CN114085730A - Microfluidic cell culture - Google Patents

Abstract

The present application relates to microfluidic cell culture, and in particular, describes systems, methods, and kits for culturing one or more biological cells in a microfluidic device, including providing nutrients and gas components configured to enhance cell growth, viability, portability, or any combination thereof. In some embodiments, culturing a single cell can produce a clonal population in a microfluidic device.

Description

Microfluidic cell culture

The application is a divisional application of Chinese patent application with the application number of 2016800363322, the application date of 2016, 4 and 22, and the name of 'microfluidic cell culture'.

Background

In the field of bioscience and related fields, it may be useful to culture one or more cells. Some embodiments of the invention include devices and methods for culturing cells or cell populations in a microfluidic device.

Disclosure of Invention

In one aspect, there is provided a microfluidic device for culturing one or more biological cells, comprising: a flow region (flow region) configured to contain a flow of a first fluidic medium; and at least one growth chamber comprising a separation region and a connection region, the separation region in fluidic connection with the connection region, and the connection region comprising a proximal opening (proximal exposing) to the flow stream region, wherein the at least one growth chamber further comprises at least one surface conditioned (conditioned to) to support cell growth, viability, portability (portability) or any combination thereof within the microfluidic device. In some embodiments, the separation region of the microfluidic device may be configured to contain a second fluid medium, and wherein when the flow region and the at least one growth chamber are substantially filled with the first and second fluid media, respectively, components of the second fluid medium may diffuse into the first fluid medium and/or components of the first fluid medium may diffuse into the second fluid medium, and the first medium may not substantially flow into the separation region. In some embodiments, the microfluidic device may further comprise a microfluidic channel having at least a portion of the flow region, and wherein the connection region of the at least one growth chamber may open directly into the microfluidic channel.

In some embodiments, at least one conditioned surface may be conditioned with one or more reagents that support cell portability within the microfluidic device. In some embodiments, at least one conditioned surface may be conditioned by a polymer comprising alkylene ether moieties. In other embodiments, at least one of the conditioned surfaces may be conditioned by a polymer comprising a sugar moiety. In some embodiments, the polymer comprising a saccharide moiety may comprise dextran. In other embodiments, at least one conditioned surface may be conditioned by a polymer comprising amino acid moieties. In some embodiments, the polymer may be Bovine Serum Albumin (BSA) or DNase 1. In other embodiments, at least one conditioned surface of a microfluidic device can be conditioned by a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety. In some embodiments, at least one conditioned surface of a microfluidic device can be conditioned by a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety.

In various embodiments of the microfluidic device, the at least one conditioned surface comprises a linking group covalently attached to a surface of the microfluidic device, and the linking group can be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof within the microfluidic device. In some embodiments, the linking group may be a siloxy linking group. In other embodiments, the linking group may be a phosphonate linking group. In various embodiments, at least one conditioned surface may comprise alkyl or fluoroalkyl moieties. In some embodiments, the fluoroalkyl moiety may be a perfluoroalkyl moiety. In some embodiments, the alkyl or fluoroalkyl moiety may have a backbone chain length of greater than 10 carbons. The alkyl or fluoroalkyl moiety may have a linear structure. In various embodiments of the microfluidic device, the linking group of the at least one conditioned surface may be directly linked to a moiety configured to support cell growth, viability, portability, or any combination thereof. In other embodiments, the linking group may be indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group can be indirectly attached via a linking moiety to a moiety configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group may include a triazolylene moiety. In other embodiments, the linking group may include one or more arylene moieties. In some embodiments, the at least one conditioned surface may comprise a sugar moiety. In other embodiments, at least one of the conditioned surfaces may include an alkylene ether moiety. In other embodiments, at least one conditioned surface may comprise an amino acid moiety. Alternatively, at least one conditioned surface may comprise a zwitterion. In other embodiments, at least one conditioned surface may comprise a phosphonic acid moiety or a carboxylic acid moiety. In other embodiments, at least one of the conditioned surfaces comprises an amino or guanidine moiety. In some other embodiments, at least one conditioned surface may comprise alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid.

In various embodiments of the microfluidic device, at least one conditioned surface of the microfluidic device may comprise at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule may disrupt the formation of an agonist silk, block an integrin receptor, or reduce binding of cells to a DNA-contaminated surface. In some embodiments, the at least one cell attachment blocking molecule may be an RGD-containing peptide. In other embodiments, the at least one cell adhesion blocking molecule may be cytochalasin B, an anti-integrin antibody, an inhibitor of fibronectin, which may include a small molecule or a DNase 1 protein. In other embodiments, the at least one cell adhesion blocking molecule may comprise a combination of more than one type of cell adhesion blocking molecule.

In various embodiments of the microfluidic device, at least one conditioned surface of the microfluidic device can comprise a cleavable moiety. In some embodiments, the cleavable moiety may be configured to allow for disruption of the conditioned surface, thereby facilitating portability of the one or more biological cells after culture.

In various embodiments of the microfluidic device, at least one conditioned surface of the microfluidic device may comprise one or more components of mammalian serum. One or more components of the mammalian serum may include

Supplements, Fetal Bovine Serum (FBS) or fetal bovine serum (FCS).

In various embodiments of the microfluidic device, the microfluidic device may further include a substrate (substrate) having a Dielectrophoresis (DEP) configuration. In some embodiments, a substrate having a DEP configuration can be configured to introduce or remove one or more biological cells into or from a growth chamber. The DEP configuration may be light-actuated.

In various embodiments of the microfluidic device, the at least one conditioned surface of the microfluidic device can be configured to be stable at a temperature of at least about 30 ℃.

In various embodiments of the microfluidic device, the separation region of at least one growth chamber of the microfluidic device may have a size sufficient to support cell expansion to a range of about 100 cells. In some embodiments, it may not exceed 1 × 10²The biological cells are maintained in at least one growth chamber, and the volume of the at least one growth chamber may be less than or equal to about 2 x 10 ⁶Cubic microns. In other embodiments, it may not exceed 1 × 10²The biological cells are maintained in at least one growth chamber, and the volume of the at least one growth chamber may be less than or equal to about 1 x 10⁷Cubic microns.

In various embodiments of the microfluidic device, the device can further comprise at least one inlet configured to input the first or second fluidic medium into the flow region; and at least one outlet configured to receive the first medium as it exits the flow region. In various embodiments of the microfluidic device, the microfluidic device may further comprise a deformable cover region over the at least one growth chamber or the separation region thereof, such that depressing the deformable cover region applies a force sufficient to export the biological cells from the separation region to the flow region. In various embodiments of the microfluidic device, the microfluidic device may include a cover, wherein at least a portion of the cover may be gas permeable, thereby providing a source of gas molecules into a fluidic medium located in the microfluidic device. The gas permeable portion of the lid may be located above the at least one growth chamber. In other embodiments, the gas permeable portion of the lid may be located above the liquid flow region. In other embodiments, the at least one growth chamber may comprise a plurality of growth chambers.

In various embodiments, the one or more biological cells can include a plurality of biological cells. In various embodiments of the microfluidic device, the at least one growth chamber may comprise at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of mammalian cells. In other embodiments, at least one growth chamber may comprise at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of immune cells. In other embodiments, the immune cell may be a lymphocyte or a leukocyte. In some other embodiments, the immune cell may be a B cell, a T cell, an NK cell, a macrophage, or a dendritic cell.

In various embodiments of the microfluidic device, the at least one growth chamber may comprise at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of adherent cells.

In various embodiments of the microfluidic device, the at least one growth chamber may comprise at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of the hybridoma cells.

In various embodiments of the microfluidic device, at least one growth chamber may comprise at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of individual cells of the biological cells and corresponding clonal colonies.

In another aspect, a system for culturing one or more biological cells on a microfluidic device is provided, the system comprising a microfluidic device having a flow region configured to contain a flow of a first fluidic medium; and at least one growth chamber, wherein the growth chamber has at least one surface conditioned to support cell growth, viability, portability, or any combination thereof within the microfluidic device. The at least one growth chamber may comprise a separation region and a connection region, said separation region being in fluid connection with said connection region, and said connection region having a proximal opening to said flow region. In some embodiments, the separation region of the microfluidic device may be configured to contain a second fluid medium, and when the flow region and the at least one growth chamber are substantially filled with the first and second fluid media, respectively, components of the second fluid medium may diffuse into the first fluid medium and/or components of the first fluid medium may diffuse into the second fluid medium, and the first medium may not substantially flow into the separation region. In some embodiments, the microfluidic device may further comprise a microfluidic channel comprising at least a portion of the flow region, and wherein the connection region of the at least one growth chamber may open directly into the microfluidic channel. The microfluidic device may be any of the microfluidic devices described herein, having any of the elements in any combination.

In various embodiments of the system, the system can further include a flow controller configured to perfuse at least the first fluid medium. The controller is configured to non-continuously perfuse at least the first fluid medium.

In various embodiments of the system, the microfluidic device of the system can further include a substrate having a Dielectrophoresis (DEP) configuration, the substrate configured to introduce or remove one or more biological cells into or from the growth chamber. The DEP configuration may be light-actuated.

In various embodiments of the system, the system can further comprise a reservoir configured to contain the first fluidic medium, wherein the reservoir is fluidically connected to the microfluidic device. The reservoir may be configured to be contacted by a gaseous environment that is capable of saturating the first fluid medium with dissolved gas molecules.

In various embodiments of the system, the system can further comprise a sensor coupled to at least one inlet of the microfluidic device, wherein the sensor can be configured to detect a pH of the first fluidic medium. In various embodiments of the system, the system can further comprise a sensor connected to the at least one outlet, wherein the sensor is configured to detect a pH of the first fluid medium as the first fluid medium exits the microfluidic device. In some embodiments, the sensor may be a light sensor.

In various embodiments of the system, the system may further comprise a detector configured to capture an image of the at least one growth chamber and any biological cells contained therein. In some embodiments, the one or more biological cells may comprise one or more mammalian cells. In other embodiments, the one or more biological cells can include one or more hybridoma cells. In other embodiments, the one or more biological cells may include one or more lymphocytes or leukocytes. Or the one or more biological cells can comprise one or more adherent cells.

In various embodiments of the system, the one or more biological cells in the growth chamber can be a single cell and the colony can be a clonal colony of biological cells.

In another aspect, a composition is provided, comprising a substrate having a Dielectrophoresis (DEP) configuration and a surface; and a conditioned surface of oxide moieties covalently attached to the surface of the substrate. The composition may have the structure of formula 1 or formula 2, and may have any value for the elements of formula 1 or formula 2 as defined herein:

in some embodiments of the composition, the conditioned surface may include a linking group covalently attached to an oxide moiety of the surface, and the linking group may be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group may be a siloxy linking group. In other embodiments, the linking group may be a phosphonate group. In some embodiments, the linking group may be directly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group can be indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, the linking group can be indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via attachment to the linking moiety. In some embodiments, the linking group can be indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via attachment to the first end of the linking moiety. The linking moiety may also comprise a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the linking moiety may also include a triazolylene moiety. In some embodiments, the triazolylene moiety may interrupt the linear portion of the linking moiety or may be linked to the linear portion of the linking moiety at the second end. In other embodiments, the backbone of the linear moiety may include an arylene moiety.

