Displacement of micro-objects in a microfluidic environment

Abstract

A method for displacing a micro-object from a chamber of a microfluidic device is provided. The present disclosure relates to systems, methods, kits, and computer-readable media for assaying and controlling micro-objects in microfluidic devices, including in situ generated hydrogel barriers within a microfluidic chamber that subdivide the chamber into areas capable of generating forces suitable for displacing micro-objects from cells without interfering with the cells themselves.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 378,183, filed October 3, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background

[0002] Microfluidic devices allow researchers to manipulate and sort minute objects, such as biological cells. This disclosure relates to systems, methods, kits, and computer-readable media for moving minute objects in microfluidic devices. Summary of the Invention [Means for solving the problem]

[0003] overview In a first aspect, provided herein is a method for displacing a micro-object from a chamber of a microfluidic device. In some embodiments, the method for displacing a micro-object from a chamber of a microfluidic device includes: (a) providing a microfluidic device, the microfluidic device including a microfluidic circuit including a substrate, a flow region, and a chamber, the chamber including an opening to the flow region, the micro-object being disposed in the chamber; (b) forming an in situ generation piston in a region of the chamber remote from the micro-object, the in situ generation piston defining a target region in the chamber remote from the in situ generation piston; and (c) illuminating the target region, thereby generating a displacement force, thereby displacing the micro-object from the chamber.

[0004] In some embodiments, the micro-object further comprises a plurality of micro-objects. In some embodiments, prior to forming the in situ piston, the method further comprises moving a portion of the plurality of micro-objects away from a region of the chamber farthest from the opening of the chamber. In some embodiments, moving the portion of the plurality of micro-objects further comprises moving the portion of the plurality of micro-objects toward the opening of the chamber. In some embodiments, the target region defined by the in situ generated piston is substantially free of micro-objects. In some embodiments, providing a microfluidic device further comprises disposing a micro-object in the chamber. In some embodiments, the method further comprises culturing the micro-object in the chamber.

[0005] In some embodiments, illuminating the target area includes illuminating the target area with a laser. In some embodiments, illuminating the target area results in the formation of a bubble within the target area, which generates a displacement force. In some embodiments, the displacement force pushes the in situ generated piston away from its original position. In some embodiments, the displacement force pushes the in situ generated piston toward an opening of the chamber. In some embodiments, illuminating the target area includes directing illumination toward a substrate, a microfluidic circuit material on a wall, or a thermal target. In some embodiments, the thermal target includes a metal deposit, a pattern of metal deposits, or a microstructure patterned on a surface. In some embodiments, illuminating the target area includes illuminating with illumination having an incident power in a range from about 1 mW to about 1000 mW.

[0006] In some embodiments, the in situ generated piston has porosity that substantially prevents the micro-objects from traversing the in situ generated piston. In some embodiments, displacing the micro-objects includes transporting the micro-objects into a flow region, and optionally, transporting the micro-objects out of the microfluidic device. In some embodiments, transporting the micro-objects out of the microfluidic device further includes flowing a medium into the flow region. In some embodiments, the flow region includes a microfluidic channel, and the opening of the chamber is near the microfluidic channel and oriented substantially parallel to the direction of flow of the fluid medium in the microfluidic channel (e.g., when the fluid medium is flowing through the microfluidic channel). In some embodiments, the chamber includes a separation region and a connection region fluidly connecting the separation region to the flow region, and the connection region includes an opening to the flow region. In some embodiments, the micro-objects are disposed within the separation region. In some embodiments, the target region is within the separation region. In some embodiments, the in situ generated piston includes a first solidified polymer network.

[0007] In some embodiments, the methods described herein further include forming an in situ generated guide element comprising a second solidified polymer network in an area near the opening of the chamber. In some embodiments, the area near the opening of the chamber is within the chamber. In some embodiments, the in situ generated guide element comprises at least one gap configured to allow displacement of micro-objects and prevent re-entry of micro-objects from the flow region into the chamber. In some embodiments, the first solidified polymer network (e.g., piston) and the second solidified polymer network (e.g., guide element) independently comprise a synthetic polymer, a modified synthetic polymer, or a biopolymer. In some embodiments, the first solidified polymer network and the second solidified polymer network independently comprise at least one of polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, denatured collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or copolymers thereof in any combination. In some embodiments, the solidified polymer network comprises polyethylene glycol acrylamide polymers. In some embodiments, the polyethylene glycol acrylamide polymers comprise linear polyethylene glycol diacrylamide polymers, two-arm polyethylene glycol diacrylamide polymers, star-shaped polyethylene glycol diacrylamide polymers, or any combination thereof. In some embodiments, the polyethylene glycol acrylamide polymers are other than linear polyethylene glycol acrylamide polymers, and all termini comprise acrylamide moieties. In some embodiments, when the polyethylene glycol acrylamide polymer is other than a linear polyethylene glycol acrylamide polymer, fewer than all of the termini comprise acrylamide moieties.

[0008] In some embodiments, forming the in situ generated piston and / or in situ generated guide element comprises flowing a first fluid medium including a flowable polymer solution through a flow region of the microfluidic device and allowing the flowable polymer solution to diffuse into the chamber. In some embodiments, forming the in situ generated piston and / or in situ generated guide element further comprises solidifying the flowable polymer solution within the chamber using photopatterning. In some embodiments, the micro object is a biological cell (e.g., a eukaryotic or prokaryotic cell) or a bead. In some embodiments, the biological cell is an animal cell, a plant cell, or a bacterial cell.

[0009] In an additional aspect, provided herein is a kit for displacing micro-objects from a chamber of a microfluidic device. In some embodiments, the kit for displacing micro-objects from a chamber of a microfluidic device includes: a) a flowable polymer configured to be controllably activated to form an in situ generated barrier comprising a solidified polymer network; and b) an inhibitor.

[0010] In some embodiments, the kit further comprises a photoinitiator. In some embodiments, the photoinitiator is a photoactivatable photoinitiator. In some embodiments, the kit further comprises a microfluidic device including a microfluidic circuit including a flow region and a chamber, wherein the chamber comprises an opening to the flow region. In some embodiments, the flow region comprises a microfluidic channel, and the opening of the chamber is proximate to the microfluidic channel and oriented substantially parallel to the flow of the fluid medium in the microfluidic channel when the fluid medium is flowing through the microfluidic channel. In some embodiments, the chamber comprises a separation region and a connection region fluidly connecting the separation region to the flow region, wherein the connection region comprises an opening to the flow region. In some embodiments, the microfluidic device comprises a plurality of chambers. In some embodiments, the microfluidic device comprises a substrate configured to generate a dielectrophoretic (DEP) force in the microfluidic circuit. In some embodiments, the solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, or a biopolymer. In some embodiments, the solidified polymer network comprises at least one of polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, denatured collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or copolymer in any combination. In some embodiments, the solidified polymer network comprises polyethylene glycol acrylamide polymer. In some embodiments, the polyethylene glycol acrylamide polymer comprises a linear polyethylene glycol diacrylamide polymer, a two-arm polyethylene glycol diacrylamide polymer, a star-shaped polyethylene glycol diacrylamide polymer, or a mixture of any combination thereof.In some embodiments, the polyethylene glycol acrylamide polymer is other than a linear polyethylene glycol acrylamide polymer and all of the termini comprise acrylamide moieties. In some embodiments, the polyethylene glycol acrylamide polymer is other than a linear polyethylene glycol acrylamide polymer and fewer than all of the termini comprise acrylamide moieties. [Brief explanation of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] 1 illustrates a system having a microfluidic device and associated control equipment according to some embodiments of the present disclosure. [Figure 1B]

[0012] 1 illustrates a microfluidic device having an isolation pen according to one embodiment of the present disclosure. [Figure 2A]

[0013] 1 illustrates a microfluidic device having an isolation pen according to some embodiments of the present disclosure. [Figure 2B] 1 illustrates a microfluidic device having an isolation pen according to some embodiments of the present disclosure. [Figure 2C]

[0014] 1 illustrates an isolation pen of a microfluidic device according to some embodiments of the present disclosure. [Figure 3]

[0015] 1 illustrates an isolation pen of a microfluidic device according to some embodiments of the present disclosure. [Figure 4A]

[0016] 1 illustrates electrokinetic characteristics of a microfluidic device according to some embodiments of the present disclosure. [Figure 4B]

[0016] Figure 1 illustrates electrokinetic characteristics of a microfluidic device according to some embodiments of the present disclosure. [Figure 5A]

[0017] 1 illustrates a system for use with a microfluidic device and associated controls according to some embodiments of the present disclosure. [Figure 5B]

[0018] 1 illustrates an imaging device according to some embodiments of the present disclosure. [Figure 6A]

[0019] 1 is a diagrammatic representation of an isolation pen of a microfluidic device including an in situ generated piston and an in situ guide element, and a region formed after creation of the piston, according to some embodiments of the present disclosure. [Figure 6B]

[0020] 1 is a photographic representation of an isolation pen of a microfluidic device including an in situ generated piston and an in situ guide element, and the region formed after creation of the piston, according to some embodiments of the present disclosure. [Figure 7]

[0021] 10 is a series of photographic representations of a time-lapse displacement process involving the use of an in situ generated piston and an in situ guide element, according to some embodiments of the present disclosure. [Figure 8]

[0022] FIG. 1 is a block diagram for displacement of micro-objects from an isolation pen of a microfluidic device. [Figure 9]

[0023] FIG. 10 is another block diagram for displacement of micro-objects from an isolation pen of a microfluidic device. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description

[0024] Some of the techniques described herein involve forming in situ generated barriers to facilitate displacement of micro-objects within a microfluidic device. For example, a microfluidic device can be used to culture and / or assay biological cells separated within separate isolation pens. To separate the cells within the separate isolation pens, dielectrophoresis (DEP) (e.g., optically actuated dielectrophoretic force (OEP)) can be employed to provide a force that moves the biological cells into the isolation pens. After culturing and / or assaying, the biological cells should be displaced from the isolation pen and migrate into a microfluidic channel. However, some biological cells may adhere to the surface of the isolation pen. The force provided by DEP may not be strong enough to easily or successfully remove the biological cells from the isolation pen.

[0013]

[0025] As described herein, an in situ generation structure can be formed to divide an isolation pen into a target area and a culture area. A DEP force can then be used to position biological cells within the culture area on one side of the in situ generation structure. A laser can then illuminate the target area on the other side of the in situ generation structure to heat the fluid medium within the target area. This heating generates bubbles, which manipulate the in situ generation structure so that a displacement force can move the biological cells from the isolation pen. This displacement force can be stronger than the DEP force, thus displacing biological cells that may adhere to surfaces and be difficult to manipulate using DEP forces. Because the in situ generation structure separates the biological cells in the culture area from the laser illumination in the target area, the biological cells are not harmed by the direct application of the displacement force provided by the bubbles. Thus, the biological cells can be displaced from the isolation pen without being harmed by the displacement force.

[0014]

[0026] More particularly, this specification describes exemplary embodiments and applications of the present disclosure. However, the present disclosure is not limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Furthermore, the figures may show simplified or partial views, and the dimensions of the elements in the figures may be exaggerated or otherwise not proportional. In addition, when the terms "on," "attached to," "connected to," "coupled to," or similar terms are used herein, an element (e.g., a material, layer, substrate, etc.) can be "on," "attached to," "connected to," or "coupled to" another element, whether or not it is directly on, attached to, connected to, or coupled to another element, or whether or not one or more intervening elements are present between the element and the other element. Also, unless the context indicates otherwise, directions (e.g., above, below, top, bottom, sideways, above, below, under, over, upper, lower, horizontal, vertical, "x," "y," "z," etc.), when provided, are relative and are provided merely as examples and for illustrative and ease of discussion, and not as limitations. Additionally, when a list of elements (e.g., elements a, b, c) is referred to, such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and / or all combinations of the listed elements. Sections herein are for ease of review only and do not limit any combination of elements discussed.

[0015]

[0027] When a dimension of a microfluidic feature is described as having a width or an area, the dimension is typically described in terms of the x-axis and / or y-axis dimension, both of which are in a plane parallel to the substrate and / or cover of the microfluidic device. The height of the microfluidic feature may be described in terms of the z-axis direction, which is perpendicular to a plane parallel to the substrate and / or cover of the microfluidic device. In some cases, the cross-sectional area of ​​a microfluidic feature, such as a channel or passage, may be related to an x-axis / z-axis area, a y-axis / z-axis area, or an x-axis / y-axis area.

[0016]

[0028] As used herein, "substantially" means functioning adequately for its intended purpose. Thus, the term "substantially" allows for small, slight variations from absolute or perfect conditions, dimensions, measurements, results, etc., as would be expected by one of ordinary skill in the art, that do not appreciably affect overall performance. When used in reference to a number, parameter, or characteristic that can be expressed as a number, "substantially" means within 10 percent.

[0017]

[0029] "Ones" means two or more. As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0018]

[0030] As used herein, μm means micrometer and μm 3 means cubic micrometer, pL means picoliter, nL means nanoliter, and μL (or uL) means microliter.

[0019]

[0031] As used herein, "air" refers to the composition of gases that predominate in the Earth's atmosphere. The four most abundant gases are nitrogen (typically present at a concentration of about 78% by volume, e.g., about 70-80% by volume), oxygen (typically present at 20.95% by volume at sea level, e.g., about 10% to about 25% by volume), argon (typically present at about 1.0% by volume, e.g., 0.1% to about 3% by volume), and carbon dioxide (typically present in the range of about 0.04% by volume, e.g., about 0.01% to about 0.07% by volume). Air may contain trace amounts of other trace gases, such as methane, nitrous oxide, or ozone, as well as trace amounts of pollutants, such as pollen and diesel particulates, and organic matter. Air may also contain water vapor (typically present at about 0.25% or may be present in the range of about 10 ppm to about 5% by volume). Air may be provided for use in culture experiments as a filtered, controlled composition and may be conditioned as described herein.

[0020]

[0032] As used herein, the term "disposed" includes within its meaning "disposed."

[0021]

[0033] As used herein, a "microfluidic device" or "microfluidic apparatus" is a device that includes one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit being composed of fluidically interconnected circuit elements including, but not limited to, regions, flow paths, channels, chambers, and / or pens, and at least one port configured to flow fluid (and optionally microscopic objects suspended in the fluid) into and / or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., about 750 μL, 500 μL, 250 μL, 200 μL, 150 μL, 100 μL, 75 μL, 50 μL, 25 μL, 20 μL, 15 μL, 10 μL, 9 μL, 8 μL, 7 μL, 6 μL, 5 μL, 4 μL, 3 μL, or 2 μL. In certain embodiments, the microfluidic circuit holds approximately 1-2 μL, 1-3 μL, 1-4 μL, 1-5 μL, 2-5 μL, 2-8 μL, 2-10 μL, 2-12 μL, 2-15 μL, 2-20 μL, 5-20 μL, 5-30 μL, 5-40 μL, 5-50 μL, 10-50 μL, 10-75 μL, 10-100 μL, 20-100 μL, 20-150 μL, 20-200 μL, 50-200 μL, 50-250 μL, or 50-300 μL. The microfluidic circuit may be configured to have a first end fluidly connected to a first port (e.g., an inlet) in the microfluidic device and a second end fluidly connected to a second port (e.g., an outlet) in the microfluidic device.

[0022]

[0034] As used herein, a "nanofluidic device" or "nanofluidic apparatus" is a type of microfluidic device having a microfluidic circuit including at least one circuit element configured to hold a volume of fluid of less than about 1 μL, e.g., about 750 nL, 500 nL, 250 nL, 200 nL, 150 nL, 100 nL, 75 nL, 50 nL, 25 nL, 20 nL, 15 nL, 10 nL, 9 nL, 8 nL, 7 nL, 6 nL, 5 nL, 4 nL, 3 nL, 2 nL, 1 nL, or less than 1 nL. A nanofluidic device can include a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one circuit section are configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one circuit element are configured to hold a volume of fluid of about 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.

[0023]

[0035] A microfluidic or nanofluidic device may be referred to herein as a "microfluidic chip" or "chip" or a "nanofluidic chip" or "chip."

[0024]

[0036] As used herein, a "microfluidic channel" or "flow channel" refers to a flow region of a microfluidic device having a length that is much longer than both the horizontal and vertical dimensions. For example, a flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times that length, at least 25 times that length, at least 100 times that length, at least 200 times that length, at least 500 times that length, at least 1,000 times that length, at least 5,000 times that length, or longer. In some embodiments, the length of a flow channel is from about 100,000 μm to about 500,000 μm, including any value in between. In some embodiments, the horizontal dimension is from about 100 μm to about 1,000 μm (e.g., from about 150 to about 500 μm) and the vertical dimension is from about 25 μm to about 200 μm (e.g., from about 40 to about 150 μm). It should be noted that flow channels can have a wide variety of different spatial configurations in microfluidic devices and are therefore not limited to completely linear elements. For example, a flow channel may be or include one or more sections having the following configurations: curved, bent, spiral, inclined, descending, forked (e.g., multiple different flow paths), and any combination thereof. In addition, a flow channel may have different cross-sectional areas along its path, widening and narrowing to provide a desired flow rate therein. A flow channel may include valves, which may be of any type known in the field of microfluidics. Examples of microfluidic channels including valves are disclosed in U.S. Patent Nos. 6,408,878 and 9,227,200, which are incorporated herein by reference in their entirety.

[0025]

[0037] As used herein, the term "transparent" refers to a material that transmits visible light without substantially altering the light as it passes through.

[0026]

[0038] As used herein, "bright field" illumination and / or "bright field" image refers to white light illumination of a microfluidic field from a broad spectrum light source, where contrast is created by absorption of light by objects within the field.

[0027]

[0039] As used herein, "structured light" refers to projected light modulated to provide one or more lighting effects. A first lighting effect can be projected light that illuminates a portion of a device's surface without illuminating (or at least minimizing) adjacent portions of that surface, such as a projected light pattern used to activate DEP forces within a DEP substrate, as described more fully below. When using a structured light pattern to activate DEP forces, varying the intensity, e.g., duty cycle, of a structured light modulator such as a DMD can be used to modify the optical power applied to the photoactivated DEP actuators, and thus the DEP forces, without changing the nominal voltage or frequency. Another lighting effect that can be produced by structured light includes projected light that can compensate for surface irregularities and irregularities associated with the light projection itself, such as dropoff at the edges of the illumination field. Structured light is typically produced by a structured light modulator, such as a digital mirror device (DMD), a microshutter array system (MSA), a liquid crystal display (LCD), or the like. Illuminating a small area of ​​a surface using structuring, e.g., illumination of a selected area of ​​interest, improves the signal-to-noise ratio (SNR) because illumination of only the selected area of ​​interest reduces stray / scattered light, thereby lowering the image darkness level. An important aspect of structured light is its ability to be rapidly varied over time. Light patterns from a structured light modulator, e.g., a DMD, can be used to focus on different targets, such as a clean mirror or surface far out of focus. Using a clean mirror allows for reproducing some self-test features, such as modulation transfer function and field curvature / tilt measurements, without the need for more expensive Shack-Hartmann sensors. Another application of structured light patterns is measuring the spatial power distribution at a sample surface using a simple power meter instead of a camera. Structured light patterns can also be used as a reference feature for aligning optical modules / system components and as a manual readout for manual focusing. Another lighting effect enabled by the use of structured light patterns is the selective curing, e.g., solidification, of hydrogels in microfluidic devices.

[0028]

[0040] As used herein, the term "microscopic object" generally refers to any microscopic object that can be separated and / or manipulated according to the present disclosure. Non-limiting examples of microscopic objects include inanimate microparticles; microbeads (e.g., polystyrene beads, glass beads, amorphous substrates, Luminex™ beads, etc.); magnetic beads; microrods; microwires; quantum dots; cells; biological organelles; vesicles or complexes; synthetic vesicles; liposomes (e.g., synthetic or membrane preparation-derived); biological microscopic objects such as lipid nanorafts; or combinations of inanimate and biological microscopic objects (e.g., microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, etc.). Beads may contain covalently or non-covalently bound moieties / molecules such as fluorescent labels, proteins (including receptor molecules), carbohydrates, antigens, small molecule signaling moieties, or other chemical / biological species that can be used in assays. In some variations, the bead / solid substrate containing the moiety / molecule may be a capture bead configured to selectively or non-selectively bind, for example, nearby small molecules, peptides, proteins, or nucleic acids. In one non-limiting example, the capture bead may contain a nucleic acid sequence configured to bind a nucleic acid having a specific nucleic acid sequence, or the nucleic acid sequence of the capture bead may be configured to bind a set of nucleic acids having related nucleic acid sequences. The type of binding may be understood to be selective. The capture bead containing the moiety / molecule may non-selectively bind structurally distinct but physicochemically similar molecules, such as size-exclusion beads or zeolites configured to capture molecules of a selected size or charge, if binding is performed. Lipid nanorafts are described, for example, in Ritchie et al. (2009) "Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs," Methods Enzymol., 464:211-231.

[0029]

[0041] As used herein, the term "cell" is used synonymously with the term "biological cell." Non-limiting examples of biological cells include animal cells such as eukaryotic cells, plant cells, mammalian cells, reptilian cells, avian cells, and fish cells; prokaryotic cells, bacterial cells, fungal cells, and protozoan cells; cells dissociated from tissues such as muscle, cartilage, fat, skin, liver, lung, and nervous tissue; immune cells such as T cells, B cells, natural killer cells, and macrophages; embryos (e.g., zygotes), oocytes, eggs, sperm cells, hybridomas, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and / or transformed cells, reporter cells, and the like. Mammalian cells can be, for example, cells from humans, mice, rats, horses, goats, sheep, cattle, primates, and the like.

[0030]

[0042] A colony of living cells is a "clone" if all of the living cells in the reproductive colony are daughter cells derived from a single parent cell. In certain embodiments, all daughter cells in a clonal colony are from a single parent cell within 10 or fewer cell divisions. In other embodiments, all daughter cells in a clonal colony are from a single parent cell within 14 or fewer cell divisions. In other embodiments, all daughter cells in a clonal colony are from a single parent cell within 17 or fewer cell divisions. In other embodiments, all daughter cells in a clonal colony are from a single parent cell within 20 or fewer cell divisions. The term "clonal cells" refers to cells of the same clonal colony.

[0031]

[0043] As used herein, a "colony" of biological cells refers to two or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or more than about 1000 cells).

[0032]

[0044] As used herein, the term "sustaining cells" refers to providing an environment, including both fluid and gaseous components, and optionally surfaces, that provides the conditions necessary for the continued survival and / or proliferation of cells.

[0033]

[0045] As used herein, the term "proliferation," when referring to cells, refers to an increase in cell number.

[0034]

[0046] As used herein, "gas permeable" means that a material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, a gas permeable material or structure may be permeable to more than one of oxygen, carbon dioxide, and nitrogen, and may even be permeable to all three gases.

[0035]

[0047] A "component" of a fluid medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, etc.

[0036]

[0048] As used herein in reference to a fluid medium, "diffuse" and "diffusion" refer to the thermodynamic movement of components of the fluid medium down a concentration gradient.

[0037]

[0049] The phrase "medium flow" refers to bulk movement of a fluid medium primarily due to any mechanism other than diffusion. For example, medium flow can include movement of a fluid medium from one point to another due to a pressure difference between the points. Such flow can include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. When one fluid medium flows into another, turbulence and mixing of the medium can occur. Flowing can include pushing solutions through and out of microfluidic channels (e.g., aspiration) or pushing fluids into and through microfluidic channels (e.g., perfusion).

[0038]

[0050] The phrase "substantially no flow" means a flow rate of the fluid medium that is less than the time-averaged diffusion rate of a component of the material (e.g., an analyte of interest) into or through the fluid medium. The diffusion rate of a component of such a material may depend, for example, on temperature, size of the component, and strength of interaction of the component with the fluid medium.

[0039]

[0051] As used herein with respect to different regions within a microfluidic device, the phrase "fluidically connected" means that the fluids in each region are connected to form a single fluid when the different regions are substantially filled with fluid, such as a fluid medium. This does not mean that the fluids (or fluid media) in the different regions are necessarily of identical composition. More precisely, fluids in different fluidly connected regions of a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, other molecules, etc.) as solutes move down their respective concentration gradients and / or in flux as they flow through the microfluidic device.

