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WO2025034867A1 - Procédés et appareils microfluidiques pour la dénudation d'ovocytes utilisant un flux oscillatoire - Google Patents

Procédés et appareils microfluidiques pour la dénudation d'ovocytes utilisant un flux oscillatoire Download PDF

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Publication number
WO2025034867A1
WO2025034867A1 PCT/US2024/041298 US2024041298W WO2025034867A1 WO 2025034867 A1 WO2025034867 A1 WO 2025034867A1 US 2024041298 W US2024041298 W US 2024041298W WO 2025034867 A1 WO2025034867 A1 WO 2025034867A1
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Prior art keywords
flow
complex
sub
oocyte
channels
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English (en)
Inventor
Baris R. MUTLU
Ismail Emre OZKUMUR
Sabrina Carla CIVALE
Mehmet Toner
Thomas L. Toth
Dionisios Sakkas
Ravi Kapur
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Autoivf Inc
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Autoivf Inc
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions

Definitions

  • Certain aspects of the present disclosure are generally directed to microfluidic methods and apparatuses for denudation of oocytes utilizing oscillatory flow.
  • BACKGROUND Oocytes contained within biological fluid oftentimes need to be denuded before they can be used in various applications, such as in vivo fertilization (IVF) applications.
  • a typical biological fluid typically contain cumulus oocyte complex (COC), which are oocytes surrounded by layers of various secondary cells (e.g., non-oocyte cells such as cumulus cells, granulosa cells, etc.).
  • COC cumulus oocyte complex
  • oocytes need to be initially denuded.
  • Several systems and/or methods for denuding oocytes are available. However, the systems and/or methods can be complicated and inefficient, and thus, more effective systems and/or methods of denuding oocytes are still needed.
  • Certain aspects of the present disclosure are generally directed to microfluidic methods and apparatuses for denudation of oocytes utilizing oscillatory flow.
  • the subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
  • One aspect is generally drawn to a microfluidic device.
  • the device comprises a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, wherein at least some of the sub-channels have a minimum cross-sectional dimension of between about 80 micrometers and about 350 micrometers, and wherein the sub-channels define a denuder region having a length within the fluidic channel of no more than 100 millimeters; a first pressure source upstream of the plurality of posts; and a second pressure source downstream of the plurality of posts.
  • the device comprises a fluidic channel having an inlet and an outlet, wherein the fluidic channel has a minimum cross-sectional dimension of between about 80 micrometers and about 350 micrometers; a plurality of posts downstream of the fluidic channel, the posts spaced apart at a distance of no more than 120 micrometers; a first pressure source upstream of the plurality of posts; and a second pressure source downstream of the plurality of posts.
  • Another aspect is generally drawn to a microfluidic device to process fluid and to isolate and denude oocytes for egg banking, egg freezing, and/or for in-vitro fertilization.
  • the device comprises a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts; a first pressure source upstream of the plurality of posts; and a second pressure source downstream of the plurality of posts.
  • the method is a method of preparing a fluid to isolate and denude an oocyte complex comprising an oocyte and bounded non-oocyte cells for egg banking, egg freezing, and/or in-vitro fertilization.
  • the method comprises passing the fluid through a fluidic channel comprising a plurality of posts such that the complex flows through sub-channels defined between neighboring pairs of posts.
  • Another set of embodiments is generally drawn to a method of denuding an oocyte complex comprising an oocyte and bounded non-oocyte cells to separate the oocyte from the non-oocyte cells.
  • the method comprise passing the complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, at least one of the sub-channels having a minimum cross-sectional dimension of between about 50 micrometers and 350 micrometers, the sub-channels defining a denuder region having a length within the fluidic channel of no more than 100 millimeters, such that at least 10% of the non-oocyte cells are removed when the complex flows through sub-channels.
  • Still another set of embodiments is directed to a method of manipulating an oocyte complex comprising an oocyte and bounded non-oocyte cells.
  • the method comprises causing a flow of fluid containing the complex in a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, such that the complex passes the plurality of posts at least 2 times.
  • Yet another set of embodiments is directed to a method of denuding an oocyte complex comprising an oocyte and non-oocyte cells to separate the oocyte from the non-oocyte cells.
  • the method comprises causing a first flow of a fluid containing the complex in a first direction through a fluidic channel; removing at least some of the non-oocyte cells from the complex as the complex flows through the fluidic channel in the first direction; causing a second flow of the fluid containing the complex in a second direction through the fluidic channel; and removing at least some of the non-oocyte cells from the complex as the complex flows through the fluidic channel in the second direction.
  • the method comprises repeatedly causing flow of a fluid containing the complex through a fluidic channel, wherein at least 10% of the non-oocyte cells in the complex are removed during repeated flow of the complex through the fluidic channel.
  • the method in another set of embodiments, is a method of manipulating an oocyte complex comprising an oocyte and non-oocyte cells.
  • the method comprises causing a flow of fluid containing the complex in a fluidic channel such that the complex flows across an imaginary line within the fluidic channel at least 2 times, wherein the imaginary line is defined in a direction orthogonal to a direction of bulk fluid flow within the fluidic channel.
  • the method is a method of manipulating an oocyte complex comprising an oocyte and non-oocyte cells.
  • the method in certain cases, comprises causing a flow of fluid containing the complex in a channel dimensioned to allow the oocyte to flow through only if not bound to the non-oocyte cells; and repeating the causing step at least 2 times.
  • the method is a method of denuding an oocyte from bounded non-oocyte cells in a fluid.
  • the method in one embodiment, comprises inducing a first flow of the fluid containing one or more non-oocyte oocyte complex in a first direction through a microfluidic channel, such that at least a portion of the bounded non-oocyte cells is removed from the complex as the oocyte flows through the microfluidic channel in the first direction; and inducing a second flow of the fluid containing the one or more cumulous oocyte complex in a second direction opposite the first direction through the microfluidic channel, such that at least a portion of the bounded non-oocyte cells is removed as the oocyte flows through the microfluidic channel in the second direction.
  • Still another set of embodiments is drawn to a method of preparing a fluid to isolate and denude a target entity-secondary entity complex comprising a target entity and one or more secondary entities to separate the target entity from the secondary entities.
  • the method comprises passing the fluid through a fluidic channel comprising a plurality of posts such that the complex flows through sub-channels defined between neighboring pairs of posts.
  • Yet another set of embodiments is directed to a method of denuding a target entity- secondary entity complex comprising a target entity and one or more secondary entities to separate the target entity from the secondary entities.
  • the method comprises passing the complex through a fluidic channel comprising a plurality of posts defining sub- channels between neighboring pairs of posts, at least one of the sub-channels having a minimum cross-sectional dimension of between about 1 micrometer and about 1 mm, the sub- channels defining denuder region having a length within the fluidic channel of no more than 100 millimeters, such that at least 10% of the secondary entities are removed when the complex flows through sub-channels.
  • Another set of embodiments is directed to a method of manipulating a target entity- secondary entity complex comprising an target entity and one or more secondary entities.
  • the method comprises causing a flow of fluid containing the complex in a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, such that the complex passes the plurality of posts at least 2 times.
  • the method in one set of embodiments, is a method of denuding an target entity- secondary entity complex comprising an target and one or more secondary entities to separate the target from the one or more secondary entities.
  • the method comprises causing a first flow of a fluid containing the complex in a first direction through a fluidic channel; removing at least some of the one or more secondary entities from the complex as the complex flows through the fluidic channel in the first direction; causing a second flow of the fluid containing the complex in a second direction through the fluidic channel; and removing at least some of the one or more secondary entities from the complex as the complex flows through the fluidic channel in the second direction.
  • the method is a method of denuding an target entity- secondary entity complex comprising an target entity and one or more secondary entities to separate the target entity from the one or more secondary entities.
  • the method comprises repeatedly causing flow of a fluid containing the complex through a fluidic channel. In some embodiments, at least 10% of the one or more secondary entities in the complex are removed during repeated flow of the complex through the fluidic channel. In yet another set of embodiments, the method is a method of manipulating an target entity-secondary entity complex comprising an target entity and one or more secondary entities. In certain embodiments, the method comprises causing a flow of fluid containing the complex in a fluidic channel such that the complex flows across an imaginary line within the fluidic channel at least 2 times, wherein the imaginary line is defined in a direction orthogonal to a direction of bulk fluid flow within the fluidic channel.
  • the method is a method of manipulating an target entity-secondary entity complex comprising an target entity and one or more secondary entities.
  • the method comprises causing a flow of fluid containing the complex in a channel dimensioned to allow the target entity to flow through only if not bound to the one or more secondary entities; and repeating the causing step at least 2 times.
  • Another set of embodiments is drawn to a method of denuding an target entity from bounded one or more secondary entities in a fluid.
  • the method comprises inducing a first flow of the fluid containing one or more target entity-secondary entity complex in a first direction through a microfluidic channel, such that at least a portion of the bounded one or more secondary entities is removed as the target entity flows through the microfluidic channel in the first direction; and inducing a second flow of the fluid containing the one or more target entity-secondary entity complex in a second direction opposite the first direction through the microfluidic channel, such that at least a portion of the bounded one or more secondary entities is removed as the target entity flows through the microfluidic channel in the second direction.
  • FIG.1A is a schematic illustration showing fluid flow in a fluidic channel in a first direction, in accordance with some embodiments
  • FIG.1B is a schematic illustration showing fluid flow in a fluidic channel in a second direction, in accordance with some embodiments
  • FIGS.2A-2C are schematic illustrations showing a microfluidic device for denuding a target entity (e.g., an oocyte) and/or for generating oscillatory flow, in accordance with some embodiments
  • FIGS.2D-2E are schematic illustrations showing fluid flows through sub-channels between neighboring pairs of posts, in accordance with some embodiments
  • FIG.3A is a schematic illustration showing a fluidic channel formed by concentric walls, in accordance with some embodiments
  • FIG.3B is a schematic illustration showing a fluidic channel formed by concentric walls and comprising a plurality of posts therein, in accordance with some embodiments
  • FIG.4A is a schematic illustration showing a microfluidic device for denuding a target
  • the present disclosure is generally directed to microfluidic methods and apparatuses for separating cells utilizing various techniques, such as oscillatory flow.
  • One example application is the denudation of oocytes.
  • Oocytes may need to be denuded before they can be effectively used in various applications, such as in vivo fertilization (IVF) applications.
  • IVVF in vivo fertilization
  • Certain aspects of the disclosure are thus directed to systems and/or methods that allow for more efficient cell separation. For instance, in some cases, the use of oscillatory flow through a channel may advantageously increase the efficiency of the separation process, while minimizing harmful effects on the cells.
  • the microfluidic device may comprise various components, such as posts which optionally have extruded portions (e.g., teeth), multiple pressure sources to control fluid flow, or the like.
  • Certain aspects as discussed herein is generally directed to systems and methods for cell separation.
  • One example application of cell separation is the denudation of oocytes.
  • Oocytes contained within biological fluid oftentimes need to be denuded before they can be used in various applications, such as in vivo fertilization (IVF) procedures.
  • IVF in vivo fertilization
  • certain aspects of the disclosure are directed to systems, devices and/or methods that collectively allow for a highly efficient oocyte denudation process.
  • Certain devices and methods described herein may have various advantages compared to conventional devices and methods for oocyte denudation.
  • an example microfluidic device comprises a fluidic channel having an inlet, and outlet, and a sub-channel (e.g., a constriction portion) between the inlet and outlet as shown in FIGS.1A-1B.
  • the microfluidic device comprises a fluidic channel 110a having an inlet 150a, an outlet 170a, and a sub-channel 130a between the inlet 150a and the outlet 170a.
  • a oocyte complexes may be repeatedly flowed across the fluidic channel.
  • the oocyte may be flowed back-and-forth, e.g., in an oscillatory fashion through the channel, and/or the oocyte may be flowed through the same channel in a certain direction a plurality of times.
  • an oocyte may flow from the inlet 150a to the outlet 170a in a first direction 12 (e.g., from left to right) as shown in FIG.1A, and then from the outlet 170a to the inlet 150a in a second direction 14 (e.g., from right to left) as shown in FIG.1B.
  • Such an oscillatory flow across the fluidic channel may be repeated multiple times within the fluidic channel.
  • the oocyte may be caused to flow in a single direction multiple times repeatedly through the microfluidic channel (e.g., in the direction shown in FIG.1A).
  • the device may be configured for repeated flow (e.g., oscillatory flow) of a fluid containing one or more oocyte complexes through a plurality of parallel microfluidic channels sized slightly larger than or about the size of an oocyte.