In various embodiments, the moieties configured to support cell growth, viability, portability, or any combination thereof can include alkyl moieties, fluoroalkyl moieties, mono-or polysaccharides, alcohol moieties, polyol moieties, alkylene ether moieties, polyelectrolyte moieties, amino moieties, carboxylic acid moieties, phosphonic acid moieties, sulfonate anion moieties, carboxylic acid betaine moieties, sulfobetaine moieties, sulfamic acid moieties, or amino acid moieties. In some embodiments, the at least one conditioned surface may comprise amino acids, alkyl moieties, perfluoroalkyl moieties, dextran moieties, and/or alkylene ether moieties. In some embodiments, at least one conditioned surface may comprise alkyl or perfluoroalkyl moieties. In some embodiments, the alkyl or perfluoroalkyl moiety has a backbone chain length of more than 10 carbons. In various embodiments, the conditioned surface can further comprise one or more cleavable moieties. The cleavable moiety can be configured to allow disruption of the conditioned surface, thereby facilitating portability of the cultured one or more biological cells.

In another aspect, a method is provided for culturing at least one biological cell in a microfluidic device having a fluid flow region configured to contain a flow of a first fluid medium; and at least one growth chamber, the method comprising the steps of: introducing at least one biological cell to the at least one growth chamber, wherein the at least one growth chamber is configured with at least one surface conditioned to support cell growth, viability, portability, or any combination thereof; and incubating the at least one biological cell for at least a period of time sufficient to expand the at least one biological cell to produce a colony of biological cells. The at least one growth chamber may comprise a separation region and a connection region, said separation region being in fluid connection with said connection region, and said connection region having a proximal opening to said flow region. In some embodiments, the separation region of the microfluidic device may be configured to contain a second fluid medium, and wherein when the flow region and the at least one growth chamber are substantially filled with the first and second fluid media, respectively, components of the second fluid medium may diffuse into the first fluid medium and/or components of the first fluid medium may diffuse into the second fluid medium, and the first medium may not substantially flow into the separation region. In some embodiments, the microfluidic device may further comprise a microfluidic channel having at least a portion of the flow region, and wherein the connection region of the at least one growth chamber may open directly into the microfluidic channel. The microfluidic device may be any of the microfluidic devices described herein, having any of the elements in any combination.

In some embodiments of the method, the at least one conditioned surface may comprise a linking group covalently attached to the surface, further wherein the linking group is attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device. In some other embodiments, the moiety configured to support cell growth, viability, portability, or any combination thereof may comprise an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid. In some embodiments, at least one conditioned surface may comprise alkyl or perfluoroalkyl moieties. In other embodiments, at least one conditioned surface may comprise an alkylene ether moiety or a dextran moiety.

In some embodiments of the method, the method may comprise the step of conditioning at least one surface of the at least one growth chamber. In some embodiments, conditioning may comprise treating at least one surface of the at least one growth chamber with a conditioning agent comprising a polymer. In other embodiments, conditioning may comprise treating at least one surface of the at least one growth chamber with one or more components of mammalian serum. In other embodiments, conditioning may comprise treating at least one surface of the at least one growth chamber with at least one cell adhesion blocking molecule.

In some embodiments of the method, introducing at least one biological cell into the at least one growth chamber may comprise moving the at least one biological cell using a Dielectrophoresis (DEP) force of sufficient strength. In some embodiments, using the DEP force may include optically actuating the DEP force.

In some embodiments of the method, the method may further comprise the step of perfusing the first fluid medium during the incubating step, wherein the first fluid medium is introduced via at least one inlet of the microfluidic device and output via at least one outlet of the microfluidic device, wherein upon output, the first fluid medium optionally comprises components from the second fluid medium.

In some embodiments of the method, the method may further comprise a step of lysing the one or more cleavable moieties of the conditioned surface after the incubating step, thereby facilitating export of the one or more biological cells from the growth chamber or the separation region thereof and into the liquid flow region.

In some embodiments of the method, the method may further comprise the step of transporting the one or more biological cells out of the growth chamber or the separation region thereof and into the flow region.

In some embodiments of the method, the at least one biological cell may comprise a mammalian cell. In other embodiments of the method, the at least one biological cell may comprise at least one immune cell. In other embodiments of the method, the at least one immune cell may comprise a lymphocyte or a leukocyte. In some other embodiments of the method, the at least one immune cell may comprise a B cell, a T cell, an NK cell, a macrophage, or a dendritic cell. In other embodiments, the at least one biological cell may comprise an adherent cell. Alternatively, the at least one biological cell may comprise a hybridoma cell.

In some embodiments of the method, the step of introducing at least one biological cell into at least one growth chamber may comprise introducing a single cell into the growth chamber, and the colony of biological cells produced by the incubating step may be a clonal colony.

In another aspect, a kit for culturing biological cells is provided, comprising a microfluidic device having: a flow region configured to contain a flow of a first fluid medium; and at least one growth chamber comprising at least one surface conditioned to support cell growth, viability, portability, or any combination thereof within the microfluidic device. At least one growth chamber may comprise a separation region and a connection region, the separation region being in fluid connection with the connection region, and the connection region having a proximal opening to the flow region. The microfluidic device may be any of the microfluidic devices described herein, having any combination of elements. In some embodiments, at least one conditioned surface of a microfluidic device can include an alkyl moiety, a fluoroalkyl moiety, a mono-or polysaccharide moiety, an alcohol moiety; a polyol moiety; an alkylene ether moiety; polyelectrolyte moieties, amino moieties, carboxylic acid moieties, phosphonic acid moieties, sulfonate moieties; a carboxylic acid betaine moiety, a sulfobetaine moiety; (ii) a sulfamic acid moiety; or an amino acid moiety. In some embodiments, at least one conditioned surface of the microfluidic device comprises at least one of a sugar moiety, an alkylene ether moiety, an alkyl moiety, a fluoroalkyl moiety, or an amino acid moiety. In some embodiments, the alkyl or fluoroalkyl moiety has a backbone chain length of greater than 10 carbons.

In various embodiments of the kit, at least one conditioned surface of the microfluidic device may comprise a linking group covalently attached to a surface of the microfluidic device, and the linking group may be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device. In some embodiments, the linking group may be a siloxy linking group. In other embodiments, the linking group may be a phosphonate linking group. In some embodiments, the linking group may be directly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In other embodiments, the linking group may be indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly attached via a linking moiety to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via attachment to the first end of the linking moiety. In various embodiments, the linking moiety may also include a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In some embodiments, the linking moiety may include a triazolylene moiety.

In various embodiments of the kit, the kit can further comprise a surface conditioning agent. In some embodiments, the surface conditioning agent can include a polymer comprising at least one of an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or a sugar moiety. In some embodiments, the surface conditioning agent may comprise a polymer comprising at least one of an alkylene ether moiety, an amino acid moiety, and/or a sugar moiety.

In other embodiments, the surface conditioning agent may comprise at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule may disrupt agonist filament formation, block integrin receptors, or reduce binding of cells to DNA-contaminated surfaces. In some embodiments, the at least one cell adhesion blocking molecule may be cytochalasin B, an RGD-containing peptide, an inhibitor of fibronectin, an anti-integrin antibody, or a DNase 1 protein. In some embodiments, the surface conditioning agent may comprise a combination of more than one cell adhesion blocking molecule.

In other embodiments, the surface conditioning agent may comprise one or more components of mammalian serum. In some embodiments, the mammalian serum can be Fetal Bovine Serum (FBS) or fetal bovine serum (FCS).

In various embodiments of the kit, the kit may further comprise a media supplement comprising reagents configured to supplement the conditioning of at least one surface of the growth chamber. The media supplement may include

A polymer.

In various embodiments of the kit, at least one conditioned surface of the microfluidic device may comprise a cleavable moiety. In some embodiments, the kit may further comprise a reagent configured to cleave a cleavable moiety of the conditioned surface.

In various embodiments of the kit, the kit can further include at least one reagent that detects the status of the biological cell.

Brief description of the drawings

Fig. 1 illustrates an example of a system for use with a microfluidic device and associated control apparatus according to some embodiments of the present invention.

Fig. 2A and 2B illustrate a microfluidic device according to some embodiments of the present invention.

Fig. 2C and 2D illustrate growth chambers according to some embodiments of the invention.

Figure 2E shows a detailed growth chamber according to some embodiments of the present invention.

Fig. 2F shows a microfluidic device according to an embodiment of the present invention.

Fig. 3A illustrates a specific example of a system for use with a microfluidic device and associated control apparatus according to some embodiments of the present invention.

Fig. 3B illustrates an imaging device according to some embodiments of the inventions.

Fig. 4A-C show another embodiment of a microfluidic device, including another example of a growth chamber for use herein.

Fig. 5A through 5E each represent an embodiment of a system component capable of providing conditioned media to a microfluidic device to support cell growth, viability, portability, or any combination thereof.

Fig. 6 is a representation of a microfluidic device having one or more sensors capable of detecting pH of a medium entering and/or exiting the microfluidic device.

FIG. 7 is an example of one embodiment of a method for perfusing a fluid medium at a microfluidic device.

Fig. 8 is an example of another embodiment of a method for perfusing a fluid medium in a microfluidic device.

Fig. 9 is a schematic representation of a conditioned surface providing enhanced support for cell growth, viability, portability, or any combination thereof.

FIGS. 10A-10E are photographic representations of one embodiment of a culture experiment according to the methods described herein.

Fig. 11A is a photographic representation of another embodiment of a culture experiment according to the methods described herein, showing a microfluidic device prior to placing cells in a growth chamber of the device.

Fig. 11B is a photographic representation of an embodiment of the culture experiment of fig. 11A at a later time when a cell is placed in a growth chamber of a microfluidic device.

Figures 12A-12C are photographic representations of an embodiment of the culture experiment of figures 11A and B, at a later point in time, showing cell expansion of the cells of figure 11B during incubation.

FIGS. 13A-13C are photographic representations of embodiments of the culture experiments of FIGS. 11A-B and 12A-12C, at later time points, showing the output of expanded cells after the end of the incubation period.

Fig. 14A and 14B are photographic representations of another embodiment of a culture experiment in a microfluidic device having at least one conditioned surface.

Detailed Description

The microfluidic environment provides the opportunity to provide a localized environment for a cell or group of cells to provide nutrients and/or soluble cell growth signaling substances to the cell or group of cells in a time-dependent manner and in a location-dependent concentration. These conditions may represent a growth environment more similar to that in vivo, or allow deviations from such typical conditions to allow for the study of non-standard conditions and growth under non-standard conditions. These requirements cannot be met using standardized large-scale cell culture methods. However, improvements are needed to more easily control one or more cells in order to: a) placing the cells in a microfluidic environment that helps support cell growth, viability, portability, or any combination thereof; b) successfully maintaining the cells and/or expanding the population of cells; and/or c) defining conditions that result in successful growth and/or maintenance. The systems and methods described herein allow for more precise cell handling, environmental control, and cell isolation techniques for microfluidic cell culture, and can be used to produce, for example, clonal cell populations.