[0040]

[0052] As used herein, a "flow path" refers to one or more fluidly connected circuit elements (e.g., channels, regions, chambers, etc.) that define and are subject to the flow of media. As such, a flow path is an example of a sweep region of a microfluidic device. Other circuit elements (e.g., non-sweep regions) may be fluidly connected to circuit elements that contain flow paths that are not subject to media flow in the flow path.

[0041]

[0053] As used herein, "separation of micro-objects" refers to confining micro-objects to defined areas within a microfluidic device.

[0042]

[0054] As used herein, "pen transfer" or "pen transfer" refers to placing a micro-object into a chamber (e.g., an isolation pen) in a microfluidic device. The force used to transfer the micro-object into the pen can be any suitable force described herein, such as dielectrophoresis (DEP), e.g., optically actuated dielectrophoretic force (OEP), gravity, magnetic force, or tilt. In some embodiments, transferring multiple micro-objects into the pen can reposition substantially all of the micro-objects. In some other embodiments, a selected number of micro-objects of a plurality of micro-objects can be transferred into the pen, while the remainder of the plurality may not be transferred into the pen. In some embodiments, when selected micro-objects are transferred into the pen, a DEP force, e.g., an optically actuated DEP force or a magnetic force, can be used to reposition the selected micro-objects. Typically, the micro-objects are introduced into a flow region of the microfluidic device, e.g., a microfluidic channel, and can be introduced into a chamber by pen transfer.

[0043]

[0055] As used herein, "removal from pen" or "removal from pen" refers to repositioning a micro-object from a chamber, e.g., an isolation pen, to a new location, e.g., a microfluidic channel, within a flow region of a microfluidic device. The force used to remove the micro-object from the pen can be any suitable force as described herein, such as, but not limited to, dielectrophoresis, e.g., an optically actuated dielectrophoretic force, gravity, an optically driven bubble, a displacing fluid flow, a magnetic force, or a tilt. In some embodiments, removal of a plurality of micro-objects from the pen can reposition substantially all of the micro-objects. In some other embodiments, a selected number of the plurality of micro-objects can be removed from the pen, while the remainder of the plurality may not be removed from the pen. In some embodiments, when selected micro-objects are removed from the pen, a DEP force, e.g., an optically actuated DEP force or a magnetic force, can be used to reposition the selected micro-objects.

[0044]

[0056] As used herein, "displace," "displacing," or "displacement" refers to repositioning a micro-object from a first position to a second position within a microfluidic device. The first and second positions are independently within a chamber (e.g., an isolation pen) or a flow region (e.g., a microfluidic channel) of the microfluidic device. The force used to remove the micro-object from the pen can be any suitable force as described herein, such as, but not limited to, dielectrophoresis, e.g., an optically actuated dielectrophoretic force, gravity, an optically driven bubble, a displacing fluid flow, a magnetic force, or a gradient. In some embodiments, displacing a plurality of micro-objects can reposition substantially all of the micro-objects. In some other embodiments, a selected number of micro-objects of a plurality of micro-objects can be displaced, while the remainder of the plurality can be undisplaced. In some embodiments, when selected micro-objects are displaced, a DEP force, e.g., an optically actuated DEP force or a magnetic force, can be used to reposition the selected micro-objects.

[0045]

[0057] As used herein, "transfer" or "transferring" refers to repositioning a micro-object from a position within a flow region of a microfluidic device, e.g., a microfluidic channel, to a location outside the microfluidic device, such as a 96-well plate or other receiving vessel. The orientation of the chamber with an opening to the microfluidic channel allows for easy removal of a micro-object that has been positioned or repositioned (e.g., pen-removed from the chamber) to be placed within the microfluidic channel. Micro-objects within a microfluidic channel can be removed without requiring disassembly (e.g., removal of the device cover) or insertion of a tool into the chamber or microfluidic channel to remove the micro-object for further processing.

[0046]

[0058] A microfluidic (or nanofluidic) device can include "sweep" regions and "non-sweep" regions. As used herein, a "sweep" region is comprised of one or more fluidly interconnected circuit elements of a microfluidic circuit, through which medium flows when fluid is flowing through the microfluidic circuit. The circuit elements of a sweep region can include, for example, all or part of a region, channel, and chamber. As used herein, a "non-sweep" region is comprised of one or more fluidly interconnected circuit elements of a microfluidic circuit, through which medium flows when fluid is flowing through the microfluidic circuit. A non-sweep region can be fluidly connected to a sweep region if the fluid connection is structured to allow diffusion but to prevent substantial medium flow between the sweep region and the non-sweep region. Thus, a microfluidic device can be structured to substantially isolate the non-sweep region from medium flow in the sweep region, while allowing substantially only diffusional communication between the sweep region and the non-sweep region. For example, a flow channel of a microfluidic device is an example of a sweep region, while a separation region of a microfluidic device (discussed in more detail below) is an example of a non-sweep region.

[0047]

[0059] As used herein, a "non-sweeping" rate of fluid medium flow means a flow rate sufficient to cause components of a second fluid medium in a separation region of an isolation pen to diffuse into a first fluid medium in the flow region and / or cause components of a first fluid medium to diffuse into a second fluid medium in the separation region, wherein further, the first medium does not substantially flow into the separation region.

[0048] Methods for promoting cell displacement

[0060] Micro-objects can move in local environments, such as within a microfluidic device, due to several forces, including, but not limited to, gravity, fluid flow induced by mechanical pumps, electrowetting, and / or dielectrophoresis (DEP). In some situations, micro-objects may adhere to the surface of the microfluidic device, and cells may adhere to the surface through interactions with surface-fouling proteins produced by themselves or other cells present within the microfluidic device. Such adhesion can reduce the portability of the micro-object. Optical illumination of selected, discrete regions on or within the microfluidic device can heat portions of the fluid medium within the microfluidic circuitry of the microfluidic device, providing a wide variety of displacement forces within the microfluidic device, thereby facilitating the displacement of micro-objects. However, optical illumination and the resulting displacement forces can be harmful to the micro-objects if applied directly to them.

[0049]

[0061] Provided herein are methods and compositions for microfluidic workflows and processes that result in improvements to micro-object displacement processes. Provided herein are piston structures with in situ generated hydrogels, the use of which can increase cell displacement speed and rate and reduce (or minimize) contact between the micro-object of interest and the displacement force. In some embodiments, the piston defines a target region within the chamber configured to generate a displacement force. In some embodiments, the piston divides the chamber, e.g., an isolation pen, into two areas, one closer to the pen opening that opens into the flow region and the second area away from the edges and the micro-object of interest. The farther area, in some embodiments, is used exclusively as the target region (displacement force generation region), while the closer area can be used to house or culture cells prior to removal from the pen.

[0050]

[0062] In some embodiments, an assay can be performed within the area of ​​interest suitable for a selected assay before, during, and / or after the formation of the in situ generated barrier. In some embodiments, the assay is performed to facilitate identification and selection of pens containing cells of interest. The assay can be of any type, including, but not limited to, assays for determining cell biological productivity, cell viability, cell proliferation rate, etc. The present disclosure is not limited by whether or not an assay is performed, the type of assay performed, or the selection of cells of interest as a result of such assays. Assays can be those described in U.S. Patent Publication No. 2020 / 0408751, filed April 15, 2020, and U.S. Patent No. 11,203,018, filed October 15, 2018, and issued December 21, 2021, each of which is incorporated herein by reference for purposes described herein. In some embodiments, the hydrogel barrier (the in situ generated piston or in situ generated guide element described herein) may be porous, allowing selective permeability to allow one, some, or all of one or more reagents associated with the assay and / or cell culture to pass through the piston and reach the tip area of ​​the chamber, and vice versa.

[0051] Transfer of cells into the pen

[0063] In some embodiments, disposing the cells in the chamber of the microfluidic device includes obtaining a microfluidic device including a microfluidic circuit including a flow region and a chamber fluidly connected to the flow region, introducing a fluid medium containing the cells into the flow region, and disposing the cells in the chamber. In some embodiments, the cells are disposed in the chamber of the microfluidic device by gravity or OEP, as described herein. In some embodiments, a positive or negative OEP can be selected depending on the surface charge of the cells to be moved.

[0052]

[0064] In some embodiments, when cells placed in the microfluidic device (e.g., pen-transfected cells) are small (e.g., non-mammalian cells, which tend to be smaller than mammalian cells), e.g., smaller than 10 μm in diameter, OEP is performed at a higher voltage. In some embodiments, the voltage is greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 V. In some embodiments, the cells have a diameter of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 μm.

[0053]

[0065] The process of pen-transferring cells can be automated using image recognition software, as described in U.S. Patent Publication No. 11,170,200 (Kim et al.), filed May 31, 2019, and granted November 9, 2021, and U.S. Patent Application Publication No. 2021 / 0209752 (Tenney et al.), filed November 24, 2020, the disclosures of each of which are incorporated herein by reference. In various embodiments, cells are transferred into separate chambers, such as NanoPen® chambers, and cultured as individual colonies. Pen-transferring single cells into individual chambers allows expansion into cell populations, providing clonal populations. The ability to observe, test, and selectively export specific clonal populations of cells exhibiting desired properties, provided by the methods described herein, is a significant improvement over macroscale techniques currently used to develop recombinant cell lines capable of producing desired bioproducts. To more efficiently utilize the potential for assay processes performed within each of the many individual chambers within a microfluidic device, barriers can be introduced within the chambers for several purposes, which may improve the assay by enhancing the assay's accuracy, sensitivity, or reproducibility, among other assay properties. For example, barriers can be introduced, e.g., generated in situ, to isolate cells away from or within an area, such as, but not limited to, isolating cells outside a target area, isolating cells in or outside a pen, isolating cells in a culture area, and / or isolating cells in or outside an assay observation area (e.g., a target area, e.g., so as not to artificially enhance signal detection throughout the chamber by being present in the target area as a point source). Barriers can also be introduced to prevent secreted analytes bound to reporter molecules (RMSA complexes) from diffusing out of the target area. Barriers can also prevent molecules that are too large (molecular weight) to pass through the barrier and that may interfere with the assay mechanism from reaching the target area of ​​the assay.

[0054] In situ generation barrier

[0066] As disclosed herein, an in situ generated barrier is formed within a chamber of a microfluidic device. Generally, one function of the in situ generated barrier in the disclosed methods is to contain cells within a specific chamber, region, or area of ​​the microfluidic device. The term "in situ generated barrier" refers to a barrier formed in a selected area while the microfluidic device is operating. During microfluidic device fabrication, the barrier generally does not form or exist before the microfluidic device is used for experimentation or research. The term "barrier" refers to a physical structure that forms and fixes in a selected area for at least a specific period of time and can prevent or block microscopic objects (such as, but not limited to, cells) from crossing the barrier. As a result, a barrier formed in situ within a chamber can divide its interior space into two areas on either side of the barrier. In some embodiments, the barrier defines a culture area and a culture exclusion area within the chamber.

[0055]

[0067] In some embodiments, the in situ generated barrier is a piston. In some embodiments, the piston is formed in situ within a chamber included in the microfluidic device. In some embodiments, following formation of the piston, two areas are defined on either side of the piston, which are distinct regions of the chamber. The first region is configured to contain a plurality of micro-objects (such as, but not limited to, cells) as an enclosed culture area, and in some embodiments, the first region is near the opening of the chamber to the flow. The second region is configured to be substantially free of micro-objects (such as, but not limited to, cells) and is configured to receive displacement force generation as a target region. In some embodiments, the second region is away from the opening of the chamber.

[0056]

[0068] In some embodiments, the in situ generated barrier is a guide element. In some embodiments, the guide element is formed in a region of the chamber near the microfluidic channel (i.e., near the opening of the chamber) to prevent or block micro-objects (such as, but not limited to, cells) from easily traversing the guide element without the application of a sufficient force, such as a displacement force. In some embodiments, such a barrier can be utilized to create an enclosed culture area. For example, in some embodiments, the in situ generated guide element can be formed in a connection region between the chamber and the microfluidic channel. In some embodiments, such a guide element in the connection region can be configured to allow the micro-object to be removed from the pen when the micro-object is subjected to a sufficient displacement force and to prevent the movement of the removed micro-object back into the chamber. In some embodiments, the in situ generated barrier can be any suitable shape, such as, but not limited to, a circular, oval, square, teardrop, or the like.

[0057]

[0069] As used herein, "culture area" refers to a predetermined area for maintaining or culturing cells for any desired period of time, although the methods of the present disclosure are not limited to maintaining or culturing cells in a culture area.

[0058]

[0070] In some embodiments, the inhibition or blocking caused by the introduction of a barrier is size-dependent. Particles can be inhibited, blocked, or allowed to traverse the barrier depending on their size. In some embodiments, the in situ generated barrier has a porosity that substantially prevents microscopic objects (e.g., cells) from traversing the in situ generated barrier. In some embodiments, the in situ generated barrier can include gaps having a width or diameter that allows microscopic objects (e.g., cells) to pass through the barrier. Nevertheless, movement of microscopic objects (e.g., cells) through the barrier via the gaps can still be prevented.

[0059] Hydrogel in situ generated barrier

[0071] In certain embodiments, the in situ generated barrier is a hydrogel. In certain embodiments, the in situ generated barrier comprises a solidified polymer network. In some embodiments, the solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, or a biopolymer. In certain embodiments, the solidified polymer network comprises at least one of polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, denatured collagen, laminin, modified laminin, a polysaccharide, a modified polysaccharide, or a copolymer in any combination. In some embodiments, the solidified polymer network does not comprise a silicone polymer.

[0060]

[0072] The physical and chemical properties that determine the suitability of a polymer for use in a solidified polymer network can include molecular weight, hydrophobicity, solubility, diffusivity, viscosity (e.g., of a medium), excitation and / or emission range (e.g., of an immobilized fluorescent reagent), known background fluorescence, properties that affect polymerization, and pore size of the solidified polymer network. The solidified polymer network is formed upon polymerization or thermal gelation of a fluid polymer solution containing at least one of polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, denatured collagen, laminin, modified laminin, polysaccharides, modified polysaccharides, or copolymers in any combination. Various copolymer classes may be used, including, but not limited to, any of the polymers listed above or biopolymers such as fibronectin, collagen, or laminin. Polysaccharides such as dextran or denatured collagen may be used. Flowable polymers may alternatively be referred to herein as prepolymers, meaning that the flowable polymer is crosslinked in situ. Biopolymers with photoactivatable functionality for polymerization may also be used.

[0061]

[0073] In some cases, the polymer may include a cleavage moiety. The cleavage moiety may include a peptide sequence inserted into the polymer that is a substrate for one or more proteases, including, but not limited to, matrix metalloproteinases, collagenases, or serine proteases such as proteinase K. Another category of cleavage moiety may include a photocleavable moiety, such as a nitrobenzyl photocleavable linker, which may be inserted at a selected position in the prepolymer. In some embodiments, the nitrobenzyl photocleavable linker may include a 1-methynyl, 2-nitrobenzyl moiety configured for photocleavage. In other embodiments, the photocleavable linker may include a benzoin moiety, a 1,3 nitrophenyl moiety, a coumarin-4-ylmethyl moiety, or a 1-hydroxy-2-cinnamoyl moiety. The cleavage moiety may be utilized to remove the solidified polymer network of the isolated structure. In other embodiments, the polymer may include a cell recognition moiety, including, but not limited to, an RGD peptide moiety recognized by integrins.

[0062]

[0074] One polymer among many that can be used is polyethylene glycol diacrylate (PEGDA) or polyethylene glycol acrylamide (diacrylamide, multiarm acrylamide, or substituted versions as described herein).

[0063]

[0075] Light-activated polymerization can be achieved using the free radical photoinitiator Igracure® 2959 (BASF). A highly efficient, non-yellowing, radical, alpha-hydroxyketone photoinitiator is typically used for initiation at wavelengths in the UV region (e.g., 365 nm), although other photoinitiators may be used. Another photoinitiator class useful for polymerization reactions is the lithium acylphosphinate family, of which lithium phenyl 2,4,6-trimethylbenzoylphosphinate is particularly useful due to its more efficient absorption at longer wavelengths (e.g., 405 nm) than the alpha-hydroxyketone class. Another photoinitiator that can be used is a water-soluble azo photoinitiator, such as 2,2-azobis[2-methyl-N-(2-hydroxyethyl)propionamide]. The photoinitiator can be present in the fluid polymer solution at a concentration of about 5 millimolar, about 8 millimolar, about 10 millimolar, about 12 millimolar, about 15 millimolar, about 18 millimolar, about 20 millimolar, about 22 millimolar, about 25 millimolar, about 28 millimolar, about 30 millimolar, about 35 millimolar, or about 40 millimolar.

[0064]

[0076] Crosslinking can be achieved by photopatterning of linear or branched PEG polymers, free radical polymerization of PEG acrylates or PEG acrylamides, and specifically tailored chemical reactions such as Michael addition, condensation, click chemistry, native chemical ligation, and / or enzymatic reactions. In particular, photopatterning of crosslinks can be used to obtain precise extent control of the physical extent of the hydrogel barrier and the degree of crosslinking as described in the following sections and examples.

[0065]

[0077] Inhibitors can be included in the fluid polymer solution to ensure precise control of photopatterning and to avoid excessive or undesired polymerization. One useful inhibitor is hydroquinone monomethyl ether (MEHQ), although other suitable inhibitors may be used. The inhibitor can be present in the fluid polymer solution at concentrations of about 1 millimolar, about 2 millimolar, about 5 millimolar, about 10 millimolar, about 15 millimolar, about 20 millimolar, about 25 millimolar, about 30 millimolar, about 35 millimolar, about 40 millimolar, or higher, as needed to provide the desired photopatterning control.

[0066]

[0078] Tunable permeability. One aspect of performing assays using in situ generated hydrogel barriers is determining which species are desirable for gaining access to an area of ​​interest. The polymer chemistry (e.g., molecular weight range, number of crosslinkable moieties per polymer unit (linear, two-arm, four-arm, eight-arm, star, or comb polymer), polymer blend), amount of photoinitiator, and choice of polymerization mode are variables that can be altered to tailor the hydrogel barrier formed. Generally, photoinitiators are photoinitiators. Photopatterning provides precise control over the geometry and degree of polymerization, and altering exposure time and illumination power can also provide further control to achieve the desired type of porosity and robustness of the polymerization characteristics.

[0067]

[0079] In many variations, polymer selection may depend on the biocompatibility of the polymer species and may be related to the particular application in which the hydrogel in situ generated barrier will be used.

[0068]

[0080] In some variations, the hydrogel may be a polyethylene glycol polymer or a modified polyethylene glycol polymer.

[0069]

[0081] Depending on the structure of the polymer, a wide range of molecular weights of flowable polymers may be suitable. In some embodiments, the flowable polymer may have a molecular weight of about 500 Da to about 20 kDa, or about 500 Da, about 1 kDa, about 3 kDa, about 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa, about 20 kDa, or any value therebetween. Useful star polymers may have a Mw (weight average molecular weight) ranging from about 500 Da to about 20 kDa (e.g., a four-arm polymer) or about 5 kDa for each arm, or any value therebetween. In some embodiments, polymers with a higher molecular weight range may be used at lower concentrations in the flowable polymer and still provide an in situ generated piston or other type of in situ generated barrier that can be used in the methods described herein.

[0070] Geometry of cell export and in situ generated barriers

[0082] The in situ generated barrier, such as a piston, of the disclosed methods provides the advantage of isolating cells from a displacement force-generating region within the chamber in which the cells are placed or cultured. In some embodiments, the disclosed methods include removing a pen from the cell chamber. In some embodiments, the cells are further transported from the microfluidic device. In some embodiments, various mechanisms can be used to remove, reduce, or bypass an in situ generated hydrogel barrier, such as a piston and / or connection region barrier, so that the cells can be removed from the pen. In some embodiments, displacing the cells includes directing laser illumination to a selected area of ​​the chamber (e.g., the displacement force-generating region) to generate a bubble that pushes toward the piston; the piston can then be deformed, displaced, flipped, slipped, and / or otherwise pushed toward the cell by a displacement force associated with the bubble; the piston, expanding fluid, and / or bubble can subsequently push the cell toward the opening of the chamber. In other embodiments, removing the cell from the pen includes directing laser illumination to a selected area of ​​the chamber (e.g., a displacement force generating region) to create a bubble that propels toward a piston, which can then be deformed, displaced, flipped, slipped, and / or otherwise propelled toward the cell by a displacement force associated with the bubble; the piston, expanding fluid, and / or bubble subsequently pushes the cell toward an opening in the chamber, and a dielectrophoretic force may then be used to remove the cell from the pen. In some embodiments, the displacement of the piston is proportional to the power and / or duration of the illumination.

[0071]

[0083] Without intending to be bound by theory, laser illumination can be projected onto a selected site on the surface of the chamber, such as a site within a displacement force-generating region, to generate heat in the fluid medium surrounding the selected site on the surface of the chamber, which can nucleate and propagate through the displacement force-generating region of the piston. In some embodiments, the selected site on the surface of the chamber is within the displacement force-generating region defined by the piston, e.g., in an area away from the chamber (e.g., pen) opening. In some embodiments, the bubble can grow with continued illumination, creating a shear flow of fluid directed toward a nearby substrate, such as toward the piston. The force and / or flow can push the in situ-generated piston, fluid medium, and micro-objects away from the cell culture area, typically in a direction toward the chamber opening. As a result, when the in situ-generated piston no longer holds its original position, the micro-objects can be displaced from the chamber. In some embodiments, the micro-objects (e.g., cells) are displaced near the opening of the chamber to the microfluidic channel and / or into the microfluidic channel by the piston, the expanding fluid (e.g., through heat), the bubble, and / or forces generated by the bubble. In some embodiments, the cells are mobilized by the bubbles to a position near the opening of the chamber to the microfluidic channel, and an OEP force is applied to further migrate the cells into the microfluidic channel, where they are subsequently flushed out of the microfluidic device by the flow introduced into the microfluidic channel. In some other embodiments, when the laser illumination pulse is terminated, the introduced bubbles collapse, creating cavitation forces that draw the fluid back toward the tip of the chamber. In some embodiments, in situ generated guiding elements can be fabricated and used to prevent micro-objects from flowing back into the chamber after the bubbles collapse and / or the fluid cools.

[0072]

[0084] In some embodiments, the illumination site can be selected to be any selected discrete site on the surface of the chamber within the target region. In some embodiments, the selected discrete illumination site can be a location within a chamber (e.g., an isolation pen) of a microfluidic device. In various embodiments, the selected discrete illumination region is within a separate region of an isolation pen, which can be configured like any of the isolation pens described herein. In some embodiments, laser illumination is projected onto a cell-free area of ​​the chamber, such as an area defined by a piston as the target region. In some embodiments, laser illumination is projected onto a target region area near the tip of the chamber. In certain embodiments, an OEP force is first applied to move cells cultured within the chamber away from the tip to create a cell-free area, and then an in situ generated piston is formed, after which laser illumination can be applied to a substantially cell-free or cell-free target region to generate a bubble. One method of using laser illumination to transport cells in the presence of a hydrogel piston is discussed further below and illustrated in Figures 6A, 6B, and 7.

[0073]

[0085] The geometry of the in situ generated piston can be selected to facilitate removal of cells from the pen. Without intending to be bound by any theory, in some embodiments, the in situ generated piston is designed to have a structural weakness such that upon application of a threshold pressure (e.g., the force generated by bubbles generated by laser illumination), the in situ generated barrier can deform, displace, invert, slip, or otherwise be pushed or repositioned from its original position, transmitting force to the cells and thus displacing them. In other words, in some embodiments, the in situ generated piston will facilitate the movement of cells into the microfluidic channel after laser illumination.

[0074]

[0086] Thus, various exemplary shapes of in situ generated pistons can be formed within the chamber. These include, but are not limited to, a bar (extending from both sides of the wall or with one or more gaps between the bar and the wall), a rectangular half or three-quarters bar, a simple or convex polygon, a dart polygon, a concave polygon, a compound polygon, an irregular polygon, a rectangular polygon with additional thickness at both ends, a side bar (two discrete side rectangular bars separated by a gap and extending from both sides of the wall), a single triangular bar, a V-shaped bar, and a V-cap (a rectangular bar with a V-shaped cap on the side of the piston facing the opening to the channel). In some embodiments, the in situ generated piston can have one or more gaps.

