπι in diameter or more, 150 μπι in diameter or more, 175 μπι in diameter or more, 200 μπι in diameter or more, 225 μπι in diameter or more, 250 μπι in diameter or more, 275 μπι in diameter or more, 300 μπι in diameter or more, etc.

In some instances, formation of a hanging drop on the end of a dispense tip may be a result of the configuration of the tip, including combinations of the size, shape, hydrophobicity of tip surfaces, hydrophilicity of tip surfaces, the presence of a channel in the tip, etc. In such instances, contacting the dispense tip with the liquid or insertion and withdrawal of the dispensing tip into and out of the liquid may be sufficient to form the hanging drop on the dispensing tip.

In other instances, the hanging drop may be formed on the tip through some action of a fluid movement component of the dispenser. For example, where a pump or aspirator is employed, the hanging drop may be formed through use of the pump or aspirator, e.g., by employing the pump or aspirator to hold the drop on the dispensing tip, by employing the pump or aspirator to extrude the drop from a channel within the dispensing tip, and the like. Such uses of a pump or aspirator to position, extrude and/or hold the hanging drop on the dispense tip may be employed in any convenient context, including e.g., where immersion (i.e., "dipping") application of the liquid to the dispense tip is employed, wherein back-filling of the dispense tip is employed, and the like.

Dispense tip configurations may vary and may include dispensers with a single dispensing tip as well as dispensers with multiple dispensing tips. Accordingly, the number of dispensing tips present on a dispenser may range from one to 1000 or more, including but not limited to e.g., 2, 4, 6, 8, 10, 12, 14, 16, 20, 24, 25, 28, 30, 32, 35, 38, 40, 48, 50, 55, 60, 64, 70, 75, 80, 90, 96, 100, 144, 196, 225, 250, 256, 300, 384, 400, 500, etc. Where multiple dispensing tips are present, the tips may be arrayed in any convenient format, including equally or unequally spaced. In some instances, the dispensing tips may be arrayed with spacing corresponding to a multi-well device into which droplets are dispensed, i.e., the space between tips may correspond to the space between some integer (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.) of wells of a multi-well device. Corresponding spacing between dispensing tips and wells of a multi-well device may allow for parallel dispensing, i.e., the simultaneous dispensing of multiple hanging drops, each present on a dispensing tip, into corresponding wells of a multi-well device.

The vertical movement of dispensing tips may be controlled in a variety of ways. For example, in some instances, individual dispensing tips of a dispenser having a single or multiple dispensing tips may be configured with independent Z-axis control such that the individual dispensing tips may be lowered and/or raised independently. In such instances, each dispensing tip may be connected to an independent Z actuator, including where each independent Z actuator is linked to an electronic controller that allows for independent Z-axis control of each dispensing tip. In some instances, groups of dispensing tips, including all or a portion of the dispensing tips of a multi-tip dispenser, may be configured with group Z control such that the individual dispensing tips of the group may be lowered and/or raised together. In such instances, the group of dispensing tips may be connected to a Z actuator, including where the Z actuator is linked to an electronic controller that allows for Z-axis control of the group of dispensing tips.

A dispenser, having a single or multiple dispensing tips, may include an attached imaging device. For example, in some instances, a subject dispenser may include an attached digital camera which may or may not include attached microscopic components (e.g., objective lenses, illuminators, mirrors (including dichroic mirrors), filters (including long, short and band pass filters, etc.), collimators, polarizers, prisms, phase rings, phototubes, etc. In some instances, a imaging device attached to the dispenser may be sufficient for imaging and/or detecting whether a cell or a desired number of cells has been deposited into a well of a multi-well device. Imaging devices attached to dispensers may serve other functions, including e.g., one or more machine vision functions, including e.g., machine vision controlled alignment of a dispense tip (e.g., alignment with a well of a multi-well device, alignment with a source device or a well of a multi- well source device, etc.) machine vision assisted insertion of a dispense tip into a well of a multi- well device, and the like. Analysis of images generated by such imaging devices may be performed, in some instances, by a computing device connected to the imaging device and having programing that, when executed by the computing device, causes the computing device to perform one or more functions, including e.g., those described herein. For example, in some instances, detection of cell(s) or the absence thereof using images obtained by an imaging device may be performed by a computing device having instructions that analyze the acquired image to determine whether a well is empty of whether a cell or a desired number of cells is present.

The dispenser and/or components of the dispenser may be computer controlled (i.e., robotic) with or without the integration of an imaging device. Accordingly, the subject methods and systems may employ a processor connected to or otherwise in communication with one or more electrical components of the dispenser to control one or more actions of the components. Such a processor may, in some instances, have instructions that, when executed by the processor, cause the connected or controlled component of the dispenser to perform one or more actions required of it. Computer controlled components of the dispenser may include but are not limited to e.g., the dispenser itself, a z actuator of the dispenser, a z actuator of one or more dispense tips (e.g., a z actuator controlling a group of dispense tips, a z actuator controlling an individual dispense tip, etc.), a fluid movement component of the dispenser (e.g., a pump, an aspirator, etc.), a conveyor attached to the dispenser (e.g., for lateral conveyance of the dispenser, e.g., between a cell suspension source and a multi-well device), an imaging device, an environmental control element of the system, and the like. A processor may be programmed with instructions for performing any function of a method described herein where applicable. A subject processor may include a computer memory, e.g., for storing instructions, and such memories may, in some instance include a non-transitory computer readable medium.

In some embodiments, the dispense tip or tips are mounted adjacent to the existing tip mount of the BioDot dispense system (or similar systems) with an independent Z actuator, thereby allows such dispense systems to dispense cells, image the multi-well device (e.g., to determine which wells still need a cell or additional cells) and dispense reagents into appropriate wells. The liquid dispensing systems may have multiple dispense channels that match the pitch, depth, width, length, etc., of the wells in a multi-well device. Accordingly, the angle of dispense channels and/or dispense tips may vary and may range from vertical to nearly horizontal, including but not limited to e.g., vertical (i.e., 0° from vertical), from 0° to 45°, from 0° to 40°, from 0° to 35°, from 0° to 30°, from 0° to 25°, from 0° to 20°, from 0° to 15°, from 0° to 10°, from 0° to 5°, from 10° to 45°, from 10° to 40°, from 10° to 35°, from 10° to 30°, from 10° to 25°, from 10° to 20°, from 10° to 15°, from 20° to 45°, from 20° to 40°, from 20° to 35°, from 20° to 30°, from 20° to 25°, from 20° to 80°, from 20° to 70°, from 20° to 60°, from 20° to 50°, from 40° to 80°, from 40° to 70°, from 40° to 60° or from 40° to 50° from vertical.

The depth of a z step conferred from a z actuator may also vary and may range from less than 1 μιη to 10 cm or more, including but not limited to e.g., from 1 μιη to 10 cm, from 1 μιη to 5 cm, from 1 μιη to 1 cm, 1 μιη to 9 mm, from 1 μιη to 5 mm, from 1 μιη to 1 mm, from 1 μιη to 500 μιη, from 1 μιη to 100 μιτι, from 1 μιη to 50 μιτι, from 1 μιη to 10 μιτι, from 5 μιη to 10 cm, from 5 μιη to 5 cm, from 5 μιη to 1 cm, 5 μιη to 9 mm, from 5 μιη to 5 mm, from 5 μιη to 1 mm, from 5 μιη to 500 μιτι, from 5 μιη to 100 μιτι, from 5 μιη to 50 μιτι, from 5 μιη to 10 μιτι, from 10 μιη to 10 cm, from 10 μιη to 5 cm, from 10 μιη to 1 cm, 10 μιη to 9 mm, from 10 μιη to 5 mm, from 10 μιη to 1 mm, from 10 μιη to 500 μιτι, from 10 μιη to 100 μιτι, from 10 μιη to 50 μιτι, from 100 μιη to 10 cm, from 100 μιη to 5 cm, from 100 μιη to 1 cm, 100 μιη to 9 mm, from 100 μιη to 5 mm, from 100 μιη to 1 mm, from 100 μιη to 500 μιτι, from 1 mm to 10 cm, from 1 mm to 5 cm, from 1 mm to 1 cm, 1 mm to 9 mm, from 1 mm to 5 mm, from 1 cm to 10 cm, from 1 cm to 5 cm, and the like.

In some embodiments, such as shown in Figure 2, a robotic liquid dispensing system (70) enclosed in a hood is employed for the contact dispensing disclosed herein. Hood enclosed systems may include various features of environmental control facilitated by hood containment including but not limited to e.g., temperature control, humidity control, light exposure control, static control, etc. In some instances, hood containment provides for air flow control, including e.g., preventing air flow within the hood, laminar or directional air flow, and the like. In some instances, hood enclosure provides for biocontainment of samples processed within the hood.

B. Poisson Dispensing

In some embodiments, the contact dispensing disclosed herein employs a method that allows a certain number of cells to be present in the hanging drops, such that the average over many such dispenses results in a single cell being dispensed. A statistical description of this phenomenon is known as the Poisson distribution. In theory, dispensing a single cell per well (n= exactly 1 cell, but not 0, 2, 3, 4, 5, 6 etc. cells) is constrained by theta theoretical maxima = of 36.8% of wells will contain exactly 1 cell. However, the Poisson distribution can be leveraged to alter the input cell concentration to a very wide range of occupancy rates. Methods for achieving a Poisson distribution are described in application number 15/049,056 (US Patent Application Pub. No. 2016/0245813 Al), which is herein incorporated by reference in its entirety. As described, a source of cells is diluted using Poisson statistics such that on average 1 cell per dispense volume is dispensed. In some embodiments, microscopy (e.g., magnifying optics) is used to visualize each well and directly determine if the imaged well contains a single cell. Upon determining whether a well contains a desired number of cells (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) further actions may be carried out. For example, when a well is determined not to contain a desired number of cells (e.g., a well is empty of contains fewer or more than the desired number of cells) the well may be skipped in further processing, e.g., further reagents may not be added to the well. In some instances, when a well is determined not to contain a desired number of cells, e.g., is empty or contains less than the desired number of cells, a further volume of cell suspension may be added to the well, e.g., to increase the probability that the well will contain a cell or the desired number of cells. In some instances, when a well is determined to contain a desired number of cells additional reagents may be added to the well, e.g., for performing a biochemical process or assay.