This specification describes exemplary embodiments and applications of the invention. However, the invention is not limited to these exemplary embodiments and applications, nor to the manner in which the exemplary embodiments and applications operate or are described herein. Also, the figures may show simplified or partial views, and the sizes of elements in the figures may be exaggerated or not to scale for clarity. Further, when the terms "on …," "connected to," or "coupled to" are used herein, one element (e.g., material, layer, substrate, etc.) can be "on," "connected to," or "coupled to" another element, whether directly on, connected, or coupled to the other element, or with one or more intervening elements between the one element and the other element. Additionally, if directions are provided (e.g., above, below, top, bottom, side, up, down, below, over, upper, lower, horizontal, vertical, "x," "y," "z," etc.), then these are relative and are provided by way of example only and for ease of illustration and discussion, not by way of limitation. Further, where a list of elements (e.g., elements a, b, c) is recited, such recitation is intended to include any one of the recited elements by themselves, any combination of less than all of the recited elements, and/or combinations of all of the recited elements. The paragraph divisions in the specification are for ease of viewing only and do not limit any combination of the elements discussed.

As used herein, "substantially" means sufficient to achieve the intended purpose. Thus, the term "substantially" allows for small, unimportant changes based on absolute or perfect states, dimensions, measurements, results, etc., such as would be expected by one of ordinary skill in the art, but without significant impact on overall performance. "substantially" when used with respect to a numerical value or a parameter or feature that may be represented as a numerical value means within ten percent.

The term "plurality" means more than one. As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

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

As used herein, the term "disposed" encompasses its meaning "located".

As used herein, a "microfluidic device" or "microfluidic apparatus" is a device that: comprising one or more independent microfluidic circuits configured to hold fluid, each microfluidic circuit comprising fluidically interconnected circuit elements including, but not limited to, one or more regions, one or more flow paths, one or more channels, one or more chambers, and/or one or more docks (pens) and at least two ports (ports) configured to allow fluid (and optionally micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device will comprise at least one microfluidic channel and at least one chamber, and will hold a volume of fluid of less than about 1mL, for example, less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 microliters. In certain embodiments, the microfluidic circuit retains about 1-2, 1-3, 1-4, 1-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-30, 5-40, 5-50, 10-75, 10-100, 20-150, 20-200, 50-250, or 50-300 microliters of fluid.

As used herein, a "nanofluidic device" or "nanofluidic apparatus" is a type of microfluidic device having a microfluidic circuit comprising at least one circuit element configured to hold a volume of fluid of less than about 1 microliter, for example, less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1nL or less. Typically, the nanofluidic device will include a plurality of piping 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 piping component is configured to hold the following volumes of fluid: about 100pL to 1nL, 100pL to 2nL, 100pL to 5nL, 250pL to 2nL, 250pL to 5nL, 250pL to 10nL, 500pL to 5nL, 500pL to 10nL, 500pL to 15nL, 750pL to 10nL, 750pL to 15nL, 750pL to 20nL, 1 to 10nL, 1 to 15nL, 1 to 20nL, 1 to 25nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one piping component is configured to hold the following volumes of fluid: about 100 to 200nL, 100 to 300nL, 100 to 400nL, 100 to 500nL, 200 to 300nL, 200 to 400nL, 200 to 500nL, 200 to 600nL, 200 to 700nL, 250 to 400nL, 250 to 500nL, 250 to 600nL, or 250 to 750 nL.

As used herein, "microfluidic channel" or "flow channel" refers to a flow region of a microfluidic device having a length that is significantly longer than the horizontal and vertical dimensions. For example, the flow channel can be at least 5 times the length of the horizontal or vertical dimension, such as at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 300 times the length, at least 400 times the length, at least 500 times the length, or more. In some embodiments, the length of the flow channel ranges from about 20,000 micrometers to about 100,000 micrometers, including any range therebetween. In some embodiments, the horizontal dimension ranges from about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns), and the vertical dimension ranges from about 25 microns to about 200 microns, e.g., about 40 to about 150 microns. It should be noted that the flow channel may have a variety of different spatial configurations in the microfluidic device, and is therefore not limited to an ideal linear element. For example, the flow channel may be or include one or more portions having the following configurations: bends, spirals, inclines, declines, bifurcations (e.g., multiple distinct flow paths), and any combination thereof. In addition, the flow channel may have different cross-sectional areas (expanding and contracting) along its path to provide the desired fluid flow therein.

As used herein, the term "blocking" generally refers to a bump or similar type of structure that is large enough to partially (but not completely) prevent a target micro-object from moving between two different regions or tubing elements in a microfluidic device. The two different regions/tubing elements may be, for example, a microfluidic incubation chamber and a microfluidic channel, or a connection region and a separation region of a microfluidic incubation chamber.

As used herein, the term "constriction" generally refers to a narrowing of the width of a conduit element (or the interface between two conduit elements) in a microfluidic device. The constriction may be located, for example, at the interface between the microfluidic incubation chamber and the microfluidic channel, or at the interface between the separation region and the connection region of the microfluidic incubation chamber.

As used herein, the term "transparent" refers to a material that allows visible light to pass through but does not substantially alter the light as it passes through.

As used herein, the term "micro-object" generally refers to any microscopic object that can be separated and collected according to the present invention. Non-limiting examples of micro-objects include: inanimate micro-objects, such as microparticles; microbeads (e.g., polystyrene beads, Luminex) ^TMBeads, etc.); magnetic beads; micron rods (microrods); microwires (microwires); quantum dots, and the like; biological micro-objects, such as cells (e.g., embryos, oocytes, sperm cells, cells isolated from tissue, eukaryotic cells, protozoa, animal cells, mammalian cells, human cells, immune cells (including but not limited to T cells, B cells, natural killer cells, macrophages, dendritic cells, and the like), hybridomas, cultured cells, cells from cell lines, cancer cells (including but not limited to circulating tumor cells), infected cells, transfected and/or transformed cells (including but not limited to CHO cells), reporter cells, prokaryotic cells, and the like); biological organelles (e.g., nuclei); a vesicle or complex; synthesizing vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts (nanorafts) (as described in ritchaie et al (2009) conservation of Membrane Proteins in Phospholipid Bilayer Nanodiscs, Method enzymol.,464:211-231 (richi et al (2009), recombination of Membrane Proteins in Phospholipid Bilayer Nanodiscs, methodological, 464:211-231), etc.); or a combination of inanimate and biological micro-objects (e.g., microbeads attached to cells, liposome-coated microbeads, liposome-coated magnetic beads, etc.). The beads may also have other moieties/molecules, covalently or non-covalently attached, such as fluorescent labels, proteins, Small molecule signaling moieties, antigens, or chemical/biological substances.

As used herein, the term "cell" refers to a biological cell, which can be a plant cell, an animal cell (e.g., a mammalian cell), a bacterial cell, a fungal cell, and the like. Mammalian cells can be, for example, from humans, mice, rats, horses, goats, sheep, cows, primates, and the like.

A colony of biological cells is "clonal" if all living cells in the colony that are capable of multiplying are daughter cells derived from a single parent cell. The term "cloned cells" refers to cells of the same clonal colony.

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

As used herein, the term "maintaining the cell(s)" refers to providing an environment that includes both fluid and gaseous components that provide the conditions necessary to keep the cells viable and/or expanded.

As used herein, the term "expand" when referring to cells refers to increasing the number of cells.

By "breathable" as referred to herein is meant that the material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, the gas permeable material or structure is permeable to one or more of oxygen, carbon dioxide, and nitrogen, and may also be permeable to all three gases.

The "component" of the 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, carbohydrates, lipids, fatty acids, cholesterol, metabolites, and the like.

As used herein with respect to a fluid medium, "diffusing …" and "diffusion" refer to the thermodynamic movement of components of the fluid medium along a concentration gradient.

The phrase "flow of the medium" refers to the bulk movement of the fluid medium, which is primarily due to any mechanism other than diffusion. For example, the flow of media may include movement of fluid media from one point to another due to a pressure differential between the points. Such flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. Turbulence and mixing of the media can result when one fluid media flows into another fluid media.

The phrase "substantially no flow" means that the flow rate of the fluid medium averages over time less than the rate at which a component of the material (e.g., an analyte of interest) diffuses into or within the fluid medium. The rate of diffusion of the components of such materials may depend on, for example, the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

As used herein with respect to different regions within a microfluidic device, the phrase "fluidically connected" refers to the fluids in each region being connected to form a single body of fluid when the different regions are substantially filled with a liquid (such as a fluidic medium). This does not mean that the fluids (or fluid medium) in the different zones must be identical in composition. In contrast, fluids in different fluidically connected regions of a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) that vary as the solutes move along their respective concentration gradients and/or as the fluid flows through the device.

Microfluidic (or nanofluidic) devices may include "swept" (swept) regions and "unswept" (unswept) regions. As used herein, a "swept-out" region includes one or more fluidically interconnected tubing elements of a microfluidic tubing, each tubing element undergoing a flow of media as fluid flows through the microfluidic tubing. The conduit elements that sweep the area may include, for example, all or part of the area, channel, and chamber. As used herein, an "unswept" region includes one or more fluidically interconnected tubing elements of a microfluidic tubing, each tubing element experiencing substantially no fluid flow as fluid flows through the microfluidic tubing. The unswept region may be in fluidic connection with the swept region, provided that the fluidic connection is configured to enable diffusion of the medium between the swept region and the unswept region, but substantially no flow of the medium. Thus, the microfluidic device may be configured to substantially separate the unswept region from the flow of the medium in the swept region, while substantially only enabling diffusive fluid communication between the swept region and the unswept region. For example, the flow channels of a microfluidic device are examples of swept areas, while the separation areas of a microfluidic device (described in further detail below) are examples of unswept areas.

As used herein, the "unswept" rate of fluid medium flow refers to the flow rate: sufficient to allow diffusion of a component of the second fluid medium in the separation region of the growth chamber into the first fluid medium in the flow region and/or diffusion of a component of the first fluid medium into the second fluid medium in the separation region; and wherein the first culture medium does not substantially flow into the separation region.

As used herein, "fluid flow path" refers to one or more fluidically connected tubing elements (e.g., one or more channels, one or more regions, one or more chambers, etc.) that define and are subject to a media flow trajectory. Thus, a fluid flow path is an example of a swept area of a microfluidic device. Other tubing elements (e.g., unswept areas) may be in fluidic connection with the tubing elements comprising the fluid flow path without undergoing flow of the culture medium in the fluid flow path.