[0075]

[0087] Furthermore, the size of the components of the hydrogel piston can vary considerably. In some embodiments, a non-uniform piston can have a variety of non-uniform dimensions. The non-uniformity can be in the width (pen wall-pen wall dimension), the "thickness" dimension (the dimension from the opening away from the pen to the opening close to the pen), and / or the "height" dimension (from the inner surface of the substrate to the inner surface of the cover of the microfluidic device, e.g., the z-dimension).

[0076]

[0088] In some embodiments, the in situ generated guide element can be a variety of shapes, but includes a gap of an appropriate size to allow removal of a micro-object from the pen under a pen removal force, but to prevent the micro-object from entering the pen after the pen removal force is removed. In some embodiments, the shape of the in situ generated guide element can be, but is not limited to, a bar (extending from both sides of the wall of the connection region, but with one or more gaps between the bar and the wall), a rectangular 1 / 2 or 3 / 4 bar, a simple or convex polygon, an arrowhead polygon, a concave polygon, a compound polygon, a trapezoid, an irregular polygon, a triangle, a wedge, and / or a teardrop.

[0077]

[0089] As shown in the pictorial example of FIG. 6A, an isolation pen 610 of a microfluidic device prepared for removing micro-objects from the pen includes at least one in situ generation barrier, i.e., piston 602. The piston generation defines two regions of the pen 610: a culture region 604 closer to the opening of the pen 610 to the microfluidic channel 612 and configured to contain a plurality of micro-objects (such as, but not limited to, cells), and a target region 606 further from the opening of the pen 610 to the microfluidic channel 612 and configured to be substantially free or devoid of micro-objects. Also shown is an optional additional in situ generation barrier, in situ generation guide element 614, established toward the proximal end of the pen 610 with an opening to the microfluidic channel 612. The generation of the in situ generation guide element 614 can help prevent re-entry of micro-objects from the microfluidic channel 612 into the pen after removal from the pen. The piston and in situ generation guide element 614 can be implemented alone or in combination. That is, piston 602 may or may not be used in conjunction with in situ guide element 614. Similarly, in situ generated guide element 614 may or may not be used in conjunction with piston 602.

[0078]

[0090] As shown in the example photograph in Figure 6B, the isolation pen 610 of a microfluidic device, prepared for removing a micro-object 616 from the pen, includes at least one in situ generated barrier, i.e., piston 602. As shown in Figure 6B, the piston 602 is concave in the center on both sides so that the side facing the tip of the isolation pen 610 and the side facing the base of the isolation pen 610 provide structurally weaker sections. That is, the thickness of the piston 602, when viewed from above, varies, being thicker along the wall of the isolation pen 610 extending from the base opening to the tip and thinner in the middle between the two thicker sections. This geometry can aid in displacing a micro-object 616 toward or into a microfluidic channel 612, as described herein. However, in some embodiments, the depressions may only be on the side of the piston 602 facing or adjacent to the target area 606, and the opposite side of the piston 602 may have a different shape (e.g., no depressions or may be substantially flat, with the flat side perpendicular to the axis defined by the proximal and distal openings of the pen, or alternatively, the flat side slopes toward the proximal opening of the pen). The depressions on the side facing the target area 606 may be useful to promote improved bubble formation and corresponding direction of displacement force as described herein.

[0079]

[0091] Furthermore, the creation of the piston defines two regions of the pen 610: a culture region 604 closer to the opening of the pen 610 to the microfluidic channel 612 and configured to contain a plurality of micro-objects 616 (such as, but not limited to, cells, as shown in the photograph); and a target region 606 further from the opening of the pen 610 to the microfluidic channel 612 and configured to be free or substantially free of micro-objects 616. Also shown is an optional additional in situ generation barrier, the in situ generation guide element 614. The in situ generation guide element 614 is "teardrop" shaped, with a thicker portion closer to the opening of the isolation pen 610 (or closer to the microfluidic channel) and tapering to a thinner end closer to the tip of the isolation pen 610 (or further from the microfluidic channel). This geometry enables a funnel-like effect that aids in the displacement of the micro-objects 616 into the microfluidic channel. Furthermore, the generation of the in situ generated guide element 614 acts as a connection region barrier that can help prevent the micro-object 616 from re-entering the isolation pen from the microfluidic channel 612 following removal or displacement from the pen.

[0080]

[0092] As shown in the series of time-lapse photographic representations of an example of pen removal in Figure 7, an isolation pen 610 of a microfluidic device prepared for removal of a micro-object 616 from the pen includes at least one in situ generated barrier, i.e., piston 602. Time-lapse photographs 750, 751, 752, 753, 754, 755, 756, 757, 758, and 759 were taken over a 10-second period and illustrate the removal of a micro-object 616 (such as, but not limited to, a cell, as shown here) from pen 610 through the generation of a displacement force initiated by laser 702 generated in displacement force generation region 606. As shown in the photograph at 750, in situ generated piston 602 separates the pen into two distinct regions: a culture region containing the micro-object 616 and a target region 606 that is free of the micro-object.

[0081]

[0093] As shown in the photographs at 751, 752, and 753, a displacement force is generated at the target area, in this example through laser illumination 702 and the generation of a bubble 704, which heats the fluid and / or causes the bubble to expand, pushing toward piston 602 and facilitating the removal of micro object 616 from the pen into microfluidic channel 612. As shown in the photograph at 754, as bubble 704 expands, piston 602 is forced to deform, displace, and / or otherwise move (706) in response to bubble expansion 704 (piston 602 deformed, displaced, and / or otherwise moved).

[0082]

[0094] While the laser illumination 702 is applied, operating parameters related to how the laser operates can also be adjusted to further facilitate movement of the micro-object 616 toward the microfluidic channel. For example, as shown in the photographs at 755, 756, and 757 in Figure 7, the position of the laser illumination 702 can be moved over a period of time from a starting point within the target area 606 to closer to the opening of the pen 610 to further expand the bubble throughout the isolation pen 610 and incubation area 604.

[0083]

[0095] In other implementations, the laser illumination 702 (e.g., the power per unit area delivered by the laser illumination 702 incident on the microfluidic device) can also be adjusted. As another example, the speed and / or acceleration at which the laser illumination 702 moves from the distal end of the isolation pen 610 toward the proximal end, which is closer to the opening to the microfluidic channel, can be adjusted. Additionally, any of the described operating parameters can be adjusted alone or in combination with any other operating parameter. For example, any combination of adjusting the position, intensity, speed, or acceleration of the laser illumination can be adjusted together.

[0084]

[0096] As shown in the photographs at 758 and 759, following cessation of laser illumination 702, bubble 710 begins to contract, drawing the removed micro-objects 708 back toward the opening of isolation pen 610, and an optional additional in situ generated barrier, such as in situ generated guide element 614, can prevent the removed micro-objects 708 from migrating back into isolation pen 610. Thus, over time, the micro-objects 616 that were in the isolation pen move toward the opening to the microfluidic channel and exceed the in situ generated guide element 614. When laser illumination 702 is stopped, some of the fluid medium quickly returns to the isolation pen, which may again pull some of the micro-objects 616 back toward the opening of the isolation pen. In some cases, some of the micro-objects 616 may move from the microfluidic channel back into the isolation pen. However, because the in situ generated guide element 614 is formed at the isolation pen's opening to the microfluidic channel, the majority of the micro-objects 616 remain in the microfluidic channel and do not return to the isolation pen. The flow of fluid medium within the microfluidic channel can then be resumed, sweeping away the micro-objects 616 for collection or removal for further analysis or use. Thus, the in situ generated guide element 614 can prevent or reduce the number of micro-objects 616 that may return into the isolation pen, thereby providing more micro-objects 616 for further analysis.

[0085]

[0097] Figure 8 is a block diagram of the displacement of a micro-object from an isolation pen of a microfluidic device. In Figure 8, a light source is directed at a target area of ​​the isolation pen (810). For example, in Figure 6A, a light source (e.g., a laser) illuminates the surface of the isolation pen 610, which is within target area 606. Target area 606 is at the tip of the isolation pen from the opening to the microfluidic channel and is separated from incubation area 604 (within which the micro-object is placed) by piston 602.

[0086]

[0098] Next, illumination of the light source at the target region generates a displacement force toward the in situ generated barrier (820). For example, in FIG. 6A, illumination using the light source heats the fluid medium in the target region 606. This forms a bubble, which expands and pushes, deforms, displaces, and / or moves the piston 602 in response to the bubble expansion. The micro-objects in the incubation region are then displaced toward the microfluidic channel (830). For example, in FIG. 6A, the micro-objects are pushed from the isolation pen 610 into the microfluidic channel 612.

[0087]

[0099] FIG. 9 is another block diagram of the displacement of micro-objects from an isolation pen of a microfluidic device. In FIG. 9, an in situ generated barrier is formed in the isolation pen (910). For example, as discussed in this disclosure, a flowable hydrogel polymer and a photoinitiator can be introduced into the microfluidic device and the isolation pen. A light source (e.g., a DMD-based light source) is used to photopattern the flowable hydrogel polymer and the photoinitiator, generating a solidified hydrogel barrier having a shape based on the photopatterning. An example is shown in FIG. 6B, where piston 602 is the generated hydrogel barrier. A micro-object can then be positioned in the isolation pen (920). For example, micro-object 616 can be positioned in the isolation pen between piston 602 and the opening of the isolation pen to the microfluidic channel. In FIG. 6B, this is incubation region 604. Thus, the piston 602 is positioned within the isolation pen 610 to separate the target region 606, which is on the side of the piston 602 closer to or facing the distal end of the isolation pen 610, from the incubation region 604, which is on the side of the piston 602 closer to or facing the proximal end of the isolation pen 610 to the opening to the microfluidic channel 612. The micro-object is then displaced from the isolation pen (930). For example, in Figure 6B, the micro-object 616 can be displaced as discussed with respect to Figure 8 and elsewhere in this disclosure.

[0088]

[0100] 6B is formed, thus defining a target region 606 and an incubation region 604 on opposite sides of the piston 602. Subsequently, a minute object 616 is placed within the incubation region 604. The minute object 616 can then be displaced from the isolation pen 610 using techniques described herein with respect to bubble formation.

[0089]

[0101] 6B is formed, thus defining the target region 606 and the incubation region 604 on opposite sides of the piston 602. Subsequently, a micro-object 616 is placed within the incubation region 604. Next, an in situ generated guide element 614 is formed at the opening of the isolation pen 610 to the microfluidic channel 612. Finally, the micro-object 616 can be displaced from the isolation pen 610 using the techniques described herein for bubble formation with the in situ guide element 614.

[0090]

[0102] A device and method for displacing and / or removing one or more micro-objects from a pen using optically driven convection and micro-object displacement.

[0091]

[0103] Other information regarding optical illumination, laser power, and bubble movement is described, for example, in U.S. Pat. No. 10,829,728 (Kurz et al., published November 10, 2020) and U.S. Patent Publication No. 2022 / 0033758 (Sackmann et al., published February 3, 2022), the contents of which are incorporated herein by reference in their entirety.

[0092]

[0104] Micro-objects, such as biological cells or embryos, can be moved to a local environment, such as within a microfluidic device, by several forces, including, but not limited to, gravity, fluid flow induced by a mechanical pump, electrowetting, and / or dielectrophoresis (DEP). To more efficiently move a micro-object from one location (e.g., a specific location where the micro-object has been cultured within the microfluidic device, e.g., a pen) to another location (e.g., another area of ​​the same microfluidic device, e.g., a microfluidic channel), a variable force vector can be applied to achieve cell repositioning. While dielectrophoresis (DEP), fluid displacement, etc., may be sufficient to move cells as desired, forces applied in different ways (e.g., convective forces, shear flow forces, impact forces such as cavitation or contact with a bubble meniscus, or any combination thereof) and / or on different time scales (e.g., from a few milliseconds to a few minutes) and / or at different magnitudes (e.g., larger or more localized forces) can also be employed to assist the movement of cells from their current location and / or to a selected location. In one non-limiting example, application of forces in addition to and / or other than DEP can be useful for displacing biological cells that have been cultured within a microfluidic device for a period of time. In some embodiments, the cells may be attached to the surface of the microfluidic device, and the DEP force may be sufficient to displace the cells from their attached location. In other embodiments, the DEP force or gravity may not be sufficient to displace the cells from their attached location.

[0093]

[0105] Optical illumination of selected discrete regions on or within a microfluidic device can heat a portion of a fluid medium within the microfluidic circuit of the microfluidic device, providing a variety of displacement forces of different magnitudes, physical types, and / or time scales that can displace micro-objects (including, but not limited to, biological cells) and / or mix the fluid medium (which may contain micro-objects, including biological cells) within the microfluidic device. As described herein, in situ generated barriers, such as pistons, can be generated to facilitate separation of a culture region from the force-generation region and facilitate displacement of micro-objects while maintaining a high level of viability. Such displacement force generation can be applied more than once at the same selected discrete region or adjacent regions (e.g., selected sites within the force-generation region), thereby allowing repeated application of forces to remove micro-objects from the pen and / or mix the medium (which may contain micro-objects) while remaining sufficiently non-destructive to the micro-objects. Transpositioning cells from one area, which in some embodiments may be a chamber, an isolation pen, or other microfluidic circuit element of another microfluidic device, to another area and / or location within the microfluidic device can be achieved by applying pulses of optical illumination to selected discrete regions within the microfluidic device (e.g., selected sites within a force-generation region). The applied force vector is a function of the energy, duration, and location of the pulse of optical illumination. In some embodiments, the pulse of optical illumination can be used to locally heat the surrounding cell medium (i.e., fluid medium), thereby increasing the local vapor pressure and creating a vapor-fluid interface that creates bubbles. The effect of heat-induced bubble generation on the surrounding fluid medium and / or cells can vary depending on the duration and configuration of the microfluidic device and / or thermal target. Some types of effects can include:

[0094] Cavitation

[0106] A short pulse of light can be used to heat a thermal target and generate a transient bubble, which creates a cavitation force that acts to propel a piston that can displace cells placed in the proximal region of the chamber.

[0095] shear flow

[0107] In other embodiments, the bubble may grow with continued illumination, creating a shear flow of fluid directed at the piston, thereby displacing and / or removing minute objects from the pen.

[0096] Meniscus Contact

[0108] Alternatively, bubbles created by heating the fluid medium at the site of the thermal target can be directed toward the piston. As the bubble moves, the meniscus of the bubble can propel the piston, displacing cells located in the proximal region of the chamber.

[0097]

[0109] In other embodiments, the bubbles are allowed to grow until they become thermodynamically favorable for stability and survival within the fluid medium, and the bubbles can then displace the surrounding liquid phase, propelling the piston and displacing cells disposed in the proximal region of the chamber.

[0098] optical lighting

[0110] The optical illumination may be a coherent light source (e.g., a laser) or a non-coherent light source. The coherent light source may be a laser characterized by a wavelength in the visible light spectrum (e.g., red wavelengths such as 662 nm), or a laser characterized by a wavelength in the infrared portion of the spectrum (e.g., near-infrared wavelengths such as 785 nm), or a laser having any other suitable wavelength. The non-coherent light source may include light having a wavelength in the visible range and / or may include light having a wavelength in the ultraviolet (UV) or infrared range. The light source may provide structured or unstructured light. The temperature gradient introduced by illumination with the light source may be modulated by increasing or decreasing the intensity of the light source. The structured light source may be modulated in several ways to control the properties of the structured light source (e.g., using a DMD to spatially modulate the light source or using an aperture and objective lens to modulate the light source).

[0099]

[0111] Without being bound by theory, the incident optical illumination may be transmitted through a transparent, substantially transparent, and / or translucent cover or base of the enclosure microfluidic device. After transmitting through the enclosure cover or base, the incident illumination can be directed to a thermal target configured to convert the optical illumination into thermal energy, as described below.

[0100] electric power

[0112] The non-coherent light can be projected in the range of about 1 milliwatt (mW) to about 1,000 milliwatts (mW), but is not limited to this range. In some embodiments, the power of the non-coherent light, whether structured or not, can be in the range of about 1 mW to about 500 mW, about 1 mW to about 100 mW, about 1 mW to about 50 mW, about 1 mW to about 20 mW, about 10 mW to about 500 mW, about 10 mW to about 200 mW, about 10 mW to about 100 mW, about 50 mW to about 800 mW, about 50 mW to about 500 mW, about 50 mW to about 200 mW, about 75 mW to about 700 mW, about 75 mW to about 400 mW, about 75 mW to about 175 mW, or any value therebetween. Depending on the area the light is focused onto and the duration of illumination, the power of the incoherent light may be greater or less than any of the power levels mentioned above.

[0101]

[0113] Coherent light can be projected in a range of about 1 mW to about 1000 mW, but is not limited to this range. In some embodiments, the power of the coherent light can be greater or less than any of the power levels listed above, depending on the area the light is focused onto and the duration of illumination. In some embodiments, the power of the coherent light can be in the range of about 1 mW to about 500 mW, about 1 mW to about 100 mW, about 1 mW to about 50 mW, about 1 mW to about 20 mW, about 10 mW to about 500 mW, about 10 mW to about 200 mW, about 10 mW to about 100 mW, about 50 mW to about 800 mW, about 50 mW to about 500 mW, about 50 mW to about 200 mW, about 75 mW to about 700 mW, about 75 mW to about 400 mW, about 75 mW to about 175 mW, or any value therebetween.

[0102]

[0114] The power of the incident light can be selected differently based on the desired movement force and / or the type of force to be removed from the pen. For example, if a perfusion, which may incorporate Marangoni effect flow, is desired, the power of the incident light can be selected as low as 1 mW and modulated as the perfusion is established and / or maintained. If micro-objects are to be moved by the use of cavitation forces, shear flow forces, or bubble contact forces, the power can be selected to be in a higher range, for example, from about 10 mW to about 100 mW. The power can also be adjusted based on the desired duration of illumination.

[0103] Lighting Site

[0115] The illumination sites may be selected to be any selected discrete region of the microfluidic device as may be useful. In most embodiments, at least one illumination site is within the displacement force generation site. In some embodiments, the illumination sites may move over time. In some embodiments, the selected discrete region of illumination may be a location within an isolation pen of the microfluidic device, particularly within a target region described herein. In various embodiments, the selected discrete illumination region is within an isolation region of the isolation pen. In various embodiments, the selected discrete illumination region is contained within the target region of the isolation pen.

[0104] Thermal Target

[0116] In some embodiments, the thermal target is a microfluidic feature of a microfluidic device, which may be a separate feature designed for this purpose. Alternatively, in some embodiments, the thermal target may be a location within a microfluidic circuit where optical illumination is applied. The thermal target is a passive microfluidic feature and does not include any self-activating resistors or electric heaters. The passive nature of the thermal target simplifies fabrication of the microfluidic device. For thermal targets including metals or microstructures, fabrication is much less complex than active thermal targets such as resistors, as described below. Active thermal targets such as resistors, unlike the passive thermal targets of the present disclosure, must have fixed electrical connections and are fabricated in fixed locations. When the thermal target is a selected location of the microfluidic circuit material or base, the flexibility to specifically and selectively generate forces when needed without additional structural features is particularly advantageous compared to fixed active thermal targets. In some embodiments, the thermal target is within a target area defined by an in situ generated piston.

[0105] Lighting period

[0117] The illumination step may be performed using any light source as described herein, and may be coherent or non-coherent. The light may be structured or unstructured. For simplicity, the following description will refer to laser illumination, although the invention is not so limited.

[0106]

[0118] In various embodiments, illuminating selected discrete sites (e.g., sites within the displacement force-generating region) with laser illumination can include illuminating the selected discrete sites with a laser. The laser can illuminate with light having a wavelength ranging from about 450 nm to about 800 nm. The laser can have a current of about 0.5 amps, 0.7 amps, 0.9 amps, 1.1 amps, 1.4 amps, 1.6 amps, 1.6 amps, 2.0 amps, 2.2 amps, 2.5 amps, 2.7 amps, 3.0 amps, or any value therebetween.

[0107]

[0119] The laser illumination may have an incident power in the range of about 1 mW to about 1000 mW, about 100 mW to about 1000 mW, about 100 mW to about 800 mW, about 100 mW to about 600 mW, about 100 mW to about 500 mW, or any range or individual value therebetween.

[0108]

[0120] In various embodiments, illuminating selected discrete sites (e.g., sites within the displacement force-generating region) with laser illumination may be performed for a time period ranging from about 10 microseconds to about 8000 milliseconds, and any value therebetween, hi some other embodiments, illuminating selected discrete sites (e.g., sites within the displacement force-generating region) may be performed for a time period ranging from about 100 milliseconds to about 2 minutes.

[0109]

[0121] In various embodiments, laser illumination can be directed to selected discrete sites (e.g., sites within a displacement force-generating region) for about 50 milliseconds, about 75 milliseconds, about 100 milliseconds, about 150 milliseconds, about 250 milliseconds, about 500 milliseconds, about 750 milliseconds, or about 1000 milliseconds. In various embodiments, laser illumination can be directed to selected discrete sites (e.g., sites within a displacement force-generating region) for a time period ranging from about 50 milliseconds to about 2000 milliseconds, from about 50 milliseconds to about 1000 milliseconds, from about 50 milliseconds to about 500 milliseconds, from about 50 milliseconds to about 300 milliseconds, from 100 milliseconds to about 1000 milliseconds, from about 200 milliseconds to about 1000 milliseconds, from about 200 milliseconds to about 700 milliseconds, from about 300 milliseconds to about 600 milliseconds, or any value therebetween. In other embodiments, laser illumination may be directed at selected discrete sites (e.g., sites within the displacement force-generating region) for a time period ranging from about 1 millisecond to about 200 milliseconds, about 1 millisecond to about 150 milliseconds, about 1 millisecond to about 100 milliseconds, about 1 millisecond to about 50 milliseconds, about 1 millisecond to about 30 milliseconds, about 25 milliseconds to about 200 milliseconds, about 25 milliseconds to about 100 milliseconds, about 25 milliseconds to about 75 milliseconds, about 50 milliseconds to about 200 milliseconds, about 50 milliseconds to about 125 milliseconds, about 50 milliseconds to about 90 milliseconds, or any value therebetween. An illumination period selected in one of these ranges may be sufficient to optically drive the generation of a bubble that can contact the piston and / or the micro-object, thereby displacing the micro-object and / or facilitating its removal from the pen.

[0110]

[0122] In various other embodiments, the laser illumination is for a period of time ranging from about 500 milliseconds to about 3000 milliseconds, from about 1000 milliseconds to about 2700 milliseconds, from about 1000 milliseconds to about 2500 milliseconds, from about 1000 milliseconds to about 2000 milliseconds, from about 1000 milliseconds to about 1500 milliseconds, from about 1300 milliseconds to about 3000 milliseconds, from about 1300 milliseconds to about 2700 milliseconds, from about 1300 milliseconds to about 2300 milliseconds, from about 1300 milliseconds to about 2000 milliseconds, from about 1300 milliseconds to about The illumination may be directed at selected discrete sites (e.g., sites within the displacement force-generating region) for a time period ranging from about 1700 milliseconds, about 1500 milliseconds to about 3000 milliseconds, about 1500 milliseconds to about 2600 milliseconds, about 1500 milliseconds to about 2300 milliseconds, about 1500 milliseconds to about 2000 milliseconds, about 1700 milliseconds to about 3000 milliseconds, about 1700 milliseconds to about 2600 milliseconds, about 1700 milliseconds to about 2000 milliseconds, or any value therebetween. Illumination periods chosen within one of these ranges may be suitable for optically driven shear flow or bubble flow contact forces.

[0111]

[0123] In still other embodiments, the step of illuminating selected discrete sites (e.g., sites within the displacement force-generating region) with laser illumination may be performed for about 10 μsec to about 200 milliseconds, about 10 μsec to about 100 milliseconds, about 10 μsec to about 1 millisecond, about 10 μsec to about 1 millisecond, about 10 μsec to about 1 millisecond, about 10 μsec to about 500 μsec, about 50 μsec to about 1 millisecond, about 50 μsec to about 500 μsec, about 50 μsec to about 300 μsec, about 1 millisecond to about 200 milliseconds, about 1 millisecond to about 150 milliseconds, about 1 millisecond to about 100 milliseconds, about 1 millisecond to about 50 milliseconds, about 1 millisecond to about 30 milliseconds, about 25 milliseconds to about 200 milliseconds, about 25 milliseconds to about 100 milliseconds, about 25 milliseconds to about 75 milliseconds, about 50 milliseconds to about 200 milliseconds, about 50 milliseconds to about 125 milliseconds, about 50 milliseconds to about 90 milliseconds, or any value between any ranges thereof. The illumination period within one such illumination range may be sufficient to generate cavitation forces within discrete selected sites adjacent to the micro-objects (e.g., sites within the displacement force generating region), thereby dislodging one or more of the micro-objects and thus facilitating their future removal from the pen. In some embodiments, the illumination period may range from about 10 μsec to about 500 μsec or from about 10 μsec to about 100 ms.