In some embodiments, multi-sample dispensers (e.g., as shown in Figures 2-4) are programmed to perform a variety of series of biochemical steps, including bioprocessing steps and/or bio-assay steps, including e.g., lysis, DNA or RNA amplification, and sample barcoding, specifically in wells only bearing a desired number of cells, e.g., a single cell. Non-limiting examples of processes and analyses that can be performed include whole genome amplification (WGA), PCR (including e.g., sequence specific PCR, random primed PCR, qPCR, multiplex PCR, etc.), reverse transcription, cDNA preparation, template switching, tagmentation, Next Generation Sequencing (NGS), library preparation (e.g., for NGS) and the like, e.g., as described in more detail below.

In some embodiments, when wells are identified as having received zero cells, a second (and third) optional Recursive Poisson Distribution (RPD) step may be employed to circumvent the statistical limitations of the Poisson distribution, thereby raising single cell occupancy rates on-chip from a theoretical maxima of 37% to > 50%. The RPD in this disclosure refers to the iterative cycle of, (a) dispensing cell-containing solutions into reaction vessels (wells, chambers, etc.) in a chip, (b) visualization of cells on-chip in individual wells, (c) identifying the on-chip cell counts (equal to zero, equal to one, and greater than one) in individual wells by software- aided microscopy, and, (d) performing additional dispense cycles of cell-containing solutions into individual wells specifically identified in the previous round as having a cell count of zero. The objective of RPD is to maximize the number of occupied reaction vessels (wells, chambers, etc.) containing a single-cell (or some other desired number of cells) above the theoretical limitations Poisson distribution for a single dispense. This disclosure does not place a limit on the number of iterative cycles.

C. Cells

The present disclosure is not limited by the type of cells that are employed in the contact dispensing.

As summarized above, the present methods may include dispensing a volume of cell suspension into a well of a multi-well device. Essentially any cell suspension, containing any cells of any source, may be employed. Cells of interest may include a cell from any organism (e.g. a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, a cell from a multicellular organism, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal, a cell from a rodent (e.g., a mouse cell, a rat cell, etc.), a cell from a human, a cell from a non-human primate, etc.).

Any type of cell may be of interest (e.g. a pluripotent progenitor cell, a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell; a somatic cell (e.g., a somatic cell of mesodermal lineage, a somatic cell of endodermal lineage, a somatic cell of ectodermal lineage, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, an epithelial cell, etc.), a progenitor cell (e.g., a progenitor cell of mesodermal lineage, a progenitor cell of endodermal lineage, a progenitor cell of ectodermal lineage), a cell of an extraembryonic lineage; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage (e.g., a nematode embryo, a fly embryo, a xenopus embryo, a zebrafish embryo, a mouse embryo; a rat embryo, a non-human primate embryo, etc.), immune cells (e.g., primary or progenitor derived immune cells such as e.g., lymphocytes (T cells (immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg) and gamma-delta T cells), B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells)), and the like. Also of interest are modified cells such as e.g., genetically modified cells, including but not limited to e.g., genetically modified stem cells, genetically modified immune cells (e.g., engineered immune cells such as those employed in: antibody production/screening, engineered immune receptor (e.g., TCR) production/screening, adoptive immunotherapies (e.g., chimeric antigen receptor expressing immune cells), etc.) and the like.

Cells may be from established cell lines or they may be primary cells, where "primary cells", "primary cell lines", and "primary cultures" are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in vitro. Primary cells, in many instances, are not cultured and may, e.g., be utilized in a method of the present disclosure following isolation and/or dissociation directly, i.e., without undergoing cell culture.

Primary cells may be harvest from an individual by any convenient method. For example, leukocytes may be conveniently harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc., are conveniently harvested by biopsy. An appropriate solution may be used for dispersion, dissociation and/or suspension of harvested cells. Such solution may be a balanced salt solution, e.g. normal saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, etc., with or without supplementation with serum (e.g., fetal calf serum) or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration (e.g., from 5-25 mM). Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. Cells may be used immediately, or they may be stored, frozen, for some period of time, being thawed and capable of being reused. In such cases, the cells may be frozen in a freezing medium, including e.g., 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in any convenient manner for thawing frozen cells.

In some instances, cells of interest may include pluripotent progenitor cells. The terms "pluripotent progenitor cells", "pluripotent progenitors", "pluripotent stem cells", "multipotent progenitor cells" and the like, as used herein refer to cells that are capable of differentiating into two or more different cell types and proliferating. Non limiting examples of pluripotent precursor cells include but are not limited to embryonic stem cells, blastocyst derived stem cells, fetal stem cells, induced pluripotent stem cells, ectodermal derived stem cells, endodermal derived stem cells, mesodermal derived stem cells, neural crest cells, amniotic stem cells, cord blood stem cells, adult or somatic stem cells, neural stem cells, bone marrow stem cells, bone marrow stromal stem cells, hematopoietic stem cells, lymphoid progenitor cells, myeloid progenitor cells, mesenchymal stem cells, epithelial stem cells, adipose derived stem cells, skeletal muscle stem cells, muscle satellite cells, side population cells, intestinal stem cells, pancreatic stem cells, liver stem cells, hepatocyte stem cells, endothelial progenitor cells, hemangioblasts, gonadal stem cells, germline stem cells, and the like. Pluripotent progenitor cells may be acquired from public or commercial sources or may be newly derived.

In some instances, cells of interest may include cancer cells, circulating cancer cells, stem cells, and cancer stem cells. The term "cancer cells" may include primary cancer cells (i.e., cancer cells derived from a primary source such as e.g., a cancer or tumor biopsy) as well as cultured cancer cells (i.e., cancer cell lines, including e.g., immortalized cancer cell lines such as e.g., 3T3 cells, A549 cells, Fl 1 cells, HeLa cells, HEK 293 cells, Jurkat cells, Vero cells, and the like). Cancer cells of interest include primary cancer cells isolated from a cancer (e.g., a carcinoma, a sarcoma, a myeloma, a leukemia, a lymphoma, a cancer of mixed cell types) from an individual, including but not limited to e.g., cancer cells isolated from any of the following cancers: Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML),

Adrenocortical Carcinoma, AIDS-Related Cancers (e.g., Kaposi Sarcoma, Lymphoma, etc.), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer (Extrahepatic), Bladder Cancer, Bone Cancer (e.g., Ewing

Sarcoma, Osteosarcoma and Malignant Fibrous Histiocytoma, etc.), Brain Stem Glioma, Brain Tumors (e.g., Astrocytomas, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma, etc.), Breast Cancer (e.g., female breast cancer, male breast cancer, childhood breast cancer, etc.), Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (e.g., Childhood, Gastrointestinal, etc.), Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System (e.g., Atypical Teratoid/Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor, Lymphoma, etc.), Cervical Cancer, Childhood Cancers, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer,

Craniopharyngioma, Cutaneous T-Cell Lymphoma, Duct (e.g., Bile Duct, Extrahepatic, etc.), Ductal Carcinoma In Situ (DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer (e.g., Intraocular Melanoma, Retinoblastoma, etc.), Fibrous Histiocytoma of Bone (e.g., Malignant, Osteosarcoma, ec ), Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor,

Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor (e.g., Extracranial, Extragonadal, Ovarian, Testicular, etc.), Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis (e.g., Langerhans Cell, etc.), Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors (e.g., Pancreatic Neuroendocrine Tumors, etc.), Kaposi Sarcoma, Kidney Cancer (e.g., Renal Cell, Wilms Tumor, Childhood Kidney Tumors, etc.), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (e.g., Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell, etc.), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma In Situ (LCIS), Lung Cancer (e.g., Non-Small Cell, Small Cell, etc.), Lymphoma (e.g., AIDS-Related, Burkitt, Cutaneous T- Cell, Hodgkin, Non-Hodgkin, Primary Central Nervous System (CNS), etc.), Macroglobulinemia (e.g., Waldenstrom, etc.), Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia (e.g., Chronic (CML), etc.), Myeloid Leukemia (e.g., Acute (AML), etc.), Myeloproliferative Neoplasms (e.g., Chronic, etc.), Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer (e.g., Lip, etc.), Oropharyngeal Cancer,

Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer (e.g., Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor, etc.), Pancreatic Cancer, Pancreatic

Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer,

Pheochromocytoma, Pituitary Tumor, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (e.g., Ewing, Kaposi, Osteosarcoma, Rhabdomyosarcoma, Soft Tissue, Uterine, etc.), Sezary Syndrome, Skin Cancer (e.g., Childhood, Melanoma, Merkel Cell

Carcinoma, Nonmelanoma, etc.), Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer (e.g., with Occult Primary, Metastatic, etc.), Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Ureter and Renal Pelvis Cancer, Urethral Cancer, Uterine Cancer (e.g., Endometrial, etc.), Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom

Macroglobulinemia and Wilms Tumor.

Most cancer deaths appear to be caused by metastatic spread and growth by circulating tumor cells at distant organs. Circulating tumor cells (CTCs), CTC clusters (two or more individual CTCs bound together), and cancer stem cells (CSCs) may be initially localized, latent systemic, or post-adjuvant treatment depleted. Consequently, CTCs and the relevant stem cells are frequently present at low numbers within a large background of normal non-cancerous cells. The low frequency of these cells generates a complex "needle in a haystack" analysis problem for detecting the required cancer cell signal within the large 'noise" background. Detection of cancer cell specific cell surface markers and analysis of these cells is deeply relevant to understanding the biology of metastatic spread. The methods and systems provided herein allow isolation and analysis of such important cancer cells.

Single-cell, multiple-cell and cell clusters may initially be either enriched or depleted from a cell or tissue milieu or population, based on the presence of antigenic / phenotypic cell- surface or intra-cellular markers including but not restricted to: protein, lipid, carbohydrate (i.e. glycosylation) post-translational modifications of those moieties, nucleic acids and their modifications, or varying combinations of these moieties. Detection of cell surface markers in single cells -including cancer cells- and transferring those cells into discrete individual wells of a microfluidic device (e.g., Wafergen's SmartChip wells) is performed with the methods and systems described herein. In other embodiments, labelled cells may be dispensed directly into wells and antigenic moieties detected directly in chip via standard or automated microscopy using a variety of widely available fluorescence filters.