As used herein, "arylene" refers to an aromatic group having six to ten ring atoms (e.g., a C6-C10 aromatic group or a C6-C10 aryl group) having at least one ring with a conjugated pi-electron system that is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl) and has one or two points of attachment to the rest of the molecule. Whenever it appears herein, a numerical range such as "6 to 10" refers to each integer in the given range; for example, "6 to 10 ring atoms" means that the aryl group can consist of 6 ring atoms, 7 ring atoms, and the like, up to and including 10 ring atoms. The term includes monocyclic and fused-ring polycyclic (i.e., rings that share adjacent pairs of ring atoms) groups. Examples of arylene include, but are not limited to, phenylene, naphthylene, and the like. The arylene moiety may be further substituted or may have no other substituents other than one or two points of attachment to other parts of the molecule.

As used herein, "heteroarylene" refers to a 5-to 18-membered aromatic group (e.g., C5-C13 heteroaryl) that includes one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur, and that may include monocyclic, bicyclic, tricyclic, or tetracyclic ring systems, and the modifier "ene" indicates that the heteroaryl ring system has one or two points of attachment to the rest of the molecule. Whenever it appears herein, a numerical range such as "5 to 18" refers to each integer in the given range; for example, "5 to 18 ring atoms" means that the heteroaryl group can consist of 5 ring atoms, 6 ring atoms, and the like, up to and including 18 ring atoms. An N-containing "heteroaromatic" or "heteroaryl" moiety refers to an aromatic group: wherein at least one backbone atom of the ring is a nitrogen atom. The polycyclic heteroaryl group may be fused or non-fused. The heteroatoms in the heteroaryl group may be optionally oxidized. One or more nitrogen atoms, if present, may be optionally quaternized. The heteroaryl group is attached to the rest of the molecule through any atom of the ring. Examples of heteroarylenes include, but are not limited to, benzimidazolylene, benzindolylene, isoxazolylene, thiazolyl, triazolylene, tetrazolylene, and thienylene (i.e., thienylene). The heteroarylene moiety may be further substituted or may have no other substituents except at one or both points of attachment to other parts of the molecule.

As used herein, the term "heterocyclyl" refers to a substituted or unsubstituted 3-, 4-, 5-, 6-, or 7-membered saturated or partially saturated ring containing one, two, or three heteroatoms, preferably one or two heteroatoms, independently selected from oxygen, nitrogen, and sulfur; or a bicyclic ring system containing up to 10 atoms including at least one heteroatom independently selected from oxygen, nitrogen and sulfur, wherein the heteroatom containing ring is saturated. Examples of heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrofurfuryl, pyrrolidinyl, piperidinyl, 4-pyranyl, tetrahydropyranyl, tetrahydrothienyl, morpholinyl, piperazinyl, dioxolanyl, dioxanyl, indolinyl, and 5-methyl-6-chromanyl. Heterocyclic groups may have one or two points of attachment to the rest of the molecule and may or may not be further substituted.

Provided is a system. A system for culturing one or more biological cells in a microfluidic device is provided, comprising a microfluidic device comprising: a flow region configured to contain a flow of a first fluid medium; and at least one growth chamber, wherein the growth chamber has at least one surface conditioned to support cell growth, viability, portability, or any combination thereof.

Microfluidic devices and systems for operating and viewing such devices. Fig. 1 shows an example of a microfluidic device 100 and system 150 that may be used in the practice of the present invention. A perspective view of the microfluidic device 100 is shown with the cover 110 partially cut away to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally includes microfluidic circuit 120 having a flow path 106 through which a fluid medium 180 (optionally carrying one or more micro-objects (not shown)) may flow into and/or through the microfluidic circuit 120. Although a single microfluidic circuit 120 is shown in fig. 1, a suitable microfluidic device may include a plurality (e.g., 2 or 3) of such microfluidic circuits. Regardless, the microfluidic device 100 may be configured as a nanofluidic device. In the embodiment shown in fig. 1, microfluidic circuit 120 includes a plurality of microfluidic growth chambers 124, 126, 128, and 130, each having one or more openings in fluidic communication with fluid flow path 106. As discussed further below, the microfluidic growth chamber includes various characteristics and structures that have been optimized for retaining micro-objects in a microfluidic device (e.g., microfluidic device 100) even when media 180 is flowing through fluid flow path 106. However, before the above is described, a brief description of the microfluidic device 100 and system 150 is provided.

As shown generally in fig. 1, microfluidic circuit 120 is defined by housing 102. Although the housing 102 may be physically configured in different configurations, in the example shown in fig. 1, the housing 102 is depicted as including a support structure 104 (e.g., a base), a microfluidic conduit structure 108, and a cover 110. The support structure 104, the microfluidic circuit structure 108 and the cover 110 may be attached to each other. For example, the microfluidic circuit structure 108 may be arranged on an inner surface 109 of the support structure 104, and the cover 110 may be arranged over the microfluidic circuit structure 108. The microfluidic circuit structure 108, together with the support structure 104 and the cover 110, may define elements of a microfluidic circuit 120.

As shown in fig. 1, the support structure 104 may be located at the bottom of the microfluidic circuit 120 and the lid 110 may be located at the top of the microfluidic circuit 120. Alternatively, the support structure 104 and the cover 110 may be configured in other orientations. For example, the support structure 104 may be located at the top of the microfluidic circuit 120 and the lid 110 may be located at the bottom of the microfluidic circuit 120. In any event, there may be one or more ports 107, each of which includes access into or out of the housing 102. Examples of passageways include valves, gates, through-holes, and the like. As shown, the port 107 is a through hole formed by a gap in the microfluidic conduit structure 108. However, the port 107 may be located in other components of the housing 102, such as the cover 110. Only one port 107 is shown in fig. 1, but the microfluidic circuit 120 may have two or more ports 107. For example, there may be a first port 107 that serves as an inlet for fluid into the microfluidic circuit 120, and there may be a second port 107 that serves as an outlet for fluid out of the microfluidic circuit 120. Whether the port 107 serves as an inlet or an outlet may depend on the direction of fluid flow through the flow path 106.

The support structure 104 may include one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 may include one or more semiconductor substrates, each semiconductor substrate being electrically connected to an electrode (e.g., all or a portion of a semiconductor substrate may be electrically connected to a single electrode). The support structure 104 may also include a printed circuit board assembly ("PCBA"). For example, the semiconductor substrate may be mounted on a PCBA.

The microfluidic circuit structure 108 may define a circuit element of a microfluidic circuit 120. Such tubing elements may include spaces or regions, such as flow channels, chambers, docks, wells (traps), etc., that may be fluidically interconnected when microfluidic tubing 120 is filled with a fluid. In the microfluidic circuit 120 shown in fig. 1, the microfluidic circuit structure 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 may partially or completely surround the microfluidic circuit material 116. For example, the frame 114 may be a relatively rigid structure that substantially encloses the microfluidic circuit material 116. For example, the frame 114 may comprise a metallic material.

The microfluidic circuit material 116 may be patterned with cavities or the like to define circuit elements and interconnections of the microfluidic circuit 120. The microfluidic circuit material 116 may include a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane ("PDMS"), etc.), which may be gas permeable. Other examples of materials from which microfluidic circuit material 116 may be constructed include molded glass; etchable materials such as siloxanes (e.g., photo-patternable siloxanes or "PPS"); photoresist (e.g., SU8), and the like. In some embodiments, such materials (and thus the microfluidic circuit material 116) may be rigid and/or substantially gas impermeable. Regardless, the microfluidic circuit material 116 may be disposed on the support structure 104 and inside the frame 114.

The cover 110 may be an integral (integral) component of the frame 114 and/or the microfluidic circuit material 116. Alternatively, the cover 110 may be a structurally different element, as shown in FIG. 1. The cover 110 may comprise the same or different material as the frame 114 and/or the microfluidic circuit material 116. Similarly, the support structure 104 may be a separate structure from the frame 114 or the microfluidic circuit material 116, as shown, or may be an integral part of the frame 114 or the microfluidic circuit material 116. Similarly, the frame 114 and microfluidic circuit material 116 may be separate structures as shown in fig. 1 or integral components of the same structure.

In some embodiments, the cover 110 may comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover 110 may include a deformable material. The deformable material may be a polymer, such as PDMS. In some embodiments, the cover 110 may include both rigid and deformable materials. For example, one or more portions of the lid 110 (e.g., one or more portions located above the growth chambers 124, 126, 128, 130) may include a deformable material that interfaces with the rigid material of the lid 110. In some embodiments, the cover 110 may also include one or more electrodes. The one or more electrodes may comprise a conductive oxide, such as Indium Tin Oxide (ITO), which may be coated on glass or similar insulating material. Alternatively, the one or more electrodes may 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 may be used in microfluidic devices have been described, for example, in US 2012/0325665 (Chiou et al), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 may be modified (e.g., by conditioning all or part of the surface facing inward toward the microfluidic channel 120) to support cell attachment, viability, and/or growth. Such modifications may include coatings of synthetic or natural polymers. In some embodiments, the cover 110 and/or the support structure 104 may be optically transparent. The cap 110 may also include at least one gas permeable material (e.g., PDMS or PPS).

Fig. 1 also shows a system 150 for operating and controlling a microfluidic device, such as the microfluidic device 100. As shown, the system 150 includes a power source 192, an imaging device 194, and a tilting device 190.

The power source 192 may provide power to the microfluidic device 100 and/or the tilting device 190 to provide a bias voltage or current as desired. For example, the power supply 192 may include one or more Alternating Current (AC) and/or Direct Current (DC) voltage or current sources. The imaging device 194 may include a device for capturing images within the microfluidic circuit 120, such as a digital camera. In some cases, the imaging device 194 also includes a detector with a fast frame rate and/or high sensitivity (e.g., for low light applications). The imaging device 194 may also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beam may be in the visible spectrum and may, for example, include fluorescent emissions. The reflected light beam may comprise reflections originating from the emission of an LED or a broad spectrum lamp such as a mercury lamp (e.g. a high pressure mercury lamp) or a xenon arc lamp. As discussed with respect to fig. 3, the imaging device 194 may also include a microscope (or optical system) that may or may not include an eyepiece.

The system 150 may also include a tilting device 190 configured to rotate the microfluidic device 100 about one or more axes of rotation. In some embodiments, the tilting device 190 is configured to support and/or hold the housing 102 including the microfluidic circuit 120 about at least one axis such that the microfluidic device 100 (and thus the microfluidic circuit 120) can be held in a horizontal orientation (i.e., 0 ° with respect to the x-axis and y-axis), a vertical orientation (i.e., 90 ° with respect to the x-axis and/or y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and microfluidic circuit 120) relative to the axis is referred to herein as the "tilt" of the microfluidic device 100 (and microfluidic circuit 120). For example, the tilting device 190 may tilt the microfluidic device 100 relative to the x-axis by 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 angle therebetween. The horizontal orientation (and thus the x-axis and y-axis) is defined as being perpendicular to the vertical axis defined by gravity. The tilting device may also tilt the microfluidic device 100 (and the microfluidic circuit 120) by an angle greater than 90 ° with respect to the x-axis and/or the y-axis, or tilt the microfluidic device 100 (and the microfluidic circuit 120) by 180 ° with respect to the x-axis or the y-axis, in order to completely invert the microfluidic device 100 (and the microfluidic circuit 120). Similarly, in some embodiments, the tilting device 190 tilts the microfluidic device 100 (and the microfluidic circuit 120) about an axis of rotation defined by the flow path 106 or some other portion of the microfluidic circuit 120.