[0112]

[0124] In some other embodiments, illuminating selected discrete sites (e.g., sites within the displacement force-generating region) with laser illumination may be performed for about 100 milliseconds to about 3 minutes, about 100 milliseconds to about 2 minutes, about 100 milliseconds to about 1 minute, about 100 milliseconds to about 10,000 milliseconds, about 100 milliseconds to about 5,000 milliseconds, about 100 milliseconds to about 1,000 milliseconds, about 500 milliseconds to about 3 minutes, about 500 milliseconds to about 1 minute, about 500 milliseconds to about 10,000 milliseconds, about 500 milliseconds to about 3,000 milliseconds, or any value therebetween.

[0113]

[0125] These ranges are merely examples and are not intended to limit the present disclosure: illumination periods outside the ranges described for each type of convection or displacement force may be identified and used and still fall within the scope of the present disclosure.

[0114] kit

[0126] In view of the above, kits for carrying out the methods of the present disclosure can be provided. In some embodiments, the kit includes a flowable polymer, e.g., a prepolymer configured to be controllably activated to form at least one in situ generated barrier, which is a piston comprising a solidified polymer network, and, optionally, an additional prepolymer configured to be controllably activated to form one or more additional in situ generated barriers comprising a solidified polymer network, the in situ generated barriers having porosity that substantially prevents cells from crossing the in situ generated barriers. The kit may further include instructions on how to create a shape suitable for in situ generated barrier formation and appropriate chamber locations for generating the shape. The kit may further include an inhibitor. The inhibitor may be packaged integrally with the flowable polymer or separately packaged. In some other embodiments, the kit may include a first instance of the inhibitor packaged integrally with the flowable polymer and a second instance of the inhibitor packaged separately. In some other embodiments, the kit may further include a photoinitiator, such as a photoinitiator suitable for inducing and / or catalyzing polymerization of the prepolymer.

[0115]

[0127] In some embodiments, the kit further comprises a microfluidic device comprising a microfluidic circuit including a flow region and a chamber, the chamber including an opening to the flow region. The microfluidic device can be any microfluidic device as described herein.

[0116] Characteristics and interoperability of microfluidic devices and systems

[0128] It should be understood that various features of the microfluidic devices, systems, and driving techniques described herein may be combined or interchangeable. For example, features described herein with reference to microfluidic devices 100, 175, 200, 300, 320, 400, 450, 520, and system attributes as depicted in Figures 1A-5B may be combined or interchangeable.

[0117] Microfluidic Devices

[0129] 1A shows an example microfluidic device 100. A perspective view of the microfluidic device 100 is shown with a cover 110 partially cut away to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally includes a microfluidic circuit 120 including a flow channel 106 through which a fluid medium 180 can flow and, optionally, which can transport one or more micro-objects (not shown) into and / or through the microfluidic circuit 120.

[0118]

[0130] As generally shown in FIG. 1A , the microfluidic circuit 120 is defined by an enclosure 102. While the enclosure 102 can be physically structured in different configurations, in the example shown in FIG. 1A , the enclosure 102 is shown to include a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110. The support structure 104, the microfluidic circuit structure 108, and the cover 110 can be attached to one another. For example, the microfluidic circuit structure 108 can be disposed on an inner surface 109 of the support structure 104, and the cover 110 can be disposed above the microfluidic circuit structure 108. Together with the support structure 104 and the cover 110, the microfluidic circuit structure 108 can define elements of the microfluidic circuit 120, forming a three-layer structure.

[0119]

[0131] As shown in FIG. 1A , the support structure 104 can be at the bottom of the microfluidic circuit 120, and the cover 110 can be at the top of the microfluidic circuit 120. Alternatively, the support structure 104 and the cover 110 can be configured in other orientations. For example, the support structure 104 can be at the top of the microfluidic circuit 120, and the cover 110 can be at the bottom of the microfluidic circuit 120. Regardless, there can be one or more ports 107, each comprising a passageway into or out of the enclosure 102. Examples of passageways include valves, gates, through-holes, etc. As shown, the port 107 is a through-hole created by a gap in the microfluidic circuit structure 108. However, the port 107 can also be in other components of the enclosure 102, such as the cover 110. While only one port 107 is shown in FIG. 1A , the microfluidic circuit 120 can have two or more ports 107. For example, there may be a first port 107 that functions as an inlet for fluid to enter the microfluidic circuit 120, and there may be a second port 107 that functions as an outlet for fluid to exit the microfluidic circuit 120. Whether a port 107 functions as an inlet or an outlet may depend on the direction in which the fluid flows through the channel 106.

[0120]

[0132] The support structure 104 can include one or more electrodes (not shown) and a substrate or multiple interconnected substrates. For example, the support structure 104 can include one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates can be electrically connected to a single electrode). The support structure 104 can further include a printed circuit board assembly ("PCBA"). For example, the semiconductor substrates can be mounted on a PCBA.

[0121]

[0133] The microfluidic circuit structure 108 can define circuit elements of the microfluidic circuit 120. Such circuit elements, such as flow regions (which may or may not include one or more flow channels), chambers (this class of circuit elements may also include a subclass including isolation pens), traps, and the like, can include spaces or regions that can be fluidically interconnected when the microfluidic circuit 120 is filled with fluid. Circuit elements can also include barriers, etc. In the microfluidic circuit 120 shown in FIG. 1A , the microfluidic circuit structure 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 can partially or completely surround the microfluidic circuit material 116. The frame 114 can be, for example, a relatively rigid structure that substantially surrounds the microfluidic circuit material 116. For example, the frame 114 can include a metallic material. However, the microfluidic circuit structure need not include a frame 114. For example, the microfluidic circuit structure can consist of (or consist essentially of) the microfluidic circuit material 116.

[0122]

[0134] The microfluidic circuit material 116 can be patterned with cavities and the like to define the circuit elements and interconnections of the microfluidic circuit 120, such as chambers, pens, and microfluidic channels. The microfluidic circuit material 116 can include a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”)), which can be gas-permeable. Other examples of materials from which the microfluidic circuit material 116 can be formed include etchable materials, such as molded glass, silicone (e.g., photopatternable silicone or “PPS”), photoresist (e.g., SU8), and the like. In some embodiments, such materials—and thus the microfluidic circuit material 116—can be rigid and / or substantially gas-impermeable. Nevertheless, the microfluidic circuit material 116 can be disposed on the support structure 104 and within the frame 114.

[0123]

[0135] The microfluidic circuit 120 can include a flow region, and one or more chambers can be disposed in and / or fluidly connected to the flow region. The chambers can have one or more openings fluidly connecting the chambers with one or more flow regions. In some embodiments, the flow region includes or corresponds to a microfluidic channel 122. While a single microfluidic circuit 120 is shown in FIG. 1A, a suitable microfluidic device can include multiple (e.g., two or three) such microfluidic circuits. In some embodiments, the microfluidic device 100 can be configured to be a nanofluidic device. As shown in FIG. 1A, the microfluidic circuit 120 can include multiple microfluidic isolation pens 124, 126, 128, and 130, each of which can have one or more openings. In some embodiments of the isolation pens, the isolation pen can have only one opening in communication with the flow path 106. In some other embodiments, the isolation pen can have more than two openings, e.g., n openings, in communication with the flow channel 106, but n-1 openings are valved such that all but one opening is closable. When all valved openings are closed, the isolation pen limits exchange of material from the flow region into the isolation pen to occur solely by diffusion. In some embodiments, the isolation pen includes various features and structures (e.g., separation regions) optimized to retain micro-objects within the isolation pen (and thus within a microfluidic device such as microfluidic device 100) even when medium 180 is flowing through the flow channel 106.

[0124]

[0136] The cover 110 can be an integral part of the frame 114 and / or the microfluidic circuit material 116. Alternatively, the cover 110 can be a structurally separate element, as shown in FIG. 1A. The cover 110 can comprise the same material as the frame 114 and / or the microfluidic circuit material 116, or a different material. In some embodiments, the cover 110 can be an integral part of the microfluidic circuit material 116. Similarly, the support structure 104 can be a separate structure from the frame 114 or the microfluidic circuit material 116, as shown, or an integral part of the frame 114 or the microfluidic circuit material 116. Similarly, the frame 114 and the microfluidic circuit material 116 can be separate structures, as shown in FIG. 1A, or can be integral parts of the same structure. Despite various possible integrations, the microfluidic device can retain a three-layer structure including a cover layer, a base layer, and a cover layer sandwiching a central layer on which the microfluidic circuit 120 is disposed.

[0125]

[0137] In some embodiments, the cover 110 can include a rigid material. The rigid material can be glass or a material having similar properties. In some embodiments, the cover 110 can include a deformable material. The deformable material can be a polymer such as PDMS. In some embodiments, the cover 110 can include both a rigid material and a deformable material. For example, one or more portions of the cover 110 (e.g., one or more portions located above the isolation pens 124, 126, 128, 130) can include a deformable material that interacts with the rigid material of the cover 110. Microfluidic devices having covers including both rigid and deformable materials are described, for example, in U.S. Pat. No. 10,058,865 (Breinlinger et al.), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cover 110 can further include one or more electrodes. The one or more electrodes can include a conductive oxide, such as indium tin oxide (ITO), which can be coated on glass or a similar insulating material. Alternatively, one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices are described, for example, in U.S. Pat. No. 9,227,200 (Chiou et al.), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cover 110 and / or the support structure 104 can be optically transparent. The cover 110 can also include at least one material (e.g., PDMS or PPS) that is gas permeable.

[0126]

[0138] In the example shown in FIG. 1A , the microfluidic circuit 120 is shown including a microfluidic channel 122 and isolation pens 124, 126, 128, and 130. Each pen includes an opening to the channel 122 but is otherwise enclosed such that the pen can substantially isolate micro-objects within the pen from the fluid medium 180 and / or micro-objects within the flow path 106 of the channel 122 or within other pens. The walls of the isolation pen extend from the inner surface 109 of the base to the inner surface of the cover 110, providing an enclosure. The isolation pen's opening to the microfluidic channel 122 is oriented at an angle to the flow 106 of the fluid medium 180 such that the flow 106 is not directed into the pen. The bulk fluid flow vector within the channel 122 may be tangential or parallel to the plane of the isolation pen's opening and is not directed into the pen's opening. In some cases, the pens 124, 126, 128, and 130 are configured to physically isolate one or more micro-objects within the microfluidic circuit 120. Isolation pens according to the present disclosure can include a variety of shapes, surfaces, and features optimized for use in conjunction with DEP, OET, OEW, fluid flow, magnetic force, centripetal force, and / or gravity, as discussed and illustrated in detail below.

[0127]

[0139] Microfluidic circuit 120 can include any number of microfluidic isolation pens. While five isolation pens are shown, microfluidic circuit 120 may have fewer or more isolation pens. As shown, microfluidic isolation pens 124, 126, 128, and 130 of microfluidic circuit 120 each have different features and configurations that may provide one or more advantages useful for maintaining, isolating, assaying, or culturing biological micro-objects. In some embodiments, microfluidic circuit 120 includes multiple identical microfluidic isolation pens.

[0128]

[0140] In the embodiment shown in FIG. 1A, a single flow path 106 is shown including a single channel 122. However, other embodiments may include multiple channels 122 within the single flow path 106, as shown in FIG. 1B. The microfluidic circuit 120 further includes an inlet valve or port 107 in communication with the flow path 106, thereby allowing the fluid medium 180 to access the flow path 106 (and the channel 122). In some cases, the flow path 106 includes a substantially straight path. In other cases, the flow path 106 is arranged in a nonlinear or serpentine manner, such as in a zigzag pattern, such that the flow path 106 moves across the microfluidic device 100 more than once, e.g., in alternating directions. Flow in the flow path 106 may proceed from the inlet to the outlet or may reverse and proceed from the outlet to the inlet.

[0129]

[0141] A multichannel device, an example microfluidic device 175, is shown in FIG. 1B, which may otherwise resemble microfluidic device 100. Microfluidic device 175 and its constituent circuit elements (e.g., channels 122 and isolation pen 128) may have any of the dimensions discussed herein. The microfluidic circuit shown in FIG. 1B has two inlet / outlet ports 107 and a flow path 106 including four separate channels 122. The number of channels into which the microfluidic circuit is subdivided may be selected to reduce fluidic resistance. For example, the microfluidic circuit may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more channels to provide a selected range of fluidic resistance. Microfluidic device 175 further includes multiple isolation pen openings from each channel 122, each isolation pen being similar to isolation pen 128 of FIG. 1A and may have any of the dimensions or functionality of any isolation pen as described herein. However, the isolation pens of microfluidic device 175 can have different shapes, such as any of the shapes of isolation pens 124, 126, or 130 in FIG. 1A or as described anywhere else herein. Additionally, microfluidic device 175 can include isolation pens with a mix of different shapes. In some cases, multiple isolation pens are configured (e.g., relative to channel 122) such that multiple isolation pens can be loaded with target micro-objects in parallel.

[0130]

[0142] 1A , the microfluidic circuit 120 may further include one or more optional micro-object traps 132. The optional traps 132 may be formed in walls that bound the channel 122 and may be located opposite one or more openings of the microfluidic isolation pens 124, 126, 128, 130. The optional traps 132 may be configured to receive or capture a single micro-object from the flow path 106 or may be configured to receive or capture multiple micro-objects from the flow path 106. In some cases, the optional traps 132 have a volume approximately equal to the volume of a single target micro-object. In some cases, the traps 132 include side passages 134 that are smaller than the target micro-objects to facilitate flow through the traps 132.

[0131] Isolation pen

[0143] The microfluidic devices described herein may include one or more isolation pens, each suitable for holding one or more microscopic objects (e.g., biological cells, groups of cells associated together). The isolation pens may be positioned within and open to the flow region, which in some embodiments is a microfluidic channel. Each of the isolation pens may have one or more openings for communicating with one or more microfluidic channels. In some embodiments, an isolation pen may have only one opening to a microfluidic channel.

[0132]

[0144] 2A-2C show isolation pens 224, 226, and 228 of microfluidic device 200, which may be like isolation pen 128 of FIG. 1A. Each isolation pen 224, 226, and 228 may include a separation region 240 and a connection region 236 that fluidly connects the separation region 240 to a flow region, which in some embodiments may include a microfluidic channel, such as channel 122. Connection region 236 may include a proximal opening 234 to the flow region (e.g., microfluidic channel 122) and a distal opening 238 to separation region 240. Connection region 236 may be configured such that the maximum penetration depth of the flow of fluid medium (not shown) flowing through microfluidic channel 122 beyond isolation pens 224, 226, and 228 does not extend into separation region 240, as discussed below with respect to FIG. 2C. In some embodiments, streamlines from the flow in the microfluidic channel do not enter the separation region. Therefore, due to the connection region 236, micro-objects (not shown) or other materials (not shown) placed within the separation region 240 of the isolation pens 224, 226, and 228 can be isolated from and substantially unaffected by the flow of the fluid medium 180 in the microfluidic channel 122.

[0133]

[0145] Each of the isolation pens 224, 226, and 228 in Figures 2A-2C has a single opening that opens directly into the microfluidic channel 122. The isolation pen opening may open laterally from the microfluidic channel 122, as shown in Figure 2A, which shows a vertical cross-section of the microfluidic device 200. Figure 2B shows a horizontal cross-section of the microfluidic device 200. An electrode-activated substrate 206 may underlie both the microfluidic channel 122 and the isolation pens 224, 226, and 228. The top surface of the electrode-activated substrate 206 within the isolation pen enclosure, which forms the floor of the isolation pen, may be positioned at the same height or substantially the same height as the top surface of the electrode-activated substrate 206 within the microfluidic channel 122 (or flow region, if no channel is present), which forms the floor of the flow channel (or flow region, if no channel is present) of the microfluidic device. The electrode-activated substrate 206 may be featureless or may have an irregular or patterned surface that varies from its greatest height to its smallest depression by less than about 3 micrometers (μm), less than about 2.5 μm, less than about 2 μm, less than about 1.5 μm, less than about 1 μm, less than about 0.9 μm, less than about 0.5 μm, less than about 0.4 μm, less than about 0.2 μm, less than about 0.1 μm, or even less. The elevation variation of the top surface of the substrate across both the microfluidic channel 122 (or flow region) and the isolation pen may be about 10% or less, about 7% or less, about 5% or less, about 3% or less, about 2% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.5% or less, about 0.3% or less, or about 0.1% or less of the height of the isolation pen wall. Alternatively, the elevation variation of the top surface of the substrate across both the microfluidic channel 122 (or flow region) and the isolation pen can be about 2% or less, about 1% or less, about 0.9% or less, about 0.8% or less, about 0.5% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less of the substrate height. While described in detail with respect to microfluidic device 200, this can apply to any microfluidic device described herein.

[0134]

[0146] The microfluidic channel 122 and the connection region 236 can be examples of sweep regions, and the separation region 240 of the isolation pens 224, 226, and 228 can be examples of non-sweep regions. Isolation pens such as 224, 226, and 228 have separation regions, each with only one opening to the connection region of the isolation pen. Exchange of fluid media into and out of the separation regions can be substantially limited to occur only by diffusion. As noted, the microfluidic channel 122 and the isolation pens 224, 226, and 228 can be configured to contain one or more fluid media 180. In the example shown in FIGS. 2A and 2B, the port 222 is connected to the microfluidic channel 122, allowing the fluid media 180 to be introduced into or removed from the microfluidic device 200. Prior to the introduction of the fluid media 180, the microfluidic device can be primed with a gas, such as carbon dioxide gas. Once the microfluidic device 200 contains the fluid medium 180, a flow 242 (see FIG. 2C ) of the fluid medium 180 in the microfluidic channel 122 can be selectively generated and stopped. For example, as shown, the ports 222 can be located at different locations (e.g., at opposite ends) of the flow region (microfluidic channel 122), and the flow 242 of the fluid medium can be created from one port acting as an inlet to another port 222 acting as an outlet.

[0135]

[0147] 2C shows a detailed view of an example of an isolation pen 224 that may contain one or more micro-objects 246, according to some embodiments. A flow 242 of fluid medium 180 in the microfluidic channel 122 beyond the proximal opening 234 of the connection region 236 of the isolation pen 224 can cause a secondary flow 244 of the fluid medium 180 into and out of the isolation pen 224. To isolate the micro-objects 246 from the secondary flow 244 in the isolation region 240 of the isolation pen 224, the length L of the connection region 236 of the isolation pen 224 (i.e., from the proximal opening 234 to the distal opening 238) must be greater than or equal to the length L of the connection region 236 of the isolation pen 224. con is the penetration depth D of the secondary flow 244 into the connection region 236 p The penetration depth D should be greater than pdepends on several factors, including the shape of the microfluidic channel 122, which in turn depends on the width W of the connection region 236 at the proximal opening 234. con , the width W of the microfluidic channel 122 at the proximal opening 234 ch , the height H of the channel 122 at the proximal opening 234 ch , and the width of the distal opening 238 of the connection region 236. Among these factors, the width W of the connection region 236 at the proximal opening 234 con and the height H of the channel 122 at the proximal opening 234 ch tends to be the most important. In addition, the penetration depth D p can be affected by the velocity of the fluid medium 180 in the channel 122 and the viscosity of the fluid medium 180. However, these factors (i.e., velocity and viscosity) affect the penetration depth D p For example, the width W of the connecting region 236 at the proximal opening 234 can be varied widely without dramatically changing the width W of the connecting region 236. con is about 50 μm, and the height H ch is about 40 μm, and the width W of the microfluidic channel 122 at the proximal opening 122 ch For a microfluidic chip 200 having a penetration depth D of the secondary flow 244 of about 100 μm to about 150 μm, p is W at a flow rate of 0.1 μL / sec con (i.e., less than 50 μm) at a flow rate of 20 μL / s con (i.e., about 100 μm), and even if the velocity of the fluid medium 180 is increased by 200 times, p This represents an increase of only about 2.5 times.

[0136]

[0148] In some embodiments, the walls of the microfluidic channel 122 and the isolation pens 224, 226, or 228 can be oriented relative to the vector of the flow 242 of the fluid medium 180 in the microfluidic channel 122 as follows: ch(or the cross-sectional area of ​​the microfluidic channel 122) can be substantially perpendicular to the flow 242 of the medium 180; con (or cross-sectional area) can be substantially parallel to the flow 242 of the medium 180 in the microfluidic channel 122; and / or the length L con can be substantially perpendicular to the flow 242 of the medium 180 within the microfluidic channel 122. The above is merely an example, and the relative positions of the microfluidic channel 122 and the isolation pens 224, 226, and 228 may be other orientations with respect to each other.

[0137]

[0149] In some embodiments, in a given microfluidic device, the configuration of the microfluidic channels 122 and openings 234 may be fixed, while the rate of flow 242 of the fluid medium 180 within the microfluidic channels 122 may be variable. Thus, in each isolation pen 224, the penetration depth D of the secondary flow 244 p is the length L of the connection area 236 con the maximum velocity V of the flow 242 of the fluid medium 180 in the channel 122, ensuring that max V max , the resulting secondary flow 244 can be contained entirely within the connection region 236 and will not enter the separation region 240. Thus, the flow 242 of fluid medium 180 within the microfluidic channel 122 (sweep region) is prevented from drawing the micro-objects 246 out of the separation region 240, which is a non-sweep region of the microfluidic circuit, resulting in the micro-objects 246 being retained within the separation region 240. Therefore, by selecting the dimensions of the microfluidic circuit and further selecting the opening parameters (e.g., velocity of the fluid medium 180), the separation region 240 of the isolation pen 224 can be prevented from being contaminated by materials from the microfluidic channel 122 or another isolation pen 226 or 228. However, the chip may be configured such that V max In many microfluidic chip configurations, the V maxNote that you don't need to worry about the

[0138]

[0150] Components (not shown) in the first fluid medium 180 in the microfluidic channel 122 can mix with the second fluid medium 248 in the separation region 240 substantially only by diffusion of the components of the first medium 180 from the microfluidic channel 122 through the connection region 236 and into the second fluid medium 248 in the separation region 240. Similarly, components (not shown) of the second medium 248 in the separation region 240 can mix with the first medium 180 in the microfluidic channel 122 substantially only by diffusion of the components of the second medium 248 from the separation region 240 through the connection region 236 and into the first medium 180 in the microfluidic channel 122. In some embodiments, the degree of fluid medium exchange between the separation region and the flow region of the isolation pen by diffusion is greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% of the fluid exchange.

[0139]

[0151] In some embodiments, first medium 180 may be the same medium or a different medium than second medium 248. In some embodiments, first medium 180 and second medium 248 can start out the same and then become different (e.g., by adjusting second medium 248 by one or more cells in separation region 240 or by varying medium 180 flowing through microfluidic channel 122).

[0140]

[0152] As shown in FIG. 2C, the width W of the connection region 236 con The width W of the connecting region 236 at the distal opening 238 can be uniform from the proximal opening 234 to the distal opening 238. con is the width W of the connection region 236 at the proximal opening 234 conIn some embodiments, the width of separation region 240 at distal opening 238 is greater than or equal to the width W of connection region 236 at proximal opening 234. con Alternatively, the width W of the connection region 236 at the tip opening 238 may be substantially the same as con is the width W of the connection region 236 at the proximal opening 234 con In some embodiments, the width W of the connection region 236 can be different from (e.g., larger or smaller than) con The connection region 236 may narrow or widen between the proximal opening 234 and the distal opening 238. For example, the connection region 236 may narrow or widen between the proximal and distal openings using a variety of different geometries (e.g., chamfering the connection region, beveling the connection region). Additionally, any portion or subportion of the connection region 236 (e.g., the portion of the connection region adjacent the proximal opening 234) may narrow or widen.