  • the use of such repeated flows through a plurality of parallel channels of the device may allow for effective denudation of oocytes from a variety of fluids (e.g., follicular fluid, ovarian tissue, inseminated oocyte complexes), while preventing chip-clogging issues, and in some cases, it may reduce the duration of the denudation process and/or damage to the denuded oocytes.
  • the devices and/or methods described herein may result in effective cleaning of debris contained within biological fluids from the channels.
  • the devices and/or methods described herein may allow for control of the degree of oocyte denudation by monitoring and/or controlling a variety of device parameters, e.g., such as the number of interactions between the oocytes and certain denudation features (e.g. teeth/jagged edges), direction of flow, flow rate, local velocity profile, and the like.
  • Debris may include any organic (e.g., granulose cells, cell clumps, tissue, red blood cell clusters, blood clots, etc.) or inorganic material other than the targeted cells or cell complexes (e.g., cumulus-oocyte-complex, oocyte, etc.).
  • Certain devices may advantageously allow for visual temporal analysis of the denudation process within a localized section of the device and allow for closed-loop control of the denudation process based on one or more measured parameters.
  • certain embodiments of the present disclosure may allow for denudation of oocytes within microfluidic channels having a relatively small size (e.g., length and footprint) compared to conventional microfluidic devices. Because the oocytes may be passed through the same channel repeatedly, longer channels and/or larger devices are not necessary for denudation of oocytes.
  • various other cells may be treated, e.g., in addition to or instead of oocytes. Non-limiting examples are discussed herein.
  • the microfluidic device may comprise one or more particularly advantageous features, e.g., such a plurality of posts and certain denudation features (e.g., extruded portions such as teeth) thereon, a plurality of microfluidic channels (i.e., sub-channel) between the posts, one or more pressure source and/or fluid controls, that allow for effective denudation of oocytes, as described in more detail below.
  • microfluidic systems and/or devices are generally described.
  • a microfluidic system may include at least one fluid conduit or channel (i.e., a microfluidic channel) having a cross-sectional dimension of less than about 1 millimeter (mm).
  • the disclosure herein is not limited to only microfluidic systems.
  • larger channels may be used.
  • the microfluidic devices described herein may be employed for processing any of a variety of biological fluids to isolate and denude target entities from bounded secondary entities contained within the fluids.
  • the microfluidic devices described herein may be employed to process follicular fluid and to isolate and denude oocytes for egg banking, egg freezing, and/or for in-vitro fertilization.
  • the device comprises a fluidic channel comprising one or more posts (e.g., a plurality of posts) therein.
  • the plurality of posts define sub-channels between neighboring pairs of posts.
  • the plurality of posts may define sub-channels between post(s) and adjacent wall(s) of the fluidic channel.
  • FIG.2A can be used to illustrate one such embodiment.
  • a microfluidic device 100 comprises a fluidic channel 110 comprising a plurality of posts 120 therein.
  • the plurality of posts 120 may define (parallel) sub-channels 130 between neighboring pairs of posts 120.
  • neighboring posts 120a and 120b may together define sub-channel 130b therebetween.
  • sub-channel 130a may be defined by post 120a and wall 113 of fluidic channel 110.
  • the use of parallel sub-channels may advantageously provide a plurality of paths for a target entity to pass through, which may reduce the risk of chip clogging and increase denudation efficiency.
  • the plurality of posts within the fluidic channel may be arranged in any appropriate fashion.
  • the plurality of posts are arranged in an array across the fluidic channel.
  • the array of posts in some cases, may comprise one or more rows of posts, where each row comprises one or more posts.
  • the posts may be arranged in any suitable arrangement, for example, a rectangular grid or in a staggered fashion, e.g., as shown in FIG. 2A.
  • the plurality of posts is not limited to being arranged in an ordered fashion, and that in some cases, at least one of the plurality of posts may be arranged randomly across the fluidic channel.
  • the array of posts may comprise n number of rows of post(s) and/or sub-channels, where n is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and/or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less.
  • the array of posts comprises a single row of post and/or sub-channels.
  • the array of posts 120 comprises multiple rows of posts and sub-channels, e.g., as exemplified by row numbers n i , n i+1, n i+2 , etc.
  • the array of posts may comprise m number of post(s) and/or sub- channels per row, where m is at least 1, at least 2, at least 3, at least 5, at least 10, at least 25, at least 50, at least 100, at least 250, at least 500, at least 750, and/or no more than 1000, no more than 750, no more than 500, no more than 250, no more than 100, no more than 50, no more than 25, no more than 10, no more than 5, no more than 3, no more than 2, or less. Combinations of the above-referenced ranges are possible (e.g., at least 1 and no more than 1000, at least 2 and no more than 1000). Other ranges are also possible.
  • the array of posts comprises a single post per row.
  • FIG.2A illustrates an embodiment in which the fluidic channel 110 comprises one or more posts
  • a fluidic channel in a device does not necessarily include a post therein.
  • FIG.1A can be used to illustrate one such embodiment.
  • fluidic channel 110a does not contain any posts therein.
  • the fluidic channel 110a comprise a single sub-channel 130d therein formed between extruded wall portions of the fluidic channel.
  • the sub-channel 130d may have any properties (e.g., minimum cross-sectional dimension) described respective to the sub-channels shown in FIG.2A.
  • the one or more posts within the fluidic channel may individually have any of a variety of appropriate shapes and forms. If more than one post is present, the posts may have the same or different shapes and/or sizes.
  • at least one of the plurality of posts comprises a shape such as kite-shaped, a hexagon, rectangular, circular, triangular, polygonal, etc.
  • FIG.2A can be used to illustrate a plurality of posts within the fluidic channel 110 having a kite-shaped structure.
  • FIG.6 can be used to illustrate a plurality of posts having hexagonal shapes. Other shapes are also possible.
  • At least some of the sub-channel(s) may have an inlet portion, an outlet portion, and a constriction portion between the inlet portion and outlet portion.
  • the constriction portion may of the sub-channel(s) may have a cross-sectional dimension that is less than those of the inlet portion and outlet portion.
  • FIG.2A can be used to illustrate one such embodiment.
  • a sub-channel defined by neighboring posts may include an inlet portion a, an outlet portion c, and a constriction portion b therebetween, with the constriction portion b having a cross-sectional dimension less than the inlet and outlet portions.
  • At least one or more (e.g., some) of the sub-channel(s) have a minimum cross- sectional dimension w in the constriction portion of the sub-channel(s), e.g., as shown in FIG. 2D.
  • the minimum cross-sectional dimension of the sub-channel(s) may be sized so as to allow denudation of a target entity of interest (e.g., an oocyte), e.g., such as having size(s) comparable to (or slightly larger) than the size of the target entity (e.g., oocyte).
  • some (or all) of the sub-channel(s) within the denuder region may have a minimum cross-sectional dimension w of greater than or equal to 20 micrometers, greater than or equal to 50 micrometers, greater than or equal to 70 micrometers, greater than or equal to 80 micrometers, greater than or equal to 100 micrometers, greater than or equal to 120 micrometers, greater than or equal to 125 micrometers, greater than or equal to 130 micrometers, greater than or equal to 140 micrometers, greater than or equal to 150 micrometers, greater than or equal to 160 micrometers, greater than or equal to 170 micrometers, greater than or equal to 180 micrometers, greater than or equal to 200 micrometers, greater than or equal to 250 micrometers, greater than or equal to 300 micrometers, and/or less than or equal to 350 micrometers, less than or equal to 300 micrometers, less than or equal to 250 micrometers, less than or equal to 200 micrometers, less than or equal to 180 micrometers, less than or
  • the minimum cross-sectional dimension of the sub-channels in the denuder region may depend, at least in part, on the type of target entity that is being denuded in the denuder region. As described elsewhere herein, the target entity may be an oocyte.
  • the size of the oocyte may also vary.
  • at least some of the sub- channels in the denuder region may have a minimum cross-sectional dimension that is at least 1.01 times (at least 1.1 times, at least 1.15 times, at least 1.2 times, at least 1.25 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, or more, and/or up to 3 times, up to 2.5 times, up to 2 times, up to 1.5 times, up to 1.4 times, up to 1.3 times, up to 1.25 times, up to 1.2 times, up to 1.15 times, up to 1.1 times, etc.) the size (e.g., diameter) of the target entity (e.g., a bare oocyte).
  • the minimum cross-sectional dimension of the sub-channels may also be slightly smaller than the target entity (e.g. a bare oocyte).
  • the target entity e.g. a bare oocyte
  • at least some of the sub-channels in the denuder region may have a minimum cross-sectional dimension that is at least 0.8 times, at least 0.82 times, at least 0.85 times, at least 0.87 times, at least 0.9 times, at least 0.92 times, at least 0.94 times, at least 0.95 times, at least 0.96 times, at least 0.97 times, at least 0.98 times, at least 0.99 times, etc.
  • the dimensions referenced above may be particularly advantageous for applications directed to oocyte denudation.
  • the minimum cross- sectional dimension of the sub-channels in the denuder region may be scaled relative to the size of the target entity (e.g., a cell, a tissue, etc.).
  • the target entity e.g., a cell, a tissue, etc.
  • the minimum cross-sectional dimension of the sub-channels be in the size range of micrometers, such as less than 100 microns, less than 50 microns, less than 25 microns, less than 20 microns, less than 15 microns, or less and/or down to 10 microns, down to 5 microns, or less.
  • the minimum cross- sectional dimension of the sub-channels be in the size range of millimeter, such as at least 0.5 mm, at least 1 mm, at least 2 mm, at least 10 mm, or more.
  • some of the sub-channels may have dimensions that differ from the rest of the sub-channels.
  • the sub-channels may be arranged such that the sub-channel(s) adjacent an inlet of the microfluidic device may have minimum cross-sectional dimension(s) larger than the minimum cross-sectional dimension(s) of sub-channel(s) further away from the inlet of the device.
  • the sub-channels 130 adjacent an inlet 150 of the device 100 may have minimum cross-sectional dimension(s) larger than cross-sectional dimension(s) of sub- channel(s) further away from the inlet 150 of the device 100 (such as those in row ni+5).
  • the minimum cross-sectional dimension of the sub-channels furthest away from the inlet 201 (adjacent outlet 202) may have the smallest minimum cross-sectional dimension amongst the other sub-channels in the denuder region, such as having a minimum cross-section dimension that is comparable to the size of the target entity.
  • each subsequent row or set of sub-channels may have minimum cross-sectional dimensions that decrease by an increment of at least 5 micrometers (e.g., at least 10 micrometers, at least 15 micrometers, or more, and/or up to 20 micrometers, up to 25 micrometers) from the minimum cross-sectional dimensions of the preceding row or set of sub-channels.
  • such an arrangement in the denuder region may allow for gradual removal (e.g., denudation) of secondary entities from the target entities, which would lead to an overall more efficient denudation process.
  • the incremental decrease in sub-channel dimensions may allow for gradual removal of non-oocyte cells (e.g., cumulus cells, corona cells, etc.) from the oocyte as the oocyte complex passes through each row or set of sub-channels.
  • the sub-channels in the denuder region may be sized that a desired level of denudation can be achieved, in some embodiments.
  • At least some of the sub-channels may have minimum cross-sectional dimensions comparable or approximately equal to the size of the target entity.
  • at least some of the sub-channels may have minimum cross-sectional dimensions that are slightly larger than the size of the target entity.
  • the plurality of sub-channels may be divided into one or more stages, with some or all of the stages containing sub-channels having different minimum cross- sectional dimensions from the those in the other stage(s).
  • the sub-channels may comprise a first stage of sub-channels having a minimum cross-sectional dimension of greater than 140 micrometers and less than or equal to 160 micrometers, a second stage of sub-channels having a minimum cross-sectional dimension of greater than 120 micrometers and less than or equal to 140 micrometers, a third stage of sub-channels having a minimum cross-sectional dimension of greater than 50 micrometers and less than or equal to 120 micrometers, etc.
  • the plurality of posts and/or sub-channels may be arranged such that a row of posts and/or sub-channels is offset from a subsequent row of posts and/or sub- channels, e.g., as shown in FIG.2A.
  • the sub-channel may be arranged such that the longitudinal axis of sub-channel(s) in one row (such as those in row n i ) of the fluidic channel passes through (or coincides with) the longitudinal axis of post(s) in a subsequent row (such as those in row ni+1).
  • the disclosure is not so limited, and that any appropriate arrangement/order of the posts and/sub- channels are possible.
  • At least a portion of the walls forming the sub-channels may comprises a plurality of extruded features (e.g., teeth).