In some cases, microfluidic device 100 is tilted into a vertical orientation such that fluid flow path 106 is located above or below one or more growth chambers. The term "above" as used herein means that the liquid flow path 106 is positioned higher than the one or more growth chambers on a vertical axis defined by gravity (i.e., objects in the growth chambers above the liquid flow path 106 will have a higher gravitational potential energy than objects in the liquid flow path). The term "below" as used herein means that the liquid flow path 106 is positioned below the one or more growth chambers on a vertical axis defined by gravity (i.e., objects in the growth chambers below the liquid flow path 106 will have a lower gravitational potential energy than objects in the liquid flow path).

In some cases, the tilting device 190 tilts the microfluidic device 100 about an axis parallel to the flow path 106. Furthermore, the microfluidic device 100 may be tilted at an angle of less than 90 ° such that the fluid flow path 106 is located above or below the one or more growth chambers, rather than directly above or below the growth chambers. In other cases, the tilting device 190 tilts the microfluidic device 100 about an axis perpendicular to the flow path 106. In still other cases, the tilting device 190 tilts the microfluidic device 100 about an axis that is neither parallel nor perpendicular to the flow path 106.

The system 150 may also include a media source 178. Media source 178 (e.g., container, reservoir, etc.) may include multiple portions or containers, each for holding a different fluid media 180. Thus, as shown in fig. 1, the media source 178 may be a device that is external to the microfluidic device 100 and separate from the microfluidic device 100. Alternatively, the media source 178 may be located wholly or partially within the housing 102 of the microfluidic device 100. For example, the media source 178 may include a reservoir that is part of the microfluidic device 100.

Fig. 1 also shows a simplified block diagram depicting an example of a control and monitoring apparatus 152 that forms part of the system 150 and that may be used in conjunction with the microfluidic device 100. As shown, an example of such a control and monitoring device 152 includes a master controller 154 including: a media module 160 for controlling a media source 178; a motion module 162 for controlling movement and/or selection of micro-objects (not shown) and/or media (e.g., droplets of media) in the microfluidic circuit 120; an imaging module 164 for controlling an imaging device 194 (e.g., a camera, a microscope, a light source, or any combination thereof) used to capture an image (e.g., a digital image); and a tilt module 166 for controlling the tilt device 190. The control apparatus 152 may also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the apparatus 152 may also include a display device 170 and an input/output device 172.

The main controller 154 may include a control module 156 and digital memory 158. The control module 156 may include, for example, a digital processor configured to operate in accordance with 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 hard-wired digital circuitry and/or analog circuitry. The media module 160, motion module 162, imaging module 164, tilt module 166, and/or other modules 168 may be similarly configured. Accordingly, the functions, processes, actions, acts, or steps of the processes 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 media module 160, the motion module 162, the imaging module 164, the tilt module 166, and/or the other modules 168 configured as described above. Similarly, the master controller 154, the media module 160, the motion module 162, the imaging module 164, the tilt module 166, and/or the other modules 168 may be communicatively coupled to send and receive data used in any of the functions, processes, actions, or steps discussed herein.

Media module 160 controls media source 178. For example, the media module 160 may control the media source 178 to input a selected fluid media 180 into the housing 102 (e.g., through the inlet port 107). Media module 160 may also control the removal of media from housing 102 (e.g., through an outlet port (not shown)). Thus, one or more media may be selectively input into and removed from the microfluidic circuit 120. The media module 160 may also control the flow of fluid media 180 in the fluid flow path 106 inside the microfluidic circuit 120. For example, in some embodiments, the media module 160 prevents the flow of media 180 in the flow path 106 and through the housing 102 before the tilt module 166 causes the tilt device 190 to tilt the microfluidic device 100 to a desired tilt angle.

The motion module 162 may be configured to control the selection, trapping, and movement of micro-objects (not shown) in the microfluidic circuit 120. As discussed below with respect to fig. 2A and 2B, the enclosure 102 can include a Dielectrophoresis (DEP), optoelectronic tweezers (OET), and/or optoelectronic wetting (OEW) configuration (not shown in fig. 1), and the motion module 162 can control activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects (not shown) and/or media droplets (not shown) in the flow path 106 and/or growth chambers 124, 126, 128, 130.

The imaging module 164 may control an imaging device 194. For example, the imaging module 164 may receive and process image data from the imaging device 194. The image data from the imaging device 194 may include any type of information captured by the imaging device 194 (e.g., the presence or absence of micro-objects, media drops, accumulation of markers (e.g., fluorescent markers, etc.)). Using the information captured by the imaging device 194, the imaging module 164 may also calculate the position of objects (e.g., micro-objects, media drops) within the microfluidic device 100 and/or the rate of motion of those objects.

The tilt module 166 may control the tilting motion of the tilting device 190. Alternatively or additionally, the tilt module 166 may control the tilt rate and time to optimize transfer of micro-objects to one or more growth chambers via gravity. Tilt module 166 is communicatively coupled with imaging module 164 to receive data describing the movement of micro-objects and/or media droplets in microfluidic circuit 120. Using this data, tilt module 166 may adjust the tilt of microfluidic circuit 120 in order to adjust the rate at which micro-objects and/or media droplets move within microfluidic circuit 120. Tilt module 166 may also use this data to iteratively adjust the position of micro-objects and/or media droplets in microfluidic circuit 120.

In the example shown in fig. 1, microfluidic circuit 120 is shown to include a microfluidic channel 122 and growth chambers 124, 126, 128, 130. Each chamber includes an opening to the channel 122, but is otherwise enclosed such that the chamber can substantially separate micro-objects within the chamber from the fluidic medium 180 and/or micro-objects in the flow path 106 of the channel 122 or other chambers. In some cases, chambers 124, 126, 128, 130 are configured to physically enclose one or more micro-objects within microfluidic circuit 120. Growth chambers according to the present invention may include various shapes, surfaces, and characteristics, which are optimized for use with DEP, OET, OEW, and/or gravity, as will be discussed and illustrated in detail below.

Microfluidic circuit 120 may include any number of microfluidic growth chambers. Although five growth chambers are shown, microfluidic circuit 120 may have fewer or more growth chambers. In some embodiments, microfluidic circuit 120 comprises a plurality of microfluidic growth chambers, wherein two or more growth chambers comprise different structures and/or features.

In the embodiment shown in fig. 1, a single channel 122 and fluid flow path 106 are shown. However, other embodiments may comprise a plurality of channels 122, each configured to include a flow path 106. Microfluidic circuit 120 also includes an inlet valve or port 107 in fluid communication with flow path 106 and fluid medium 180, whereby fluid medium 180 may enter channel 122 via inlet port 107. In some cases, the fluid flow path 106 comprises a single path. In some cases, the single paths are arranged in a zigzag pattern such that the fluid flow path 106 passes through the microfluidic device 100 two or more times in alternating directions.

In some cases, microfluidic circuit 120 includes a plurality of parallel channels 122 and flow paths 106, wherein fluidic medium 180 within each flow path 106 flows in the same direction. In some cases, the fluidic medium within each flow path 106 flows in at least one of a forward or reverse direction. In some cases, multiple growth chambers are configured (e.g., relative to channel 122) such that they can be loaded with target micro-objects in parallel.

In some embodiments, microfluidic circuit 120 further comprises one or more micro-object wells 132. The wells 132 are generally formed in the walls bounding the channels 122 and may be disposed opposite the openings of one or more of the microfluidic growth chambers 124, 126, 128, 130. In some embodiments, the trap 132 is configured to receive or capture a single micro-object from the fluid flow path 106. In some embodiments, the trap 132 is configured to receive or capture a plurality of micro-objects from the fluid flow path 106. In some cases, well 132 includes a volume that is substantially equal to the volume of a single target micro-object.

The well 132 may also include an opening configured to assist the flow of the target micro-object into the well 132. In some cases, well 132 includes an opening having a height and width substantially equal to the dimensions of a single target micro-object, thereby preventing larger micro-objects from entering the micro-object well. The well 132 may also include other features configured to help retain the target micro-object within the well 132. In some cases, well 132 is aligned with respect to the opening of the microfluidic growth chamber and is located on the opposite side of channel 122 such that when microfluidic device 100 is tilted about an axis parallel to channel 122, trapped micro-objects exit well 132 in a trajectory that causes the micro-objects to fall into the opening of the growth chamber. In some cases, well 132 includes side passages 134 that are smaller than the target micro-object in order to facilitate flow through well 132, thereby increasing the likelihood of micro-objects being trapped in well 132.

In some embodiments, Dielectrophoretic (DEP) forces are exerted on the fluidic medium 180 (e.g., in the flow path and/or in the growth chamber) by one or more electrodes (not shown) to manipulate, transport, separate, and sort micro-objects located therein. For example, in some embodiments, DEP forces are applied to one or more portions of microfluidic circuit 120 in order to transfer individual micro-objects from fluid flow path 106 into a desired microfluidic growth chamber. In some embodiments, DEP forces are used to prevent micro-objects within a growth chamber (e.g., growth chambers 124, 126, 128, or 130) from being displaced from the growth chamber. Further, in some embodiments, DEP forces are used to selectively remove micro-objects previously collected according to the teachings of the present invention from the growth chamber. In some embodiments, the DEP force comprises an optoelectronic tweezers (OET) force.

In other embodiments, an electro-optical wetting (OEW) force is applied to one or more locations (e.g., locations that help define a flow path and/or growth chamber) in the support structure 104 (and/or lid 110) of the microfluidic device 100 by one or more electrodes (not shown) to manipulate, transport, separate, and sort droplets located in the microfluidic circuit 120. For example, in some embodiments, an OEW force is applied to one or more locations in the support structure 104 (and/or the lid 110) to transfer individual droplets from the fluid flow path 106 into a desired microfluidic growth chamber. In some embodiments, OEW forces are used to prevent droplets within a growth chamber (e.g., growth chamber 124, 126, 128, or 130) from being displaced from the growth chamber. Additionally, in some embodiments, OEW forces are used to selectively remove droplets previously collected according to the teachings of the present invention from the growth chamber.

In some embodiments, DEP and/or OEW forces are combined with other forces (e.g., flow and/or gravity) in order to manipulate, transport, separate, and sort micro-objects and/or droplets within the microfluidic circuit 120. For example, housing 102 can be tilted (e.g., by tilting device 190) to position fluid flow path 106 and micro-objects located therein above the microfluidic growth chamber, and gravity can transport the micro-objects and/or droplets into the chamber. In some embodiments, DEP and/or OEW forces may be applied before other forces are applied. In other embodiments, DEP and/or OEW forces may be applied after other forces are applied. In other cases, DEP and/or OEW forces may be applied simultaneously with or alternating with other forces.