Any convenient methods of cell labeling and/or detection may be employed. For example, in some instances, cellular markers (including intracellular markers and cell surface markers) may be bound by a specific binding member that is detectable. Detectable specific binding members may be directly detectable (e.g., coupled to a detectable moiety, such as e.g., a fluorescent molecule) or may be indirectly detectable (e.g., coupled to a binding site (e.g., a biotin, a streptavidin, an immunoglobulin domain, an affinity tag, etc.) bound by a second specific binding member that is detectable (e.g., fluorescent secondary antibody). Specific binding members also include nucleic acids including but not limited to e.g., aptamers, oligonucleotide probes (e.g., RNA probes, DNA probes, LNA probes, etc.) that bind or hybridize with a specific target (e.g., a protein or nucleic acid target). Nucleic acid specific binding members may be directly detectable (e.g., conjugated to a fluorophore) or indirectly detectable (e.g., through binding of a second specific binding member).

Labeling of cells may be performed on live cells (e.g., through binding a specific binding member to a cell surface marker) or fixed cells, where permeabilization may or may not be employed depending on whether a subject marker is accessible on the surface or the cell or intracellular. Useful methods of labeling include immunohistochemistry, in situ hybridization, and the like. In some instances, cells may be labeled with an expressed detectable molecule such as e.g., an expressed fluorescent protein, an expressed bioluminescent protein, and the like.

Where fixed and/or permeabilized cells are employed any convenient method of fixing and/or permeabilizing may be employed including cross-linking and non-crosslinking fixatives including but not limited to e.g., formaldehyde, paraformaldehyde, formaldehyde/acetone, methanol/acetone, ethanol, methanol, Carnoy's, and the like. Permeabilization may be facilitated by any convenient method including e.g., one or more chemical or enzymatic methods including e.g., protease digestion, mild detergent exposure (e.g., Triton X-100, NP-40, saponin, etc.). In some instances, cells may be unfixed.

In some instances, cells may be labeled with one or more nucleic acid or cytoplasm dyes and/or viability dyes including but not limited to e.g., DNA dyes, DNA intercalating dyes, vital dyes, propidium iodide, calcein, Hoechst dyes, etc. Non-limiting examples of viability dyes, for detecting live and/or dead cells, include e.g., propidium iodide (PI), 7-amino-actinomycin D (7- AAD), and those available from commercial distributors such as Fixable Viability Dye eFluor 455UV/450/506/520/660/780 (Affymetrix eBioscience, San Diego, CA), LIVE/DEAD Fixable Blue/Violet/ Aqua/Yellow stain (Life Technologies, Grand Island, NY), Zombie

Aqua/Green/NIR/RED/UV/Violet/Yellow (BioLegend, San Diego, CA), and the like. Non- limiting examples of nucleic acid dyes include e.g., Hoechst 33342 (2'-(4-Ethoxyphenyl)-5-(4- methyl-l-piperazinyl)-lH,l'H-2,5'-bibenzimidazole trihydrochloride) and Hoechst 33258 (4-[6- (4-Methyl-l-piperazinyl)-r,3'-dihydro-lH,2'H-2,5'-bibenzimidazol-2'-ylidene]-2,5- cyclohexadien-l-one trihydrochloride) and others of the Hoechst series; SYTO 40, SYTO 11, 12, 13, 14, 15, 16, 20, 21, 22, 23, 24, 25 (green); SYTO 17, 59 (red), DAPI, DRAQ5™ (an anthraquinone dye with high affinity for double stranded DNA), YOYO-1, propidium iodide,

YO-PRO-3, TO-PRO-3, YOYO-3 and TOTO-3, SYTOX Green, SYTOX, methyl green, acridine homodimer, 7-aminoactinomycin D, 9-amino-6-chloro-2-methoxyacridine, and the like. As a non-limiting example, methods of circulating tumor cell (CTC) enrichment and visualization are known in the art and may be employed for generating (and later visualizing) the initial cell suspension employed in the methods and systems described herein. For example, Table 1 of Krebs et al. Nat Rev Clin Oncol. 2014 Mar; 11(3): 129-44 (herein incorporated by reference, and specifically with respect to Table 1). Examples of markers that can be employed to enrich and visualize CTCs include, but are not limited to: CD45, EpCAM, MUC1, and HER2. Antibodies to such markers may be employed to label and visualize such cells. Any type of suitable method may be employed for isolating and enriching CTCs, such as flow cytometry, column binding, etc.

In some instances, the sample from which cells are derived may be a biopsy or swab, e.g., a biopsy or swab collected to diagnose, monitor, or otherwise evaluate a subject, e.g., diagnose the subject for a cellular deficiency or disease, e.g., cancer. In some instances, a sample from which the cells are derived may be a previously collected and stored sample, e.g., a banked tissue sample, from the subject to be treated, including but not limited to e.g., stored cardiac tissue or cells, stored musculoskeletal tissue or cells, stored reproductive tissue or cells, stored skin tissue or cells, stored bone tissue or cells, stored bone marrow tissue or cells, stored vascular tissue or cells, stored umbilical cord blood tissue or cells, and the like. In some instances, a sample from which the cells are derived is fresh, i.e., not previously stored or frozen.

Following the collection of a cell or tissue or organ sample or biopsy or swab the cells may be processed. For example, in the case of solid and/or semi-solid tissues (e.g., solid tumors, skin tissue, brain tissue, muscle tissue, liver tissue, adipose tissue, etc.) the tissue may be dissociated into a single cell suspension. Any convenient method of cell dissociation may be employed including e.g., enzymatic (e.g., protease) dissociation, non-enzymatic (e.g., chemical or physical) dissociation, and the like. The cells of a dissociated solid or semi-solid tissue sample may be further processed, including e.g., through fractionation, enrichment, sorting, staining, etc., or may not be further processed. Cells of liquid cellular samples (e.g., blood, amniotic fluid, etc.) may be processed, including e.g., through fractionation, enrichment, sorting, staining, etc., or may not be processed. Any convenient technique or device may be employed to facilitate such processing steps including but not limited to e.g., density gradients, centrifuges, tissue culture dishes/flasks, filters, syringes, blood separation tubes, FACS, and the like.

Prepared cell suspensions, whether or not involving dissociation and/or one or more processing steps (e.g., as described above), may be prepared in or transferred to a suitable container for cell dispensing. Suitable containers for cell dispensing may be referred to herein as "source containers" or "source devices" which may have one or more "source compartments" or "source device wells". Suitable source containers include but are not limited to e.g., tubes, flasks, dishes, bottles, troughs, multi-well devices (e.g., multi-well plates, including e.g., 6-, 12-, 24-, 36-, 48-, 96-, 384- and 1536-well plates, and the like). In some instances, a subject source container may be configured such that the dispense tip may contact cell suspension present in the source container, e.g., for extracting cell suspension from the source container. In some instances, a source container may be connected, e.g., by a tube or other liquid transfer device, to the dispenser to facilitate filling of the dispense tip, e.g., by back-filling the dispense tip.

Configurations of source containers may vary and may include where the source container and the dispense tip are configured to be compatible.

D. Multi-well Device

The present disclosure is not limited by the type of multi-well testing devices (e.g., plates or chips) employed in the contact dispensing. The top surface of the multi-well devices will have, at least in part, a hydrophobic surface (e.g., near where the hydrophilic wells are formed). In general, such devices have a plurality of hydrophilic wells that contain, or are dimensioned to contain, liquid (e.g., liquid that is trapped in the wells such that gravity alone cannot make the liquid flow out of the wells). One exemplary chip is WAFERGEN's 5184-well SMARTCHIP, where the wells have a hydrophilic coating. Other exemplary chips are provided in U.S. Patents 8,252,581; 7,833,709; and 7,547,556, all of which are herein incorporated by reference in their entireties including, for example, for the teaching of chips, wells, thermocycling conditions, and associated reagents used therein). Other exemplary chips include the OPENARRAY plates used in the QUANTSTUDIO real-time PCR system (Applied Biosystems). Another exemplary multi- well device is a 96-well or 384-well plate.

The overall size of the multi-well devices may vary and it can range, for example, from a few microns to a few centimeters in thickness, and from a few millimeters to 50 centimeters in width or length. In some instances,, the size of the entire device ranges from about 10 mm to about 200 mm in width and/or length, and about 1 mm to about 10 mm in thickness. In some embodiments, the chip is about 40 mm in width by 40 mm in length by 3 mm in thickness.

The total number of wells (e.g., nanowells) on the multi-well device may vary depending on the particular application in which the subject chips are to be employed. The density of the hydrophilic wells on the chip surface may vary depending on the particular application. The density of wells, and the size and volume of wells, may vary depending on the desired application and such factors as, for example, the species of the organism for which the methods of this disclosure are to be employed.

The present disclosure is not limited by the number of hydrophilic wells in the multi-well device or the number of wells in a multi-well source device. A large number of wells may be incorporated into a device. In various embodiments, the total number of wells on the device is from about 100 to about 200,000, or from about 5000 to about 10,000. In other embodiments the device comprises smaller chips, each of which comprises about 5,000 to about 20,000 wells. For example, a square chip may comprise 125 by 125 nanowells, with a diameter of 0.1 mm.

Useful source devices, i.e., devices configured to contain the source fluid (e.g., cell suspension) for dispensing, will vary and may include single vessel devices as well as multi-well devices. For example, in some instances, a subject source device may include a single well, trough, tube, bottle, flask, dish, bowl, etc. configured to contain the source liquid for transfer into a dispenser. In some instances, a subject source device may include a plurality of wells or arrayed tubes configured to contain the source liquid for transfer into a dispenser. Source devices may be specifically configured to align with dispensers having one or multiple dispense tips. For example, a multi-well source device may include wells that are spaced to correspond with the spacing between the dispenser tips of a multi-tip dispenser such that more than one, including all, of the dispenser tips may be each simultaneously inserted into a well of the multi-well source device. Multi-well source devices may thus be configured to be compatible with the dispense tips of multi-tip dispensers, including where the multi-well source device has a number of wells equal to the number of dispenser tips or where the number of wells and the number of dispenser tips are unequal.