  • the minimum cross-sectional dimension w of a sub-channel may correspond to a distance between the outermost edges (e.g., tips) of extruded features (e.g., teeth) on opposing walls of the sub- channel(s).
  • at least a portion of the walls on the plurality of posts 120 defining the sub-channel(s)130 comprise extruded features (e.g., teeth) 140.
  • the minimum cross-sectional dimension w of the sub-channel may correspond to the distance between the outermost edges of extruded features 140 on respective walls of neighboring posts (e.g., post 120a, post 120b).
  • at least a portion of the extruded wall portions of the fluidic channel 110a may comprise teeth, and the minimum cross-sectional dimension w may correspond to the distance between the tips of teeth on opposing walls of the fluidic channel.
  • FIGS.1A-2E show one set of embodiments in which the walls forming the sub- channels comprise a plurality of extruded features (e.g., teeth), it should be understood that the disclosure is not so limited, and that in other embodiments, at least one (or both) of the walls forming the sub-channels may be substantially smooth (e.g., lack of teeth thereon).
  • the sub-channel(s) and/or post(s) define a denuder region within the fluidic channel.
  • the denuder region is configured to facilitate denuding of target entities (e.g., oocytes) from bounded and/or associated secondary entities (e.g., bounded non-oocyte cells) as the target entities pass through sub-channel(s) within the denuder region.
  • target entities e.g., oocytes
  • secondary entities e.g., bounded non-oocyte cells
  • the region of fluidic channel 110 comprising the plurality of post(s) 120 and/or subchannel(s) 130 defines a denuder region 115.
  • the region of fluidic channel 110a comprising the subchannel 130d defines a denuder region 115a.
  • the denuder region may advantageously have a relatively small length.
  • the length of denuder region may be measured horizontally across from the first (row of) sub-channel(s) or post(s) in the denuder region to the last (row of) sub-channel(s) or post(s) in the denuder region.
  • FIGS.1A-2A can be used to illustrate this.
  • the length L of the denuder region can be measured from the first row n i of post(s) or sub-channel(s) to the last row n i+5 of post(s) or sub-channel(s).
  • the length of the single denuder region 115a would the same as the length of the sub-channel 130a itself.
  • the length of denuder region within the microfluidic device may be relatively short, such as at least 50 micrometers, at least 100 micrometers, at least 500 micrometers, at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm, at least 8 mm, at least 10 mm, at least 20 mm, at least 40 mm, at least 60 mm, at least 80 mm, at least 100 mm, at least 150 mm, and/or no more than 200 mm, no more than 150 mm, no more than 100 mm, no more than 80 mm, no more than 60 mm, no more than 40 mm, no more than 20 mm, no more than 10 mm, no more than 8 mm, no more than 5 mm, no more than 3
  • Combinations of the above-referenced ranges are possible (e.g., at least 10 mm and no more than 200 mm, at least 10 mm and no more than 100 mm, at least 20 micrometers and no more than 200 mm, at least 20 micrometers and no more than 200 mm, at least 50 micrometers and no more than 200 mm, or at least 50 micrometers and no more than 10 mm).
  • Other ranges are also possible.
  • the length of the denuder region can be relatively short, as the effective length of the denuder region that the entities (e.g., cells) actually experience is the product of the length of the denuder region and the number of times that the entities pass therethrough (e.g., in one direction, or in more than one direction).
  • the entities e.g., cells
  • only a single pass of the entities through the denuder region is contemplated.
  • each of the sub-channel(s) in the denuder region described above may have a length of at least 50 micrometers, at least 100 micrometers, 200 micrometers, at least 500 micrometers, at least 1 mm, at least 2 mm, at least 4 mm, at least 10 mm, at least 20 mm, at least 50 mm, at least 100 mm, or more, and/or no more than 200 mm, no more than 100 mm, no more than 50 mm, no more than 20 mm, no more than 10 mm, no more than 5 mm, no more than 2 mm, no more than 1 mm, no more than 500 micrometers, no more than 200 micrometers, no more than 100 micrometers, or less.
  • the sub-channel(s) comprise inlet portions and/or outlet portions that are tapered towards the constriction portions of the sub-channel(s).
  • the inlet portion a and outlet portion b may be tapered into the constriction portion b of the sub-channel.
  • the presence of such tapered inlet and/or outlet portions may effectively direct bulk fluid flow into the sub-channel(s) from the inlet and/or outlet portion into the constriction portion, thereby facilitating denudation of target entities (e.g., oocyte) as they travel through the sub-channel(s).
  • target entities e.g., oocyte
  • the post may have any of a variety of shapes (e.g., hexagonal, kite-like, etc. or other shapes such as those described herein) that can lead to formation of sub- channel(s) having tapered inlet and/or outlet portions, as described above.
  • the present disclosure is not so limited, and that in certain embodiments, at least one or more of the inlet and/or outlet portions may not be tapered.
  • the sub-channel(s) may optionally be formed by one or more wall(s) (of neighboring posts) comprising teeth.
  • the presence of teeth on the wall(s) of the sub-channels may advantageously aid denudation of target entities (e.g., oocyte complexes) and they pass through the sub-channel(s).
  • target entities e.g., oocyte complexes
  • the wall(s) of the sub-channel(s) are defined by neighboring pairs of posts, e.g., as shown in FIGS.2A-2E.
  • both opposing walls forming the sub-channel may comprise teeth. In other cases, only one of the opposing walls forming the sub-channel may comprise teeth. In some cases, at least a portion on the posts 120 forming the walls of inlet portion a, constriction portion b, and outlet portion c of the sub-channel(s) comprises teeth. In some cases, the constriction portion b of the sub-channel(s) comprises teeth. In some cases, the inlet portion a and/or outlet portion c of the sub-channel(s) comprises teeth.
  • the teeth on the post(s) and/or wall(s) of the sub-channel(s) may have any of a variety of appropriate shapes, including, but not limited to, round, triangular, rectangular, saw-teeth, quadrilaterals, and/or other polygonals. While FIG.2A illustrate one embodiment of posts comprising teeth that are saw-teethed, it should be understood that the disclosure is not so limited, and that in other embodiments, the teeth may have other shapes. For example, FIGS.
  • FIG. 7A-7B can be used to illustrate various shapes of teeth on the post(s) and/or wall(s) of the sub- channel(s), such as saw-teeth (FIG.7A), rectangular (FIG.7B), triangular (FIG.7C), and circular (FIG.7D).
  • the teeth on the post(s) may be particularly advantageous for the teeth on the post(s) to have one or more sharp features (e.g., an edge, a corner, a point, a tip, etc.). The presence of these sharp features in the teeth may allow for more efficient denudation of secondary entities from the target entity (by inducing tear and disruption of the secondary entities) and as the target entity gains contact with these features.
  • Non-limiting example of teeth on posts and/or walls having one or more sharp features include saw-teeth, rectangular, triangular, as shown in FIG.2D and/or FIGS.7A-7C.
  • the teeth on the post(s) and wall(s) may have any of a variety of appropriate dimensions and/or orientations.
  • the teeth may comprises a first length (e.g., L FEAT-1 ), a second length (e.g., L FEAT-3 ), a first height (e.g., L FEAT-2 ), a second height (e.g., L FEAT-4 ), a first angle ( ⁇ FEAT-1 ), and a second angle (e.g., ⁇ FEAT-2 ).
  • the first length (e.g., LFEAT-1), may have any of a variety of values, such at between 5 micrometers and 30 micrometers, between 10 micrometers and 20 micrometers, or between 15 micrometers and 30 micrometers.
  • the second length (e.g., L FEAT-3 ) may have any of a variety of values, such at between 0 micrometers and 30 micrometers, between 10 micrometers and 20 micrometers, or between 15 micrometers and 30 micrometers.
  • the first height (e.g., LFEAT-2) may have any of a variety of appropriate values, such as between 30 micrometers and 80 micrometers, between 40 micrometers and 60 micrometers, or between 50 micrometers and 80 micrometers.
  • the second height may have any of a variety of appropriate values, such as between 10 micrometers and 50 micrometers, between 20 micrometers and 40 micrometers, or between 30 micrometers and 50 micrometers.
  • the angles ( ⁇ FEAT-1,2 ) can be between 20 degrees and 80 degrees, or 30 degrees and 60 degrees, Other ranges are possible.
  • the length of the feature sides (LFEAT-1,2,3,4) can be as small as 1 ⁇ m and as large as 1000 ⁇ m.
  • the angles ( ⁇ FEAT-1,2 ) can be as small as 0.1° and as large as 179.9°.
  • the extruded featured may be circular shaped and the features may be defined by the circle radius (R FEAT ), the offset of its center from the wall (L OFFSET ), and the distance between features (LFEAT), as shown in FIG.7D.
  • the teeth on the one or more wall(s) of the sub-channel(s) may be oriented in any of a variety of appropriate directions.
  • the teeth may be oriented at any angle, such as being saw- teethed, e.g., as shown in FIGS.2A-2E.
  • the teeth on one or more wall(s) of the sub-channel may be unidirectional.
  • the teeth 140 on the posts 120a and/or 120b may be oriented facing a first direction 12 of bulk fluid flow through the sub-channel(s).
  • the teeth on one or more wall(s) of the sub-channel may be bi-directional, such as comprising a first set of teeth that is oriented facing a first direction of bulk fluid flow across the sub-channel, and a second set of teeth that is oriented facing a second direction of bulk fluid flow across the sub-channel, e.g., as shown in FIG.6.
  • each of the opposing walls comprise a first set of teeth that is oriented facing a forward fluid flow through the sub-channel and a second set of teeth that is oriented facing a backward fluid flow through the sub-channel.
  • the first set of teeth may be oriented at any of a variety of appropriate angles facing a first direction of bulk fluid flow across the sub-channel.
  • the first set of teeth may be oriented at an angle between 0.1° and 90°, between 5° and 80°, between 20° and 70°, or between 30° and 60° facing the bulk fluid flow across the sub-channel.
  • the second set of teeth may be oriented in a direction that is substantially opposite to the first set of teeth.
  • the second set of teeth may have a tip that is oriented in a direction that is at least 5 degrees, at least 10 degrees, at least 20 degrees, at least 30 degrees, at least 35 degrees, at least 40 degrees, at least 45 degrees, at least 60 degrees, at least 70 degrees, at least 90 degrees, or more, and/or up to 120 degrees, up to 140 degrees, up to 160 degrees, or up to 179 degrees, or up to 179.9 degrees clockwise from the tip of the first set of teeth. Combinations of the above-referenced ranges are possible (e.g., at least 5 degrees and up to 179.9 degrees, or at least 30 degrees and up to 160 degrees). Other ranges are also possible.
  • the microfluidic device comprises a first pressure source upstream of the denuder region containing the plurality of posts and a second pressure source downstream of the denuder region. In some embodiments, the device further comprises a first inlet adjacent and connected to the first pressure source and a second inlet adjacent and connected to the second pressure source.
  • the first pressure source and/or second pressure source independently comprises one or more pumps (e.g., syringe pumps, peristaltic pumps, positive displacement pumps, etc.).
  • the presence of at least two pressure sources may advantageously allow for oscillatory flow across the fluidic channel, such as causing flow across the denuder region containing the plurality of posts and sub-channels in the first direction and in the second direction.
  • the first pressure source may be configured exert pressure in a first direction, such that a fluid within the fluidic channel may be caused or induced to flow in the first direction within the device.
  • the second pressure source may be configured to exert a pressure in a second direction, such that the fluid within the fluidic channel may be caused or induced to flow in the second direction.
  • FIG.2A can be used to illustrate one such embodiment.
  • the microfluidic device 100 comprises a first inlet 150 and a second inlet 160.
  • the first inlet 150 may be positioned adjacent and connected to a first pressure source 155 and the second inlet 160 may be positioned adjacent and connected to a second pressure source 165.
  • the first pressure source 155 may be configured exert pressure in a first direction, such that a fluid within the fluidic channel (e.g., denuder region) may flow in the first direction 12 (e.g., a forward direction).
  • the second pressure source 165 may be configured to exert a pressure in a second direction, such that the fluid within the fluidic channel may flow in the second direction 14 (e.g., a reverse direction).
  • the microfluidic device comprises a capture region positioned downstream of a denuder region of the fluidic channel containing the post(s) and/or sub- channel(s).
  • the capture region may be disposed between the first inlet and the second inlet of the microfluidic device.
  • the microfluidic device 100 may further comprise a capture region 172 positioned downstream of the denuder region 115 containing the posts 120 and/or sub-channels 130 and between the first inlet 150 and the second inlet 160.
  • the capture region may comprise a plurality of posts, in some embodiments.