[0141]

[0153] FIG. 3 shows another exemplary embodiment of a microfluidic device 300 including a microfluidic circuit structure 308, which includes a channel 322 and an isolation pen 324, and which has features and properties like any isolation pen described herein for microfluidic devices 100, 175, 200, 400, 520 and any other microfluidic device described herein.

[0142]

[0154] The exemplary microfluidic device of FIG. 3 has a width W ch , and includes a microfluidic channel 322 containing a stream 310 of a first fluid medium 302, and one or more isolation pens 324 (only one is shown in FIG. 3). The isolation pens 324 each have a length L s 3, a connection region 336, and a separation region 340, the separation region 340 containing the second fluid medium 304. The connection region 336 is open to the microfluidic channel 322 and has a width W con1 a proximal opening 334 having a width W con2and a tip opening 338 having a width W con1 is, as described herein, W con2 The wall of the isolation pen 324 may be formed of a microfluidic circuit material 316, which may further form a connection region wall 330. The connection region wall 330 may correspond to a structure that flanks the proximal opening 334 and extends at least partially within the enclosed portion of the isolation pen 324. In some embodiments, the length L of the connection region 336 may be 1 / 2 . con is the length L of the connection area wall 330 wall The connection region wall 330 is at least partially defined by a penetration depth D of the secondary flow 344. p The length L is chosen to be greater than wall Thus, secondary flow 344 may be contained entirely within the connection region without extending into separation region 340.

[0143]

[0155] The connection region wall 330 may define a hook region 352, which is a subregion of the separation region 340 of the isolation pen 324. Because the connection region wall 330 extends into the isolation pen's internal cavity, the connection region wall 330 contributes to the extent of the hook region. wall By selecting the length L of the connection region wall 330, the hook region 352 can function as a physical barrier to protect the secondary flow 344. In some embodiments, the length L of the connection region wall 330 can be selected to function as a physical barrier to protect the secondary flow 344. wall The longer the hook area 352 is, the more protected it is.

[0144]

[0156] In an isolation pen configured as in Figures 2A-2C and 3, the separation region can have any type of shape and size and can be selected, for example, to regulate the diffusion of nutrients, reagents, and / or media into the isolation pen to reach the far wall of the isolation pen opposite the proximal opening of the connection region to the flow region (or microfluidic channel). The size and shape of the separation region can further be selected to regulate the diffusion of waste products and / or secretion products of biological micro-objects from the separation region through the proximal opening of the connection region of the isolation pen. In general, the shape of the separation region is not critical to the ability of the isolation pen to separate micro-objects from the direct flow in the flow region.

[0145]

[0157] In some other embodiments of the isolation pen, the separation region may have two or more openings fluidically connecting the separation region with the flow region of the microfluidic device. However, for a separation region having n openings fluidically connecting the separation region to the flow region (or two or more flow regions), n-1 openings may be valved. When the n-1 valved openings are closed, the separation region has only one active opening, and exchange of materials into and out of the separation region occurs only by diffusion.

[0146]

[0158] Examples of microfluidic devices having pens capable of depositing, culturing, and / or monitoring biological micro-objects are described, for example, in U.S. Pat. No. 9,857,333 (Chapman et al.), U.S. Pat. No. 10,010,882 (White et al.), and U.S. Pat. No. 9,889,445 (Chapman et al.), each of which is incorporated herein by reference in its entirety.

[0147] Microfluidic circuit element dimensions

[0159] Various dimensions and / or features of the isolation pen and the microfluidic chamber into which it opens can be selected, as described herein, to limit the introduction of contaminants or unwanted micro-objects from the flow region / microfluidic channel to the separation region of the isolation pen, limit the exchange of components in the fluid medium from the channel or from the separation region to substantially only diffusional exchange, facilitate the transport of micro-objects to and / or from the isolation pen, and / or facilitate the growth or proliferation of biological cells. The microfluidic channel and isolation pen in any embodiment described herein can have any suitable combination of dimensions, which can be selected by one of skill in the art from the teachings of the present disclosure.

[0148]

[0160] In any of the microfluidic devices described herein, a microfluidic channel can have a uniform cross-sectional height along its length, which can be a substantially uniform cross-sectional height and can be any cross-sectional height as described herein. At any point along a microfluidic channel, a substantially uniform cross-sectional height of a channel whose upper surface is defined by the inner surface of the cover and whose lower surface is defined by the inner surface of the base can be substantially the same as the cross-sectional height at any other point along the channel, e.g., can have a cross-sectional height that differs from the cross-sectional height at any other location within the channel by no more than about 10%, no more than about 9%, no more than about 8%, no more than about 7%, no more than about 6%, no more than about 5%, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, or less than about 1%.

[0149]

[0161] Furthermore, the chambers of the microfluidic devices described herein, e.g., isolation pens, can be oriented substantially flush with the microfluidic channels into which they open. That is, the enclosed volume of the chamber is formed by an upper surface defined by the inner surface of the cover, a lower surface defined by the inner surface of the base, and walls defined by the microfluidic circuit material. Thus, the lower surface of the chamber can be flush with, e.g., substantially flush with, the lower surface of the microfluidic channel. The upper surface of the chamber can be flush with, e.g., substantially flush with, the upper surface of the microfluidic channel. Thus, the chamber can have the same, e.g., substantially the same, cross-sectional height as the chamber, which can have any value as described herein, and the chambers and microfluidic channels in the microfluidic device can have a substantially uniform cross-sectional height throughout the flow region of the microfluidic device and can be substantially flush throughout the microfluidic device.

[0150]

[0162] The coplanarity of the undersides of the chambers and microfluidic channels can provide distinct advantages in conjunction with repositioning of micro-objects within microfluidic devices using DEP or magnetic forces: Transferring and removing micro-objects from pens, especially selective transfer / removal, can be greatly facilitated if the undersides of the chambers and the microfluidic channels into which they open have a coplanar orientation.

[0151]

[0163] The proximal opening of the connection region of the isolation pen has a width (e.g., W ) that is at least as large as the largest dimension of the micro-object (e.g., biological cell, which may be a plant cell, such as a plant protoplast) for which the isolation pen is intended. con or W con1 In some embodiments, the proximal opening can have a width (e.g., W ) of about 20 μm, about 40 μm, about 50 μm, about 60 μm, about 75 μm, about 100 μm, about 150 μm, about 200 μm, or about 300 μm. con or W con1 ) The above is merely an example, and the width of the proximal opening (e.g., W con or Wcon1 ) can be selected to be a value between any of the values ​​listed above (e.g., about 20 to 200 μm, about 20 to 150 μm, about 20 to 100 μm, about 20 to 75 μm, about 20 to 60 μm, about 50 to 300 μm, about 50 to 200 μm, about 50 to 150 μm, about 50 to 100 μm, about 50 to 75 μm, about 75 to 150 μm, about 75 to 100 μm, about 100 to 300 μm, about 100 to 200 μm, or about 200 to 300 μm).

[0152]

[0164] In some embodiments, the connection region of the isolation pen is approximately equal to the width of the proximal opening (e.g., W con or W con1 ), which is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, at least 3.5 times, at least 4.0 times, at least 4.5 times, at least 5.0 times, at least 6.0 times, at least 7.0 times, at least 8.0 times, at least 9.0 times, or at least 10.0 times the length from the proximal opening to the distal opening to the separation region of the isolation pen (e.g., L con Thus, for example, the proximal opening of the connection region of the isolation pen may have a width (e.g., W ) of about 20 μm to about 200 μm (e.g., about 50 μm to about 150 μm). con or W con1 ), and the connection region may have a length L that is at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening. con As another example, the proximal opening of the connection region of the isolation pen may have a width (e.g., W con or W con1 ), and the connection region may have a length L that is at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening. con may have:

[0153]

[0165] The microfluidic channel of the microfluidic device that the isolation pen opens into can have a specified size (e.g., width or height). In some embodiments, the height of the microfluidic channel at its proximal opening to the connection region of the isolation pen (e.g., H ch ) can be within any of the following ranges: 20 to 100 μm, 20 to 90 μm, 20 to 80 μm, 20 to 70 μm, 20 to 60 μm, 20 to 50 μm, 30 to 100 μm, 30 to 90 μm, 30 to 80 μm, 30 to 70 μm, 30 to 60 μm, 30 to 50 μm, 40 to 100 μm, 40 to 90 μm, 40 to 80 μm, 40 to 70 μm, 40 to 60 μm, or 40 to 50 μm. The above are merely examples, and the height (e.g., H ch ) can be selected to be between any of the values ​​listed above. Additionally, the height of the microfluidic channel 122 (e.g., H ch ) can be selected to be any of these heights in regions of the microfluidic channel other than the region at the proximal opening of the isolation pen.

[0154]

[0166] The height of the microfluidic channel at its proximal opening to the connection region of the isolation pen (e.g., W ch) can be within any of the following ranges: about 20-500 μm, 20-400 μm, 20-300 μm, 20-200 μm, 20-150 μm, 20-100 μm, 20-80 μm, 20-60 μm, 30-400 μm, 30-300 μm, 30-200 μm, 30-150 μm, 30 ~100μm, 30~80μm, 30~60μm, 40~300μm, 40~200μm, 40~150μm, 40~100μm, 40~80μm , 40~60μm, 50~1,000μm, 50~500μm, 50~400μm, 50~300μm, 50~250μm, 50~200μm, 50 ~150μm, 50~100μm, 50~80μm, 60~300μm, 60~200μm, 60~150μm, 60~100μm, 60~80μm m, 70~500μm, 70~400μm, 70~300μm, 70~250μm, 70~200μm, 70~150μm, 70~100μm, 80 100 μm, 90 to 400 μm, 90 to 300 μm, 90 to 250 μm, 90 to 200 μm, 90 to 150 μm, 100 to 300 μm, 100 to 250 μm, 100 to 200 μm, 100 to 150 μm, 100 to 120 μm, 200 to 800 μm, 200 to 700 μm, or 200 to 600 μm. The above are merely examples, and the width (e.g., W ch ) can be a value selected to be between any of the values ​​listed above. Additionally, the width of the microfluidic channel (e.g., W ch ) can be selected to be any of these widths in regions of the microfluidic channel other than the region at the proximal opening of the isolation pen. In some embodiments, the width W of the microfluidic channel at the proximal opening to the connection region of the isolation pen is ch (e.g., transverse to the direction of overall fluid flow through the channel) is the width of the proximal opening (e.g., W con or W con1 ) can be substantially perpendicular to

[0155]

[0167] The cross-sectional area of ​​the microfluidic channel at its proximal opening to the connection region of the isolation pen is approximately 500-50,000 square micrometers, 500-40,000 square micrometers, 500-30,000 square micrometers, 500-25,000 square micrometers, 500-20,000 square micrometers, 500-15,000 square micrometers, 500-10,000 square micrometers, 500-7,500 square micrometers, 500-5,000 square micrometers, 1,000-25,000 square micrometers, 1,000-20,000 square micrometers, 1,000-15,000 square micrometers, 1,000-25,000 square micrometers, 1,000-25,000 square micrometers, 1,000-15,000 square micrometers, 1,000-25,000 square micrometers, 1,000-20,000 square micrometers, 1,000-15,000 square micrometers, 1,000-25,000 square micrometers, 1,000-25,000 square micrometers, 1,000-30,000 square micrometers, 1,000-30,000 square micrometers, 1,000-40,000 square micrometers, 1,000-40,000 square micrometers, 1,000-50,000 square micrometers, 1,000-60,000 square micrometers, 1,000-60,000 square micrometers, 1,000-70,000 square micrometers The cross-sectional area of ​​the microfluidic channel at the proximal opening can be between 00 and 10,000 μm, 1,000 and 7,500 μm, 1,000 and 5,000 μm, 2,000 and 20,000 μm, 2,000 and 15,000 μm, 2,000 and 10,000 μm, 2,000 and 7,500 μm, 2,000 and 6,000 μm, 3,000 and 20,000 μm, 3,000 and 15,000 μm, 3,000 and 10,000 μm, 3,000 and 7,500 μm, or 3,000 to 6,000 μm. The above are merely examples, and the cross-sectional area of ​​the microfluidic channel at the proximal opening can be selected to be between any of the values ​​listed above. In various embodiments, the cross-sectional area of ​​the microfluidic channel in regions of the microfluidic channel other than the region at the proximal opening can also be selected to be between any of the values ​​listed above, hi some embodiments, the cross-sectional area is selected to be a substantially uniform value along the entire length of the microfluidic channel.

[0156]

[0168] In some embodiments, the microfluidic chip has a proximal opening (e.g., 234 or 334) of the isolation pen connection region that has a width (e.g., W con or W con1 ), wherein the connecting region has a length L that is at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening. con (e.g., 236 or 336), and the microfluidic channel may have a height at the proximal opening (e.g., H chAs another example, the proximal opening (e.g., 234 or 334) of the connection region of the isolation pen may have a width (e.g., W ) of about 20 μm to about 100 μm (e.g., about 20 μm to about 60 μm). con or W con1 ), and the connection region may have a length L that is at least 1.0 times (e.g., at least 1.5 times or at least 2.0 times) the width of the proximal opening. con (e.g., 236 or 336), and the microfluidic channel may have a height at the proximal opening (e.g., H ch The above are merely examples, and the width (e.g., W con or W con1 ), the length of the connection region (e.g., L con ), and / or the width (e.g., W ch ) can be a value selected to be between any of the values ​​listed above. Generally, however, the width (W con or W con1 ) is the width of the microfluidic channel (W ch In some embodiments, the width of the proximal opening (W con or W con1 ) is the width of the microfluidic channel (W ch ), or about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, or about 30% of the width (W ch ) is the width of the base opening of the connection area of ​​the isolation pen (W con or W con1 ) may be at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, or at least 10.0 times the

[0157]

[0169] In some embodiments, the size W of the channel 122, 322, 618, 718 C (For example, cross-sectional width W ch, diameter, area, etc.) is the size W of the chamber opening, e.g., isolation pen opening 234, 334, etc. O (For example, cross-sectional width W con The diffusivity of the material can be about 1.25, about 1.5, about 2, about 2.5, about 3, or more than 3 times the diameter, area, etc., of the selected chamber (e.g., isolation pens 224, 226 in FIG. 2B ) through opening 234, 334 into channel 122, 322, 618, 718, and subsequently re-enter a downstream or adjacent chamber (e.g., isolation pen 228). The diffusivity of a molecule (e.g., an analyte of interest such as an antibody) depends on several factors, including (but not limited to) temperature, viscosity of the medium, and the diffusion coefficient, D, of the molecule. For example, the D of an IgG antibody in aqueous solution at about 20° C. is about 4.4×10 -7 cm 2 / sec, whereas the dynamic viscosity of cell culture medium is approximately 9 x 10 -4 m 2 / sec. Thus, an antibody in cell culture medium at about 20°C can have a diffusion rate of about 0.5 μm / sec. Thus, in some embodiments, the time period for diffusion from biological micro-objects in isolation pens such as 224, 226, 228, 324 to channels 122, 322, 618, 718 can be about 10 minutes or less (e.g., about 9, 8, 7, 6, 5, or less minutes). The time period for diffusion can be manipulated by changing parameters that affect the diffusion rate. For example, the temperature of the medium can be increased (e.g., to a physiological temperature such as about 37°C) or decreased (e.g., to about 15°C, 10°C, or 4°C), thereby increasing or decreasing the diffusion rate, respectively. Alternatively or additionally, the concentration of solutes in the medium can be increased or decreased as discussed herein to separate selected pens from solutes from other upstream pens.

[0158]

[0170] Thus, in some variations, the width of the microfluidic channel at its proximal opening to the connection region of the isolation pen (e.g., W ch) is about 50 to 500 μm, about 50 to 300 μm, about 50 to 200 μm, about 70 to 500 μm, about 70 to 300 μm, about 70 to 250 μm, about 70 to 200 μm, about 70 to 150 μm, about 70 to 100 μm, about 80 to 500 μm, about 80 to 300 μm, about 80 to 250 μm, about 80 to The width W of the microfluidic channel at its proximal opening to the connection region of the isolation pen can be about 100 to 200 μm, about 80 to 150 μm, about 90 to 500 μm, about 90 to 300 μm, about 90 to 250 μm, about 90 to 200 μm, about 90 to 150 μm, about 100 to 500 μm, about 100 to 300 μm, about 100 to 250 μm, about 100 to 200 μm, or about 100 to 150 μm. ch The width W of the opening of the chamber (e.g., isolation pen) can be about 70 to 250 μm, about 80 to 200 μm, or about 90 to 150 μm. con can be about 20 to 100 μm, about 30 to 90 μm, or about 20 to 60 μm. ch is approximately 70 to 250 μm, and W con is about 20 to 100 μm; ch is approximately 80 to 200 μm, and W con is about 30 to 90 μm; ch is approximately 90 to 150 μm, and W con is about 20 to 60 μm; or their W ch and W con Any combination of widths.

[0159]

[0171] In some embodiments, the proximal opening of the connection region of the isolation pen (e.g., 234 or 334) is smaller than the height of the flow region / microfluidic channel at the proximal opening (e.g., H ch ) is 2.0 times or less (e.g., 2.0, 1.9, 1.8, 1.5, 1.3, 1.0, 0.8, 0.5, or 0.1 times) the width (e.g., W con or W con1 ) or has a value within a range defined by any one of the above values.

[0160]

[0172] In some embodiments, the width W of the proximal opening (e.g., 234 or 334) of the connection region of the isolation pen con1 is the width W of the tip opening (e.g., 238 or 338) to the separation region con2 In some embodiments, the width W of the proximal opening may be equal to con1 is the width of the tip opening W con2 May be different from W con1 and / or W con2 is W con or W con1 The proximal and distal openings may be selected from any of the values ​​described above. In some embodiments, the walls defining the proximal and distal openings (including the connecting region walls) may be substantially parallel to one another. In some embodiments, the walls defining the proximal and distal openings may be selected so that they are not parallel to one another.

[0161]

[0173] The length of the connection area (e.g., L con ) is approximately 1 to 600 μm, 5 to 550 μm, 10 to 500 μm, 15 to 400 μm, 20 to 300 μm, 20 to 500 μm, 40 to 400 μm, 60 to 300 μm, 80 to 200 μm, approximately 100 to 150 μm, approximately 20 to 300 μm, approximately 20 to 250 μm, approximately 20 to 200 μm, approximately 20 to 150 μm, approximately 20 to 100 μm, approximately 30 to The length of the connection region (e.g., L con ) can be selected to be a value between any of the values ​​listed above.

[0162]

[0174] The connection region wall of the isolation pen may be formed by a width (e.g., W con or W con1), at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, or at least 3.5 times the length (e.g., L wall In some embodiments, the connection region wall may have a length L of about 20 to 200 μm, about 20 to 150 μm, about 20 to 100 μm, about 20 to 80 μm, or about 20 to 50 μm. wall The above are merely examples, and the connection region walls may have a length L selected to be between any of the values ​​listed above. wall may have:

[0163]

[0175] The isolation pen has a length L of about 40 to 600 μm, about 40 to 500 μm, about 40 to 400 μm, about 40 to 300 μm, about 40 to 200 μm, about 40 to 100 μm, or about 40 to 80 μm. s The above are merely examples, and the isolation pen may have a length L selected to be between any of the values ​​listed above. s may have:

[0164]

[0176] According to some embodiments, the isolation pen may be positioned at a specified height (e.g., H s In some embodiments, the isolation pen may have a height H of about 20 μm to about 200 μm (e.g., about 20 μm to about 150 μm, about 20 μm to about 100 μm, about 20 μm to about 60 μm, about 30 μm to about 150 μm, about 30 μm to about 100 μm, about 30 μm to about 60 μm, about 40 μm to about 150 μm, about 40 μm to about 100 μm, or about 40 μm to about 60 μm). s The above is merely an example, and the isolation pen may have a height H selected to be between any of the values ​​listed above. s It can have:

[0165]

[0177] Height H of the connection area at the base opening of the isolation pen concan be any of the following heights: 20 to 100 μm, 20 to 90 μm, 20 to 80 μm, 20 to 70 μm, 20 to 60 μm, 20 to 50 μm, 30 to 100 μm, 30 to 90 μm, 30 to 80 μm, 30 to 70 μm, 30 to 60 μm, 30 to 50 μm, 40 to 100 μm, 40 to 90 μm, 40 to 80 μm, 40 to 70 μm, 40 to 60 μm, or 40 to 50 μm. con can be selected to be between any of the values ​​listed above. Typically, the height H of the connection region con is the height H of the microfluidic channel at the proximal opening of the connection region ch Furthermore, the height of the isolation pen, H s is typically the height of the connection area H con and / or the height H of the microfluidic channel ch In some embodiments, H s , H con , H ch can be selected to be the same as any of the values ​​listed above for the selected microfluidic device.

[0166]

[0178] The separation region can be configured to contain only 1, 2, 3, 4, 5, or a similarly relatively small number of micro-objects. In other embodiments, the separation region can contain more than 10, more than 50, or more than 100 micro-objects. Thus, the volume of the separation region can be, for example, at least 1×10 4 cubic μm, 1×10 5 cubic μm, 5×10 5 cubic μm, 8×10 5 cubic μm, 1×10 6 cubic μm, 2×10 6 cubic μm, 4×10 6 cubic μm, 6×10 6 cubic μm, 1×10 7 cubic μm, 3×10 7 cubic μm, 5×10 7 cubic μm, 1×10 8 cubic μm, 5×10 8 cubic μm, 8×108 The above are merely examples, and the separation area can be between any of the values ​​listed above (e.g., 1×10 5 cubic μm and 5×10 5 Between 5 x 10 cubic micrometers 5 cubic μm and 1×10 6 Between cubic μm and 1 x 10 6 cubic μm and 2×10 6 Between cubic μm, or 2 x 10 6 cubic μm and 1×10 7 The microscopic object can be configured to contain a selected number of microscopic objects and a volume between 1000 and 1000 cubic micrometers.

[0167]

[0179] According to some embodiments, the isolation pen of the microfluidic device can have a designated volume. The designated volume of the isolation pen (or isolation region of the isolation pen) can be selected to allow a single cell or a small number of cells (e.g., 2-10 or 2-5) to quickly adjust the medium and thereby obtain favorable (or optimal) growth conditions. In some embodiments, the isolation pen can accommodate approximately 5×10 5 cubic μm, 6×10 5 cubic μm, 8×10 5 cubic μm, 1×10 6 cubic μm, 2×10 6 cubic μm, 4×10 6 cubic μm, 8×10 6 cubic μm, 1×10 7 cubic μm, 3×10 7 cubic μm, 5×10 7 cubic μm, or approximately 8×10 7 In some embodiments, the isolation pen has a volume of about 1 nL to about 50 nL, 2 nL to about 25 nL, 2 nL to about 20 nL, about 2 nL to about 15 nL, or about 2 nL to about 10 nL. The above are merely examples, and the isolation pen can have a volume selected to be any value between any of the values ​​listed above.

[0168]

[0180] According to some embodiments, the flow of fluid medium within a microfluidic channel (e.g., 122 or 322) is controlled at a specified maximum velocity (e.g., V max ) in some embodiments. max ) can be set to approximately 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, or 25 μL / sec. The above are merely examples, and the flow of fluid medium within the microfluidic channel may have a maximum velocity (e.g., V) selected to be any value between the values ​​listed above. max The flow of the fluid medium in the microfluidic channel can typically be expressed as V max Although Vmax may be highly dependent on the particular size and number of channels and isolation pens opening into the channels, the fluid medium may flow at a rate less than V max may flow at about 0.1 μL / sec to about 20 μL / sec, about 0.1 μL / sec to about 15 μL / sec, about 0.1 μL / sec to about 12 μL / sec, about 0.1 μL / sec to about 10 μL / sec, or about 0.1 μL / sec to about 7 μL / sec, without exceeding In some portions of a typical workflow, the flow rate of the fluid medium can be about 0.1 μL / sec, about 0.5 μL / sec, about 1.0 μL / sec, about 2.0 μL / sec, about 3.0 μL / sec, about 4.0 μL / sec, about 5.0 μL / sec, about 6.0 μL / sec, about 7.0 μL / sec, about 8.0 μL / sec, about 9.0 μL / sec, about 10.0 μL / sec, about 11.0 μL / sec, or any range bounded by two of the above values, e.g., 1-5 μL / sec or 5-10 μL / sec. The flow rate of the fluid medium in the microfluidic channel can be about 12 μL / sec or less, about 10 μL / sec or less, about 8 μL / sec or less, or about 6 μL / sec or less.