The hydrophilic wells (e.g., nanowells) in a multi-well device may be fabricated in any convenient size, shape or volume. In some instances, the well may be about 100 μπι to about 1 mm in length, about 100 μπι to about 1 mm in width, and about 100 μπι to about 1 mm in depth. The length, width (or diameter) and height of the wells may vary and may range from less than 50 μπι to more than 5 mm, including but not limited to e.g., 50 μπι to 5 mm, 75 μπι to 5 mm, 100 μπι to 5 mm, 200 μπι to 5 mm, 300 μπι to 5 mm, 400 μπι to 5 mm, 500 μπι to 5 mm, 600 μπι to 5 mm, 700 μπι to 5 mm, 800 μπι to 5 mm, 900 μπι to 5 mm, 1 mm to 5 mm, 2 mm to 5 mm, 3 mm to 5 mm, 4 mm to 5 mm, 50 μπι to 2 mm, 75 μπι to 2 mm, 100 μπι to 2 mm, 200 μπι to 2 mm, 300 μπι to 2 mm, 400 μπι to 2 mm, 500 μπι to 2 mm, 600 μπι to 2 mm, 700 μπι to 2 mm, 800 μπι to 2 mm, 900 μπι to 2 mm, 1 mm to 2 mm, 50 μπι to 1 mm, 75 μπι to 1 mm, 100 μπι to 1 mm, 200 μπι to 1 mm, 300 μπι to 1 mm, 400 μπι to 1 mm, 500 μπι to 1 mm, 50 μπι to 500 mm, 75 μηι to 500 mm, 100 μιη to 500 mm, 200 μιη to 500 mm, 300 μιη to 500 mm, 400 μιη to 500 mm, etc. In various embodiments, each nanowell has an aspect ratio (ratio of depth to width) of from about 1 to about 4, including e.g., 1 to 4, 1 to 3, 1 to 2, 1, 2 to 4, 2 to 3, 2, 3 to 4, 3, and 4. In one embodiment, each nanowell has an aspect ratio of about 2. The transverse sectional area may be circular, elliptical, oval, conical, rectangular, triangular, polyhedral, or in any other shape. The transverse area at any given depth of the well may also vary in size and shape.

In some embodiments, the wells have a volume of from about 0.1 nl to about 1 ul. A nanowell may have a volume of less than 1 ul, in some instances less than 500 nl. The volume may be less than 200 nl, or less than 100 nl. In some embodiments, the volume of the nanowell is about 100 nl. In some embodiments, the volume of the nanowell is about 150 nl. The volume of a well of a multi-well device may vary and may range from less than 0.1 nl to 100 μΐ or more, including but not limited to e.g 0.1 nl to 100 μΐ, 0.1 nl to 90 μΐ, 0.1 nl to 80 μΐ, 0.1 nl to 70 μΐ, 0.1 nl to 60 μΐ, 0.1 nl to 50 μΐ, 0.1 nl to 40 μΐ, 0.1 nl to 30 μΐ, 0.1 nl to 20 μΐ, 0.1 nl to 15 μΐ, 0.1 nl to 10 μΐ, 0.1 nl to 5 μΐ, 0.1 nl to 1 μΐ, 0.1 nl to 900 μΐ, 0.1 nl to 800 μΐ, 0.1 nl to 700 μΐ, 0.1 nl to 600 μΐ, 0.1 nl to 500 μΐ, 0.1 nl to 450 μΐ, 0.1 nl to 400 μΐ, 0.1 nl to 350 μΐ, 0.1 nl to 300 μΐ, 0.1 nl to 250 μΐ, 0.1 nl to 200 μΐ, 0.1 nl to 150 μΐ, 0.1 nl to 100 μΐ, 0.1 nl to 50 μΐ, etc. Where desired, a nanowell can be fabricated to increase the surface area to volume ratio, thereby facilitating heat transfer through the unit, which can reduce the ramp time of a thermal cycle. The cavity of each well (e.g., nanowell) may take a variety of configurations. For instance, the cavity within a well may be divided by linear or curved walls to form separate but adjacent compartments, or by circular walls to form inner and outer annular compartments.

An exemplary multi-well device (e.g., chip) may have a thickness of about 0.625 mm, with a well have having dimensions of about 0.25 mm (250 um) in length and width. The nanowell depth can be about 0.525 mm (525 um), leaving about 0.1 mm of the chip beneath a given well. A nanowell opening can include any shape, such as round, square, rectangle or any other desired geometric shape. By way of example, a nanowell can include a diameter or width of between about 100 μπι and about 1 mm, a pitch or length of between about 150 μπι and about 1 mm and a depth of between about 10 μπι to about 1 mm. The cavity of each well may take a variety of configurations. For instance, the cavity within a nanowell may be divided by linear or curved walls to form separate but adjacent compartments.

The wells (e.g., nanowells) of the multi-well device may be formed using, for example, commonly known photolithography techniques. The nanowells may be formed using a wet KOH etching technique, an anisotropic dry etching technique, mechanical drilling, injection molding and or thermo forming (e.g., hot embossing). The wells may be coated with a hydrophilic polymer or other coating.

The hydrophilicity of a hydrophilic multi-well device material or coatings will vary and may range from a water contact angle of less than 120°, including but not limited to e.g., less than 110°, less than 100°, less than 90°, less than 85°, less than 80°, less than 75°, less than 70°, less than 65°, etc. In some instances, the hydrophilicity of a material or coating, expressed as contact angle, may range from less than 20° to more than 90°, including but not limited to e.g., from 20° to 90°, from 25° to 90°, from 30° to 90°, from 35° to 90°, from 40° to 90°, from 45° to 90°, from 50° to 90°, from 55° to 90°, from 60° to 90°, from 65° to 90°, from 70° to 90°, from 75° to 90°, from 80° to 90°, from 85° to 90°, from 20° to 80°, from 25° to 80°, from 30° to 80°, from 35° to 80°, from 40° to 80°, from 45° to 80°, from 50° to 80°, from 55° to 80°, from 60° to 80°, from 65° to 80°, from 70° to 80°, from 75° to 80°, from 40° to 85°, from 40° to 80°, from 40° to 75°, from 40° to 70°, from 40° to 65°, from 40° to 60°, from 40° to 55°, from 40° to 50°, from 40° to 45°, from 50° to 85°, from 50° to 80°, from 50° to 75°, from 50° to 70°, from 50° to 65°, from 50° to 60°, from 50° to 55°, from 60° to 85°, from 60° to 80°, from 60° to 75°, from 60° to 70°, from 60° to 65°, from 65° to 85°, from 65° to 70°, etc.

As summarized above, the top surface of a multi-well device may be composed of a hydrophobic material or coated with a hydrophobic material. Examples of such materials include, for example, polyimide, polyester, polyethylene, polyurethane, TEFLON PTFE, fluorosilane, NEVERWET coatings (from Rust-Oleum), coatings from HYDROBEAD,

NANOMYTE coatings (from NEI corp.), ACULON's hydrophobic and superhydrophobic surface treatments, or combinations of any two, any three, any four, any five, or any six or more of such polymers and coatings).

The hydrophobicity of a hydrophobic multi-well device material or coatings will vary and may range from a water contact angle of 120° or more, including but not limited to e.g., 125° or more, 130° or more, 135° or more, 140° or more, 145° or more, 150° or more, 155° or more, 160° or more, 165° or more, 170° or more or 175° or more. In some instances, the

hydrophobicity of a material or coating, expressed as contact angle, may range from less than 120° to more than 175°, including but not limited to e.g., from 120° to 175°, from 125° to 175°, from 130° to 175°, from 135° to 175°, from 140° to 175°, from 145° to 175°, from 150° to 175°, from 155° to 175°, from 160° to 175°, from 165° to 175°, from 170° to 175°, from 120° to 170°, from 120° to 165°, from 120° to 160°, from 120° to 155°, from 120° to 150°, from 120° to 145°, from 120° to 140°, from 120° to 135°, from 120° to 130°, from 120° to 125°, from 140° to 170°, from 140° to 165°, from 140° to 160°, from 140° to 155°, from 140° to 150°, from 140° to 145°, from 150° to 170°, from 150° to 165°, from 150° to 160°, from 150° to 155°, from 150° to 150°, from 150° to 145°, from 155° to 170°, from 155° to 165°, from 155° to 160°, from 160° to 170°, from 165° to 170°, and the like.

The hydrophobicity and/or hydrophilicity, of a material or surface, including coated surfaces, may be expressed using various conventions, including e.g., contact angle as employed herein, and may be calculated or measured, depending on the context, using various methods including but not limited to e.g., static sessile drop method (e.g., using a contact angle goniometer), pendant drop method, dynamic sessile drop method, dynamic Wilhelmy method, single-fiber Wilhelmy method, Washburn's equation capillary rise method, and the like. Contact angles discussed herein may be contact angles as measured by any one of the static sessile drop method, the pendant drop method, the dynamic sessile drop method, the dynamic Wilhelmy method, the single-fiber Wilhelmy method or the Washburn's equation capillary rise method or a combination of such methods. Contact angles may be measured using various probe liquids including but not limited to e.g., water (e.g., deionized water), diiodomethane, formamide, and the like.

Components of the multi-well devices, e.g., surfaces, wells, etc., and/or components of the dispensers, e.g., dispensing tips and surfaces thereof, may be described herein as having relative hydrophobicity and/or relative hydrophilicity, i.e., being relatively hydrophobic or being relatively hydrophilic. By "relatively hydrophobic" is meant that the subject component is more hydrophobic than the component to which it is compared in the relevant context.

Correspondingly, by "relatively hydrophilic" is meant that the subject component is more hydrophilic than the component to which it is compared in the relevant context. A relatively hydrophobic component need not necessarily have a contact angle of greater than 90°, provided the subject component is more hydrophobic relative to the component to which is it compared. Likewise, a relatively hydrophilic component need not necessarily have a contact angle of less than 90°, provided the subject component is more hydrophilic relative to the component to which is it compared.

As such, a relatively hydrophobic component may have a contact angle that is 1° greater or more than the component to which it is compared, including but not limited to e.g., at least 1° greater, at least 2° greater, at least 3° greater, at least 4° greater, at least 5° greater, at least 6° greater, at least 7° greater, at least 8° greater, at least 9° greater, at least 10° greater, at least 15° greater, at least 20° greater, at least 25° greater, at least 30° greater, at least 35° greater, at least 40° greater, at least 45° greater, at least 50° greater, at least 55° greater, at least 60° greater, at least 65° greater, at least 70° greater, at least 75° greater or at least 80° greater than the component to which it is compared.

A relatively hydrophilic component may have a contact angle that is 1° less than the component to which it is compared, including but not limited to e.g., at least 1° less, at least 2° less, at least 3° less, at least 4° less, at least 5° less, at least 6° less, at least 7° less, at least 8° less, at least 9° less, at least 10° less, at least 15° less, at least 20° less, at least 25° less, at least 30° less, at least 35° less, at least 40° less, at least 45° less, at least 50° less, at least 55° less, at least 60° less, at least 65° less, at least 70° less, at least 75° less or at least 80° less than the component to which it is compared.