Fig. 2A-2F illustrate various embodiments of microfluidic devices that can be used in the practice of the present invention. Fig. 2A depicts an embodiment of an electrokinetic device in which the microfluidic device 200 is configured to be optically actuated. A variety of optically actuated electrokinetic devices are known in the art, including devices having an opto-electronic tweezers (OET) configuration and devices having an opto-electronic wetting (OEW) configuration. Examples of suitable OET configurations are shown in the following U.S. patent documents, all of which are incorporated herein by reference in their entirety: U.S. Pat. No. RE 44,711 (Wu et al) (originally issued in U.S. Pat. No. 7,612,355); and U.S. Pat. No. 7,956,339 (Ohta et al). Examples of OEW configurations are shown in U.S. patent No. 6,958,132 (Chiou et al) and U.S. patent application publication No. 2012/0024708 (Chiou et al), both of which are incorporated herein by reference in their entirety. Another example of a light actuated electrodynamic device includes a combined OET/OEW configuration, examples of which are shown in U.S. patent publication nos. 20150306598 (Khandros et al) and 20150306599 (Khandros et al) and their corresponding PCT publications WO2015/164846 and WO2015/164847, which are incorporated herein by reference in their entirety.

A moving microfluidic device configuration. As mentioned above, the control and monitoring apparatus of the system may comprise a motion module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of the microfluidic device. Microfluidic devices may have a variety of motion configurations depending on the type of object being moved and other considerations. For example, a Dielectrophoresis (DEP) configuration can be utilized to select and move micro-objects in a microfluidic circuit. Accordingly, the support structure 104 and/or the lid 110 of the microfluidic device 100 may include a DEP configuration for selectively inducing DEP forces on micro-objects in the fluid medium 180 in the microfluidic circuit 120 to select, capture, and/or move individual micro-objects or groups of micro-objects. Alternatively, the support structure 104 and/or the cover 110 of the microfluidic device 100 can comprise an Electrowetting (EW) configuration for selectively inducing EW forces on droplets in the fluidic medium 180 in the microfluidic circuit 120 to select, capture, and/or move individual droplets or groups of droplets.

One example of a microfluidic device 200 including a DEP configuration is shown in fig. 2A and 2B. While fig. 2A and 2B show, for simplicity, a side cross-sectional view and a top cross-sectional view, respectively, of a portion of the housing 102 of a microfluidic device 200 having an open region/chamber 202, it is to be understood that the region/chamber 202 may be part of a fluid conduit element having a more detailed structure, such as a growth chamber, a flow region, or a flow channel. In addition, the microfluidic device 200 may include other fluid conduit elements. For example, the microfluidic device 200 may include multiple growth chambers or growth chambers and/or one or more flow regions or flow channels, such as those described herein with respect to the microfluidic device 100. The DEP configuration can be incorporated into, or select a portion of, any such fluid conduit element of the microfluidic device 200. It should also be understood that any of the microfluidic device components and system components described above or below may be incorporated into the microfluidic device 200 and/or used in conjunction with the microfluidic device 200. For example, the system 150 described above including the control and monitoring device 152 may be used with a microfluidic device 200 that includes one or more of a media module 160, a motion module 162, an imaging module 164, a tilt module 166, and other modules 168.

As shown in fig. 2A, the microfluidic device 200 includes a support structure 104 having a bottom electrode 204 and an electrode activation substrate 206 covering the bottom electrode 204, and a lid 110 having a top electrode 210, wherein the top electrode 210 is spaced apart from the bottom electrode 204. The top electrode 210 and the electrode activation substrate 206 define opposing surfaces of the region/chamber 202. Thus, the medium 180 contained in the region/chamber 202 provides a resistive connection between the top electrode 210 and the electrode activation substrate 206. Also shown is a power supply 212 configured to be connected to the bottom electrode 204 and the top electrode 210 and to generate a bias voltage between these electrodes as required to generate DEP forces in the region/chamber 202. The power source 212 may be, for example, an Alternating Current (AC) power source.

In certain embodiments, the microfluidic device 200 shown in fig. 2A and 2B can have a light-actuated DEP configuration. Accordingly, the varying pattern of light 222 from the light source 220, which may be controlled by the motion module 162, may selectively activate and deactivate the varying pattern of DEP electrodes at the region 214 of the inner surface 208 of the electrode activation substrate 206. (hereinafter, the region 214 of the microfluidic device having the DEP configuration is referred to as the "DEP electrode region") as shown in fig. 2B, a light pattern 222 directed at the inner surface 208 of the electrode activation substrate 206 may illuminate a selected DEP electrode region 214a (shown in white) in a pattern such as a square. The unirradiated DEP electrode regions 214 (cross-hatched) are referred to hereinafter as "dark" DEP electrode regions 214. The relative electrical impedance through the DEP electrode activation substrate 206 (i.e., from the bottom electrode 204 up to the inner surface 208 of the electrode activation substrate 206 at the intersection with the media 180 in the flow stream region 106) is greater than the relative electrical impedance through the media 180 in the region/chamber 202 at each dark DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the lid 110). However, the illuminated DEP electrode regions 214a exhibit a reduced relative impedance through the electrode activation substrate 206 that is less than the relative impedance through the medium 180 in the region/chamber 202 at each illuminated DEP electrode region 214 a.

With the power supply 212 activated, the aforementioned DEP configuration creates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode region 214a and the adjacent dark DEP electrode region 214, which in turn creates 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 fluidic medium 180 can be selectively activated and deactivated at many different such DEP electrode regions 214 at the inner surface 208 of the region/chamber 202 by varying the light pattern 222 projected from the light source 220 into the microfluidic device 200. Whether DEP forces attract or repel nearby micro-objects may depend on parameters such as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or micro-objects (not shown).

The square pattern 224 of illuminated DEP electrode regions 214a shown in fig. 2B is merely an example. Any pattern of DEP electrode regions 214 can be illuminated (and thus activated) by a light pattern 222 projected onto the device 200, and the pattern of illuminated/activated DEP electrode regions 214 can be repeatedly changed by changing or moving the light pattern 222.

In some embodiments, the electrode activation substrate 206 may include or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode activation substrate 206 may be featureless. For example, the electrode activation substrate 206 may include or be composed of a hydrogenated amorphous silicon (a-Si: H) layer. H may comprise, for example, about 8% to 40% hydrogen (calculated as 100 x the number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si: H layer may have a thickness of about 500nm to about 2.0 microns. In such embodiments, DEP electrode regions 214 may be formed in any pattern anywhere on the inner surface 208 of the electrode activation substrate 208, according to the light pattern 222. Thus, the number and pattern of DEP electrode regions 214 need not be fixed, but may be made to correspond to the light pattern 222. Examples of microfluidic devices having DEP configurations comprising a photoconductive layer (such as those described above) have been described, for example, in U.S. patent No. RE 44,711 (Wu et al), initially issued as U.S. patent No. 7,612,355, the entire contents of which are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 may comprise a substrate having a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers forming a semiconductor integrated circuit, such as is known in the semiconductor arts. For example, the electrode activation substrate 206 may include a plurality of phototransistors, including, for example, lateral bipolar phototransistors, each corresponding to a DEP electrode region 214. Alternatively, the electrode activation substrate 206 can include electrodes (e.g., conductive metal electrodes) controlled by the phototransistor switches, wherein each such electrode corresponds to a DEP electrode region 214. The electrode activation substrate 206 may include a pattern of such phototransistors or phototransistor control electrodes. For example, the pattern may be an array of substantially square phototransistor or phototransistor control electrodes arranged in rows and columns, as shown in figure 2B. Alternatively, the pattern may be an array of substantially hexagonal phototransistors or phototransistor control electrodes forming a hexagonal lattice. Regardless of the pattern, the circuit elements can form electrical connections between the DEP electrode region 214 and the bottom electrode 210 at the inner surface 208 of the electrode activation substrate 206, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 222. When not activated, each electrical connection may have a high impedance such that the relative impedance through the electrode activation substrate 206 (i.e., from the bottom electrode 204 to the inner surface 208 of the electrode activation substrate 206 that borders the medium 180 in the region/chamber 202) is greater than the relative impedance through the medium 180 at the respective DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the lid 110). However, when activated by light in the light pattern 222, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 at each illuminated DEP electrode region 214, thereby activating the DEP electrode at the respective DEP electrode region 214, as described above. DEP electrodes that attract or repel micro-objects (not shown) in the medium 180 can thus be selectively activated and deactivated at a number of different DEP electrode regions 214 at the inner surface 208 of the electrode activation substrate 206 in the region/chamber 202 in a manner determined by the light pattern 222.

Examples of microfluidic devices having electrode-activated substrates including phototransistors have been described, for example, in U.S. patent No. 7,956,339 (Ohta et al) (see, e.g., device 300 shown in fig. 21 and 22 and the description thereof), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode-activated substrates with electrodes controlled by phototransistor switches have been described, for example, in U.S. patent publication No. 2014/0124370 (Short et al) (see, e.g., devices 200, 400, 500, 600, and 900 and descriptions thereof shown in the various figures), the entire contents of which are incorporated herein by reference.

In some embodiments of DEP configured microfluidic devices, the top electrode 210 is part of a first wall (or lid 110) of the housing 102, and the electrode activation substrate 206 and the bottom electrode 204 are part of a second wall (or support structure 104) of the housing 102. The region/chamber 202 may be between the first wall and the second wall. In other embodiments, the electrode 210 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 206 and/or the electrode 210 is part of the first wall (or cover 110). Further, the light source 220 may alternatively be used to illuminate the housing 102 from below.

With the microfluidic device 200 of fig. 2A-2B having a DEP configuration, the motion module 162 can select micro-objects (not shown) in the medium 180 in the region/chamber 202 by projecting a light pattern 222 into the device 200 to activate a first set of one or more DEP electrodes at the DEP electrode region 214a of the inner surface 208 of the electrode activation substrate 206 in a pattern (e.g., a square pattern 224) that surrounds and captures the micro-objects. The motion module 162 can then move the captured micro-objects by moving the light pattern 222 relative to the device 200 to activate the second set of one or more DEP electrodes at the DEP electrode region 214. Alternatively, the device 200 may be moved relative to the light pattern 222.

In other embodiments, the microfluidic device 200 can have a DEP configuration that does not rely on photo-activation of DEP electrodes at the inner surface 208 of the electrode activation substrate 206. For example, the electrode activation substrate 206 can include selectively addressable and energizable electrodes opposite a surface (e.g., the cover 110) containing at least one electrode. A switch (e.g., a transistor switch in a semiconductor substrate) can be selectively opened and closed to activate or deactivate the DEP electrode at the DEP electrode region 214, thereby creating a net DEP force on a micro-object (not shown) in the region/chamber 202 near the activated DEP electrode. Depending on characteristics such as the frequency of the power source 212 and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber 202, DEP forces may attract or repel nearby micro-objects. By selectively activating and deactivating a set of DEP electrodes (e.g., at a set of DEP electrode regions 214 forming a square pattern 224), one or more micro-objects in the region/chamber 202 can be trapped and moved within the region/chamber 202. The motion module 162 of fig. 1 can control such switches to activate and deactivate the various DEP electrodes to select, trap, and move specific micro-objects (not shown) around the area/chamber 202. Microfluidic devices having DEP configurations comprising selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. patent nos. 6,294,063 (Becker et al) and 6,942,776 (Medoro), the entire contents of which are incorporated herein by reference.