[0169]

[0181] In various embodiments, the microfluidic device has isolation pens configured as in any embodiment discussed herein, the microfluidic device may have from about 5 to about 10 isolation pens, from about 10 to about 50 isolation pens, from about 25 to about 200 isolation pens, from about 100 to about 500 isolation pens, from about 200 to about 1000 isolation pens, from about 500 to about 1500 isolation pens, from about 1,000 to about 2500 isolation pens, from about 2,000 to about 5,000 isolation pens, from about 3,500 to about 700 The isolation pens may have 0 isolation pens, about 5,000 to about 10,000 isolation pens, about 7,500 to about 15,000 isolation pens, about 12,500 to about 20,000 isolation pens, about 15,000 to about 25,000 isolation pens, about 20,000 to about 30,000 isolation pens, about 25,000 to about 35,000 isolation pens, about 30,000 to about 40,000 isolation pens, about 35,000 to about 45,000 isolation pens, or about 40,000 to about 50,000 isolation pens. The isolation pens need not all be the same size and can include a variety of configurations (e.g., different widths, different features within the isolation pens).

[0170] Coating solutions and materials

[0182] In some embodiments, at least one interior surface of the microfluidic device comprises a coating material that provides a layer of organic and / or hydrophilic molecules suitable for the maintenance, growth, and / or movement of biological micro-objects (i.e., biological micro-objects that exhibit improved survival, greater growth, and / or greater transportability within the microfluidic device). The conditioned surface may reduce surface fouling, participate in providing a hydration layer, and / or otherwise protect the biological micro-objects from contact with non-organic materials within the microfluidic device.

[0171]

[0183] In some embodiments, substantially all interior surfaces of the microfluidic device comprise a coating material. The coated interior surfaces can include surfaces of flow regions (e.g., channels), isolation pens, or combinations thereof. In some embodiments, each of a plurality of isolation pens has at least one interior surface coated with the coating material. In other embodiments, each of a plurality of flow regions or channels has at least one interior surface coated with the coating material. In some embodiments, at least one interior surface of each of a plurality of isolation pens and each of a plurality of chambers is coated with the coating material. The coating may be applied before or after the introduction of the biological micro-objects, or may be introduced simultaneously with the biological micro-objects. In some embodiments, the biological micro-objects may be transported to the microfluidic device in a fluid medium containing one or more coating reagents. In other embodiments, the interior surfaces of a microfluidic device (e.g., a microfluidic device having an electrode-activated substrate, such as, but not limited to, a device containing dielectrophoresis (DEP) electrodes) may be treated or "primed" with a coating solution containing a coating agent before introducing the biological micro-objects into the microfluidic device. Any convenient coating agent / coating solution can be used, including but not limited to serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof.

[0172] Synthetic polymer coating materials

[0184] At least one interior surface may include a coating material comprising a polymer. The polymer may be non-covalently bound (e.g., non-specifically attached) to at least one surface. The polymer may have a variety of structural moieties, such as those found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein. Various alkylene ether containing polymers may be suitable for use in the microfluidic devices described herein, including, but not limited to, Pluronic® polymers, such as Pluronic® L44, L64, P85, and F127 (including F127NF). Other examples of suitable coating materials are described in U.S. Patent Application Publication No. 2016 / 0312165, the entire contents of which are incorporated herein by reference.

[0173] Covalent coating material

[0185] In some embodiments, at least one interior surface comprises covalently attached molecules that provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological micro-objects within the microfluidic device, providing a conditioning surface for such cells. The covalently attached molecules comprise binding groups, which are attached to one or more surfaces of the microfluidic device, as described below. The binding groups are also covalently attached to surface-modifying moieties configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth / transfer of biological micro-objects.

[0174]

[0186] In some embodiments, covalently attached moieties configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects may include alkyl or fluoroalkyl (including perfluoroalkyl) moieties; monosaccharides or polysaccharides (which may include, but are not limited to, dextran); alcohols (including, but not limited to, propargyl alcohol); polyhydric alcohols, including, but not limited to, polyvinyl alcohol; alkylene ethers, including, but not limited to, polyethylene glycol; polyelectrolytes (including, but not limited to, polyacrylic acid or polyvinylphosphonic acid); amino groups (derivatives thereof, including, but not limited to, for example, alkylated amines, hydroxyalkylated amino groups, guanidinium, and heterocyclic groups containing a non-aromatized nitrogen ring atom, including, but not limited to, for example, morpholinyl or piperazinyl); carboxylic acids, including, but not limited to, propiolic acid (which may provide a carboxylate anion surface); phosphonic acids, including, but not limited to, ethynylphosphonic acid (which may provide a phosphonate anion surface); sulfonate anions; carboxybetaines; sulfobetaines; sulfamic acids; or amino acids.

[0175]

[0187] In various embodiments, the covalently attached moiety configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects in a microfluidic device can include a non-polymeric moiety such as an alkyl moiety, an amino acid moiety, an alcohol moiety, an amino moiety, a carboxylic acid moiety, a phosphonic acid moiety, a sulfonic acid moiety, a sulfamic acid moiety, or a saccharide moiety. Alternatively, the covalently attached moiety can include a polymeric moiety that can include any of these moieties.

[0176]

[0188] In some embodiments, the microfluidic device can have a hydrophobic layer on the interior surface of the base that includes covalently bonded alkyl moieties. The covalently bonded alkyl moieties can include carbon atoms that form a linear chain (e.g., a linear chain of at least 10 or at least 14, 16, 18, 20, 22, or more carbon atoms) and can be unbranched alkyl moieties. In some embodiments, the alkyl group can include a substituted alkyl group (e.g., some of the carbons in the alkyl group can be fluorinated or perfluorinated). In some embodiments, the alkyl group can include a first segment that can include a perfluoroalkyl group bonded to a second segment that can include an unsubstituted alkyl group, and the first and second segments can be bonded directly or indirectly (e.g., by an ether bond). The first segment of the alkyl group can be positioned away from the binding group, and the second segment of the alkyl group can be positioned near the binding group.

[0177]

[0189] In other embodiments, the covalently attached moiety can include at least one amino acid, which can include two or more amino acids. Thus, the covalently attached moiety can include a peptide or a protein. In some embodiments, the covalently attached moiety can include an amino acid that can provide an amphoteric surface to support cell growth, viability, mobility, or any combination thereof.

[0178]

[0190] In other embodiments, the covalently bound moiety may further comprise a streptavidin or biotin moiety. In some embodiments, a modified biological moiety, such as, for example, a biotinylated protein or peptide, may be introduced to the interior surface of a microfluidic device having covalently bound streptavidin and bound to the surface via the covalently bound streptavidin, thereby providing a modified surface displaying the protein or peptide.

[0179]

[0191] In other embodiments, the covalently bonded moiety may comprise at least one alkylene oxide moiety and may include any alkylene oxide polymer as described above. One useful class of alkylene ether-containing polymers is polyethylene glycol (PEG, MPEG).w <100,000 Da) or alternatively polyethylene oxide (PEO,M w In some embodiments, the PEG has an M of about 1000 Da, 5000 Da, 10,000 Da, or 20,000 Da. w In some embodiments, the PEG polymer may be further substituted with hydrophilic or charged moieties, such as, but not limited to, alcohol functionalities or carboxylic acid moieties.

[0180]

[0192] The covalently attached moiety may comprise one or more sugars. The covalently attached sugar may be a monosaccharide, disaccharide, or polysaccharide. The covalently attached sugar may be modified to introduce a reactive pair moiety that allows for coupling or processing to attach to a surface. One exemplary covalently attached moiety may include a dextran polysaccharide, which may be indirectly attached to a surface via an unbranched linker.

[0181]

[0193] The coating material providing the conditioning surface may contain only one type of covalently bonded moiety, or it may contain two or more different types of covalently bonded moieties. For example, a polyethylene glycol conditioning surface may have covalently bonded alkylene oxide moieties having a specified number of alkylene oxide units, all of which are the same, e.g., have the same binding group and covalent bond to the surface, the same overall length, and the same number of alkylene oxide units. Alternatively, the coating material may have two or more types of covalently bonded moieties attached to the surface. For example, the coating material may include molecules having a first specified number of covalently bonded alkylene oxide units, and may further include an additional set of molecules having bulky moieties, such as proteins or peptides, attached to covalently bonded alkylene oxide groups having a greater number of alkylene oxide units. The different types of molecules may be varied in any suitable ratio to obtain desired surface properties. For example, a conditioning surface having a mixture of first molecules having a chemical structure with a first specified number of alkylene oxide units and second molecules containing peptide or protein moieties that can be bound to the covalently bound alkylene moieties via a biotin / streptavidin binding pair can have a first molecule:second molecule ratio of about 99:1, about 90:10, about 75:25, about 50:50, about 30:70, about 20:80, about 10:90, or any ratio selected to lie between these values. In this case, the first set of molecules, with different, less sterically demanding termini and fewer backbone atoms, can serve to functionalize the entire substrate surface, thereby avoiding undesired adhesion or contact with the silicon / silicon oxide, hafnium oxide, or alumina that constitutes the substrate itself. The selection of the mixing ratio of the first and second molecules can also modulate the surface modification introduced by the second molecules containing peptide or protein moieties.

[0182] Adjustment surface characteristics

[0194] Various factors, such as the manner in which the conditioning surface is formed on the substrate (e.g., vapor deposition, liquid deposition, spin coating, flooding, and electrostatic coating), can affect the physical thickness of the conditioning surface. In some embodiments, the conditioning surface can have a thickness of about 1 nm to about 10 nm. In some embodiments, the covalently bonded moieties of the conditioning surface can form a monolayer when covalently bonded to the surface of a microfluidic device (which may include an electrode-activated substrate with dielectrophoresis (DEP) or electrowetting (EW) electrodes) and can have a thickness of less than 10 nm (e.g., less than 5 nm or about 1.5 to 3.0 nm). These values ​​contrast with, for example, surfaces prepared by spin coating, which can typically have a thickness of about 30 nm. In some embodiments, the conditioning surface does not need to be a fully formed monolayer to function properly for operation within a DEP-configured microfluidic device. In other embodiments, the conditioning surface formed by the covalently bonded moieties can have a thickness of about 10 nm to about 50 nm.

[0183] Single or multi-part adjustment surfaces

[0195] The covalent coating material may be formed by the reaction of molecules already containing moieties configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological micro-objects in a microfluidic device, as described below, and may have the structure of Formula I, as shown below. Alternatively, the covalent coating material may be formed in a two-part sequence, having the structure of Formula II, by attaching a moiety configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological micro-objects to a surface-modifying ligand that is itself covalently attached to the surface. In some embodiments, the surface may be formed in a two- or three-part sequence including a streptavidin / biotin binding pair to introduce proteins, peptides, or mixed-modified surfaces. [ka]

[0184]

[0196] The coating material can be covalently bonded to the oxide on the surface of the DEP- or EW-construction substrate. The coating material can be bonded to the oxide via a linking group ("LG"), which can be a siloxy or phosphonate ester group formed from the reaction of a siloxane group or a phosphonic acid group with the oxide. The moiety configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects in a microfluidic device can be any moiety described herein. The linking group LG can be directly or indirectly connected to the moiety configured to provide a layer of organic and / or hydrophilic molecules suitable for the maintenance / growth of biological microobjects in a microfluidic device. When the linking group LG is directly connected to the moiety, the optional linker ("L") is not present and n is 0. When the linking group LG is indirectly connected to the moiety, the linker L is present and n is 1. The linker L may have a linear portion, where the backbone of the linear portion may contain 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, subject to chemical bonding constraints as known in the art. It may be interrupted with any combination of one or more moieties, which may be selected from ether, amino, carbonyl, amide, and / or phosphate groups, arylene, heteroarylene, or heterocyclic groups. In some embodiments, the linking group CG may be a reactive moiety R x and the reaction pair part R px (i.e., reactive moiety R x CG represents a group generated from reaction of a reactive moiety with a moiety configured to react with CG. CG can be a carboxamidyl group, a triazolylene group, a substituted triazolylene group, a carboxamidyl, a thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or an alkenyl group, or any other suitable group that can be formed upon reaction of a reactive moiety with its respective reactive pair moiety. In some embodiments, CG can further represent a streptavidin / biotin binding pair.

[0185]

[0197] Further details of suitable coating treatments and modifications, as well as preparation methods, can be found in U.S. Patent Application Publication No. 2016 / 0312165 (Lowe, Jr. et al.), U.S. Patent Application Publication No. 2017 / 0173580 (Lowe, Jr. et al.), International Publication No. 2017 / 205830 (Lowe, Jr. et al.), and International Publication No. 2019 / 01880 (Beemiller et al.), the disclosures of each of which are incorporated herein by reference in their entirety.

[0186] Microfluidic Device Mobility Technology

[0198] The microfluidic devices described herein can be used in conjunction with any type of motive technology. As described herein, the system's control and monitoring equipment can include a motive module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of the microfluidic device. Motive technologies can include, for example, dielectrophoresis (DEP), electrowetting (EW), and / or other motive technologies. Microfluidic devices can have a variety of motive configurations depending on the type of object being moved and other conditions. Returning to FIG. 1A , for example, the support structure 104 and / or cover 110 of the microfluidic device 100 can include a DEP electrode-activated substrate for selectively inducing a motive force on micro-objects in the fluid medium 180 within the microfluidic circuit 120, thereby selecting, capturing, and / or moving individual or groups of micro-objects.

[0187]

[0199] In some embodiments, a motive force is applied across fluid medium 180 (e.g., within the channels and / or isolation pens) via one or more electrodes (not shown) to manipulate, transport, separate, and sort micro-objects therein. For example, in some embodiments, a motive force is applied to one or more portions of micro-fluidic circuit 120 to transport a single micro-object from channel 106 to a desired micro-fluidic isolation pen. In some embodiments, a motive force is used to prevent micro-objects within the isolation pen from being displaced therefrom. Additionally, in some embodiments, a motive force is used to selectively remove micro-objects from the isolation pen that have previously been collected according to embodiments of the present disclosure.

[0188]

[0200] In some embodiments, the microfluidic device is configured as an optically actuated electromechanical device, such as in an optoelectronic tweezers (OET) and / or optoelectrowetting (OEW) configuration device. Examples of suitable OET configuration devices (e.g., including an optically actuated dielectrophoretic electrode-activated substrate) can include those shown in U.S. Patent No. RE44,711 (Wu et al.) (originally issued as U.S. Patent No. 7,612,355), U.S. Patent No. 7,956,339 (Ohta et al.), U.S. Patent No. 9,908,115 (Hobbs et al.), and U.S. Patent No. 9,403,172 (Short et al.), each of which is incorporated herein by reference in its entirety. Examples of suitable OEW configuration devices can include those shown in U.S. Patent No. 6,958,132 (Chiou et al.) and U.S. Patent Application No. 9,533,306 (Chiou et al.), each of which is incorporated herein by reference in its entirety. Examples of suitable optically actuated electromechanical devices, including combination OET / OEW devices, can include those shown in U.S. Patent Application Publication No. 2015 / 0306598 (Khandros et al.), U.S. Patent Application Publication No. 2015 / 0306599 (Khandros et al.), and U.S. Patent Application Publication No. 2017 / 0173580 (Lowe et al.), each of which is incorporated herein by reference in its entirety.

[0189]

[0201] It should be understood that, for simplicity, the various examples in Figures 1-5B may show portions of a microfluidic device and not other portions. Furthermore, Figures 1-5B may be part of, or implemented as, one or more microfluidic systems. In one non-limiting example, Figures 4A and 4B show vertical and horizontal cross-sectional views, respectively, of a portion of enclosure 102 of microfluidic device 400 having region / chamber 402 that may be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, an isolation pen (which may be like any isolation pen described herein), a flow region, or a flow channel. For example, microfluidic device 400 may be similar to microfluidic devices 100, 175, 200, 300, 520, or any other microfluidic device as described herein. Additionally, microfluidic device 400 may include other fluidic circuit elements and may be part of the control and monitoring equipment 152 described above, which includes one or more of media module 160, motive module 162, imaging module 164, optional tilt module 166, and other modules 168. Microfluidic devices 175, 200, 300, 520, and any other microfluidic devices described herein may similarly have any of the features described in detail with respect to Figures 1A, 1B, 4A, and 4B.

[0190]

[0202] As shown in the example of FIG. 4A , the microfluidic device 400 includes a support structure 104 having a lower electrode 404 and an electrode-active substrate 406 overlying the lower electrode 404, and a cover 110 having an upper electrode 410 spaced apart from the lower electrode 404. The upper electrode 410 and the electrode-active substrate 406 define opposing sides of a region / chamber 402. Thus, a fluid medium 180 contained in the region / chamber 402 provides a resistive connection between the upper electrode 410 and the electrode-active substrate 406. Also shown is a power supply 412 connected to the lower electrode 404 and the upper electrode 410 and configured to generate a bias voltage between the electrodes as needed to generate a DEP force in the region / chamber 402. The power supply 412 can be, for example, an alternating current (AC) power supply.

[0191]

[0203] In certain embodiments, the microfluidic device 400 shown in FIGS. 4A and 4B can have an optically actuated DEP electrode-activated substrate. Thus, varying patterns of light 418 from a light source 416, which can be controlled by the actuator module 162, can selectively activate and deactivate varying patterns of DEP electrodes in regions 414 of the inner surface 408 of the electrode-activated substrate 406. (Regions 414 of a microfluidic device having a DEP electrode-activated substrate are hereinafter referred to as "DEP electrode regions.") As shown in FIG. 4B, the light pattern 418 directed toward the inner surface 408 of the electrode-activated substrate 406 can selectively illuminate DEP electrode regions 414a (shown in white) in a pattern such as a square. The unilluminated DEP electrode regions 414 (cross-hatched) are hereinafter referred to as "dark" DEP electrode regions 414. The relative electrical impedance through the DEP electrode-active substrate 406 (i.e., from the lower electrode 404 to the inner surface 408 of the electrode-active substrate 406 that interacts with the fluid medium 180 in the flow region 106) is greater than the relative electrical impedance through the fluid medium 180 in the region / chamber 402 (i.e., from the inner surface 408 of the electrode-active substrate 406 to the upper electrode 410 of the cover 110) at each dark DEP electrode region 414. However, the illuminated DEP electrode region 414a exhibits a reduced relative impedance through the electrode-active substrate 406 that is less than the relative impedance through the fluid medium 180 in the region / chamber 402 at each illuminated DEP electrode region 414a.

[0192]

[0204] With the power source 412 activated, the DEP configuration generates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode region 414a and the adjacent dark DEP electrode region 414, and the electric field gradient generates a local DEP force that attracts or repels nearby micro-objects (not shown) in the fluid medium 180. Thus, DEP electrodes that attract or repel micro-objects in the fluid medium 180 can be selectively activated and deactivated at many different such DEP electrode regions 414 on the inner surface 408 of the region / chamber 402 by varying the light pattern 418 projected from the light source 416 onto the microfluidic device 400. Whether the DEP force attracts or repels nearby micro-objects can depend on parameters such as the frequency of the power source 412 and the dielectric properties of the fluid medium 180 and / or the micro-objects (not shown). Depending on the frequency of the power applied to the DEP configuration and the choice of fluid medium (e.g., a highly conductive medium such as PBS or other medium suitable for sustaining biological cells), a negative DEP force can be generated. The negative DEP force can repel micro-objects away from the location of the induced non-uniform electric field. In some embodiments, a microfluidic device incorporating DEP technology can generate a negative DEP force.

[0193]

[0205] The square pattern 420 of illuminated DEP electrode regions 414a shown in Figure 4B is merely one example. Any pattern of DEP electrode regions 414 can be illuminated (and thereby activated) by a pattern of light 418 projected onto the microfluidic device 400, and the pattern of illuminated / activated DEP electrode regions 414 can be repeatedly changed by changing or moving the light pattern 418.

[0194]

[0206] In some embodiments, the electrode-activated substrate 406 can include or consist of a photoconductive material. In such embodiments, the inner surface 408 of the electrode-activated substrate 406 can be featureless. For example, the electrode-activated substrate 406 can include or consist of a layer of hydrogenated amorphous silicon (a-Si:H). a-Si:H can be, for example, a layer of hydrogenated amorphous silicon (a-Si:H) with a hydrogen content of about 8% to 40% (100 * The a-Si:H layer can have a thickness of about 500 nm to about 2.0 μm. In such embodiments, the DEP electrode regions 414 can be fabricated in any pattern at any location on the inner surface 408 of the electrode-activated substrate 406 according to the light pattern 418. Thus, the number and pattern of the DEP electrode regions 414 need not be fixed but can correspond to the light pattern 418. Examples of microfluidic devices having DEP configurations including photoconductive layers as described above are described, for example, in U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355), each of which is incorporated herein by reference in its entirety.

[0195]

[0207] In other embodiments, the electrode-activated substrate 406 can include a substrate including multiple doped layers, electrically insulating layers (or regions), and conductive layers forming a semiconductor integrated circuit, such as is known in the semiconductor arts. For example, the electrode-activated substrate 406 can include multiple phototransistors, including, for example, lateral bipolar phototransistors, each corresponding to a DEP electrode region 414. Alternatively, the electrode-activated substrate 406 can include electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, each corresponding to a DEP electrode region 414. The electrode-activated substrate 406 can include a pattern of such phototransistors or phototransistor-controlled electrodes. The pattern can be, for example, an array of substantially square phototransistors or phototransistor-controlled electrodes arranged in a matrix. Alternatively, the pattern can be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes forming a hexagonal lattice. Regardless of the pattern, the electrical circuit elements can form electrical connections between the DEP electrode regions 414 and the lower electrode 404 on the inner surface 408 of the electrode-activated substrate 406, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 418, as described above.

[0196]

[0208] Examples of microfluidic devices having electrode-activated substrates that include phototransistors are described, for example, in U.S. Patent No. 7,956,339 (Ohta et al.) and U.S. Patent No. 9,908,115 (Hobbs et al.), the entire contents of each of which are incorporated herein by reference. Examples of microfluidic devices having electrode-activated substrates that include electrodes controlled by phototransistor switches are described, for example, in U.S. Patent No. 9,403,172 (Short et al.), the entire contents of which are incorporated herein by reference.

[0197]

[0209] In some embodiments of the DEP configuration microfluidic device, the upper electrode 410 is part of the first wall (or cover 110) of the enclosure 402, and the electrode-active substrate 406 and lower electrode 404 are part of the second wall (or support structure 104) of the enclosure 102. The region / chamber 402 can be between the first and second walls. In other embodiments, the electrode 410 is part of the second wall (or support structure 104), and one or both of the electrode-active substrate 406 and / or electrode 410 are part of the first wall (or cover 110). Additionally, the light source 416 can alternatively be used to illuminate the enclosure 102 from below.

[0198]

[0210] 4A and 4B having a DEP electrode-activated substrate, as described herein with respect to FIG. 1A, the motive module 162 of the control and monitoring equipment 152 can select micro-objects (not shown) in the fluid medium 180 in the region / chamber 402 by projecting a light pattern 418 into the microfluidic device 400 and activating a first set of one or more DEP electrodes in the DEP electrode region 414a on the inner surface 408 of the electrode-activated substrate 406 in a pattern (e.g., a square pattern 420) that surrounds and traps the micro-objects. The motive module 162 can then move the light pattern 418 relative to the microfluidic device 400 to activate a second set of one or more DEP electrodes in the DEP electrode region 414, thereby moving the in situ-generated trapped micro-objects. Alternatively, the microfluidic device 400 can be moved relative to the light pattern 418.

[0199]

[0211] In other embodiments, the microfluidic device 400 can be a DEP-configured device that does not rely on photoactivation of DEP at the inner surface 408 of the electrode-activating substrate 406. For example, the electrode-activating substrate 406 can include selectively addressable and energizable electrodes positioned opposite a surface (e.g., the cover 110) that includes at least one electrode. Switches (e.g., transistor switches in a semiconductor substrate) can be selectively opened and closed to activate or deactivate DEP electrodes in the DEP electrode regions 414, thereby producing a net DEP force on micro-objects (not shown) in the region / chamber 402 near the activated DEP electrodes. Depending on the frequency of the power source 412 and the properties, such as the dielectric properties, of the medium (not shown) and / or micro-objects in the region / chamber 402, the DEP force can either attract or repel nearby micro-objects. By selectively activating and deactivating a set of DEP electrodes (e.g., in a set of DEP electrode regions 414 forming square pattern 420), one or more micro-objects in region / chamber 402 can be selected and moved within region / chamber 402. Motivation module 162 in FIG. 1A controls such switches, which in turn can activate and deactivate individual DEP electrodes of a plurality of DEP electrodes to select and move specific micro-objects (not shown) around region / chamber 402. Microfluidic devices having DEP electrode-activated substrates containing selectively addressable and energizable electrodes are known in the art and are described, for example, in U.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776 (Medoro), each of which is incorporated herein by reference in its entirety.