For example, a well may be relatively hydrophilic as compared to another surface of the multi-well device. In absolute terms, such a well may be hydrophilic (i.e., have a contact angle of less than 90°) or may be hydrophobic (i.e., have a contact angle of more than 90°). In some instances, as compared to a hydrophobic surface of a multi-well device a relatively hydrophilic well may have a contact angle of at least 1° less than the hydrophobic surface, including but not limited to e.g., at least 1° less, at least 2° less, at least 3° less, at least 4° less, at least 5° less, at least 6° less, at least 7° less, at least 8° less, at least 9° less, at least 10° less, at least 15° less, at least 20° less, at least 25° less, at least 30° less, at least 35° less, at least 40° less, at least 45° less, at least 50° less, at least 55° less, at least 60° less, at least 65° less, at least 70° less, at least 75° less or at least 80° less.

In some instances, a dispense tip may be composed of or coated with a material that is relatively hydrophobic as compared to a well of a multi-well device. For example, the contact angle of a dispense tip may be 1° greater or more than the contact angle of the well, including but not limited to e.g., at least 1° greater, at least 2° greater, at least 3° greater, at least 4° greater, at least 5° greater, at least 6° greater, at least 7° greater, at least 8° greater, at least 9° greater, at least 10° greater, at least 15° greater, at least 20° greater, at least 25° greater, at least 30° greater, at least 35° greater, at least 40° greater, at least 45° greater, at least 50° greater, at least 55° greater, at least 60° greater, at least 65° greater, at least 70° greater, at least 75° greater or at least 80° greater than the contact angle of the well.

In some instances, the combined components of a multi-well device and dispensing system may be configured, e.g., through material selection and/or coating, such that the hydrophobicities/hydrophilicities relative to one another are in order, from most hydrophobic to most hydrophilic: a top surface of the multi-well device, a surface of the dispense tip and a well of the multi-well device. Accordingly, in some instances, a well may be relatively hydrophilic as compared to the surface of the multi-well device and the dispensing tip may be relatively hydrophobic as compared to the well. Put another way, the dispensing tip may be less hydrophilic as compared to the well and the well may be more hydrophilic as compared to the surface of the multi-well plate.

The present disclosure is not limited by the method used to generate the multi-well devices herein, with hydrophobic to surfaces surrounding hydrophilic wells. Accordingly, the methods employed to generate the multi-well devices utilized in the methods and systems of the disclosure will vary. Such methods may employ, alone or in combination, various coating techniques including but not limited to e.g., spray coating, immersion coating, film coating, line- of-site deposition (e.g., physical vapor deposition, chemical vapor deposition, etc.), and the like.

In some instances, a multi-well device having a relatively hydrophobic top surface and a relatively hydrophilic well surface may be fabricated by spray coating. For example, the wells may be protected, e.g., covered (e.g., via removable protective solids, such as described in greater detail below), and the unprotected surface(s) (e.g., unprotected hydrophilic surfaces) of a multi-well device may be spray coated, e.g., with a hydrophobic coating, as to apply the coating to the unprotected surface(s). In some instances, a surface or surfaces of a multi-well device may be protected, e.g., covered, and the unprotected well surface(s) (e.g., unprotected hydrophobic surfaces) may be spray coated, e.g., with a hydrophilic coating, so as to apply the coating the unprotected surface(s). Surfaces, including surfaces of the multi-well device (e.g., a top surface) and surfaces of wells (e.g., bottom well surfaces, side well surfaces, etc.), may be protected using any convenient method, including e.g., applying a protective material or solid to the surface, e.g., a film, a tape, an object (e.g., mask) or a plurality of objects or solids sized and shaped to cover the surface or a portion of the surface, and the like. Protective materials may be removable and may be held in place by any convenient means during application of a coating including e.g., by an adhesive, by gravity, by frictional forces, by static charge, etc. The above process, as described for spray coating, may be adapted for use with other coating methods including but not limited to e.g., immersion coating, film coating, line-of-site deposition (e.g., physical vapor deposition, chemical vapor deposition, etc.), and the like.

In some instances, methods for generating such multi-well devices may include as follows. A multi-well chip, that already has hydrophilic wells, could be treated by masking off all of the wells by inserting removable surface protectors, such as removable protective solids, e.g., solids of a shape configured to match the wells, such as spheres for cylindrical wells, into the wells (e.g., spheres with a diameter slightly larger than the wells). The multi-well chip could then be sprayed with a hydrophobic or super-hydrophobic coating such that the top surface around the wells has a hydrophobic coating. Once the sprayed on coating dries, the multi-well chip is turned upside down to release the spheres (e.g., by brushing them off or by gravity pulling them off, or agitating the chip to cause the spheres to come out of the wells). In some embodiments, the microspheres employed are stainless steel microspheres from COSPHERIC LLC (Santa Barbara, CA).

The multi-well devices described herein, with hydrophobic surfaces surrounding hydrophilic wells, in some embodiments, are more forgiving and tolerate more misalignment with dispensing tips (e.g., when nano-liter sized wells are employed on chips with thousands of wells) as the difference in hydrophobicity pulls into the well a drop that is half in and half out of the well.

As summarized above, in some instances, imaging may be performed on multi-well devices utilized in the present methods. For example, a well of a multi-well device may be imaged, e.g., to detect the presence of a cell, the absence of a cell, whether a desired number of cells is present in the well, and the like. Accordingly, in some embodiment, the multi-well device may be configured to be compatible with such imaging. Configurations of imaging-compatible multi-well devices will vary and may include configurations for upright imaging (i.e., imaging from above the multi-well device) as well as for inverted imaging (i.e., imaging from beneath the multi-well device). Accordingly, in some instances, a multi-well device may be configured with sufficiently wide wells and/or sufficiently shallow wells to facilitate imaging into the well from above. In some instances, a multi-well device may be configured with a base material that is sufficient for imaging through the base material into the well, including where the base material is made of an optically clear material including e.g., glass or an optical plastic.

E. Reagents and Assays

Reagents may be pre-dispensed into the wells of the multi-well device, or added after a cell or cells are added to a well. Reagents contained within the liquid in the multi-well device (whether added before, during or after cell dispensing) depend on the reaction that is to be run with the single cell (or multiple cells) that is deposited into each well. In some embodiments, the wells contain a reagent for conducting a nucleic acid amplification reaction. Reagents can be reagents for immunoassays, nucleic acid preparation, analysis and detection assays (including but not limited to nucleic acid amplification, e.g., PCR (including e.g., sequence specific PCR, random primed PCR, qPCR, multiplex PCR, etc.), whole genome amplification (WGA), library preparation, reverse transcription, cDNA preparation, template switching, tagmentation, Next Generation Sequencing (NGS), library preparation (e.g., for NGS) and the like. Reagents can be in a dry state or a liquid state in a unit of the chip.

Non-limiting examples of reagents that may be added to and/or already present in a well of a multi-well device include but are not limited to e.g., oligonucleotides (including e.g., primers and probes, including DNA, RNA and nucleotide analog oligonucleotide primers and probes, template switch oligonucleotides, etc.), barcode containing nucleic acids, sequencing adapter containing nucleic acids, template nucleic acids (e.g., DNA templates, RNA templates, etc.), transposon nucleic acids, enzymes (e.g., polymerases (e.g., reverse transcriptase, RNA polymerase, etc.), transposases, nucleases (e.g., endonucleases (e.g., restriction endonucleases), exonucleases, Cas9 nucleases, etc.), ligases, DNA repair enzymes (e.g., uracil-DNA glycosylase, endonuclease III, IV, V, VIII, etc.), methyltransferases, phosphatases, sulfurylases,

recombinases, kinases, nuclease inhibitors (e.g., an RNase inhibitor), etc.), dNTPs (e.g., dATP, dCTP, dGTP, dTTP, and/or dUTP), dyes (e.g., DNA binding dye (e.g., DAPI, Hoechst, SYBR® Green, etc.), viability dyes, etc.), salts, metal cofactors, enzyme-stabilizing components (e.g., DTT), and the like.

In some embodiments, the wells contain at least one of the following reagents: a probe, a polymerase, and dNTPs. In other embodiments, the wells contain a solution comprising a probe, a primer and a polymerase. In various embodiments, each well comprises (1) a primer for a polynucleotide target within a standard genome, and (2) a probe associated with said primer which emits a concentration dependent signal if the primer binds with said target. In various embodiments, each well comprises a primer for a polynucleotide target within a genome, and a probe associated with the primer which emits a concentration dependent signal if the primer binds with the target. In other embodiments, at least one well of the chip contains a solution that comprises a forward PCR primer, a reverse PCR primer, and at least one FAM labeled MGB quenched PCR probe. In some embodiments, primer pairs are dispensed into a well and then dried, such as by freezing. The user can then selectively dispense, such as nano-dispense, the sample, probe and/or polymerase.

In other embodiments of the disclosure, the wells may contain any of the above solutions in a dried (e.g., lyophilized) form. In these embodiments, this dried form may be coated to the wells or be directed to the bottom of the well. The user may add a mixture of water and the captured cells to each of the wells before analysis, including e.g., where the water and/or cell suspension solution is sufficient to rehydrate the dried reagent(s). In these embodiments, the chip comprising the dried down reaction mixture may be sealed with a liner, stored or shipped to another location.

Multi-well devices, with a single cell or a desired number of cells in each well, may be used for genotyping, gene expression, or other DNA assays preformed by PCR. Assays performed in the plate are not limited to DNA assays such as TAQMAN, TAQMAN Gold, SYBR gold, and SYBR green but also include other assays such as receptor binding, enzymatic assays, and other high throughput screening assays.

In some embodiments cells are subjected (e.g., after lysis and/or other processing steps) to amplification and/or sequencing analysis. Conducting one or more amplification reactions may comprise one or more PCR-based amplifications, non-PCR based amplifications, or a combination thereof. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), nested PCR, linear amplification, multiple displacement amplification (MDA), real-time SDA, rolling circle amplification, circle-to-circle amplification transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). Those of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require that RNA be reversed transcribed to DNA prior to amplification (e.g., RT-PCR), whereas other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159 and 4,965,188, each of which is herein incorporated by reference in its entirety), commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to permit exponential increase in copy numbers of target nucleic acids. In a variation called RT-PCR, reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from RNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA. For other various permutations of PCR see, e.g., U.S. Pat. Nos. 4,683, 195, 4,683,202 and 4,800,159; Mullis et a\., Meth. Enzymol. 155: 335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which is herein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and 5,399,491, each of which is herein incorporated by reference in its entirety), commonly referred to as TMA, synthesizes multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH in which multiple RNA copies of the target sequence autocatalytically generate additional copies. See, e.g., U.S. Pat. Nos.