  • the plurality of posts in some cases, may be spaced apart such that a plurality of sub-channel(s) are defined between neighboring pairs of posts.
  • the plurality of sub-channels between the posts in the capture region may have a minimum cross-sectional dimension that is less than the minimum cross-sectional dimension of the sub-channel(s) within the denuder region of the fluidic channel, according to some embodiments.
  • the plurality of sub- channels in the capture region may be sized to only allow denuded entities (e.g., denuded or bare oocytes without the bound non-oocyte cells) to flow readily therethrough while trapping larger target complexes (e.g., oocyte complex) and/or debris.
  • the capture region 172 of the device may comprise a plurality of posts 175 defining a plurality of sub-channels 177 between neighboring pairs of posts.
  • the first pressure source 155 may be positioned upstream of the capture region 172 containing the posts 175, and the second pressure source 165 may be positioned downstream of the capture region 172.
  • One or more outlets, such as a first outlet 170, may be positioned downstream of the capture region 172.
  • a second outlet may be positioned upstream of the capture region 172, such as between the denuder region and the capture region, as shown in FIG.9B.
  • some (or all) of the sub-channel(s) within the capture region of the fluidic channel may have a minimum cross-sectional dimension d (e.g., as shown in FIG.5) of greater than or equal to 30 micrometers, greater than or equal to 40 micrometers, greater than or equal to 50 micrometers, greater than or equal to 65 micrometers, greater than or equal to 80 micrometers, greater than or equal to 90 micrometers, greater than or equal to 100 micrometers, greater than or equal to 110 micrometers, greater than or equal to 120 micrometers, greater than or equal to 130 micrometers, greater than or equal to 140 micrometers, and/or less than or equal to 150 micrometers, less than or equal to 140 micrometers, less than or equal to 130 micrometers, less than or equal to 120 micrometers, less than or equal to 110 micrometers, less than or equal to 100 micrometers, less than or equal to 90 micrometers, less than or equal to 80 micrometers, less than or equal to 65 micrometers,
  • the above-referenced ranges may be particularly advantageous for certain type of applications, e.g., denudation of oocyte complexes.
  • the disclosure is not so limited, and that in some embodiments, depending on the application, the minimum cross-sectional dimension of the sub-channels in the capture region may be scaled relative to the size of the target entity (e.g., a cell, a tissue, etc.).
  • the minimum cross-sectional dimension of the sub-channels be in the size range of micrometers, such as less than 100 microns, less than 50 microns, less than 25 microns, less than 20 microns, less than 15 microns, or less and/or down to 10 microns, down to 5 microns, or less.
  • the minimum cross-sectional dimension of the sub-channels be in the size range of millimeter, such as at least 0.5 mm, at least 1 mm, at least 2 mm, at least 10 mm, or more.
  • At least some (or all) of the sub-channels in the capture regions have a dimension sized such that a fully denuded (or dissociated) target entity (e.g., an oocyte) cannot pass therethrough when subjected to flow.
  • a fully denuded (or dissociated) target entity e.g., an oocyte
  • at least some (or all) of the sub-channels in the capture regions may have a size that is at least 10 micrometers, at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, and/or up to 50 micrometers, at least 60 micrometers, at least 80 micrometers, or up to 100 micrometers, or more, smaller than the size (e.g., diameter) of the fully denuded (or dissociated) target entity.
  • At least some (or all) of the sub-channels in the capture regions may have a size that is at least 10%, at least 20%, at least 30%, at least 40%, or more, and/or up to 50%, up to 60%, up to 70%, or up to 80% less than the size of a fully denuded (or dissociated) target entity.
  • the sub-channel(s) between the posts in the capture region may have a minimum cross-sectional dimension that is less than the minimum cross-sectional dimension of the sub-channel(s) within the denuder region of the fluidic channel, according to some embodiments.
  • the sub-channel(s) between the posts in the capture region may have minimum cross-sectional dimensions that are at least 10 micrometers (e.g., at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, and/or up to 50 micrometers, up to 70 micrometers, up to 100 micrometers, up to 120 micrometers, up to 150 micrometers, or more) less than the minimum cross-sectional dimension of the sub- channel(s) in the denuder region.
  • minimum cross-sectional dimensions that are at least 10 micrometers (e.g., at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, and/or up to 50 micrometers, up to 70 micrometers, up to 100 micrometers, up to 120 micrometers, up to 150 micrometers, or more) less than the minimum cross-sectional dimension of the sub- channel(s) in the denuder region.
  • the plurality of sub-channels in the capture region may be sized to only allow denuded entities (e.g., denuded or bare oocytes without the bound non-oocyte cells) to flow readily therethrough while trapping larger target complexes (e.g., oocyte complex) and/or debris.
  • the plurality of posts within the capture region may be arranged in any appropriate manner or combination, such as an array of posts, a row of posts, etc.
  • the capture region may comprise a row of posts that spans a cross-section of the fluidic channel, such as shown in FIG.2A.
  • the capture region 172 may have an overall width that is larger than the denuder region 115, e.g., such as at least 2 times (or at least 5 times, at least 10 times, at least 20 times) larger than the denuder region.
  • the plurality of posts within the capture region may have any of a variety of appropriate shapes, including but not limited to, triangles, polygons, and/or any other shapes described elsewhere herein with respect to the posts in the denuder region.
  • the plurality of posts may have smooth surfaces.
  • the plurality of posts may have surfaces comprising teeth, as described elsewhere with respect to the posts in the denuder region.
  • FIG.2A illustrates an embodiment in which the device comprises a capture region
  • the fluidic channel 110 does not include capture region therein.
  • FIG.9C can be used to illustrate one such embodiment.
  • the device does not contain any capture region therein.
  • the microfluidic device further comprises one or more outlets configured to output one or more denuded target entities (e.g., oocytes) from the microfluidic device.
  • the outlet may be positioned downstream of the denuder region of the fluidic channel comprising a plurality of posts and/or sub-channels, according to some embodiments.
  • one or more outlet may be positioned downstream and/or upstream of the capture region, as described in more detail below.
  • the microfluidic device 100 comprises an outlet 170 positioned downstream of the denuder region 115 in the fluidic channel 110 containing the plurality of posts 120 and/or sub-channels 130.
  • the outlet 170 may be further positioned downstream the capture region 172.
  • FIG.2A illustrates an embodiment in which the outlet is positioned downstream of the capture region, it should be understood that the disclosure is not so limited, and that in certain embodiments, the outlet may be disposed upstream of the capture region.
  • a first outlet may be disposed upstream of the capture region, while a second outlet may be disposed downstream of the capture region.
  • the method comprises causing a first flow in a first direction from an inlet (e.g., inlet 1) of the fluidic channel to one or more outlet of the fluidic channel (e.g., outlet 1 or outlet 2).
  • the method comprises further causing a second flow in a second direction from either the first and/or the second outlet to the inlet of the fluidic channel.
  • the outlet may be in fluidic communication with a collection region.
  • the collection region may be a region for collecting oocytes or other target cells.
  • the collection region may comprise one or more wells for collecting an oocyte.
  • the wells may be positioned to allow imaging, e.g., using a camera or a microscope. This may be useful, for example, for examining or selecting oocytes or other target cells.
  • the microfluidic device may further comprise one or more fluid controls, e.g., valves, switches, actuators, and the like. Specific non-limiting examples of one or more fluid controls include pinch valve, butterfly valve, gate valves, ball valves, plug valves, PDMS valve, quake valve, elastic deformation based valves, piezo valves, and/or pneumatic valves.
  • the fluid controls may be a solenoid actuated valve.
  • the one or more fluid controls may be arranged to control the one or more pressure sources, inlets and/or outlets of the device.
  • the one or more fluid controls may be disposed between the first pressure source and the first inlet and/or between the second pressure source and the second inlet.
  • the one or more fluid controls may be employed to control connectivity between the pressure source(s) and the inlet(s) and modulate flow directionality of fluids within the device.
  • FIG.2A can be used to illustrate one such embodiment.
  • a fluid control 180 may be disposed between the second inlet 160 and the second pressure source 165.
  • the fluid control 180 may allow the second pressure source 165 to connect with the second inlet 160, such that a pressure may be applied by the second pressure source 165 to cause fluid flow in the device in the second direction 14.
  • a second position e.g., closed position
  • the fluid control 180 may disconnect the second pressure source 165 from the second inlet 160, such that the fluid flow within the device is no longer be affected by the second pressure source.
  • a fluid control 182 may be disposed between the first inlet 150 and the first pressure source 155. Such a fluid control may be employed to modulate connectivity between the first inlet and the first pressure source and to control fluid flow in the first direction 12 in the channel.
  • FIG.2A illustrates an embodiment in which fluid controls are disposed between the various inlets and pressure sources
  • one or more fluid controls may be disposed adjacent the one or more outlets (e.g., outlet 170) to control the opening and closing of the outlets (based on a command received from a controller and/or processor), according to some embodiments.
  • the microfluidic device described herein may further comprise a controller (e.g., a closed loop controller), according to some embodiments.
  • the controller may be configured to selectively actuate one or more components, e.g., the one or more pressure sources, one or more inlets and/or outlets, and/or the various fluid control valves, based on one or more measured parameter within the device.
  • measured parameters include a denudation level of a target entity (e.g., oocyte) within the device, various fluid flow properties (e.g., directionality of fluid flow, flow rate), and/or number of rounds of repeated flow (e.g., oscillatory flow) across the fluidic channel (e.g., denuder region) containing the plurality of posts and/or sub-channels.
  • the device may further comprise one or more sensors and/or detectors (e.g., image detector) configured to measure the one or more parameters, and one or more processors configured to relay a signal from the sensors and/or detectors to the controller, and vice versa.
  • a controller 190 may be configured to selectively actuate one or more fluid control valves, such as the fluid controls 180 and 182 shown in FIG.2A, or the various fluid controls adjacent the one or more outlets (shown in FIG.9B).
  • the controller may selectively actuate the fluid controls 180 and/or 182 from a first position, e.g., a closed state, to a second position, e.g., an open state, such that fluids in the device may be caused to flow in the direction 14 or 12 in response to a pressure exerted by the pressure sources 165 or 155.
  • the fluidic channel described herein may have any appropriate height.
  • the fluidic channel may have a height of at least 50 micrometers, 100 micrometers, at least 350 micrometers, at least 500 micrometers, at least 1,000 micrometers, at least 2,500 micrometers, at least 5,000 micrometers, and/or no more than 10,000 micrometers, no more than 5,000 micrometers, no more than 2,5000 micrometers, no more than 1,000 micrometers, no more than 500 micrometers, no more than 350 micrometers, no more than 100 micrometers, or less. Combinations of the above-referenced ranges are possible (e.g., at least 50 micrometers and no more than 350 micrometers, at least 50 micrometers and no more than 500 micrometers, or at least 50 micrometers and no more than 10,000 micrometers ).
  • the target entity is an oocyte
  • the one or more bounded secondary entities include one or more types of secondary cells (i.e., non-oocyte cell(s)) bounded to (e.g., surrounding or attached to) the oocyte, including, but not limited to, cumulus cells, granulosa cells, mural cells, thecal cells, red blood cells, or combinations thereof.
  • the secondary or non-oocyte cells when removed, may be discarded as waste, used for diagnostic analysis, stored for later use, or the like.
  • methods of the denuding an oocyte complex comprising an oocyte and non-oocyte cells to separate the oocyte from the non-oocyte cells are described.
  • one or more methods described herein may involve the use of oscillatory fluid flow within a microfluidic device for denudation of oocytes contained within a fluid sample.
  • the fluid sample containing the oocyte may include any of a variety of types of fluids, including, but not limited to, follicular fluid, fluid containing ovarian tissue, fluid containing inseminated oocyte complexes, and/or any other fluid containing other tissues and/or debris in addition to the oocyte.
  • the fluid may also include cell culture media in some embodiments.
  • the oocyte in the fluid sample, may be surrounded by or bounded to one or more layers of secondary entities, such as one or more layers of cumulus cells and/or granulosa cells, to form secondary entity bounded oocyte complex, such as cumulus oocyte complex (COC).
  • COC cumulus oocyte complex
  • the fluid sample may be derived from any of a variety of mammals, including, but not limited to, human, mouse, bovine, porcine, goat and/or ovine.
  • the fluid may also be a type of culture media used to store, transport or hold tissues prior to the procedure.
  • the oocyte and/or cumulus oocyte complex may have any of a variety of sizes described herein, depending on the type of mammal.
  • the method comprises causing repeating or oscillatory flow within a fluid channel.
  • certain embodiments are directed to causing a first flow of the fluid containing one or more oocyte complexes in a first direction through a fluidic channel.