[0200]

[0212] Whether the microfluidic device 400 has a dielectrophoresis electrode-activating substrate, an electrowetting electrode-activating substrate, or both dielectrophoresis and electrowetting electrode-activating substrates, a power source 412 can be used to provide an electrical potential (e.g., an AC voltage potential) that powers the electrical circuitry of the microfluidic device 400. The power source 412 can be the same as or a component of the power source 192 referenced in FIG. 1A. The power source 412 can be configured to provide an AC voltage and / or current to the upper electrode 410 and the lower electrode 404. In the case of an AC voltage, the power source 412 can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate a net DEP force (or electrowetting force) strong enough to select and move individual micro-objects (not shown) in the region / chamber 402, as discussed above, and / or to alter the wettability of the inner surface 408 of the support structure 104 in the region / chamber 202, as also discussed above. Such frequency ranges and average or peak power ranges are known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.), U.S. Pat. No. RE44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355), and U.S. Patent Application Publication Nos. 2014 / 0124370 (Short et al.), 2015 / 0306598 (Khandros et al.), 2015 / 0306599 (Khandros et al.), and 2017 / 0173580 (Lowe, Jr. et al.), the disclosures of each of which are incorporated herein by reference in their entirety.

[0201]

[0213] Other forces may be used alone or in combination to move selected micro-objects within a microfluidic device. Bulk fluid flow within a microfluidic channel may move micro-objects within a flow region. Localized fluid flow, which may operate within a microfluidic channel, an isolation pen, or another type of chamber (e.g., a reservoir), may also be used to move selected micro-objects. Localized fluid flow may be used to move selected micro-objects from a flow region to a non-flow region, such as an isolation pen or reservoir, or from a non-flow region to a flow region. Localized flow may be actuated by deformation of a deformable wall of a microfluidic device, as described in U.S. Pat. No. 10,058,865 (Breinlinger et al.), which is incorporated herein by reference in its entirety.

[0202]

[0214] Gravity can be used to move micro-objects within a microfluidic channel to and / or from an isolation pen or other chamber, as described in U.S. Patent No. 9,744,533 (Breinlinger et al.), which is incorporated herein by reference in its entirety. The use of gravity (e.g., by tilting the microfluidic device and / or the support to which the microfluidic device is attached) can be useful for bulk movement of cells from an isolation pen to / from the flow region. Magnetic forces can be employed to move micro-objects containing paramagnetic materials, which can include magnetic micro-objects attached to or associated with biological micro-objects. Alternatively or additionally, centripetal forces can be used to move micro-objects within a microfluidic channel and into or out of an isolation pen or other chamber within a microfluidic device.

[0203]

[0215] In another alternative form of moving micro-objects, laser-generated displacement forces can be used to eject or assist in the ejection of micro-objects from an isolation pen or any other chamber in a microfluidic device, as described in U.S. Patent No. 10,829,728 (Kurz et al.), filed June 15, 2018, and granted November 10, 2020, which is incorporated herein by reference in its entirety.

[0204]

[0216] In some embodiments, DEP forces are combined with other forces, such as fluid flow (e.g., bulk fluid flow within a channel or localized fluid flow actuated by deformation of a deformable surface of a microfluidic device, laser-generated movement forces, and / or gravity) to manipulate, transport, separate, and sort micro-objects and / or droplets within the microfluidic circuit 120. In some embodiments, DEP forces can be applied before other forces. In other embodiments, DEP forces can be applied after other forces. In still other cases, DEP forces can be applied alternately with other forces. In the microfluidic devices described herein, repositioning of micro-objects generally may not rely on gravity or fluidic forces to position or capture micro-objects at selected locations. While gravity may be chosen as one form of repositioning force, the ability to reposition micro-objects within a microfluidic device does not rely solely on the use of gravity. While fluid flow in a microfluidic channel may be used to introduce micro-objects into the microfluidic channel (e.g., a flow region), such localized flow is not relied upon to transfer micro-objects into or remove them from the pen; localized flow (e.g., forces derived from actuation of a deformable surface) may, in some embodiments, be selected from among the other types of repositioning described herein to transfer micro-objects into or remove them from the pen or to transport them out of the microfluidic device.

[0205]

[0217] When DEP is used to reposition micro-objects, whether the micro-objects are being repositioned from a channel to an isolation pen or from an isolation pen to a channel, bulk fluid flow within the channel is typically stopped before applying DEP to the micro-objects to reposition the micro-objects within the microfluidic circuitry of the device, after which bulk fluid flow may be resumed.

[0206] system

[0218] 1A, a system 150 for operating and controlling a microfluidic device, such as for controlling microfluidic device 100, is shown. System 150 controls its various components (e.g., medium module 160, motive module 162, imaging module 164, tilt module 166, and other modules 168) to perform any of the techniques described herein. For example, master controller 154 can include control module 156 (e.g., processor or controller circuitry) configured to access digital memory 158 that stores computer instructions that, when executed by one or more computing devices (e.g., control module 156), cause the one or more computing devices to perform the techniques described herein, including forming in situ generated structures, controlling light sources to project onto the microfluidic device, and facilitating displacement of micro-objects.

[0207]

[0219] The electrical power source 192 can provide electrical power and, if necessary, bias voltage or current to the microfluidic device 100. The electrical power source 192 can include, for example, one or more alternating current (AC) and / or direct current (DC) voltage or current sources.

[0208]

[0220] The system 150 can further include a medium source 178. The medium source 178 (e.g., a container, reservoir, etc.) can include multiple sections or containers, each holding a different fluid medium 180. Thus, the medium source 178 can be external to the microfluidic device 100 and can be a separate device from the microfluidic device 100, as shown in FIG. 1A. Alternatively, the medium source 178 can be wholly or partially within the enclosure 102 of the microfluidic device 100. For example, the medium source 178 can include a reservoir that is part of the microfluidic device 100.

[0209]

[0221] 1A also shows a simplified block diagram representation of an example of control and monitoring equipment 152 that may form part of system 150 and be utilized in conjunction with microfluidic device 100. As shown, an example of such control and monitoring equipment 152 may include a master controller 154 that includes a media module 160 for controlling a media source 178, a motive module 162 for controlling the movement and / or selection of micro-objects (not shown) and / or media (e.g., droplets of media) in microfluidic circuit 120, an imaging module 164 for controlling an imaging device (e.g., a camera, a microscope, a light source, or any combination thereof) for capturing images (e.g., digital images), and an optional tilt module 166 for controlling tilt of microfluidic device 100. Control equipment 152 may also include other modules 168 for controlling, monitoring, or performing other functions related to microfluidic device 100. As shown, monitoring equipment 152 may further include a display device 170 and an input / output device 172.

[0210]

[0222] The master controller 154 may include a control module 156 and a digital memory 158. The control module 156 may include, for example, a digital processor configured to operate according to machine-executable instructions (e.g., software, firmware, source code, etc.) stored as non-transitory data or signals in the memory 158. Alternatively or additionally, the control module 156 may include hardwired digital and / or analog circuitry. The medium module 160, the motive module 162, the imaging module 164, the optional tilt module 166, and / or the other modules 168 may be similarly configured. Accordingly, the process functions, process acts, actions, or steps discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus may be performed by any one or more of the master controller 154, the medium module 160, the motive module 162, the imaging module 164, the optional tilt module 166, and / or the other modules 168 configured as discussed above. Similarly, the master controller 154, the medium module 160, the motive module 162, the imaging module 164, the optional tilt module 166, and / or other modules 168 may be communicatively coupled to send and receive data used in any function, process, act, action, or step discussed herein.

[0211]

[0223] The medium module 160 controls the medium source 178. For example, the medium module 160 can control the medium source 178 to input a selected fluid medium 180 into the enclosure 102 (e.g., through the inlet port 107). The medium module 160 can also control the removal of medium from the enclosure 102 (e.g., through an outlet port (not shown)). Thus, one or more media can be selectively input to and removed from the microfluidic circuit 120. The medium module 160 can also control the flow of fluid medium 180 in the flow channels 106 within the microfluidic circuit 120. The medium module 160 can also provide regulated gas conditions to the medium source 178, for example, an environment including 5% CO2 (or higher). The medium module 160 can also control the temperature of the medium source enclosure, for example, to provide appropriate temperature control for the support cells in the medium source.

[0212] Motive Module

[0224] The motive module 162 can be configured to control the selection and movement of micro-objects (not shown) within the microfluidic circuit 120. The enclosure 102 of the microfluidic device 100 can include one or more electromotive mechanisms, including a dielectrophoretic (DEP) electrode-activating substrate, an optoelectronic tweezers (OET) electrode-activating substrate, an electrowetting (EW) electrode-activating substrate, and / or an optoelectrowetting (OEW) electrode-activating substrate, and the motive module 162 can control the activation of electrodes and / or transistors (e.g., phototransistors) to select and move micro-objects and / or droplets within the channel 106 and / or isolation pens 124, 126, 128, and 130. The kinetic mechanisms can be any suitable single mechanism or combination of mechanisms, as described in the paragraphs describing the motive techniques used within the microfluidic device. A DEP-configured device can include one or more electrodes that apply a non-uniform electric field within the microfluidic circuit 120 sufficient to exert a dielectrophoretic force on the micro-objects within the microfluidic circuit 120. The OET component device may include light-activatable electrodes to provide selective control of the movement of micro-objects within the microfluidic circuit 120 via light-induced dielectrophoresis.

[0213]

[0225] The imaging module 164 can control the imaging device. For example, the imaging module 164 can receive and process image data from the imaging device. The image data from the imaging device can include any type of information captured by the imaging device (e.g., the presence or absence of a micro-object, a droplet of medium, the accumulation of a label such as a fluorescent label, etc.). Using the information captured by the imaging device, the imaging module 164 can further calculate the position of the object (e.g., the micro-object, the droplet of medium) and / or the speed of movement of such object within the microfluidic device 100.

[0214]

[0226] The imaging device (part of the imaging module 164, discussed below) may include a device such as a digital camera for capturing images within the microfluidic circuit 120. In some cases, the imaging device may further include a detector with a fast frame rate and / or high sensitivity (e.g., for low-light applications). The imaging device may also include mechanisms for directing simulated radiation and / or light beams toward the microfluidic circuit 120 and collecting radiation and / or light beams reflected or emitted from the microfluidic circuit 120 (or microobjects contained therein). The emitted light beam may be in the visible spectrum and may include, for example, fluorescent radiation. The reflected light beam may include reflected emissions from an LED or a broad-spectrum lamp, such as a mercury lamp (e.g., a high-pressure mercury lamp) or a xenon arc lamp. The imaging device may further include a microscope (or optical train), which may or may not include an eyepiece.

[0215] support structure

[0227] The system 150 may further include a support structure 190 configured to support and / or hold the enclosure 102 containing the microfluidic circuit 120. In some embodiments, the optional tilt module 166 may be configured to activate the support structure 190 to rotate the microfluidic device 100 about one or more axes of rotation. The optional tilt module 166 may be configured to support and / or hold the microfluidic device 100 in an elevation orientation (i.e., at 0° relative to the x-axis and y-axis), a vertical orientation (i.e., at 90° relative to the x-axis and / or y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and the microfluidic circuit 120) relative to the axes is referred to herein as the "tilt" of the microfluidic device 100 (and the microfluidic circuit 120). For example, support structure 190 can optionally be used to tilt microfluidic device 100 relative to the x-axis at 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90°, or any degree therebetween (e.g., as controlled by optional tilt module 166). When the microfluidic device is tilted at an angle greater than about 15°, tilting can be performed to effect bulk movement of micro-objects from a flow region (e.g., a microfluidic channel) to / from an isolation pen to a flow region (e.g., a microfluidic channel). In some embodiments, the support structure 190 can hold the microfluidic device 100 at an angle of 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, or 10° relative to the x-axis (horizontal), as long as DEP is an effective force to move micro-objects from the isolation pen into the microfluidic channel. Because the surface of the electrode-activated substrate is substantially flat, DEP forces can be used even when the distal end of the isolation pen, opposite the opening to the microfluidic channel, is positioned vertically lower than the microfluidic channel.

[0216]

[0228] In some embodiments, when the microfluidic device is tilted or held at an angle relative to the horizontal, the microfluidic device 100 can be oriented such that the inner surface of the base of the flow channel 106 is laterally tilted above or below the inner surface of the base of one or more isolation pen openings to the flow channel. As used herein, the term "above" indicates that the flow channel 106 is positioned higher on the vertical axis defined by gravity than one or more isolation pens (i.e., objects in the isolation pen above the flow channel 106 have higher gravitational potential energy than objects in the flow channel), and vice versa when the flow channel 106 is positioned below one or more isolation pens. In some embodiments, the support structure 190 can be held at an angle of less than about 5°, less than about 4°, less than about 3°, or less than about 2° relative to the x-axis (horizontal), thereby placing the isolation pen at a lower potential energy than the flow channel. In some other embodiments, when long-term culture (e.g., for more than about 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, more than 7 days, or more) is performed in the microfluidic device, the device can be supported on a culture support and tilted at a greater angle of about 10°, about 15°, about 20°, about 25°, about 30°, or any angle therebetween, to retain the biological micro-objects in the isolation pen during the long-term culture period. At the end of the culture period, the microfluidic device containing the cultured biological micro-objects can be returned to support 190 in system 150, where the tilt angle is reduced to a value as described above, allowing DEP to be used to remove the biological micro-objects from the isolation pen. Further examples of the use of gravity induced by tilt are described in U.S. Pat. No. 9,744,533 (Breinlinger et al.), the entire contents of which are incorporated herein by reference.

[0217] Nest

[0229] 5A , system 150 can include a structure (also referred to as a “nest”) 500 configured to hold a microfluidic device 520, which can be like microfluidic device 100, 200, or any other microfluidic device described herein. Nest 500 can include a socket 502 capable of interfacing with microfluidic device 520 (e.g., optically actuated electrokinetic device 100, 200, etc.) and providing an electrical connection from a power source 192 to microfluidic device 520. Nest 500 can further include an integrated electrical signal generation subsystem 504. Electrical signal generation subsystem 504 can be configured to supply a bias voltage to socket 502 such that, when held by socket 502, a bias voltage is applied across a pair of electrodes within microfluidic device 520. Thus, electrical signal generation subsystem 504 can be part of power source 192. The ability to apply a bias voltage to the microfluidic device 520 does not mean that the bias voltage is applied all the time the microfluidic device 520 is held by the socket 502. Rather, in most cases, the bias voltage is applied intermittently, for example, only when needed to facilitate the generation of electrokinetic forces, such as dielectrophoresis or electrowetting, in the microfluidic device 520.

[0218]

[0230] 5A, the nest 500 can include a printed circuit board assembly (PCBA) 522. The electrical signal generation subsystem 504 can be mounted on and electrically integrated with the PCBA 522. The exemplary support also includes a socket 502 mounted to the PCBA 522.

[0219]

[0231] In some embodiments, Nest 500 can include an electrical signal generation subsystem 504 configured to measure the amplified voltage at microfluidic device 520 and then adjust its own output voltage as needed to achieve the desired voltage measured at microfluidic device 520. In some embodiments, the waveform amplification circuit can have a +6.5V to −6.5V power supply generated by a pair of DC-DC converters mounted on PCBA 522, generating a signal at microfluidic device 520 of up to 13 Vpp.

[0220]

[0232] In certain embodiments, the Nest 500 further includes a controller 508, such as a microprocessor, used to sense and / or control the electrical signal generation subsystem 504. Examples of suitable microprocessors include Arduino™ microprocessors, such as the Arduino Nano™. The controller 508 may be used to perform functions and analysis or may communicate with an external master controller 154 (shown in FIG. 1A). In the embodiment shown in FIG. 5A, the controller 508 communicates with the master controller 154 (of FIG. 1A) through an interface (e.g., a plug or connector).

[0221]

[0233] As shown in FIG. 5A , the support structure 500 (e.g., a nest) can further include a thermal control subsystem 506. The thermal control subsystem 506 can be configured to regulate the temperature of the microfluidic device 520 held by the support structure 500. For example, the thermal control subsystem 506 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). In the embodiment shown in FIG. 5A , the support structure 500 includes an inlet 516 and an outlet 518 to receive cooled fluid from an external reservoir of the cooling unit (not shown) and direct the cooled fluid into a flow path 514, through a cooling block, and then back to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and / or the flow path 514 can be mounted on a case 512 of the support structure 500. In some embodiments, the thermal control subsystem 506 is configured to regulate the temperature of the Peltier thermoelectric device to achieve a target temperature of the microfluidic device 520. Temperature regulation of the Peltier thermoelectric device can be achieved by a thermoelectric power source, such as a Pololu™ thermoelectric power source (Pololu Robotics and Electronics Corp.). The thermal control subsystem 506 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit.

[0222]

[0234] The Nest 500 may include a serial port 524, which allows the microprocessor of the controller 508 to communicate with the external master controller 154 via the interface. Additionally, the microprocessor of the controller 508 may communicate with the electrical signal generation subsystem 504 and the thermal control subsystem 506 (e.g., via a Plink tool (not shown)). Thus, via the combination of the controller 508, the interface, and the serial port 524, the electrical signal generation subsystem 504 and the thermal control subsystem 506 may communicate with the external master controller 154. In this manner, the master controller 154 may assist the electrical signal generation subsystem 504 by, among other things, performing scaling calculations for output voltage adjustments. A graphical user interface (GUI) (not shown), provided via a display device 170 coupled to the external master controller 154, may be configured to plot temperature and waveform data obtained from the thermal control subsystem 506 and the electrical signal generation subsystem 504, respectively. Alternatively or additionally, the GUI may allow updates to the controller 508, the thermal control subsystem 506, and the electrical signal generation subsystem 504.

[0223] Optical Subsystem

[0235] 5B is a schematic diagram of an optical subsystem 550 having an optical device 510 for imaging and manipulating micro-objects in a microfluidic device 520, which can be any microfluidic device described herein. The optical device 510 can be configured to perform imaging, analysis, and manipulation of one or more micro-objects within the enclosure of the microfluidic device 520.

[0224]

[0236] The optical device 510 may have a first light source 552, a second light source 554, and a third light source 556. The first light source 552 may send light to a structured light modulator 560, which may include a digital mirror device (DMD) or a microshutter array system (MSA), either of which may be configured to receive light from the first light source 552 and selectively send a subset of the received light to the optical device 510. Alternatively, the structured light modulator 560 may include a device that generates its own light (thus eliminating the need for the light source 552), such as an organic light-emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD). The structured light modulator 560 may be, for example, a projector. Thus, the structured light modulator 560 may be capable of emitting both structured and unstructured light. In certain embodiments, the imaging module and / or the driving module of the system may control the structured light modulator 560 .

[0225]

[0237] In embodiments in which the structured light modulator 560 includes a mirror, the modulator can have multiple mirrors. Each mirror in the multiple mirrors can have a size of about 5 μm×5 μm to about 10 μm×10 μm, or any value therebetween. The structured light modulator 560 can include an array of mirrors (or pixels) that is 2000×1000, 2580×1600, 3000×2000, or any value therebetween. In some embodiments, only a portion of the illumination area of ​​the structured light modulator 560 is used. The structured light modulator 560 can send a selected subset of light to a first dichroic beam splitter 558, which can reflect the light to a first tube lens 562.

[0226]

[0238] First tube lens 562 can have a large opening, for example, a diameter of greater than about 40 mm to about 50 mm or larger, and can provide a large field of view. Thus, first tube lens 562 can have an aperture large enough to capture all (or substantially all) of the light beam from structured light modulator 560.

[0227]

[0239] Structured light 515 having a wavelength of about 400 nm to about 710 nm may alternatively or additionally provide fluorescence excitation illumination to the microfluidic device.

[0228]

[0240] The second light source 554 may provide unstructured bright-field illumination. The bright-field illumination light 525 may have any suitable wavelength, and in some embodiments, may have a wavelength of about 400 nm to about 760 nm. The second light source 554 may send light to a second dichroic beam splitter 564 (which may also receive illumination light 535 from a third light source 556), from which the second light, i.e., bright-field illumination light 525, may be sent to a first dichroic beam splitter 558. The second light, i.e., bright-field illumination light 525, may then be sent from the first dichroic beam splitter 558 to a first tube lens 562.

[0229]

[0241] The third light source 556 can send light through a matching relay lens pair (not shown) to a mirror 566. From there, the third illumination light 535 can be reflected to a second dichroic beam splitter 564, from which it can be sent to a first beam splitter 558 and then on to a first tube lens 562. The third illumination light 535 can be a laser and can have any suitable wavelength. In some embodiments, the laser illumination 535 can have a wavelength of about 350 nm to about 900 nm. The laser illumination 535 can be configured to heat portions of one or more isolation pens within the microfluidic device. Laser illumination 535 may be configured to heat fluid media, micro-objects, walls or portions of walls of an isolation pen, metal targets disposed within a microfluidic channel or isolation pen, or photocleavable physical barriers within a microfluidic device, as described in more detail in U.S. Patent Application Publication Nos. 2017 / 0165667 (Beaumont et al.) and 2018 / 0298318 (Kurz et al.), each of which is incorporated herein by reference in its entirety. In other embodiments, laser illumination 535 may be configured to initiate photocleavage of a surface-modified portion of a modified surface of a microfluidic device or photocleavage of a portion that provides adhesive functionality to micro-objects within an isolation pen within a microfluidic device. Further details of photocleavage using a laser can be found in International Publication No. WO 2017 / 205830 (Lowe, Jr. et al.), the disclosure of which is incorporated herein by reference in its entirety.

[0230]

[0242] Light from the first, second, and third light sources (552, 554, 556) passes through a first tube lens 562 and is sent to a third dichroic beam splitter 568 and a filter changer 572. The third dichroic beam splitter 568 may reflect a portion of the light and pass it through one or more filters in a filter changer 572 and then to an objective lens 570, which may be an objective lens changer with multiple different objective lenses that can be swapped out as needed. Some of the light (515, 525, and / or 535) passes through the third dichroic beam splitter 568 and may be terminated or absorbed by a beam block (not shown). Light reflected from the third dichroic beam splitter 568 passes through the objective lens 570 to illuminate the sample plane 574, which can be part of a microfluidic device 520 such as an isolation pen described herein.

[0231]

[0243] 5A , the nest 500 can be integrated with or part of the optical device 510. The nest 500 can provide electrical connections to the enclosure and can be further configured to provide fluid connections to the enclosure. A user can load the microfluidic device 520 into the nest 500. In some other embodiments, the nest 500 can be a separate component independent of the optical device 510.

[0232]

[0244] Light can reflect and / or emit from sample plane 574, pass through objective lens 570, filter changer 572, and third dichroic beam splitter 568 to second tube lens 576. Light can pass through second tube lens 576 (or imaging tube lens 576) and be reflected off mirror 578 to imaging sensor 580. Stray light baffles (not shown) can be placed between first tube lens 562 and third dichroic beam splitter 568, between third dichroic beam splitter 568 and second tube lens 576, and between second tube lens 576 and imaging sensor 580.

[0233] Objective lens

[0245] The optical device may include an objective lens 570 specifically designed and configured for viewing and manipulating micro-objects in the microfluidic device 520. For example, a conventional microscope objective lens is designed to view micro-objects on a slide or through 5 mm of aqueous fluid while the micro-objects in the microfluidic device 520 are inside a plurality of isolation pens within a viewing plane 674 having a depth of 20, 30, 40, 50, 60, 70, 80 μm, or any value therebetween. In some embodiments, a transparent cover 520 a, e.g., a glass or ITO cover approximately 750 μm thick, may be placed on top of the plurality of isolation pens, which are positioned above the microfluidic substrate 520 c. Therefore, images of micro-objects obtained using a conventional microscope objective lens may have large aberrations, such as spherical and chromatic aberrations, which can degrade image quality. The objective lens 570 of the optical device 510 may be configured to correct spherical and chromatic aberrations in the optical device 510. The objective lens 570 may have one or more magnification levels available, such as 4x, 10x, 20x, etc.