5,399,491 and 5,824,518, each of which is herein incorporated by reference in its entirety. In a variation described in U.S. Publ. No. 20060046265 (herein incorporated by reference in its entirety), TMA optionally incorporates the use of blocking moieties, terminating moieties, and other modifying moieties to improve TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), herein incorporated by reference in its entirety), commonly referred to as LCR, uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid. The DNA oligonucleotides are covalently linked by a DNA ligase in repeated cycles of thermal

denaturation, hybridization and ligation to produce a detectable double-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad. Sci. USA 89: 392- 396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455, 166, each of which is herein incorporated by reference in its entirety), commonly referred to as SDA, uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTPaS to produce a duplex hemi-phosphorothioated primer extension product, endonuclease- mediated nicking of a hemi-modified restriction endonuclease recognition site, and polymerase- mediated primer extension from the 3' end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking and strand displacement, resulting in geometric amplification of product. Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequence based amplification (U.S. Pat. No. 5, 130,238, herein incorporated by reference in its entirety), commonly referred to as NASB A; one that uses an RNA replicase to amplify the probe molecule itself (Lizardi et al., BioTechnol. 6: 1197 (1988), herein incorporated by reference in its entirety), commonly referred to as QP replicase; a transcription based amplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173 (1989)); and, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is herein incorporated by reference in its entirety). For further discussion of known amplification methods see Persing, David H., "In Vitro Nucleic Acid Amplification Techniques" in Diagnostic Medical

Microbiology: Principles and Applications (Persing et al., Eds.), pp. 51-87 (American Society for Microbiology, Washington, DC (1993)). In some embodiments, nucleic acid sequencing methods are utilized (e.g., for detection of amplified nucleic acids). In some embodiments, the technology provided herein finds use in a Second Generation (a.k.a. Next Generation or Next-Gen), Third Generation (a.k.a. Next-Next- Gen), or Fourth Generation (a.k.a. N3-Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), semiconductor sequencing, massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology, etc. Morozova and Marra provide a review of some such technologies in Genomics, 92: 255 (2008), herein incorporated by reference in its entirety. Those of ordinary skill in the art will recognize that because RNA is less stable in the cell and more prone to nuclease attack experimentally RNA is usually reverse transcribed to DNA before sequencing.

A number of DNA sequencing techniques are suitable, including fluorescence-based sequencing methodologies (See, e.g., Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety). In some embodiments, the technology finds use in automated sequencing techniques understood in that art. In some embodiments, the present technology finds use in parallel sequencing of partitioned amplicons (PCT Publication No: WO2006084132 to Kevin McKernan et al., herein incorporated by reference in its entirety). In some embodiments, the technology finds use in DNA sequencing by parallel oligonucleotide extension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., and U.S. Pat. No. 6,306,597 to Macevicz et al., both of which are herein incorporated by reference in their entireties). Additional examples of sequencing techniques in which the technology finds use include the Church polony technology (Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; U.S. Pat. No. 6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; herein incorporated by reference in their entireties), the 454 picotiter pyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380; US 20050130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat. No. 6,787,308; U.S. Pat. No. 6,833,246; herein incorporated by reference in their entireties), the Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat. Biotechnol. 18:630-634; U.S. Pat. No. 5,695,934; U.S. Pat. No. 5,714,330; herein incorporated by reference in their entireties), and the Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO

00018957; herein incorporated by reference in its entirety). Next-generation sequencing (NGS) methods share the common feature of massively parallel, high-throughput strategies, with the goal of lower costs in comparison to older sequencing methods (see, e.g., Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; each herein incorporated by reference in their entirety). NGS methods can be broadly divided into those that typically use template

amplification and those that do not. Amplification-requiring methods include pyrosequencing commercialized by Roche as the 454 technology platforms (e.g., GS 20 and GS FLX), Life Technologies/Ion Torrent, the Solexa platform commercialized by Illumina, GnuBio, and the Supported Oligonucleotide Ligation and Detection (SOLiD) platform commercialized by Applied Biosystems. Non-amplification approaches, also known as single-molecule sequencing, are exemplified by the Heli Scope platform commercialized by Helicos Biosciences, and platforms commercialized by VisiGen, Oxford Nanopore Technologies Ltd., and Pacific

Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated by reference in its entirety), template DNA is fragmented, end-repaired, ligated to adaptors, and clonally amplified in-situ by capturing single template molecules with beads bearing oligonucleotides complementary to the adaptors. Each bead bearing a single template type is compartmentalized into a water-in-oil microvesicle, and the template is clonally amplified using a technique referred to as emulsion PCR. The emulsion is disrupted after amplification and beads are deposited into individual wells of a picotitre plate functioning as a flow cell during the sequencing reactions. Ordered, iterative introduction of each of the four dNTP reagents occurs in the flow cell in the presence of sequencing enzymes and luminescent reporter such as luciferase. In the event that an appropriate dNTP is added to the 3' end of the sequencing primer, the resulting production of ATP causes a burst of luminescence within the well, which is recorded using a CCD camera. It is possible to achieve read lengths greater than or equal to 400 bases, and 10⁶ sequence reads can be achieved, resulting in up to 500 million base pairs (Mb) of sequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 6,833,246; U.S. Pat. No. 7, 115,400; U.S. Pat. No. 6,969,488; each herein incorporated by reference in its entirety), sequencing data are produced in the form of shorter-length reads. In this method, single-stranded fragmented DNA is end-repaired to generate 5'-phosphorylated blunt ends, followed by Klenow- mediated addition of a single A base to the 3' end of the fragments. A-addition facilitates addition of T-overhang adaptor oligonucleotides, which are subsequently used to capture the template-adaptor molecules on the surface of a flow cell that is studded with oligonucleotide anchors. The anchor is used as a PCR primer, but because of the length of the template and its proximity to other nearby anchor oligonucleotides, extension by PCR results in the "arching over" of the molecule to hybridize with an adjacent anchor oligonucleotide to form a bridge structure on the surface of the flow cell. These loops of DNA are denatured and cleaved.

Forward strands are then sequenced with reversible dye terminators. The sequence of

incorporated nucleotides is determined by detection of post-incorporation fluorescence, with each fluor and block removed prior to the next cycle of dNTP addition. Sequence read length ranges from 36 nucleotides to over 250 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No. 6,130,073; each herein incorporated by reference in their entirety) also involves fragmentation of the template, ligation to oligonucleotide adaptors, attachment to beads, and clonal amplification by emulsion PCR. Following this, beads bearing template are immobilized on a derivatized surface of a glass flow-cell, and a primer complementary to the adaptor oligonucleotide is annealed. However, rather than utilizing this primer for 3' extension, it is instead used to provide a 5' phosphate group for ligation to interrogation probes containing two probe-specific bases followed by 6 degenerate bases and one of four fluorescent labels. In the SOLiD system, interrogation probes have 16 possible combinations of the two bases at the 3' end of each probe, and one of four fluors at the 5' end. Fluor color, and thus identity of each probe, corresponds to specific color-space coding schemes. Multiple rounds (usually 7) of probe annealing, ligation, and fluor detection are followed by denaturation, and then a second round of sequencing using a primer that is offset by one base relative to the initial primer. In this manner, the template sequence can be computationally re-constructed, and template bases are interrogated twice, resulting in increased accuracy. Sequence read length averages 35 nucleotides, and overall output exceeds 4 billion bases per sequencing run.

In some embodiments, the technology finds use in nanopore sequencing (see, e.g., Astier et al., J. Am. Chem. Soc. 2006 Feb 8; 128(5): 1705-10, herein incorporated by reference). The theory behind nanopore sequencing has to do with what occurs when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it. Under these conditions a slight electric current due to conduction of ions through the nanopore can be observed, and the amount of current is exceedingly sensitive to the size of the nanopore. As each base of a nucleic acid passes through the nanopore, this causes a change in the magnitude of the current through the nanopore that is distinct for each of the four bases, thereby allowing the sequence of the DNA molecule to be determined.

In some embodiments, the technology finds use in Heli Scope by Helicos Biosciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 7, 169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No. 7,482, 120; U.S. Pat. No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Pat. No. 7,501,245; each herein incorporated by reference in their entirety). Template DNA is fragmented and

polyadenylated at the 3' end, with the final adenosine bearing a fluorescent label. Denatured polyadenylated template fragments are ligated to poly(dT) oligonucleotides on the surface of a flow cell. Initial physical locations of captured template molecules are recorded by a CCD camera, and then label is cleaved and washed away. Sequencing is achieved by addition of polymerase and serial addition of fluorescently-labeled dNTP reagents. Incorporation events result in fluor signal corresponding to the dNTP, and signal is captured by a CCD camera before each round of dNTP addition. Sequence read length ranges from 25-50 nucleotides, with overall output exceeding 1 billion nucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on the detection of hydrogen ions that are released during the polymerization of DNA (see, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appl. Pub. Nos. 20090026082, 20090127589, 20100301398,

20100197507, 20100188073, and 20100137143, incorporated by reference in their entireties for all purposes). A microwell contains a template DNA strand to be sequenced. Beneath the layer of microwells is a hypersensitive ISFET ion sensor. All layers are contained within a CMOS semiconductor chip, similar to that used in the electronics industry. When a dNTP is

incorporated into the growing complementary strand a hydrogen ion is released, which triggers a hypersensitive ion sensor. If homopolymer repeats are present in the template sequence, multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. This technology differs from other sequencing technologies in that no modified nucleotides or optics is used. The per-base accuracy of the Ion Torrent sequencer is -99.6% for 50 base reads, with -100 Mb to 100Gb generated per run. The read-length is 100-300 base pairs. The accuracy for homopolymer repeats of 5 repeats in length is -98%. The benefits of ion semiconductor sequencing are rapid sequencing speed and low upfront and operating costs.