  • a first flow of fluid in a first direction may correspond to a fluid flowing in a direction from an inlet of the fluidic channel, through the denuder region comprising post(s) and/or sub-channel(s), towards the outlet of the fluidic channel.
  • the fluidic channel may have any of a variety of properties described herein, e.g., such as the fluidic channel 110 or 110a illustrated in FIGS.1A-2.
  • the sub-channel(s) within the denuder region may comprise one of more features (e.g., teeth, constriction portion having a certain minimum cross-sectional dimension, tapered inlet and outlet portions) that aid denudation of oocyte during oscillatory flow.
  • FIG.1A and FIG.2D can be used to illustrate a fluid flow in a first direction.
  • the denuder region may comprise a single sub-channel as shown in FIG.1A, or a plurality of sub-channel(s) defined between neighboring posts as shown in FIG.2D.
  • a first flow of a fluid containing one or more oocyte complexes 18 may be caused in a first direction 12 through a fluidic channel 110a from the inlet 150a of the fluidic channel, through the sub-channel 130a in the denuder region 115a, to the outlet 170a of the fluidic channel.
  • the one or more oocyte complexes 18 may comprise an oocyte 18a surrounded at least partially by a plurality of non-targe secondary entities 18b (e.g., non-oocyte cells).
  • a first flow of a fluid containing one or more oocyte complexes 18 may be caused to flow in a first direction 12 through from an inlet 150 of the fluidic channel, through some of the sub-channels 130 defined by neighboring pairs of posts 120 in the denuder region 115, to an outlet 170 of the fluidic channel.
  • the fluid containing the oocyte complex is caused to flow through the fluidic channel in the first direction, according to some embodiments, at least a portion of the bounded non- oocyte cells is removed or separated from the complex as the complex flows through the fluidic channel in the first direction.
  • At least 1%, at least 5%, at least 10%, at least 20%, and/up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, or up to 9050%, up to 95%, up to 99%, or up to 100%, of the bounded non-oocyte cells is removed as the oocyte complex flows through the fluidic channel in the first direction.
  • the fluidic channel e.g., 110 or 110b
  • the sub- channel(s) e.g., 130 or 130a
  • the method comprises causing a second flow of the fluid containing the one or more cumulous oocyte complex in a second direction through the fluidic channel.
  • a second flow of fluid in a second direction may correspond to a flow in a reverse direction, such as from the outlet of the fluidic channel, through the denuder region comprising post(s) and/or sub-channel(s), towards the inlet of the fluidic channel.
  • a second flow of a fluid containing one or more oocyte complexes 18 may be caused to flow in a second direction 14 from the outlet 170a of the fluidic channel, through the sub-channel 130a, to the inlet 150a of the fluidic channel.
  • a second flow of a fluid containing one or more oocyte complexes 18 may be caused to flow in a second direction 14, from the outlet 170 of the fluidic channel, through some of the sub-channels 130 in the denuder portion 115, to the inlet 150 of the fluidic channel.
  • the second direction may be substantially opposite to the first direction.
  • a flow having the second direction may be oriented in a direction that is greater than 90 degrees, greater than or equal to 120 degrees, greater than or equal to 140 degrees, greater than or equal to 160 degrees, greater than or equal to 180 degrees, greater than or equal to 200 degrees, greater than or equal to 220 degrees, greater than or equal to 240 degrees, and/or less than 270 degrees, less than or equal to 240 degrees, less than or equal to 220 degrees, less than or equal to 200 degrees, less than or equal to 180 degrees, less than or equal to 160 degrees, less than or equal to 140 degrees, or less than or equal to 120 degrees from the first direction of the first fluid in the clockwise direction.
  • the second direction may be directly opposite the first direction, e.g., oriented about 180 degrees clockwise from the first direction.
  • the fluid containing the oocyte complex is caused to flow through the fluidic channel in the second direction, according to some embodiments, at least a portion of the bounded non- oocyte cells is removed or separated from the complex as the complex flows through the fluidic channel (e.g. denuder portion) in the second direction.
  • At least 1%, at least 5%, at least 10%, at least 20%, and/up to 30%, up to 40%, or up to 50% of the bounded non-oocyte cells is removed as the oocyte complex flows through the fluidic channel in the second direction.
  • the fluidic channel e.g., 110 or 110b
  • the sub-channel(s) e.g., 130 or 130a
  • the first and/or second flow may be caused by a pressure exerted by one or more pressure sources.
  • the first flow may be caused by a pressure exerted by a first pressure source to flow in a first direction
  • the second flow may be caused by a pressure exerted by a second pressure source to flow in a second direction, according to some embodiments.
  • the first flow may be caused to flow in the first direction 12 by a first pressure exerted by a first pressure source (e.g., pressure source 155 shown in FIG.2A)
  • the second flow may be caused to flow in the second direction 14 by a second pressure exerted by a second pressure source (e.g., second pressure source 165 shown in FIG.2A), according to some embodiments.
  • a first pressure source e.g., pressure source 155 shown in FIG.2A
  • a second pressure source e.g., second pressure source 165 shown in FIG.2A
  • the first pressure and/or second pressure exerted by the pressure sources may have of a variety of values.
  • the first pressure and/or second pressure may have a value of at least at least 0.01 psi, at least 0.05 psi, at least 0.1 psi, at least 0.5 psi, at least 1 psi, at least 2 psi, at least 5 psi, at least 10 psi, at least 20 psi, at least 50 psi and/or up to 100 psi, up to 200 psi, up to 300 psi, up to 400 psi, up to 500 psi, or more.
  • the fluid containing the oocyte complex may be subjected to first flow and second flow through the fluidic channel repeatedly, e.g., in an alternating and/or periodic fashion.
  • the oocyte complex subjected to a round of first and second flow may be further caused to flow through the denuder region of the fluidic channel via a second round of the first and second flow, e.g., as shown in FIGS.1A-1B or FIGS.2D-2E.
  • the above-referenced process may be repeated for any number of times, such as at least 1 round, at least 2 rounds, at least 3 rounds, 5 rounds, at least 10 rounds, at least 20 rounds, at least 30 rounds, at least 50 rounds, at least 100 rounds, and/or up to 200 rounds, up to 500 rounds, up to 1000 rounds, or more, of a first and second flow.
  • the repeating step may be carried out at a rate of at least 0.1 rounds of flow per minute, at least 1 round of flow per minute, at least 2 rounds of flow per minute, at least 4 rounds of flow per minute, at least 6 rounds of flow per minute, at least 8 rounds of flow per minute, at least 10 rounds of flow per minute, at least 20 rounds of flow per minute, at least 40 rounds of flow per minute, at least 60 rounds of flow per minute, at least 100 rounds of flow per minute, at least 500 rounds of flow per minute, at least 1,000 rounds of flow per minute, at least 5,000 rounds of flow per minute, or more, and/or no more than 10,000 rounds of flow per minute, no more than 5,000 rounds of flow per minute, no more than 1,000 rounds of flow per minute, no more than 500 rounds of flow per minute, no more than 100 rounds of flow per minute, no more than 60 rounds of flow per minute, no more than 40 rounds of flow per minute, no more than 20 rounds of flow per minute, no more than 10 rounds of flow per minute, no more than 8 rounds of flow per minute, no more
  • first flow and/or second flow may have any of a variety of appropriate mean flow velocities.
  • the first flow and/or second flow has a mean flow velocity of at least 0.01 cm/s, at least 0.1 cm/s, at least 1 cm/s, at least 5 cm/s, at least 10 cm/s, at least 12 cm/s, at least 15 cm/s, at least 20 cm/s, at least 24 cm/s, at least 30 cm/s, at least 50 cm/s, at least 100 cm/s, at least 500 cm/s, at least 1,000 cm/s, at least 5,000 cm/s, or more, and/or no more than 10,000 cm/s, no more than 5,000 cm/s, no more than 1,000 cm/s, no more than 500 cm/s, no more than 100 cm/s, no more than 50 cm/s, no more than 30 cm/s, no more than 24 cm/s, no more than 20 cm/s, no more than 15 cm/s, no more than 12 cm/s, no more than 10 cm/s, no more than 5 cm/s, no more than 1 cm/s, no more than 0.1
  • the first flow and the second flow may have mean flow velocities that are same or different, according to some embodiments.
  • the first flow and/or second flow may have any of a variety of appropriate flow rates.
  • the first flow and/or second flow has a flow rate of at least 0.01 mL/min, at least 0.1 mL/min, at least 1 mL/min, at least 2 mL/min, at least 5 mL/min, at least 10 mL/min, at least 15 mL/min, at least 50 mL/min, at least 100 mL/min, at least 500 mL/min, at least 1,000 mL/min, at least 5,000 mL/min, or more, and/or no more than 10,000 mL/min, no more than 5,000 mL/min, no more than 1,000 mL/min, no more than 500 mL/min, no more than 100 mL/min, no more than 50 mL/min, no more than 15 mL/min, no more than 10 mL/min, no more than 5 mL/min, no more than 2 mL/min, no more than 1 mL/min, no more than 0.1
  • first flow and the second flow may have flow rates that are same or different, according to some embodiments.
  • the first flow and/or second flow may have any of a variety of appropriate duty cycles.
  • the duty cycle may be measured as a percentage of time the fluid flows in one direction (e.g., first flow in a first direction or second flow in a second direction) during one round of oscillatory flow (e.g., one round of first flow and second flow).
  • a first flow in a first direction may have a duty cycle of at least 0.1%, at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 70%, at least 80%, at least 90%, and/or up to 95%, up to 97%, up to 99%, up to 99.5%, up to 99.9%.
  • a first flow in a first direction having duty cycle of 70% may correspond to a first flow moving in a first direction for 70% of the time, and a second flow moving in a second direction for 30% of the time, during one round of oscillatory flow.
  • the method comprises trapping the oocyte complex (or other entities) subjected to the first flow and/or second flow in a capture region comprising a plurality of posts defining a plurality of sub-channels between neighboring pairs of posts.
  • the oocyte complex when the oocyte complex is caused to flow through the denuder region containing the sub-channel(s) via a first flow in a first direction, the oocyte complex may become partially denuded and the partially denuded oocyte complex may be subsequently trapped by the sub-channels in the capture region.
  • the trapped oocyte complexes Upon trapping the partially denuded oocyte complexes, the trapped oocyte complexes may be caused to flow via a second flow in a second direction, such that the trapped oocyte complexes flow through the denuder region containing the sub-channel(s).
  • the above-mentioned oscillatory flow may be repeated for any appropriate number of times, as described elsewhere herein.
  • FIGS.2B-2C can be used to illustrate one such embodiment.
  • the oocyte complex as the oocyte complex is caused to flow (by a first applied pressure) through the denuder region 115 containing the sub-channel(s) 130 via the first flow in the first direction 12, the oocyte complex becomes at least partially denuded and may be subsequently trapped by the plurality of sub-channels 177 defined between neighboring pairs of posts 175 within the capture region 172.
  • the trapped oocyte complexes may be caused to flow (by a second applied pressure) via the second flow in the second direction 14, such that the trapped oocyte complexes may again flow through the denuder region 115 containing the sub-channel(s) 130 for further denudation.
  • the method further comprises releasing the denuded oocyte when a measured parameter within the device reaches a threshold value.
  • the measured parameter may be a denudation level of the oocyte within the device, and the denuded oocyte may be released from the device when the denudation level reaches a predetermined threshold value.
  • a closed-loop controller described elsewhere herein may be employed to subject the oocyte complexes within the fluid channel to additional rounds of repeated flow (e.g., oscillatory flow) for additional denudation.
  • the controller may be operationally linked to one or more components described here (e.g., one or more pressure sources, inlets and/or outlets, fluid controls) to control the flow of fluid and oocyte complexes within the device.
  • the controller upon reaching a predetermined denudation level, the controller may be operated to stop the repeated flow (e.g., oscillatory flow) through the fluidic channel.
  • the denuded oocytes may be released from device via one or more outlets.
  • the denuded oocytes may be caused to flow in the first direction through the plurality of sub-channels in the capture region via an application of a pressure by the first pressure source.
  • the sub-channels in the capture region are sized to allow denuded oocytes (e.g., bare oocytes without the bound non-oocyte cells) to flow readily through, the denuded oocytes may flow through the capture region and flow out of the first outlet.
  • the denuded oocyte may be trapped at the capture filter.
  • a pressure may be applied to cause slight deformation in shape and/or size of the denuded oocyte, such that the oocyte may be forced through sub-channels in the capture region.
  • the first outlet may be actuated to open by the controller and/or fluid controls to release the oocyte.