As yet another example, the microfluidic device 200 can have an Electrowetting (EW) configuration, which can replace the DEP configuration, or can be located in a separate portion of the microfluidic device 200 from the portion having the DEP configuration. The EW configuration can be either an electro-wetting configuration or an electro-wetting-on-dielectric (EWOD) configuration, both of which are known in the art. In some EW configurations, the support structure 104 has an electrode activation substrate 206 sandwiched between a dielectric layer (not shown) and the bottom electrode 204. The dielectric layer may include a hydrophobic material and/or may be coated with a hydrophobic material. For microfluidic devices 200 having the EW configuration, the inner surface 208 of the support structure 104 is the inner surface of the dielectric layer or hydrophobic coating thereof.

The dielectric layer (not shown) may include one or more oxide layers and may have a thickness of about 50nm to about 250 nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer may include a layer of an oxide, such as a metal oxide (e.g., aluminum oxide or hafnium oxide). In certain embodiments, the dielectric layer may comprise a dielectric material other than a metal oxide, such as silicon oxide or nitride. Regardless of the exact composition and thickness, the dielectric layer may have an impedance of about 10k Ω to about 50k Ω.

In some embodiments, the inner surface of the dielectric layer that faces inwardly toward the region/chamber 202 is coated with a hydrophobic material. The hydrophobic material may comprise, for example, carbon fluoride molecules. Examples of fluorinated carbon molecules include perfluoropolymers, such as polytetrafluoroethylene (e.g.,

) Or poly (2, 3-difluoromethylene-perfluorotetrahydrofuran) (e.g. CYTOP)^TM). Molecules constituting the hydrophobic material may be covalently attached to the surface of the dielectric layer. For example, molecules of the hydrophobic material may be covalently attached to the surface of the dielectric layer by linking moieties (e.g., siloxane groups, phosphonic acid groups, or thiol groups). Thus, in some embodiments, the hydrophobic material may comprise an alkyl-terminated siloxane, an alkyl-terminated phosphonic acid, or an alkyl-terminated thiol. The alkyl group can be a long chain hydrocarbon (e.g., a chain having at least 10 carbons, or a chain of at least 16, 18, 20, 22 or more carbons). Alternatively, fluorination (or perfluorination)Carbon chains are substituted for alkyl groups. Thus, for example, the hydrophobic material may comprise a fluoroalkyl terminated siloxane, a fluoroalkyl terminated phosphonic acid, or a fluoroalkyl terminated thiol. In some embodiments, the hydrophobic coating has a thickness of about 10nm to about 50 nm. In other embodiments, the hydrophobic coating has a thickness of less than 10nm (e.g., less than 5nm, or about 1.5nm to 3.0 nm).

In some embodiments, the cover 110 of the microfluidic device 200 having an electrowetting configuration is also coated with a hydrophobic material (not shown). The hydrophobic material may be the same hydrophobic material used to coat the dielectric layer of the support structure 104, and the hydrophobic coating may have a thickness substantially the same as the thickness of the hydrophobic coating on the dielectric layer of the support structure 104. In addition, the lid 110 may include an electrode activation substrate 206 sandwiched between a dielectric layer and a top electrode 210 in the manner of the support structure 104. The dielectric layers of the electrode activation substrate 206 and the cap 110 may have the same composition and/or dimensions as the dielectric layers of the electrode activation substrate 206 and the support structure 104. Thus, the microfluidic device 200 may have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 may include a photoconductive material, such as those described above. Thus, in some embodiments, the electrode activation substrate 206 may include or consist of a hydrogenated amorphous silicon layer (a-Si: H). H may comprise, for example, about 8% to 40% hydrogen (calculated as 100 x the number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si: H layer may have a thickness of about 500nm to about 2.0 microns. Alternatively, as described above, the electrode activation substrate 206 may include an electrode (e.g., a conductive metal electrode) controlled by a phototransistor switch. Microfluidic devices having electro-optical wetting configurations are known in the art and/or may be constructed with electrode-activated substrates known in the art. For example, U.S. Pat. No. 6,958,132 (Chiou et al), the entire contents of which are incorporated herein by reference, discloses a electrowetting configuration having a photoconductive material such as a-Si: H, while the above-referenced U.S. Pat. publication No. 2014/0124370 (Short et al) discloses an electrode activated substrate having electrodes controlled by phototransistor switches.

Thus, microfluidic device 200 can have a photo-electrowetting configuration, and light pattern 222 can be used to activate a photoconductive EW region or a photo-responsive EW electrode in electrode activation substrate 206. Such activated EW regions or EW electrodes of the electrode activation substrate 206 can generate electrowetting forces at the inner surface 208 of the support structure 104 (i.e., the inner surface that covers the dielectric layer or hydrophobic coating thereof). By varying the light pattern 222 incident on the electrode-activated substrate 206 (or moving the microfluidic device 200 relative to the light source 220), droplets (e.g., containing an aqueous medium, solution, or solvent) in contact with the inner surface 208 of the support structure 104 can be moved through an immiscible fluid (e.g., an oil medium) present in the region/chamber 202.

In other embodiments, microfluidic device 200 may have an EWOD configuration, and electrode activation substrate 206 may include selectively addressable and energizable electrodes that do not rely on light for activation. Thus, the electrode activation substrate 206 can include a pattern of such Electrowetting (EW) electrodes. For example, the pattern can be an array of substantially square EW electrodes arranged in rows and columns, as shown in figure 2B. Alternatively, the pattern can be an array of substantially hexagonal EW electrodes forming a hexagonal lattice of dots. Regardless of the pattern, the EW electrodes can be selectively activated (or deactivated) by an electrical switch, such as a transistor switch in a semiconductor substrate. By selectively activating and deactivating the EW electrodes in the electrode activation substrate 206, droplets (not shown) that are in contact with the inner surface 208 of the overlying dielectric layer or hydrophobic coating thereof can be moved within the region/chamber 202. The motion module 162 in figure 1 can control such switches to activate and deactivate individual EW electrodes to select and move a particular droplet around the region/chamber 202. Microfluidic devices having EWOD configurations with selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. patent No. 8,685,344 (Sundarsan et al), the entire contents of which are incorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, the power supply 212 may be used to provide a potential (e.g., an AC voltage potential) that powers the circuitry of the microfluidic device 200. The power supply 212 may be the same as or a component of the power supply 192 referenced in FIG. 1. The power supply 212 may be configured to provide an AC voltage and/or current to the top electrode 210 and the bottom electrode 204. For AC voltages, the power supply 212 may provide a range of frequencies and a range of average or peak powers (e.g., voltages or currents): which, as described above, is sufficient to generate a net DEP force (or electrowetting force) strong enough to trap and move individual micro-objects (not shown) in the region/chamber 202, and/or, as also described above, is sufficient to alter the wetting characteristics of the inner surface 208 of the support structure 104 (i.e., the dielectric layer and/or the hydrophobic coating on the dielectric layer) in the region/chamber 202. Such frequency ranges and average or peak power ranges are known in the art. See, for example, 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. US2014/0124370 (Short et al), US2015/0306598(Khandros et al), and US2015/0306599(Khandros et al).

A growth chamber. Non-limiting examples of generic growth chambers 244, 246, and 248 are shown within the microfluidic device 240 shown in fig. 2C and 2D. Each growth chamber 244, 246, and 248 may include a separation structure 250 defining a separation region 258 and a connection region 254 fluidly connecting the separation region 258 to the channel 122. The connecting region 254 may include a proximal opening 252 to the channel 122 and a distal opening 256 to the separating region 258. The connecting region 254 may be configured such that the maximum penetration depth of the flow of fluid medium (not shown) from the channel 122 into the growth chambers 244, 246, 248 does not extend into the separation region 258. Thus, due to the connection region 254, micro-objects (not shown) or other materials (not shown) disposed in the separation region 258 of the growth chambers 244, 246, 248 may be separated from and substantially unaffected by the flow of the medium 180 in the channel 122.

Thus, the channel 122 may be an example of a swept area, and the separation region 258 of the growth chambers 244, 246, 248 may be an example of an unswept area. It should be noted that channel 122 and growth chambers 244, 246, 248 may be configured to contain one or more fluid media 180. In the example shown in fig. 2C-2D, port 242 is connected to channel 122 and allows for the introduction or removal of fluidic media 180 into or from microfluidic device 240. The microfluidic device may be filled with a gas, such as carbon dioxide gas, prior to introduction of the fluid medium 180. Once microfluidic device 240 contains fluidic medium 180, a flow 260 of fluidic medium 180 in channel 122 may be selectively generated and stopped. For example, as shown, the ports 242 may be arranged at different locations (e.g., opposite ends) of the channel 122, and a flow 260 of the culture medium may be formed from one port 242 serving as an inlet to another port 242 serving as an outlet.

Fig. 2E shows a detailed view of an example of a growth chamber 244 according to the present invention. An example of a micro-object 270 is also shown.

As is known, the flow 260 of fluid medium 180 in the microfluidic channel 122 through the proximal opening 252 of the growth chamber 244 may allow a secondary flow 262 of medium 180 to enter and/or exit the growth chamber 244. To separate micro-objects 270 in the separation region 258 of the growth chamber 244 from the secondary flow 262, the length L of the connection region 254 of the growth chamber 244_con(i.e., from proximal opening 252 to distal opening 256) should be greater than the penetration depth D of secondary flow 262 into junction region 254_p. Depth of penetration D of secondary flow 262_pDepending on the velocity of fluid medium 180 flowing in channel 122 and various parameters related to the configuration of channel 122 and proximal opening 252 to connection region 254 of channel 122. For a given microfluidic device, the configuration of channel 122 and opening 252 will be fixed, while the rate of flow 260 of fluid medium 180 in channel 122 will be variable. Thus, for each growth chamber 244, the maximum velocity V of flow 260 of fluid medium 180 in channel 122 may be identified_maxEnsuring the penetration depth D of the secondary flow 262_pNot exceeding the length L of the connecting region 254 _con. As long as the flow 260 rate of fluid medium 180 in channel 122 does not exceed the maximum velocity V_maxThe resulting secondary flow 262 may be confined to the passage 122 and the connecting region 254 and remain outside of the separation region 258. Thus, the flow 260 of medium 180 in channel 122 will not drag micro-objects 270 out of separation region 258. In contrast, micro-objects 270 located in the separation region 258 will stay in the separation region 258 whileRegardless of the flow 260 of fluid medium 180 in channel 122.

In addition, so long as the flow 260 rate of medium 180 in channel 122 does not exceed V_maxThe flow 260 of fluid medium 180 in the channel 122 does not move various particles (e.g., microparticles and/or nanoparticles) from the channel 122 to the separation region 258 of the growth chamber 244. Thus, the length L of the connection region 254 is made_conGreater than the maximum penetration depth D of the secondary flow 262_pOne growth chamber 244 may be prevented from being contaminated by a wide variety of particles from channel 122 or another growth chamber (e.g., growth chambers 246, 248 in fig. 2D).