The technology finds use in another nucleic acid sequencing approach developed by Stratos Genomics, Inc. and involves the use of Xpandomers. This sequencing process typically includes providing a daughter strand produced by a template-directed synthesis. The daughter strand generally includes a plurality of subunits coupled in a sequence corresponding to a contiguous nucleotide sequence of all or a portion of a target nucleic acid in which the individual subunits comprise a tether, at least one probe or nucleobase residue, and at least one selectively cleavable bond. The selectively cleavable bond(s) is/are cleaved to yield an Xpandomer of a length longer than the plurality of the subunits of the daughter strand. The Xpandomer typically includes the tethers and reporter elements for parsing genetic information in a sequence corresponding to the contiguous nucleotide sequence of all or a portion of the target nucleic acid. Reporter elements of the Xpandomer are then detected. Additional details relating to

Xpandomer-based approaches are described in, for example, U.S. Pat. Pub No. 20090035777, entitled "High Throughput Nucleic Acid Sequencing by Expansion," filed June 19, 2008, which is incorporated herein in its entirety.

Other single molecule sequencing methods include real-time sequencing by synthesis using a VisiGen platform (Voelkerding et al., Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. Pat. App. Ser. No. 11/671956; U.S. Pat. App. Ser. No. 11/781166; each herein incorporated by reference in their entirety) in which immobilized, primed DNA template is subjected to strand extension using a fluorescently-modified polymerase and fl orescent acceptor molecules, resulting in detectible fluorescence resonance energy transfer (FRET) upon nucleotide addition.

Reagents for any suitable type of assay may be added to the wells of the multi-well chip (e.g., using a multi-well dispenser, such as the one from WAFERGEN BIOSYSTEMS). Such reagents may be added to the wells before or after a cell (e.g., a single cell) is added to a well. In some embodiments, protein detection assay components (e.g., anti-body based assays) are added to the wells. In other embodiments, SNP detection assay components are added to the wells. In other embodiments, nucleic acid sequencing assay components are added to the wells. In some embodiments, nucleic acid sequence assay components that employ barcoding for labelling individual mRNA molecules, and/or for labeling for cell/well source (e.g., if wells pooled before sequencing analysis), and/or for labeling particular multi-well chips (e.g., if wells from two or more multi-well chips are pooled prior to sequencing) are employed. Examples of such barcoding methodologies and reagents are found in Pat. Pub. US2007/0020640, Pat. Pub.

2012/0010091, U.S. Pat. 8,835,358, U.S. Pat. 8,481,292, Qiu et al. (Plant. Physiol., 133, 475- 481, 2003), Parameswaran et al. (Nucleic Acids Res. 2007 Oct; 35(19): el30), Craig et al.

reference (Nat. Methods, 2008, October, 5(10):887-893), Bontoux et al. (Lab Chip, 2008, 8:443- 450), Esumi et al. (Neuro. Res., 2008, 60:439-451), Hug et al., J. Theor., Biol., 2003, 221 :615- 624), Sutcliffe et al. (PNAS, 97(5): 1976-1981; 2000), Hollas and Schuler (Lecture Notes in Computer Science Volume 2812, 2003, pp 55-62), and WO201420127; all of which are herein incorporated by reference in their entireties, including for reaction conditions and reagents related to barcoding and sequencing of nucleic acids.

In some embodiments, the barcode tagging and sequencing methods of WO2014201272

("SCRB-seq" method) are employed. The necessary reagents for the SCRB-seq method (e.g., modified as necessary for small volumes) are added to the wells of the multi-well chips (e.g., where the single cell in the well has been lysed). Briefly, the SCRB-seq method amplifies an initial mRNA sample from a single cell in multi-well plates (as described above), where each well has a single cell. Initial cDNA synthesis uses a first primer with: i) N6 or Nl 1 for cell/well identification, ii) N10 for particular molecule identification, iii) a poly T stretch to bind mRNA, and iv) a region that creates a region where a second template-switching primer will hybridize. The second primer is a template switching primer with a poly G 3' end, and 5' end that has iso- bases. After cDNA amplification, the tagged cDNA single cell/well samples are pooled. Then full-length cDNA synthesis occurs with two different primers, and full-length cDNA is purified. Next, a NEXTERA sequencing library is prepared using an i7 primer (adds one of 12 i7 tags to identify particular multi-well plates) and P5NEXTPT5 to add P5 tag for NEXTERA sequencing (P7 tag added to other end for NEXTERA). The library is purified on a gel, and then NEXTERA sequencing occurs. As a non-liming example, with twelve i7 plate tags, and 384 cell/well- specific barcodes, this allows total of 4,608 single cell transciptomes to be done at once. This method allows for quantification of mRNA transcripts in single cells and allows users to count the absolute number of transcript molecules/cell to remove any variables from normalization.

In further embodiments image and chip mapped wells within the chip are dynamically and/or statically selected for further analysis by a combination of single or multiple addition of reagents for detection and/or resolution of nucleic acids or lipids or carbohydrates or protein cell components reagents. F. Computer Related Embodiments

As summarized above, components, e.g., dispenser components and components thereof, of the subject systems and employed in the subject methods may be computer controlled (i.e., robotic). Accordingly, the subject methods and systems may employ a processor connected to or otherwise in communication with one or more electrical components of the dispenser to control one or more actions of the components. In some instances, the components of the systems as described herein may be connected by a wired data connection. Any suitable and appropriate wired data connection may find use in connecting the components of the described systems, e.g., as described herein, including but not limited to e.g., commercially available cables such as a USB cable, a coaxial cable, a serial cable, a C2G or Cat2 cable, a Cat5/Cat5e/Cat6/Cat6a cable, a Token Ring Cable (Cat4), a VGA cable, a HDMI cable, a RCA cable, an optical fiber cable, and the like. In some instances, wireless data connections may be employed including but not limited to e.g., radio frequency connections (e.g., P AN/L AN/MAN/W AN wireless networking, UHF radio connections, etc.), an infrared data transmission connection, wireless optical data connections, and the like.

As summarized above, the devices and systems of the instant disclosure may further include a "memory" that is capable of storing information such that it is accessible and retrievable at a later date by a computer. Any desired information may be stored on such a memory, including but not limited to e.g., instructions for performing one or more steps of a method, and the like. Any convenient data storage structure may be chosen, based on the means used to access the stored information. In certain aspects, the information may be stored in a "permanent memory" (i.e., a memory that is not erased by termination of the electrical supply to a computer or processor) or "non-permanent memory". Computer hard-drive, CD-ROM, floppy disk, portable flash drive and DVD are all examples of permanent memory. Random Access Memory (RAM) is an example of non-permanent memory. A file in permanent memory may be editable and re-writable.

Substantially any circuitry can be configured to a functional arrangement within the devices and systems for performing the methods disclosed herein. The hardware architecture of such circuitry, including e.g., a specifically configured computer, is well known by a person skilled in the art, and can comprise hardware components including one or more processors (CPU), a random-access memory (RAM), a read-only memory (ROM), an internal or external data storage medium (e.g., hard disk drive). Such circuitry can also comprise one or more graphic boards for processing and outputting graphical information to display means. The above components can be suitably interconnected via a bus within the circuitry, e.g., inside a specific- use computer. The circuitry can further comprise suitable interfaces for communicating with general-purpose external components such as a monitor, keyboard, mouse, network, etc. In some embodiments, the circuitry can be capable of parallel processing or can be part of a network configured for parallel or distributive computing to increase the processing power for the present methods and programs. In some embodiments, the program code read out from the storage medium can be written into a memory provided in an expanded board inserted in the circuitry, or an expanded unit connected to the circuitry, and a CPU or the like provided in the expanded board or expanded unit can actually perform a part or all of the operations according to the instructions of the programming, so as to accomplish the functions described.

The instant disclosure includes computer readable medium, including non-transitory computer readable medium, which stores instructions for methods, or portions thereof, described herein. Aspects of the instant disclosure include computer readable medium storing instructions that, when executed by a computing device, cause the computing device to perform one or more steps of a method as described herein.

In certain embodiments, instructions in accordance with the methods described herein can be coded onto a computer-readable medium in the form of "programming", where the term "computer readable medium" as used herein refers to any storage or transmission medium that participates in providing instructions and/or data to a computer for execution and/or processing. Examples of storage media include a floppy disk, hard disk, optical disk, magneto-optical disk, CD-ROM, CD-R, magnetic tape, non-volatile memory card, ROM, DVD-ROM, Blue-ray disk, solid state disk, and network attached storage (NAS), whether or not such devices are internal or external to the computer. A file containing information can be "stored" on computer readable medium, where "storing" means recording information such that it is accessible and retrievable at a later date by a computer.

The computer-implemented method described herein can be executed using programming that can be written in one or more of any number of computer programming languages. Such languages include, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as any many others. Notwithstanding the appended claims, the disclosure is also defined by the following clauses:

1. A method comprising:

dispensing a hanging drop of liquid from a dispense tip onto a hydrophobic top surface of a multi-well device,

wherein said multi-well device comprises a plurality of hydrophilic wells formed in said hydrophobic top surface,

wherein said dispensing comprises contacting said hanging drop with said hydrophobic surface while said hanging drop is still in contact with said dispense tip, and

wherein said dispensing causes said hanging drop to separate from said dispense tip and move along said hydrophobic surface and into one of said hydrophilic wells.

2. The method of Clause 1, wherein said hydrophobic surface of said multi-well device has a water contact angle greater than about 140 degrees.

3. The method of Clauses 1 or 2, wherein said hydrophilic surface of said wells has a water contact angle of about 65-80 degrees.

4. The method of any of the preceding clauses, wherein said dispense tip is composed of a material that is less hydrophilic than said hydrophilic wells.

5. The method of any of the preceding clauses, wherein the method further comprises detecting which wells of the multi-well device contain one or more cells after the dispensing.

6. The method of Clause 5, wherein the detecting comprises imaging the wells.

7 The method of Clauses 5 or 6, wherein the one or more cells are detected based on dye labeling of the cells.

8. The method of any of Clauses 5 to 7, wherein the one or more cells are labeled with a dye selected from the group consisting of: a nucleic acid dye, a cytoplasm dye and a viability dye. 9. The method of any of the preceding clauses, wherein said hanging drop of liquid comprises a dispense volume of a cell suspension, wherein said cell suspension comprises cells present in said cell suspension at a concentration such that, on average, between 1 and 20 cells are present in said dispense volume. 10. The method of Clause 9, wherein between 1 and 10 cells are present in said dispense volume.

11. The method of Clause 9, wherein on average one cell is present in said dispense volume.

12. The method of any of Clauses 9 to 11, further comprising: dispensing a first additional volume into a well determined to contain one or more cells.