  • FIG.2B can be used to illustrate one such embodiment.
  • the controller 190 may be operated to stop the repeated flow (e.g., oscillatory flow) through the fluidic channel upon detecting a measured parameter of the oocyte complex (e.g., denudation level) and trigger release of the denuded oocyte from the device.
  • a measured parameter of the oocyte complex e.g., denudation level
  • the denuded oocytes may be caused to flow in the first direction 12 through the plurality of sub- channels 177 in the capture region 172 via a pressure applied by the first pressure source 155.
  • the denuded oocyte may readily flow through the sub-channels 177 and flow out of the outlet 170.
  • a slight pressure may be applied by the first pressure source to cause the denuded oocytes to deform in shape and/or size, such that the denuded oocytes may squeeze through sub-channels 177 in the capture region 155 and flow out of the outlet 170 downstream of the capture region 172.
  • the denuded oocytes trapped at the capture region may be caused to flow in the second direction (e.g., a reverse direction) via an application of a pressure by the second pressure source.
  • the second outlet may be actuated to open by the controller and/or fluid controls and the denuded oocyte may be caused to flow out of the second outlet, according to some embodiments.
  • FIG.9B One such embodiment is illustrated in FIG.9B.
  • the denuded oocytes trapped at the capture region may be caused to flow in the second direction (e.g., a backward flow) via an application of a pressure by the second pressure source, and subsequently released via the second outlet.
  • one or more chemicals and/or enzymatic solutions may be flowed through the fluidic channel to facilitate denudation of the oocytes.
  • the one or more chemical and/or biological solutions may include an enzyme capable of removing one or more non-oocyte cells bounded to an oocyte.
  • Non-limiting examples of chemicals and/or biological disassociating enzymes may include hyaluronidase, cumulase, trypsin, and/or pronase.
  • the one or more chemical and/or enzymatic solutions may be flowed into the device via the one or more inlets before, during, and/or after the oscillatory flow steps, according to some embodiments.
  • the method comprises repeatedly causing flow of a fluid containing an oocyte complex comprising an oocyte and non-oocyte cells through a fluidic channel, such that the oocyte separates from the non-oocyte cell.
  • the fluid containing the complex flowing through the fluidic channel may be repeated for a plurality of times, such as at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, and/or up to 100 times, up to 200 times, up to 500 times, up to 1000 times, or more. Combinations of the above-referenced ranges are possible (e.g., at least 2 times and up to 500 times, or at least 10 times and up to 1000 times). Other ranges are also possible.
  • the flow of fluid may be repeating, or oscillatory in some embodiments.
  • the repeated flows described herein may lead to a relatively high denudation level.
  • at least 50%, at least 60%, at least 70%, 80%, at least 85%, at least 90%, or more, and/or up to 95%, up to 97%, up to 99%, up to 99.5%, up to 99.9%, or 100% of the non-oocyte cells in the complex are removed during repeated flow of the complex through the fluidic channel.
  • the method comprises repeatedly flowing a fluid containing an oocyte complex comprising an oocyte and non-oocyte cells through a fluidic channel to remove non-oocyte cells until a predetermined (and desirable) amount of non-oocyte cells is left behind on (or bounded to) the oocyte.
  • a method may be employed to produce an oocyte complex containing a desirable amount of non-oocyte cells for use in non-ICSI IVF applications.
  • the fluidic channel may comprise a plurality of posts defining sub-channels between neighboring pairs of posts, e.g., as shown in FIGS.2A-2E and FIG.3B.
  • the flows may be repeated such that the complex passes the plurality of posts at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, and/or up to 100 times, up to 200 times, up to 500 times, up to 1000 times, or more. Combinations of the above-referenced ranges are possible (e.g., at least 2 times and up to 500 times, or at least 10 times and up to 1000 times).
  • the oocyte may repeatedly flow across an imaginary line within the fluidic channel multiple times, such as at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, and/or up to 100 times, up to 200 times, up to 500 times, up to 1000 times, or more. Combinations of the above-referenced ranges are possible (e.g., at least 2 times and up to 500 times, or at least 10 times and up to 1000 times).
  • the oocyte complex 18 may repeatedly flow across an imaginary axis 20 of the microfluidic channel.
  • the imaginary line is defined in a direction orthogonal to a direction of bulk fluid flow (e.g., direction 12 or 14) within the fluidic channel.
  • the repeated flows may be oscillatory or non- oscillatory in nature.
  • the repeated flows may be oscillatory in nature, e.g., such as capable of oscillating between a first flow in a first direction and a second flow in the second direction (e.g., as shown in FIGS.1A-1B or FIGS. 2D-2E).
  • the repeated flows through the fluidic channel may be non- oscillatory in nature.
  • at least some of the repeated flows through the channel may be in the same direction, e.g., either in a first direction or a second direction.
  • the first direction may be a clockwise direction and the second direction may be counterclockwise direction.
  • FIGS.3A-3B can be used to illustrate one such embodiment.
  • a microfluidic device 200 may comprise a fluidic channel 210 formed between two concentric walls, e.g., an outer wall and an inner wall.
  • the fluidic channel may comprise one or more sub-channels 230 defined by extruded portions on the inner and outer wall, in some embodiments.
  • the sub-channels 230 in some cases, may be sized to have a minimum cross- sectional dimension comparable to or slightly larger than a target entity (e.g., an oocyte) flowing through sub-channel.
  • the sub-channels 230 may have any of a variety of properties (e.g., teeth, minimum cross-sectional dimension) described elsewhere herein.
  • a fluid containing an oocyte complex 18 comprising an oocyte 18a and non-oocyte cells 18b may be caused to repeatedly flow in the same direction (e.g., clockwise or counterclockwise) through the sub-channels 230, such that the oocyte 18a separates from the non-oocyte cells 18b.
  • an outlet 250 may be actuated to open and the denuded oocyte may exit from the outlet 250.
  • the outer and/or inner walls of the fluid channel may be actuated to rotate in a particular direction.
  • FIG.3B can be used to illustrate another embodiment in which the fluidic channel shown in FIG.3A comprises a plurality of posts 220 therein.
  • the plurality of posts 220 may define a plurality of sub-channels between neighboring pairs of posts.
  • the plurality of posts and/or sub-channels may have any of a variety of properties (e.g., shapes, sizes, teeth) described elsewhere herein.
  • the fluidic channel in FIG.3B may be employed for repeated fluid flow in the same manner as the fluidic channel in FIG.3A.
  • a fluid containing an oocyte complex comprising an oocyte and non-oocyte cells may be repeatedly flowed in the same direction (e.g., clockwise or counterclockwise) through some of the sub-channels defined by the plurality of posts 220, such that the complex is denuded and the oocyte separates from the non- oocyte cells.
  • the method comprises passing one or more oocyte complexes through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, such that at least 50%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and/or up to 95%, up to 99%, up to 99.5%, up to 99.9%, or up to 100% of a total amount of the bounded non-oocyte cells are removed when the complex flows through the sub-channels. In some embodiments, at least 90% of the non-oocyte cells are removed when the complex flows through the sub-channels described herein.
  • the oocyte complex may be passed through fluidic channels having any of a variety of configurations, e.g., as shown in FIGS.1-3B.
  • a method of manipulating an oocyte complex comprising an oocyte and non-oocyte cells is described herein.
  • the method comprises causing a flow of fluid containing the complex in a channel dimensioned to allow the oocyte to flow through only if not bound to the non-oocyte cells.
  • one or more of the sub-channels 130 in the denuder region 115 of the fluidic channel 110 may be sized to have a minimum cross-sectional dimension comparable to bare oocytes (e.g., oocytes not bounded to non-oocyte cells), such that only bare oocytes may readily pass therethrough.
  • the sub-channel described herein may have any of a variety of minimum cross- sectional dimensions described elsewhere herein.
  • oocytes complexes having sizes larger than the minimum cross-sectional dimension of the sub-channel may be temporarily deformed and forced through some of the sub-channels, e.g., as shown in FIGS.2A-2C.
  • pressure source 155 may be employed to exert a sufficient pressure to cause a flow in the first direction 12 at a sufficient flow rate, such that oocyte complexes having sizes larger than the minimum cross-sectional dimensions of some of the sub-channels 130 may be temporarily deformed and forced through the sub-channels 130.
  • causing oocyte complexes to flow through sub-channels dimensioned to have sizes comparable to bare oocytes may facilitate denudation of the oocyte from bounded non-oocyte cells.
  • the fluid containing the complex may be caused to flow repeatedly (via either the oscillatory or non-oscillatory flow described elsewhere herein) through the sub-channel(s) dimensioned to have minimum cross-sectional dimensions comparable to bare oocyte, e.g., for at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, and/or up to 100 times, up to 200 times, up to 500 times, up to 1000 times, or more.
  • the trapped debris may be subjected to oscillatory flows within the device when the fluid is caused to flow in a first direction, e.g., as shown in FIG.2B, followed by a flow in a second direction, e.g., as shown in FIG.2C.
  • the use of repeated oscillatory flow may mechanically break up larger debris originally trapped in the sub-channels into small pieces, thus causing the small pieces of debris to flow through the sub-channels and subsequently be removed from the channel.
  • a method of preparing follicular fluid to isolate and denude an oocyte complex comprising an oocyte and non-oocyte cells for egg banking, egg freezing, and/or in-vitro fertilization is disclosed here.
  • the method may comprise, according to some embodiments, passing the follicular fluid through a fluidic channel comprising a plurality of posts such that the complex flows through sub-channels defined between neighboring pairs of posts.
  • the fluid channel, the plurality of posts, and/or the sub-channels may have any of a variety of properties (e.g., dimensions, shapes, configurations, arrangements, amounts, etc.) as described elsewhere herein and/or with respect to FIGS.2A-3B.
  • FIGS.2A-3B may be used to illustrate one such embodiment.
  • a follicular fluid may be passed through a fluidic channel (e.g., channel 110 or 210) comprising a plurality of posts (e.g., posts 120 or 220) such that the complex flows through sub-channels (e.g., sub-channels 130 or 230) defined between neighboring pairs of posts.
  • the complex may be repetitively passed through the fluidic channel via an oscillatory motion, e.g., as illustrated in FIGS.2A-3B.
  • the complex may be passed through the fluidic channel in a first direction through some of the sub- channels from an inlet to an outlet of the fluidic channel, e.g., as illustrated by FIGS.2B or 2D, followed by a flow through the fluidic channel in a second direction through some of the sub- channels from the outlet to the inlet of the fluidic channel, e.g., as illustrated by FIGS.2C or 2E.
  • the complex may be repetitively passed through the fluidic channel in a non-oscillatory motion, e.g., as shown in FIGS.3A-3B.
  • At least a portion e.g., at least 10%, at least 25%, at least 50%, at least 90%, and/or up to 95%, up to 99%, or up to 100%
  • the above-referenced embodiments are generally directed to methods of denuding oocyte complex comprising an oocyte and non-oocyte cells to separate the oocyte from the non- oocyte cells, it should be understood that the disclosure is not so limited, and that in other embodiments, the methods may be generalized to denuding any target entity-secondary entity complex other than the oocyte complex.
  • any of the above-reference methods and/or devices may be employed to denude target entity-secondary entity complex comprising a target entity and one or more secondary entities to separate the target entity from the secondary entities.
  • the target entity may include any of a variety of cell types, including, but not limited to tumor cells.
  • certain embodiments comprises passing the target entity-secondary entity complex through a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, such that at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, up to 100%) of the secondary entities are removed when the complex flows through sub-channels.
  • the target entity- secondary entity complex may be passed repeatedly through a device having any of a variety of configurations noted herein, such as the devices illustrated in FIGS.2A-3B, via either an oscillatory flow (FIGS.2A-2C) or a non-oscillatory flow (FIGS.3A-3B).
  • a device having any of a variety of configurations noted herein, such as the devices illustrated in FIGS.2A-3B, via either an oscillatory flow (FIGS.2A-2C) or a non-oscillatory flow (FIGS.3A-3B).
  • the method comprises causing a flow of fluid containing the target entity-secondary entity complex in a fluidic channel comprising a plurality of posts defining sub-channels between neighboring pairs of posts, such that the complex passes the plurality of posts at least 2 times (e.g., at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least at least 20 times, at least 30 times, at least 50 times, and/or up to 100 times, up to 200 times, up to 500 times, up to 1000 times).
  • the fluid containing the target entity-secondary entity complex may be caused to flow in an oscillatory fashion, e.g., as shown in FIGS.2A-2E.
  • a first flow of a fluid containing the complex may be caused in a first direction 12 through a fluidic channel, followed by a second flow of the fluid containing the complex in a second direction 14 through the fluidic channel.