[0234] Lighting Mode

[0246] In some embodiments, the structured light modulator 560 can be configured to modulate the light beam received from the first light source 552 and transmit a plurality of illumination light beams 515, which are structured light beams, to an enclosure of the microfluidic device, e.g., an area including an isolation pen. The structured light beam can include a plurality of illumination light beams. The plurality of illumination light beams can be selectively activated to generate a plurality of illumination patterns. In some embodiments, the structured light modulator 560 can be configured to generate an illumination pattern similar to that described with respect to FIGS. 4A and 4B, and the illumination pattern can be moved and adjusted. The optical device 510 can further include a control unit (not shown), which can be configured to adjust the illumination pattern to selectively activate one or more of the plurality of DEP electrodes of the substrate 520c to generate a DEP force and move one or more micro-objects within the plurality of isolation pens in the microfluidic device 520. For example, the plurality of illumination patterns can be controlled and adjusted over time to manipulate micro-objects in the microfluidic device 520. Each of the multiple illumination patterns can be shifted to shift the position of the generated DEP force and move the structured light from one position to another, thereby moving micro-objects within the enclosure of the microfluidic device 520.

[0235]

[0247] In some embodiments, the optical device 510 can be configured such that each of the multiple isolating pens at the sample plane 574 within the field of view is simultaneously focused onto the image sensor 580 and the structured light modulator 560. In some embodiments, the structured light modulator 560 can be positioned at a conjugate plane of the image sensor 580. In various embodiments, the optical device 510 can have a confocal configuration or confocality. The optical device 510 can be further configured such that only each interior area of ​​the flow region and / or each of the multiple isolating pens at the sample plane 574 within the field of view is imaged onto the image sensor 580 to reduce overall noise and thereby increase image contrast and resolution.

[0236]

[0248] In some embodiments, the first tube lens 562 can be configured to generate a collimated light beam and send the collimated light beam to the objective lens 570. The objective lens 570 can receive the collimated light beam from the first tube lens 562 and focus the collimated light beam on each interior area of ​​the flow region and on each of a plurality of isolated pens at a sample plane 574 within a field of view of the image sensor 580 or the optical device 510. In some embodiments, the first tube lens 562 can be configured to generate a plurality of collimated light beams and send the plurality of collimated light beams to the objective lens 570. The objective lens 570 can receive the plurality of collimated light beams from the first tube lens 562 and focus the plurality of collimated light beams on each of a plurality of isolated pens at a sample plane 574 within a field of view of the image sensor 580 or the optical device 510.

[0237]

[0249] In some embodiments, the optical device 510 can be configured to illuminate at least a portion of the isolation pen with multiple illumination spots. The objective lens 570 can receive multiple collimated light beams from the first tube lens 562 and project multiple illumination spots, which can form an illumination pattern, onto each of the multiple isolation pens at the sample plane 574 within the field of view. For example, each of the multiple illumination spots can have a size of approximately 5 μm x 5 μm, 10 μm x 10 μm, 10 μm x 30 μm, 30 μm x 60 μm, 40 μm x 40 μm, 40 μm x 60 μm, 60 μm x 120 μm, 80 μm x 100 μm, 100 μm x 140 μm, and any value therebetween. The illumination spots can individually have a circular, square, or rectangular shape. Alternatively, the illumination spots can be grouped into multiple illumination spots (e.g., illumination patterns) to form larger polygons such as rectangles, squares, or wedges. The illumination pattern can surround (e.g., enclose) a non-illuminated space, which can be square, rectangular, or polygonal. For example, each of the plurality of illumination spots can have an area of ​​about 150 to about 3000 square microns, about 4000 to about 10000 square microns, or 5000 to about 15000 square microns. The illumination pattern can have an area of ​​about 1000 to about 8000 square microns, about 4000 to about 10000 square microns, 7000 to about 20000 square microns, 8000 to about 22000 square microns, 10000 to about 25000 square microns, and any value therebetween.

[0238]

[0250] The optical system 510 can be used to determine how to reposition micro-objects to and from an isolation pen of a microfluidic device and how to count the number of micro-objects present in the microfluidic circuitry of the device. Further details on repositioning and counting micro-objects can be found in U.S. Patent Application Publication No. 2016 / 0160259 (Du), U.S. Patent No. 9,996,920 (Du et al.), and International Publication No. 2017 / 102748 (Kim et al.). Optical system 510 may be employed in assay methods to determine the concentration of reagents / assay products, and further details can be found in U.S. Patent Nos. 8,921,055 (Chapman), 10,010,882 (White et al.), and 9,889,445 (Chapman et al.); International Application Publication No. 2017 / 181135 (Lionberger et al.); and International Application No. PCT / US2018 / 055918 (Lionberger et al.). Further details of the features of optical apparatus suitable for use in systems for observing and manipulating microscopic objects in microfluidic devices as described herein can be found in International Publication No. WO 2018 / 102747 (Lundquist et al.), the disclosure of which is incorporated herein by reference in its entirety.

[0239] Additional system components for maintaining diverse cells within the isolation pen of a microfluidic device

[0251] To promote the growth and / or proliferation of the cell population, environmental conditions conducive to maintaining functional cells can be provided by additional components of the system, such as those that provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from the cells.

[0240] experiment Systems and Devices

[0252] We employed an OPTOSELECT® device, a nanofluidic device controlled by a BEACON® optical instrument (both manufactured by PhenomeX Inc.). The instrument includes an optical train containing a mounting stage for the chip coupled to a temperature controller; pumps and fluid medium conditioning components; and a camera and a structured light source suitable for activating a phototransistor within the chip. The OPTOSELECT® device includes a substrate configured with OptoElectroPositioning (OEP®) technology, which provides an OET force that activates the phototransistor. The chip also includes multiple microfluidic channels, each with multiple NANOPEN® chambers (or chambers) fluidically connected thereto. The volume of each chamber is approximately 1×10 6 cubic μm.

[0241] Device Priming

[0253] 250 μL of 100% carbon dioxide was flowed through the OPTOSELECT® device at a flow rate of 12 μL / sec, followed by 250 μL of PBS containing 0.1% PLURONIC® F27 (LIFE TECHNOLOGIES® Catalog No. P6866) at 12 μL / sec, and finally 250 μL of PBS at 12 μL / sec. A wetting solution was then introduced, thereby introducing the conditioned surface to the surface within the microfluidic device. Details of the surface and its introduction are described in U.S. Patent Application Publication No. 2016 / 0312165, filed April 22, 2016, and U.S. Patent Application Publication No. 2019 / 0275516, filed November 20, 2018, the disclosures of each of which are incorporated herein by reference in their entirety.

[0242] Medium perfusion during culture

[0254] Media is perfused through the OPTOSELECT® device according to one of two methods: (1) perfuse at 0.01 μL / sec for 2 hours, perfuse at 2 μL / sec for 64 seconds, and repeat; (2) perfuse at 0.01 μL / sec for 100 seconds, stop flow for 500 seconds, perfuse at 2 μL / sec for 64 seconds, and repeat.

[0243] System Preparation

[0255] An OPTOSELECT® chip was loaded into the Beacon instrument for a series of pre-workflow operations. A wetting solution was introduced to the chip and then incubated to functionalize the surface. Each chip was then flushed with DI water to remove the wetting solution. After wetting and priming, the Beacon instrument automatically placed fiducial markers on the chip for xy stage and focus calibration. A reference image was then performed.

[0244] Single cell loading

[0256] Cells were loaded as previously described in U.S. Patent No. 11,170,200 (Kim et al.), filed May 31, 2019, granted November 9, 2021, and U.S. Patent Publication No. 2021 / 0209752 (Tenney et al.), filed November 24, 2020. Single cells were automatically identified by the Beacon instrument control software using a conventional neural network algorithm. A positioning strategy was then automatically implemented to maximize loading throughput using OEP technology. After loading, residual cells in the channel were flushed and discarded.

[0245]

[0257] Example 1: Cell Displacement

[0258] Cells from the cell population were suspended in PBS and introduced into an OPTOSELECT® microfluidic device, e.g., a chip, within the BEACON® optical fluidics system. A single cell was placed within each isolation pen and cultured on-chip. The cells were maintained and allowed to grow for a period of time, and cells / colonies of interest were identified. Using a suitable method (e.g., OEP® technology, etc.), cells within the isolation pen were moved from the region furthest from the isolation pen opening to a region closer to the pen opening, creating a fluid space between the cells and the furthest part of the pen.

[0246]

[0259] A fluid hydrogel polymer was introduced into a solution and allowed to diffuse into the isolation pen. A photoinitiator was also introduced into the solution containing the fluid hydrogel polymer. The hydrogel polymer was solidified by photopatterning, for example, by photoactivation of polymerization (1 to 1.5 seconds or 3.5 to 5 seconds of exposure, 10× objective, 50% power), to form a solidified hydrogel barrier. The in situ-generated hydrogel barrier was formed as a piston, and optionally, a guide element was also formed in the region near the opening of the chamber. The in situ-generated piston was used for cell displacement. The formed piston defined the isolation pen into two areas (regions). The region away from the opening was the target region (displacement force generation region) and was substantially devoid of any cells (e.g., few or no cells). The region near the opening contained multiple cells (e.g., many, substantially all, or all cells) and was a cell culture region suitable for culturing cells before removal from the pen. Laser illumination was directed toward the target region, generating a displacement force sufficient to remove cells from the pen. The cells were removed from the pen and placed into a microfluidic channel, and then removed from the microfluidic device by flowing media through the microfluidic channel.

[0247]

[0260] A selected pen was prepared to facilitate cell removal from the pen, as shown diagrammatically in Figure 6A and pictorially in Figure 6B. A series of time-lapse photographs illustrating the pen removal process is shown in Figure 7. Beginning with the removal of the cell from the pen, a light-activated dielectrophoretic bar was programmed to move toward the pen opening into the channel. As the light bar was moved closer to the pen opening, a transistor in the substrate was activated, thus moving the cell away from the tip of the isolation pen. Following the movement of the cell away from the tip of the isolation pen, a hydrogel piston was formed as described herein.

[0248]

[0261] Laser illumination was then directed toward a target region, e.g., an area of ​​the substrate within the displacement force generation region, as shown in FIG. 7 . The dielectrophoretic force had already been removed from that portion of the pen, thus avoiding direct impingement of the laser illumination on the cells. Laser illumination-induced heating and / or bubble formation in the medium displaced fluid in the target region. The heating (including medium expansion) and / or bubbles applied a force toward the base of the pen, e.g., toward the pen opening, thus applying a force to the in situ-generated piston, causing it to melt, displace, and / or deform. Positioning the piston between the cells and the target region protected the cells from the forces associated with displacement force generation, enhancing the viability of the displaced cells and the efficiency of displacing the cells for removal and transport from the pen.

Claims

1. 1. A method for displacing a micro-object from a chamber of a microfluidic device, comprising: providing the microfluidic device, the microfluidic device including a microfluidic circuit including a substrate, a flow region, and the chamber, the chamber including an opening to the flow region, and the micro-object disposed within the chamber; forming an in situ generation piston in a region of the chamber remote from the minute object, the in situ generation piston defining a target region in the chamber remote from the in situ generation piston; illuminating the target area, thereby generating a displacement force, thereby displacing the micro-object from the chamber; and A method comprising:

2. The method of claim 1 , wherein the minute object further comprises a plurality of minute objects.

3. 3. The method of claim 2, wherein before forming the in situ piston, the method further comprises moving a portion of the plurality of minute objects away from a region of the chamber farthest from the opening of the chamber.

4. The method of claim 3 , wherein moving a portion of the plurality of micro-objects further comprises moving the portion of the plurality of micro-objects toward the opening of the chamber.

5. The method of claim 1 , wherein the target area defined by the in situ generated piston is substantially free of the minute objects.

6. The method of claim 1 , wherein providing the microfluidic device further comprises disposing the micro-object in the chamber.

7. The method of claim 6 further comprising culturing the micro-objects in the chamber.

8. The method of claim 1 , wherein illuminating the target area comprises illuminating the target area with a laser.

9. 9. The method of claim 1, wherein illuminating the target area results in the formation of a bubble within the target area, the bubble generating a displacement force.

10. 10. The method of claim 1, wherein the displacement force pushes the in situ generated piston away from its original position.

11. The method of claim 10 , wherein the displacement force urges the in situ generated piston toward the opening of the chamber.

12. The method of claim 1 , wherein illuminating the target area comprises directing illumination onto the substrate, a wall microfluidic circuit material, or a thermal target.

13. The method of claim 12 , wherein the thermal target comprises a metal deposit, a pattern of metal deposits, or a microstructure patterned on a surface.

14. 14. The method of claim 1, wherein illuminating the target area comprises illuminating with illumination having an incident power in a range from about 1 mW to about 1000 mW.

15. 15. The method of claim 1, wherein the in situ generated piston has a porosity that substantially prevents the minute objects from traversing the in situ generated piston.

16. 16. The method of claim 1, wherein displacing the micro-object comprises transporting the micro-object into the flow region and, optionally, transporting the micro-object out of the microfluidic device.

17. 17. The method of claim 16, wherein transporting the micro-objects from the microfluidic device further comprises flowing a medium through the flow region.

18. 18. The method of any one of claims 1 to 17, wherein the flow region comprises a microfluidic channel, and the opening of the chamber is proximate to the microfluidic channel and oriented substantially parallel to the direction of flow of a fluid medium in the microfluidic channel (e.g., when the fluid medium is flowing through the microfluidic channel).

19. 19. The method of claim 1, wherein the chamber comprises a separation region and a connection region fluidly connecting the separation region to the flow region, the connection region comprising the opening to the flow region.

20. The method of claim 19 , wherein the minute objects are disposed within the separation region.

21. 21. The method of claim 19 or 20, wherein the target region is within the separation region.

22. 22. The method of any one of claims 1 to 21, wherein the in situ generated piston comprises a first solidified polymer network.

23. 23. The method of claim 1, further comprising forming an in situ generated guide element comprising a second solidified polymer network in an area of ​​the chamber near the opening.

24. 24. The method of claim 23, wherein the area proximate the opening of the chamber is within the chamber.

25. 25. The method of claim 23 or 24, wherein the in situ generated guide element includes at least one gap configured to allow displacement of the micro-objects and prevent re-entry of the micro-objects from the flow region into the chamber.

26. 26. The method of any one of claims 23 to 25, wherein the first solidified polymer network (e.g., piston) and the second solidified polymer network (e.g., guide element) independently comprise a synthetic polymer, a modified synthetic polymer, or a biopolymer.

27. 27. The method of claim 26, wherein the first solidified polymer network and the second solidified polymer network independently comprise at least one of polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, denatured collagen, laminin, modified laminin, polysaccharides, modified polysaccharides, or copolymers in any combination.

28. 28. The method of claim 27, wherein the solidified polymer network comprises polyethylene glycol acrylamide polymers.

29. 30. The method of claim 28, wherein the polyethylene glycol acrylamide polymer comprises a linear polyethylene glycol diacrylamide polymer, a two-arm polyethylene glycol diacrylamide polymer, a star polyethylene glycol diacrylamide polymer, or a mixture of any combination thereof.

30. 30. The method of claim 29, wherein the polyethylene glycol acrylamide polymer is other than a linear polyethylene glycol acrylamide polymer, all termini of which contain acrylamide moieties.

31. 31. The method of claim 29 or 30, wherein when the polyethylene glycol acrylamide polymer is other than a linear polyethylene glycol acrylamide polymer, less than all of the termini comprise acrylamide moieties.

32. 32. The method of any one of claims 1 to 31, wherein forming the in situ generated piston and / or the in situ generated guide element comprises flowing a first fluid medium containing a flowable polymer solution through the flow region of the microfluidic device, and flowing the flowable polymer solution to diffuse within the chamber.

33. 33. The method of claim 32, wherein forming the in situ generated piston and / or the in situ generated guide element further comprises solidifying the fluid polymer solution within the chamber using photopatterning.

34. 34. The method of any one of claims 1 to 33, wherein the microscopic object is a biological cell (e.g., a eukaryotic or prokaryotic cell) or a bead.

35. 35. The method of claim 34, wherein the biological cell is an animal cell, a plant cell, or a bacterial cell.

36. 1. A kit for displacing a micro-object from a chamber of a microfluidic device, comprising: a flowable polymer configured to be controllably activated to form an in situ generated barrier comprising a solidified polymer network; an inhibitor; Includes a kit.

37. 37. The kit of claim 36, further comprising a photoinitiator.

38. 38. The kit of claim 37, wherein the photoinitiator is a photoactivatable photoinitiator.

39. 39. The kit of any one of claims 36 to 38, further comprising a microfluidic device comprising a microfluidic circuit comprising a flow region and a chamber, the chamber comprising an opening to the flow region.

40. 40. The kit of claim 39, wherein the flow region comprises a microfluidic channel, and the opening of the chamber is proximate to the microfluidic channel and oriented substantially parallel to the flow of fluid medium in the microfluidic channel when the fluid medium is flowing within the microfluidic channel.

41. 41. The kit of claim 39 or 40, wherein the chamber comprises a separation region and a connection region fluidly connecting the separation region to the flow region, the connection region comprising the opening to the flow region.

42. 42. The kit of any one of claims 39 to 41, wherein the microfluidic device comprises multiple chambers.

43. 43. The kit of any one of claims 39 to 42, wherein the microfluidic device comprises a substrate configured to generate dielectrophoretic (DEP) forces within the microfluidic circuit.

44. 44. The kit of any one of claims 36 to 43, wherein the solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, or a biopolymer.

45. 45. The kit of any one of claims 36-44, wherein the solidified polymer network comprises at least one of polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, polyacrylic acid (PAA), modified polyacrylic acid, fibronectin, modified fibronectin, collagen, denatured collagen, laminin, modified laminin, polysaccharides, modified polysaccharides, or copolymers in any combination.

46. 46. ​​The kit of claim 44 or 45, wherein the solidified polymer network comprises polyethylene glycol acrylamide polymers.

47. 47. The kit of claim 46, wherein the polyethylene glycol acrylamide polymer comprises a linear polyethylene glycol diacrylamide polymer, a two-arm polyethylene glycol diacrylamide polymer, a star polyethylene glycol diacrylamide polymer, or a mixture of any combination thereof.

48. 48. The kit of claim 46 or 47, wherein the polyethylene glycol acrylamide polymer is other than a linear polyethylene glycol acrylamide polymer, all termini of which contain acrylamide moieties.

49. 48. The kit of claim 46 or 47, wherein when the polyethylene glycol acrylamide polymer is other than a linear polyethylene glycol acrylamide polymer, fewer than all of the termini contain acrylamide moieties.

50. 1. A method for displacing a micro-object from an isolation pen of a microfluidic device, comprising: forming an in situ generation structure within the isolation pen of the microfluidic device, thereby defining (a) a first region proximate an opening of the isolation pen to a microfluidic channel and (b) a second region remote from the opening of the isolation pen to the microfluidic channel, the in situ generation structure being located between the first region and the second region; positioning a minute object within the first region of the isolation pen; illuminating the second region of the isolation pen, thereby generating a displacement force to displace the micro-object from the first region of the isolation pen into the microfluidic channel; A method comprising:

51. 51. The method of claim 50, wherein illuminating the second region of the isolation pen comprises projecting laser illumination onto a surface of the isolation pen in the second region.

52. 52. The method of claim 50 or 51, wherein illuminating the second area of ​​the isolation pen comprises projecting light onto a surface of the isolation pen in the second area and moving a position of the light projected onto the surface.

53. 53. The method of claim 52, wherein moving the position of the light comprises moving the position toward the opening of the isolation pen.

54. The method comprises:

54. A method according to any one of claims 50 to 53, further comprising forming an in situ generated guide structure at the opening of the isolation pen to the microfluidic channel after the micro object is positioned within the first region of the isolation pen and before illuminating the second region of the isolation pen.

55. 55. The method of claim 54, wherein the in situ generated guide structure tapers as it extends toward the second region away from the opening of the isolation pen.

56. 56. The method of any one of claims 50 to 55, wherein illuminating the second region of the isolation pen includes generating a bubble in the second region, the bubble providing the displacement force.

57. 57. The method of any one of claims 50 to 56, wherein the displacement force moves the in situ generation structure towards the opening of the isolation pen.

58. 58. The method of any one of claims 50 to 57, wherein the in situ generated structure is a solidified polymer network.

59. A non-transitory computer-readable medium having stored thereon computer program instructions, the computer program instructions, when executed by one or more computing devices, causing the one or more computing devices to: forming an in situ generation structure in an isolation pen of a microfluidic device, thereby defining (a) a first region proximate an opening of the isolation pen to a microfluidic channel and (b) a second region remote from the opening of the isolation pen to the microfluidic channel, the in situ generation structure being located between the first region and the second region; positioning the minute object within the first region of the isolation pen; illuminating the second region of the isolation pen, thereby generating a displacement force to displace the micro-object from the first region of the isolation pen into the microfluidic channel; 1. A non-transitory computer-readable medium configured to cause

60. 60. The non-transitory computer-readable medium of claim 59, wherein illuminating the second region of the isolation pen comprises projecting laser illumination onto a surface of the isolation pen in the second region.

61. 61. The non-transitory computer-readable medium of claim 59 or 60, wherein illuminating the second region of the isolation pen comprises projecting light onto a surface of the isolation pen in the second region and moving a position of the light projected onto the surface.

62. 62. The non-transitory computer-readable medium of claim 61, wherein moving the position of the light comprises moving the position toward the opening of the isolation pen.

63. 63. A non-transitory computer-readable medium as described in any one of claims 59 to 62, wherein the computer program instructions cause the one or more computing devices to form an in situ generated guide structure at the opening of the isolation pen to the microfluidic channel after the micro object is positioned within the first region of the isolation pen and before illuminating the second region of the isolation pen.

64. 64. The non-transitory computer-readable medium of claim 63, wherein the in situ generated guide structure tapers as it extends toward the second region away from the opening of the isolation pen.

65. 65. The non-transitory computer-readable medium of any one of claims 59 to 64, wherein illuminating the second region of the isolation pen includes generating a bubble in the second region, the bubble providing the displacement force.

66. 66. The non-transitory computer-readable medium of any one of claims 59 to 65, wherein the displacement force moves the in situ generation structure toward the opening of the isolation pen.

69. 67. The non-transitory computer-readable medium of any one of claims 59 to 66, wherein the in situ generated structure is a solidified polymer network.

70. 1. A system comprising: one or more processors and memory; The one or more processors and memory forming an in situ generation structure in an isolation pen of a microfluidic device, thereby defining (a) a first region proximate an opening of the isolation pen to a microfluidic channel and (b) a second region remote from the opening of the isolation pen to the microfluidic channel, the in situ generation structure being located between the first region and the second region; positioning a minute object within the first region of the isolation pen; illuminating the second region of the isolation pen, thereby generating a displacement force to displace the micro-object from the first region of the isolation pen into the microfluidic channel; A system configured to:

71. 71. The system of claim 70, wherein illuminating the second region of the isolation pen comprises projecting laser illumination onto a surface of the isolation pen in the second region.

72. 72. The system of claim 70 or 71, wherein illuminating the second area of ​​the isolation pen comprises projecting light onto a surface of the isolation pen in the second area and moving a position of the light projected onto the surface.

73. 73. The system of claim 72, wherein moving the position of the light comprises moving the position toward the opening of the isolation pen.

74. one or more processors and memory; 74. The system of any one of claims 70 to 73, further configured to form an in situ generated guide structure at the opening of the isolation pen to the microfluidic channel after the micro object is positioned within the first region of the isolation pen and before illuminating the second region of the isolation pen.

75. 75. The system of claim 74, wherein the in situ generated guide structure tapers as it extends toward the second region away from the opening of the isolation pen.

76. 76. The system of any one of claims 70 to 75, wherein illuminating the second region of the isolation pen includes generating a bubble in the second region, the bubble providing the displacement force.

77. 77. The system of any one of claims 70 to 76, wherein the displacement force moves the in situ generation structure towards the opening of the isolation pen.

78. 78. The system of any one of claims 70 to 77, wherein the in situ generated structure is a solidified polymer network.

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