13. The method of Clause 12, wherein the first additional volume is free of cells. 14. The method of Clauses 12 or 13, wherein the first additional volume is equal to said dispense volume. 15. The method of any of Clauses 12 to 14, wherein the first additional volume contains at least one reagent selected from the group consisting of: a nucleic acid, an enzyme, a dNTP, a dye and a detectable specific binding member.

16. The method of any of the preceding clauses, further comprising, prior to said dispensing, contacting said dispense tip with a cell suspension such that said hanging drop of liquid is formed on said dispense tip.

17. The method of any of the preceding clauses, wherein dispensing is performed by a robotic liquid dispensing system, wherein said robotic liquid dispensing system comprises a fluid movement component, and wherein said fluid movement component comprises at least one fluidic channel that terminates in said dispense tip.

18. The method of any of the preceding clauses, wherein the wells of the multi-well device contain, prior to the dispensing, at least one reagent.

19. The method of Clause 18, wherein the at least one reagent is lyophilized.

20. The method of Clauses 18 or 19, wherein the at least one reagent is selected from the group consisting of: a nucleic acid, an enzyme, a dNTP, a dye, a detectable specific binding member and combinations thereof.

21. The method of Clause 20, wherein the nucleic acid comprises a barcode.

22. The method of Clauses 20 or 21, wherein the nucleic acid is an oligonucleotide primer.

23. The method of any of the preceding clauses, wherein the method further comprises nucleic acid amplification, reverse transcription, library preparation, sequencing or a combination thereof.

24. The method of Clause 23, wherein the method comprises library preparation comprising cDNA library preparation, next generation sequencing (NGS) library preparation, or a combination thereof. 25. The method of Clauses 23 or 24, wherein the library preparation comprises template switching, tagmentation or a combination thereof.

26. The method of any of Clauses 23 to 25, wherein method comprises sequencing comprising NGS.

27. The method of any of Clauses 23 to 26, wherein the method comprises nucleic acid amplification comprising whole genome amplification (WGS), polymerase chain reaction (PCR) or a combination thereof. 28. The method of any of Clauses 5 to 27, wherein the one or more cells are cells of a eukaryotic organism.

29. The method of Clause 28, wherein the eukaryotic organism is a plant, invertebrate animal or vertebrate animal.

30. The method of Clause 29, wherein the vertebrate animal is a mammal.

31. The method of any of Clauses 28 to 30, wherein the method further comprises dissociating a tissue from the eukaryotic organism to prepare a cell suspension containing the one or more cells.

32. A system comprising:

a) a multi-well device, wherein said multi-well devices comprises a hydrophobic top surface with a plurality of relatively hydrophilic wells formed therein; and

b) a fluid movement component comprising at least one fluidic channel, wherein said fluidic channel comprises a dispense tip configured to form a hanging drop of liquid when liquid is in said fluidic channel. 33. The method of Clause 32, wherein said hydrophobic surface of said multi-well device has a water contact angle greater than about 140 degrees.

34. The method of Clauses 32 or 33, wherein said hydrophilic surface of said wells has a water contact angle of about 65-80 degrees.

35. The method of any of Clauses 32 to 34, wherein said dispense tip is composed of a material that is less hydrophilic than said relatively hydrophilic wells.

36. The method of any of Clauses 32 to 35, wherein the wells of the multi-well device contain at least one reagent disposed thereon.

37. The method of Clause 36, wherein the at least one reagent is lyophilized.

38. The method of Clauses 36 or 37, wherein the at least one reagent is selected from the group consisting of: a nucleic acid, an enzyme, a dNTP, a dye, a detectable specific binding member and combinations thereof.

39. The method of Clause 38, wherein the nucleic acid comprises a barcode. 40. The method of Clauses 38 or 39, wherein the nucleic acid is an oligonucleotide primer.

41. A method of making a multi-well device comprising a hydrophobic top surface and a plurality of hydrophilic wells, the method comprising:

distributing a removable protective solid into each well of the plurality of hydrophilic wells to produce a plurality of protected hydrophilic wells;

applying a hydrophobic coating to the multi-well device to generate the hydrophobic top surface; removing the removable protective solids from the hydrophilic wells, wherein the removable protective solids prevent application of the hydrophobic coating to the hydrophilic wells. 42. The method of Clause 41, wherein the hydrophilic wells have a circular cross- section and the removable protective solids are spherical.

43. The method of Clauses 41 or 42, wherein the applying comprises spray coating or line-of-site deposition.

44. The method of any of Clauses 41 to 43, wherein the multi-well device is composed of a hydrophilic material.

45. The method of any of Clauses 41 to 44, wherein the method further comprises applying a hydrophilic coating to the multi-well device prior to the distributing.

46. The method of any of Clauses 41 to 45, wherein the removing comprising inverting the multi-well device to allow the removable protective solids to dislodge from the hydrophilic wells by gravity.

47. The method of any of Clauses 41 to 46, wherein the method further comprises disposing at least one reagent in the wells of the multi-well device after the removing.

48. The method of Clause 47, wherein the at least one reagent is lyophilized.

49. The method of Clauses 47 or 48, wherein the at least one reagent is selected from the group consisting of: a nucleic acid, an enzyme, a dNTP, a dye, a detectable specific binding member and combinations thereof. 50. The method of Clause 49, wherein the nucleic acid comprises a barcode.

51. The method of Clauses 49 or 50, wherein the nucleic acid is an oligonucleotide primer. The following examples are offers by way of illustration and not by way of limitation.

EXAMPLES

Example 1: Single Cell Library Preparation and RNA-Seq

Prior to cell dispensing, a multi-well chip with hydrophilic wells and a hydrophobic surface is prepared in accordance with the invention, e.g., as described above. Barcoded 3' first- strand primers are pre-distributed into each well and lyophilized. The prepared chip with first- strand primers is stored until use.

A solid tumor biopsy sample is obtained from a subject and dissociated into a cell suspension using mechanical dissociation followed by enzymatic digestion. The cell suspension is stained with Hoechst and propidium iodide (PI) and is loaded into a dispensing trough of an automated cell dispensing system. A robotic dispenser of the system, having eight dispensing tips, is used to dispense single cells according to a Poisson distribution into the wells of the prepared first-strand primer-containing chip. Specifically, the dispensing tips are dipped into and removed from the trough to contact the cell suspension and produce a hanging drop on the end of each dispense tip. The dispenser transports the dispense tips to the chip and touches off the hanging drops into the wells of the chip by a defined z-actuator motion. The hanging drops descend into the bottom of the wells, through hydrophilic attraction of the wells and hydrophobic repulsion by the surface of the chip, and re-hydrate the lyophilized 3' first strand primer.

The wells of the chip are automatically imaged by the automated cell dispensing system to detect viable cells based on Hoechst (+) and PI (-) staining. The number of viable cells present in each well is determined by the system and wells containing a single viable cell, as desired, are identified for further processing. First strand synthesis master mix is added to the identified wells containing single cells. Thermal controllers cycle the wells through the necessary incubations for reverse transcription, first strand synthesis and cDNA preparation. The prepared cDNAs are then pooled and amplified, with sequencing adapter addition, to generate a sequencing ready library barcoded at the single cell level. NGS sequencing is performed and the reads are mapped back to individual cells based on barcode identification. All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS We claim:

1. A method comprising:

dispensing a hanging drop of liquid from a dispense tip onto a hydrophobic top surface of a multi-well device,

wherein said multi-well device comprises a plurality of hydrophilic wells formed in said hydrophobic top surface,

wherein said dispensing comprises contacting said hanging drop with said hydrophobic surface while said hanging drop is still in contact with said dispense tip, and wherein said dispensing causes said hanging drop to separate from said dispense tip and move along said hydrophobic surface and into one of said hydrophilic wells.

2. The method of Claim 1, wherein said hydrophobic surface of said multi-well device has a water contact angle greater than about 140 degrees, said hydrophilic surface of said wells has a water contact angle of about 65-80 degrees or both.

3. The method of Claims 1 or 2, wherein the method further comprises detecting which wells of the multi-well device contain one or more cells after the dispensing.

4. The method of any of the preceding claims, wherein said hanging drop of liquid comprises a dispense volume of a cell suspension, wherein said cell suspension comprises cells present in said cell suspension at a concentration such that, on average, between 1 and 20 cells are present in said dispense volume.

5. The method of Claim 4, wherein on average one cell is present in said dispense volume.

6. The method of any of the preceding claims, further comprising, prior to said dispensing, contacting said dispense tip with a cell suspension such that said hanging drop of liquid is formed on said dispense tip.

7. The method of any of the preceding claims, wherein dispensing is performed by a robotic liquid dispensing system, wherein said robotic liquid dispensing system comprises a fluid movement component, and wherein said fluid movement component comprises at least one fluidic channel that terminates in said dispense tip.

8. The method of any of the preceding claims, wherein the wells of the multi-well device contain, prior to the dispensing, at least one reagent.

9. The method of Claim 8, wherein the at least one reagent is selected from the group consisting of: a nucleic acid, an enzyme, a dNTP, a dye, a detectable specific binding member and combinations thereof.

10. The method of any of the preceding claims, wherein the method further comprises nucleic acid amplification, reverse transcription, library preparation, sequencing or a combination thereof.

11. A system comprising:

a) a multi-well device, wherein said multi-well devices comprises a hydrophobic top surface with a plurality of relatively hydrophilic wells formed therein; and

b) a fluid movement component comprising at least one fluidic channel, wherein said fluidic channel comprises a dispense tip configured to form a hanging drop of liquid when liquid is in said fluidic channel.

12. The system of Claim 11, wherein said hydrophobic surface of said multi-well device has a water contact angle greater than about 140 degrees, said hydrophilic surface of said wells has a water contact angle of about 65-80 degrees or both.

13. The system of Claims 11 or 12, wherein the wells of the multi-well device contain at least one reagent disposed thereon.

14. A method of making a multi-well device comprising a hydrophobic top surface and a plurality of hydrophilic wells, the method comprising:

distributing a removable protective solid into each well of the plurality of hydrophilic wells to produce a plurality of protected hydrophilic wells;

applying a hydrophobic coating to the multi-well device to generate the hydrophobic top surface;

removing the removable protective solids from the hydrophilic wells, wherein the removable protective solids prevent application of the hydrophobic coating to the hydrophilic wells.

15. The method of Claim 14, wherein the applying comprises spray coating or line-of-site deposition.