  • at least some (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more) of the one or more secondary entities from the complex is removed as the complex flows through the fluidic channel 110 in the first direction 12 and at least some (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more) of the one or more secondary entities from the complex as the complex flows through the fluidic channel 110 in the second direction 14.
  • the fluid containing the target entity-secondary entity complex may be caused to flow repeatedly in a non-oscillatory fashion, as shown in FIGS.3A-3B.
  • the fluid containing the target entity-secondary entity complex e.g., complex 18
  • the fluid containing the target entity-secondary entity complex may be caused to flow in the fluidic channel such that the complex flows across an imaginary line (e.g., imaginary line 20 as shown in FIG.2D) within the fluidic channel at least 2 times (e.g., at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least at least 20 times, at least 30 times, at least 50 times, and/or up to 100 times, up to 200 times, up to 500 times, up to 1000 times).
  • a method of manipulating a target entity-secondary entity complex comprising a target entity and one or more secondary entities is described herein.
  • the method comprises causing a flow of fluid containing the complex in a channel dimensioned to allow the target entity to flow through only if not bound to the secondary entities.
  • one or more of the sub-channels 130 in the denuder region 115 of the fluidic channel 110 may be sized to have a minimum cross- sectional dimension comparable to a bare target entity (e.g., target entity not bounded to secondary entities), such that only denuded target entity may readily pass therethrough.
  • the sub-channel described herein may have any of a variety of minimum cross-sectional dimensions described elsewhere herein.
  • the fluid containing the complex may be caused to flow repeatedly (via either the oscillatory or non-oscillatory flow described elsewhere herein) through the sub-channel(s), e.g., for at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 50 times, and/or up to 100 times, up to 200 times, up to 500 times, up to 1000 times, or more. Combinations of the above- referenced ranges are possible (e.g., at least 2 times and up to 500 times, or at least 10 times and up to 1000 times). Other ranges are also possible.
  • the above-referenced methods or devices may be employed to dissociate conglomerates or aggregates of target cells into individual cells for use in application such as in cell sorting, single-cell sequencing, etc.
  • the devices and/or methods described herein may be used to process a fluid containing tumor tissue or circulating tumor cell clusters (CTCCs).
  • CTCCs tumor cell clusters
  • the fluid may be passed into the denuder region, where, under an application of repeated flow (e.g., oscillatory flow) and/or enzyme treatment, cell-cell interactions may be weakened and the tissue or the CTCC cluster may dissociate into individual cells in the denuder region.
  • the individual cells may be in turn captured by the capture region and released from the device for further use, according to some embodiments.
  • a variety of oocytes have been determined, e.g., in various stages of maturation (e.g., in the GV stage (Germinal Vesicle), and/or the M1 (Metaphase I) and M2 (Metaphase II) stages).
  • manual techniques are usually unable to find and isolate immature oocytes.
  • one or more devices described herein may be defined using any suitable form factor.
  • the one or more devices is defined by a rectangular form factor, for example, as shown in FIG.2. In other embodiments, however, the one or more devices is defined by a circular form factor, for example, as shown in FIG.13. In some embodiments, the circular form factor comprise one or more holes. In some embodiments, the one or more holes are concentric about the center of the device. Other form factors are also possible in other embodiments. In some embodiments, the one or more devices described herein comprise a wall angle of between 2 degrees and 10 degrees, relative to an axis that is perpendicular to a bottom surface of the microfluidic device.
  • the wall angle is greater than or equal to 1 degree, greater than or equal to 2 degrees, greater than or equal to 3 degrees, greater than or equal to 4 degrees, greater than or equal to 5 degrees, greater than or equal to 6 degrees, greater than or equal to 7 degrees, greater than or equal to 8 degrees, greater than or equal to 9 degrees, or greater than or equal to 10 degrees, relative to an axis that is perpendicular to a bottom surface of the microfluidic device.
  • the wall angle is less than or equal to 10 degrees, less than or equal to 9 degrees, less than or equal to 8 degrees, less than or equal to 7 degrees, less than or equal to 6 degrees, less than or equal to 5 degrees, less than or equal to 4 degrees, less than or equal to 3 degrees, less than or equal to 2 degrees, or less than or equal to 1 degree, relative to an axis that is perpendicular to a bottom surface of the microfluidic device. Combinations of the above recited ranges are also possible in some embodiments.
  • the wall angle is greater than or equal to 1 degree and less than or equal to 10 degrees, relative to an axis that is perpendicular to a bottom surface of the microfluidic device.
  • the wall angle is 3 degrees relative to an axis that is perpendicular to a bottom surface of the microfluidic device. In other embodiments, the wall angle is 5 degrees relative to an axis that is perpendicular to a bottom surface of the microfluidic device.
  • a device may comprise one or more regions (e.g., filter regions, denuder regions, inertial focusing regions, concentrator regions, and/or capture regions, etc.) with one or more wall angles, according to some embodiments. In some embodiments, the one or more wall angles of the one or more regions is the same. In other embodiments, the one or more wall angles of the one or more regions is different.
  • the one or more wall angles of the one or more regions may be selected from any one of the above recited ranges and/or combinations thereof.
  • a filter region has a 3 degree wall angle and a denuder region has a 5 degree wall angle.
  • the filter region and denuder region both have a 3 degree wall angle or a 3 degree wall angle.
  • devices comprising adjacent sidewalls having non-zero wall angles may define a channel with a trapezoidal-like shape, wherein a first portion of the trapezoidal-like channel will have a larger cross-sectional dimension than the second portion of the trapezoidal-like channel.
  • devices comprising a plurality of posts (e.g., filter region, denuder region, and capture region) having non-zero wall angles may in some embodiments define a plurality of trapezoidal-like sub-channels between neighboring pairs of posts (see, as examples, FIG.14A and 14B).
  • a cross-sectional dimension of a first portion of a trapezoidal-like sub-channel is larger than the cross-sectional dimension of a second portion of the trapezoidal-like sub-channel.
  • the cross-sectional dimension of the first portion of the trapezoidal-like sub-channel is smaller than the cross-sectional dimension of the second portion of the trapezoidal-like sub-channel.
  • FIG.14A illustrates exemplary wall dimensions for a 3-degree wall angle and FIG. 2141B illustrates exemplary wall dimensions for a 5-degree wall angle, as non-limiting examples. However, wall angles may vary in other embodiments, e.g., from about 1 degree to about 10 degrees, etc.
  • FIG.14C shows a scanning electron micrograph of a denudation region with a 3 degree wall angle.
  • a plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) has an average cross-sectional dimension of between 10 microns and 200 microns. In some embodiments, the average cross- sectional dimension of the plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) 10 microns and 200 microns.
  • the average cross-sectional dimension of the plurality of trapezoidal-like sub- channels is greater than or equal to 10 microns, greater than or equal to 25 microns, greater than or equal to 50 microns, greater than or equal to 75 microns, greater than or equal to 100 microns, greater than or equal to 125 microns, greater than or equal to 150 microns, greater than or equal to 175 microns, or greater than or equal to 200 microns,.
  • the average cross- sectional dimension of the trapezoidal-like sub-channels is less than or equal to 200 microns, less than or equal to 175 microns, less than or equal to 150 microns, less than or equal to 125 microns, less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 25 microns, or less than or equal to 10 microns. Combinations of the above recited ranges are also possible in some embodiments.
  • the average cross-sectional dimension of the trapezoidal-like sub-channels is greater than or equal to 10 micron and less than or equal to 200 microns.
  • a plurality of trapezoidal-like sub-channels in a filter region, a denuder region, and/or a capture region e.g., formed by neighboring pairs of posts having a non-zero wall angle
  • the average cross-sectional dimension of the plurality of trapezoidal-like sub-channels in the filter region, a denuder region, and/or a capture region is greater than or equal to 10 microns, greater than or equal to 25 microns, greater than or equal to 50 microns, greater than or equal to 75 microns, greater than or equal to 100 microns, greater than or equal to 125 microns, greater than or equal to 150 microns, greater than or equal to 175 microns, or greater than or equal to 200 microns,.
  • the average cross-sectional dimension of the trapezoidal-like sub-channels in the filter region, a denuder region, and/or a capture region is less than or equal to 200 microns, less than or equal to 175 microns, less than or equal to 150 microns, less than or equal to 125 microns, less than or equal to 100 microns, less than or equal to 75 microns, less than or equal to 50 microns, less than or equal to 25 microns, or less than or equal to 10 microns. Combinations of the above recited ranges are also possible in some embodiments.
  • the average cross-sectional dimension of the trapezoidal-like sub-channels in the filter region, denuder region, and/or a capture region is greater than or equal to 10 microns and less than or equal to 200 microns.
  • an average cross-sectional dimension of a plurality of sub- channels in a denuder region is between 10 microns and 200 microns and the average cross- sectional dimension of the sub-channels in a capture region is between 10 microns and 200 microns.
  • a plurality of trapezoidal-like sub-channels (e.g., formed by neighboring pairs of posts having a non-zero wall angle) has an average height of between 100 microns and 400 microns.
  • the average height of the plurality of trapezoidal-like sub-channels is greater than or equal to 100 microns, greater than or equal to 130 microns, greater than or equal to 160 microns, greater than or equal to 190 microns, greater than or equal to 220 microns, greater than or equal to 250 microns, greater than or equal to 280 microns, greater than or equal to 310 microns, greater than or equal to 340 microns, greater than or equal to 370 microns, or greater than or equal to 400 microns.
  • the average height of the plurality of trapezoidal-like sub-channels is less than or equal to 400 microns, less than or equal to 370 microns, less than or equal to 340 microns, less than or equal to 310 microns, less than or equal to 280 microns, less than or equal to 250 microns, less than or equal to 220 microns, less than or equal to 190 microns, less than or equal to 160 microns, less than or equal to 130 microns, or less than or equal to 100 microns. Combinations of the above recited ranges are also possible in some embodiments.
  • the average cross-sectional dimension of the trapezoidal-like sub-channel is greater than or equal to 100 micron and less than or equal to 400 microns.
  • a plurality of trapezoidal-like sub-channels in a filter region, a denuder region, and/or a capture region e.g., formed by neighboring pairs of posts having a non-zero wall angle
  • the average height of the plurality of trapezoidal-like sub-channels in the filter region, denuder region, and/or capture region is greater than or equal to 100 microns, greater than or equal to 130 microns, greater than or equal to 160 microns, greater than or equal to 190 microns, greater than or equal to 220 microns, greater than or equal to 250 microns, greater than or equal to 280 microns, greater than or equal to 310 microns, greater than or equal to 340 microns, greater than or equal to 370 microns, or greater than or equal to 400 microns.
  • the average height of the plurality of trapezoidal-like sub-channels in the filter region, denuder region, and/or capture region is less than or equal to 400 microns, less than or equal to 370 microns, less than or equal to 340 microns, less than or equal to 310 microns, less than or equal to 280 microns, less than or equal to 250 microns, less than or equal to 220 microns, less than or equal to 190 microns, less than or equal to 160 microns, less than or equal to 130 microns, or less than or equal to 100 microns. Combinations of the above recited ranges are also possible in some embodiments.
  • the average cross- sectional dimension of the plurality of trapezoidal-like sub-channels in the filter region, denuder region, and/or capture region is greater than or equal to 100 micron and less than or equal to 400 microns.
  • an average height of a plurality of sub-channels in a denuder region is between 100 microns and 400 microns and the average height of the sub-channels in a capture region is between 100 microns and 400 microns.
  • posts within the denuder region may in some cases comprise teeth that serve as strippers to denude the oocytes (see FIGS.1-3). In devices comprising non-zero wall angles, these strippers become tapered, as illustrated in FIG. 14C. Neighboring posts may be used in some instances to create trapezoidal-like sub-channels with a series of tapered strippers. (FIG.14C).
  • EXAMPLE 1 This example features microfluidic systems and methods utilizing oscillatory flow fields for the denudation of mammalian (e.g. human, mouse, bovine) cumulus/granulosa oocyte complexes (COCs) containing oocytes. An example of one such device is shown in FIG.4A.
  • mammalian e.g. human, mouse, bovine
  • COCs oocyte complexes
  • the microfluidic system contains an array of denudation constrictions in a microfluidic channel, where the widths of the constrictions are similar to the hydrodynamic size of the target oocyte and the cumulus cell layer around them, and the surface of the constriction walls have extruded features (i.e., teeth) that can be of various geometries (e.g. triangles, rectangles, saw-teeth).
  • a fluid sample which contains oocytes may be processed, such as follicular fluid collected from a patient during IVF treatment, COCs purified and suspended in media, inseminated COCs generated during standard IVF process, and/or dissociated human, bovine or mouse ovaries.
  • the sample fluid containing oocytes may be introduced to the microfluidic device through an inlet port and passed through the array of denudation constrictions via a steady or oscillatory flow field.
  • the oscillatory flow field may be applied such that the direction of flow may be reversed in a controlled periodic fashion, thereby allowing the oocytes to interact with the constriction array a multitude of times and get denuded over time.
  • parameters that characterize the oscillatory flow may include the frequency of oscillation (f), duty cycle (percent duration of flow in one flow direction) of oscillation (D), and flow-rate (q) in each flow direction.
  • the denuded oocytes may be released from the chip via a steady or oscillatory flow field.
  • a typical fluidic constriction channel and post is shown in FIG.5 and FIG.6.
  • the narrowest section of the constriction may be defined as the constriction width (w), which may be used to characterize the shortest distance between the extruded features of the constriction.
  • the extruded featured may be circular shaped and the features may be defined by the circle radius (R FEAT ), the offset of its center from the wall (L OFFSET ), and the distance between features (L FEAT ).
  • the length of the feature sides (L FEAT-1,2,3,4 ) can be as small as 1 ⁇ m and as large as 1000 ⁇ m.
  • the angles ( ⁇ FEAT-1,2 ) can be as small as 0.1° and as large as 179.9°.
  • the extruded feature may be circular, where the feature diameter can be as small as 1 ⁇ m and as large as 1000 ⁇ m.
  • Posts can have one repeating pattern of a geometrical feature, or a combination of different geometrical features.
  • the extruded features can be symmetrical with respect to the flow direction, or counter- angled towards one flow direction (Uni-directional, FIG.5) or both of the flow directions (Bi-directional, FIG.6) to improve oocyte denudation performance.
  • the extruded features are similar to the shape of saw-teeth and are angled towards both flow directions (FIG.6).
  • the constriction width (w) would be the distance between straight channel walls.
  • the denudation can be achieved by repeated encounter of oocytes with channel segments narrower or in the same order of width to the COCs.
  • a constriction array can be defined based on the number of fluidic constrictions per row (m) and the number of constriction rows (n), and the total number of fluidic constrictions N.
  • the width of the fluidic constrictions is the same within a constriction row, but can change between different rows.
  • FIG.8 shows a constriction array where rows 1 and 2 have a constriction width of 160 ⁇ m, rows 3 and 4 have a constriction width of 140 ⁇ m, and rows 5 and 6 have a constriction width of 120 ⁇ m.
  • the smallest constriction width (w) can be as small as 50 ⁇ m.
  • the largest constriction width (w) can be as large as 1000 ⁇ m.
  • both the number of fluidic constrictions per row (m) and the number of constriction rows (n) can be as small as 1.
  • m and n can be as large as 1000.
  • multiple constriction arrays can be utilized in a microfluidic chip, where they are connected to each other in series, in parallel, or used in conjunction with microfluidic sections of the microfluidic chip with other functionalities.
  • a final row of filter posts with a smaller gap size and/or reduced flow rate can be added to prevent oocytes from escaping the chip.
  • the height (H d ) of the device can be defined as the height (depth) of the features in the z-direction (normal to the plane of the Figures). In one example, the height of the device may be about 350 ⁇ m. In some cases, the height of the device is the same throughout the sections of the chip. In some cases, the height of the device is different in different sections of the chip. In some cases, the height of the device in a given section can be as small as 50 ⁇ m, and as large as 10,000 ⁇ m.
  • the flowrates during the forward (direction 1) phase of the oscillatory flow and the backward (direction 2) phase of the oscillatory flow can be defined as Q FW and Q BW .
  • the flowrates Q FW and Q BW can be the same, or different.
  • the net flowrate in the device can be adjusted towards either direction by a combination of flowrates (Q FW and Q BW ) and the duty cycle (percent time of the flow in one direction during one full period of the oscillatory flow).
  • the duty cycle may be about 70% (meaning flow is going in the forward direction 70% of the period, i.e.70 seconds in forward direction, then 30 seconds in reverse direction). In some cases, the duty cycle can be between 0.1% to 99.9%.
  • the forward and backward flowrate is about 2 mL/min in each direction.
  • the flowrate in each direction can be as low as 0.01 mL/min and as high as 1000 mL/min.
  • Oscillatory flow field in a microfluidic chip can be generated by numerous combinations of flow source units (e.g. pressure sources, syringe pumps), sample reservoirs (e.g. syringes, conical tubes), and/or flow control units (e.g. valves).
  • flow source units e.g. pressure sources, syringe pumps
  • sample reservoirs e.g. syringes, conical tubes
  • flow control units e.g. valves
  • flow may be modulated using two different pressure sources, where the pressures are set at P 1 for inlet 1 and P 2 for inlet 2, where P 2 is greater than P 1 (FIG.9A).
  • P 1 may be connected directly to the inlet 1 via tubing
  • P 2 may be connected to the inlet 2 via tubing which goes through a solenoid activated pinch valve, and the valve may be controlled by a preprogrammed microprocessor (e.g. iOS).
  • a preprogrammed microprocessor e.g. e.g. a preprogrammed microprocessor
  • the pinch valve when the pinch valve is in a closed position, no flow can pass through the valve and the flow direction may be from inlet 1 towards the outlet.
  • the pinch valve is in an open position, flow can pass through and the flow direction may be from inlet 2 towards the inlet 1, which may in turn generate the reverse flow through the constriction array. Meanwhile, there may also be flow from inlet 2 towards the outlet (which would not contribute to the oscillatory flow field).
  • the oocyte capture row may stop the oocytes from moving farther during forward flow. This may be achieved by expanding the channel to slow down the flow, and the capture row gaps may have a width slightly smaller than the oocyte.
  • the valve connected to inlet 2 may be permanently closed, and inlet pressure P1 may be increased to push the oocytes through the capture row and release them through the outlet.
  • FIG.9B Another example of the microfluidic device is illustrated in FIG.9B. As shown, in this example of the device, an oocyte release outlet may be introduced before the capture row along with the second pinch valve. In this case, the second pinch valve may stay closed during oscillatory denudation.
  • the first pinch valve may be permanently opened, inlet 1 may be closed, and the second pinch valve may be opened to release the oocytes with a backward flow to the oocyte release outlet.
  • the advantage of this design is that the oocytes do not pass through the oocyte capture row to be released.
  • FIG.9C Another example of the microfluidic device is illustrated in FIG.9C. As shown, an oocyte capture row may not be present in the device. In this case, the flow parameters may be adjusted to ensure that oocytes stay on the chip during the application of the oscillatory flow field, such that the oocytes do not flow through the outlet channel and leave the chip.
  • samples can be treated with hyaluronidase or other similar solutions that facilitate oocyte denudation before the oocytes are transferred to the device.
  • hyaluronidase or other similar solutions that facilitate oocyte denudation can be introduced to the device while the oocytes are in the device. This could be accomplished by through one of the existing inlets, or another embodiment of the system with a different inlet for hyaluronidase or similar solution. Denudation may be carried out using a microfluidic device described above.
  • oocyte denudation occurred gradually over processing duration, during which oocytes repeatedly travel through denudation segments (FIGS.10A- 10C). As shown, the oocyte at time tend became more denuded compared to the oocyte at time tstart. Denudation time may be defined as the total time oocytes have spent travelling through the denudation segments until all the oocytes are fully denuded.
  • denudation time may depend on the device geometry, such as: number of fluidic constrictions per row (m), the number of constriction rows (n), and the total number of fluidic constrictions (N), and the flow parameters such as: frequency of oscillation (f), duty cycle (percent duration of flow in one flow direction) of oscillation (D), and/or pressure (P) / flow- rate (q) in each flow direction, according to some embodiments.
  • oscillation frequency can be as low as 0.1 oscillations/min and as high as 10,000 oscillations/min.
  • a closed-loop control system may be implemented to determine the flow parameters and/or time to stop the oscillatory flow and release the oocytes, based on their denudation level (FIG.12).
  • an imaging system e.g. camera
  • the microprocessor e.g. the microprocessor
  • the microprocessor can be pre-programmed to change the flow parameters (e.g. increase the flowrate within the channel), or introduce denudation catalyzing agents (e.g.
  • the microprocessor e.g. iOS, FPGA, Raspberry-Pi
  • pressure control systems such as (e.g. Elveflow, Fluigent) for flow modulation, and also computer software (e.g. Python packages for computer vision) for image processing.
  • the device and method described above may be used to denude oocyte from various liquid samples.
  • the oocyte containing fluid may be follicular fluid collected from a patient during IVF treatment.
  • the device by itself or by integrating with an upstream or downstream process, may be used to obtain denuded oocytes from patient’s follicular fluid.
  • the denuded oocytes can then be either fertilized via intracellular sperm injection (ICSI), or frozen (vitrified) for later use.
  • the oocyte containing fluid may be IVF-inseminated oocytes which still retain their cumulus/granulosa cells after insemination.
  • This system by itself or by integrating with an upstream or downstream process, is used to obtain denuded inseminated oocytes (potential embryos) from the sample.
  • the system can also check for fertilization and identify and sort oocytes based on successful fertilization.
  • the oocyte containing fluid may be dissociated animal (mouse, bovine, or other) ovaries.
  • the device described herein, by itself or by integrating with an upstream or downstream process, may be used to obtain denuded oocytes from this sample.
  • the device and method described in this example may allow for denudation of oocytes in microfluidic channels of limited length and footprint, rather than using a long channel of serially or parallelly connected denudation segments.
  • the device and method described above further allows for denudation of oocytes when the oocytes are mixed with other tissue and debris in a complex fluid (such as follicular fluid, ovarian tissue, inseminated cumulus oocyte complexes), without chip-clogging issues.
  • Oscillatory flow through parallel channels actively can be used to clean up debris from channels, and increase the likelihood of oocytes finding and interacting with debris free denudation features during the process.
  • the device may allow for generation of flow fields during oscillation to change the 3D orientation of oocytes before they go through a denudation segment each time, thereby allowing for different surfaces of the oocytes to interact with denudation features and thus increase the efficiency and quality of denudation process. Improved efficiency in turn may allow for shorter duration of the denudation process and fewer interactions with denudation features (e.g., extruded portions). This may in turn reduce potentially harmful stress imposed on the oocytes during denudation process.
  • the device and/or method described in this example further allows for precise control over the exact number of interactions between the oocytes and the denudation features, independently from the number of denudation elements on the chip.
  • the device may also allow for visual (qualitative or quantitative) temporal analysis of the denudation process, since: (i) denudation of the oocytes can occur at a small, localized section of the device; (ii) oocytes can appear repeatedly at a region of interest for viewing with the use of oscillatory flow; and (iii) flow can be stopped and restarted at will to allow for better imaging.
  • the continuous visual feedback system associated with the device can allow for closed-loop control, where oocytes can be automatically (via image processing) or manually (by user) verified to be denuded before release.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

La présente divulgation concerne de manière générale des procédés et des appareils microfluidiques pour séparer des cellules à l'aide de diverses techniques, telles qu'un écoulement oscillatoire. La dénudation des ovocytes en est un exemple d'application. Les ovocytes peuvent avoir besoin d'être denudés avant qu'ils puissent être efficacement utilisés dans diverses applications, telles que des applications de fécondation in vivo (FIV). Certains aspects de la divulgation concernent ainsi des systèmes et/ou des procédés qui permettent une séparation cellulaire plus efficace. Par exemple, dans certains cas, l'utilisation d'un écoulement oscillatoire à travers un canal peut avantageusement augmenter l'efficacité du processus de séparation, tout en réduisant au minimum les effets nocifs sur les cellules. Le dispositif microfluidique peut comprendre divers composants, tels que des montants qui ont éventuellement des parties extrudées (par exemple, des dents), de multiples sources de pression pour réguler l'écoulement de fluide, ou similaire.
PCT/US2024/041298 2023-08-08 2024-08-07 Procédés et appareils microfluidiques pour la dénudation d'ovocytes utilisant un flux oscillatoire Pending WO2025034867A1 (fr)

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WENG LINDONG, LEE GLORIA Y., LIU JIE, KAPUR RAVI, TOTH THOMAS L., TONER MEHMET: "On-chip oocyte denudation from cumulus–oocyte complexes for assisted reproductive therapy", LAB ON A CHIP, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 18, no. 24, 4 December 2018 (2018-12-04), UK , pages 3892 - 3902, XP055804188, ISSN: 1473-0197, DOI: 10.1039/C8LC01075G *

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