Since the connection region 254 of the channel 122 and growth chambers 244, 246, 248 may be affected by the flow 260 of the medium 180 in the channel 122, the channel 122 and connection region 254 may be considered a swept (or flow) region of the microfluidic device 240. On the other hand, the separation region 258 of the growth chambers 244, 246, 248 can be considered an unswept (or non-liquid flow) region. For example, a component (not shown) in first fluid medium 180 in channel 122 may mix with second fluid medium 280 in separation region 258 substantially only by diffusion of the component of first medium 180 (from channel 122 through connecting region 254 and into second fluid medium 280 in separation region 258). Similarly, components (not shown) of second medium 280 in separation region 258 can mix with first medium 180 in channel 122 substantially only by diffusion of components of second medium 280 (from separation region 258 through junction region 254 and into first medium 180 in channel 122). First medium 180 may be the same or different medium as second medium 280. In addition, first medium 180 and second medium 280 may be initially the same and then become different (e.g., by conditioning second medium 280 by one or more cells in separation region 258, or by changing the medium 180 flowing through channel 122).

Maximum penetration depth D of secondary flow 262 caused by flow 260 of fluid medium 180 in channel 122_pMay depend on a number of parameters as described above. Examples of such parameters include: the shape of the channel 122 (e.g., the channel may beDirecting media into the junction region 254, transferring media from the junction region 254, or directing media into the channel 122 in a direction substantially perpendicular to the proximal opening 252 of the junction region 254); width W of channel 122 at proximal opening 252_ch(or cross-sectional area); and the width W of the connecting region 254 at the proximal opening 252_con(or cross-sectional area); velocity V of flow 260 of fluid medium 180 in channel 122; viscosity of first medium 180 and/or second medium 280, and so forth.

In some embodiments, the dimensions of channel 122 and growth chambers 244, 246, 248 may be oriented relative to the vector of flow 260 of fluid medium 180 in channel 122 as follows: width W of channel_ch(or the cross-sectional area of channel 122) may be substantially perpendicular to flow 260 of medium 180; width W of connecting region 254 at opening 252_con(or cross-sectional area) may be substantially parallel to the flow 260 of medium 180 in channel 122; and/or length L of the connecting region _conMay be substantially perpendicular to the flow 260 of the medium 180 in the channel 122. The foregoing are merely examples, and the relative positions of the passage 122 and the growth chambers 244, 246, 248 may be other orientations relative to one another.

As shown in FIG. 2E, the width W of the connecting region 254_conMay be uniform from proximal opening 252 to distal opening 256. Thus, the width W of the connecting region 254 at the distal opening 256_conMay be the width W of the connection region 254 at the proximal opening 252, herein_conAny range identified. Alternatively, the width W of the connecting region 254 at the distal opening 256_conMay be greater than the width W of the connecting region 254 at the proximal opening 252_con。

As shown in FIG. 2E, the width of the separation region 258 at the distal opening 256 may be the same as the width W of the connection region 254 at the proximal opening 252_conAre substantially the same. Thus, the width of the separation region 258 at the distal opening 256 may be the width W of the connection region 254 at the proximal opening 252, herein_conAny range identified. Alternatively, the width of the separation region 258 at the distal opening 256 may be greater or less than the width of the connection region 254 at the proximal opening 252Width W_con. Further, distal opening 256 may be smaller than proximal opening 252, and connecting region 254 has a width W _conMay narrow between proximal opening 252 and distal opening 256. For example, using a variety of different geometries (e.g., beveling, etc.) the connection region 254 may narrow between the proximal and distal openings. Further, any portion or sub-portion of the connecting region 254 (e.g., a portion of the connecting region adjacent the proximal opening 252) may be narrowed.

Fig. 4A-C depict another exemplary embodiment of a microfluidic device 400 comprising microfluidic tubing 432 and a flow channel 434 that is a variation of each of the microfluidic device 100, tubing 132, and channel 134 of fig. 1. The microfluidic device 400 also has a plurality of growth chambers 436 that are additional variations of the growth chambers 124, 126, 128, 130, 244, 246, or 248 described above. In particular, it should be understood that the growth chamber 436 of the apparatus 400 shown in fig. 4A-C may be substituted for any of the growth chambers 124, 126, 128, 130, 244, 246, or 248 of the described apparatuses 100, 200, 240, and 290. Similarly, the microfluidic device 400 is another variant of the microfluidic device 100, and it may also have the same or different DEP configuration as the microfluidic devices 100, 200, 240, 290 described above, as well as any of the other microfluidic system components described herein.

The microfluidic device 400 of fig. 4A-C includes a support structure (not visible in fig. 4A-C, but may be the same as or substantially similar to the support structure 104 of the device 100 depicted in fig. 1), a microfluidic conduit structure 412, and a lid (not visible in fig. 4A-C, but may be the same as or substantially similar to the lid 122 of the device 100 depicted in fig. 1). The microfluidic circuit structure 412 includes a frame 414 and a microfluidic circuit material 416, which may be the same as or substantially similar to the frame 114 and the microfluidic circuit material 116 of the device 100 depicted in fig. 1. As shown in fig. 4A, a microfluidic circuit 432 defined by microfluidic circuit material 416 may include a plurality of channels 434 (two are shown, but there may be more) to which a plurality of growth chambers 436 are fluidically connected.

Each growth chamber 436 may include a separation structure 446, a separation region 444 within separation structure 446, and a connection region 442. Connecting region 442 fluidly connects channel 434 to separation region 444 from a proximal opening 472 at channel 434 to a distal opening 474 at separation structure 436. Generally, the flow 482 of the first fluid medium 402 in the channel 434 can produce a secondary flow 484 of the first medium 402 from the channel 434 into and/or out of the respective connection region 442 of the growth chamber 436, as discussed above with respect to fig. 2D and 2E.

As shown in fig. 4B, the connection region 442 of each growth chamber 436 generally includes a region extending between a proximal opening 472 to the channel 434 and a distal opening 474 to the separation structure 446. Length L of connecting region 442_conMay be greater than the maximum penetration depth D of the secondary flow 484_pIn this case, the secondary flow 484 may extend into the union region 442 without being redirected toward the separation region 444 (as shown in FIG. 4A). Alternatively, as shown in FIG. 4C, the length L of the connecting region 442_conMay be less than the maximum penetration depth D_pIn which case the secondary flow 484 would extend through the union region 442 and be redirected toward the separation region 444. In the latter case, the length L of the connecting region 442_c1And L_c2And greater than the maximum penetration depth D_pSuch that the secondary flow 484 does not extend into the separation region 444. Regardless of the length L of the connecting region 442_conWhether greater than the penetration depth D_pOr length L of the connecting region 442_c1And L_c2Whether the sum of (A) and (B) is greater than the penetration depth D_pNo more than a maximum velocity V of first medium 402 in channel 434_max Flow 482 of (a) will result in a flow having a depth of penetration D_pAnd micro-objects (not shown, but may be the same as or substantially similar to micro-objects 270 shown in fig. 2E) in separation region 444 of growth chamber 436 are not carried away from separation region 444 by flow 482 of first medium 402 in channel 434. Flow 482 in channel 434 also does not carry a wide variety of substances (not shown) from channel 434 into separation region 444 of growth chamber 436. Thus, diffusion is the only mechanism by which components in first medium 402 in channel 434 can move from channel 434 into second medium 404 in separation region 444 of growth chamber 436. Similarly, the Diffusion is the only mechanism by which components in second medium 404 in separation region 444 of growth chamber 436 can move from separation region 444 into first medium 402 in channel 434. First medium 402 may be the same medium as second medium 404, or first medium 402 may be a different medium than second medium 404. Alternatively, first medium 402 and second medium 404 can be initially the same and then become different, such as by separating one or more cells in region 444 to condition the second medium, or by changing the medium flow through channel 434.

As shown in FIG. 4B, the width W of the channel 434 in the channel 434_ch(i.e., transverse to the direction of fluid media flow through the channel, as indicated by arrow 482 in FIG. 4A) can be substantially perpendicular to the width W of the proximal opening 472_con1And thus substantially parallel to the width W of the distal opening 474_con2. However, the width W of the proximal opening 472_con1And width W of distal opening 474_con2And need not be substantially perpendicular to each other. For example, the width W of the proximal opening 472_con1A shaft (not shown) oriented thereon and a width W of the distal opening 474_con2The angle between the further axes oriented thereon may be different from perpendicular and thus different from 90 °. Examples of alternative orientation angles include angles in any of the following ranges: about 30 ° to about 90 °, about 45 ° to about 90 °, about 60 ° to about 90 °, and so forth.

In various embodiments of the growth chamber (e.g., 124, 126, 128, 130, 244, 246, 248, or 436), the separation region (e.g., 258 or 444) is configured to contain a plurality of micro-objects. In other embodiments, the separation region may be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. Thus, for example, the volume of the separation region may be at least 3 x 10³、6×10³、9×10³、1×10⁴、2×10⁴、4×10⁴、8×10⁴、1×10⁵、2×10⁵、4×10⁵、8×10⁵、1×10⁶、2×10⁶、4×10⁶、6× 10⁶、1×10⁷、2×10⁷、4×10⁷、6×10⁷、1×10⁸Cubic microns or larger.

In various embodiments of the growth chamber, the width W of the channel 122, 434 at the proximal opening (e.g., 252, 472)_chMay be in any of the following ranges: 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, 100-100 microns, 100-120 microns. The foregoing are examples only, and the width W of the channels 122, 434_chMay be within other ranges (e.g., ranges defined by any of the endpoints listed above). Furthermore, W of the channels 122, 434 _chMay be selected as any one of the areas of the channel other than the proximal opening of the growth chamber.

In some embodiments, the height of the cross-section of the growth chamber is about 30 to about 200 microns, or about 50 to about 150 microns. In some embodiments, the cross-sectional area of the growth chamber is about 100,000 to about 2,500,000 square microns, or about 200,000 to about 2,000,000 square microns. In some embodiments, the connecting region has a cross-sectional height that matches a cross-sectional height of a corresponding growth chamber. In some embodiments, the connecting region has a cross-sectional width of about 50 to about 500 microns, or about 100 to about 300 microns.

In various embodiments of the growth chamber, the height H of the channel 122, 434 at the proximal opening 252, 472_chMay be in any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height H of the channels 122, 434 _chMay be within other ranges (e.g., by any of the endpoints described aboveA defined range). Height H of channels 122, 434_chMay be selected as any one of the areas of the channel other than the proximal opening of the growth chamber.

In various embodiments of the growth chamber, the cross-sectional area of the channel 122, 434 at the proximal opening 252, 472 can be in any of the following ranges: 500-50,000 square micron, 500-40,000 square micron, 500-30,000 square micron, 500-25,000 square micron, 500-20,000 square micron, 500-15,000 square micron, 500-10,000 square micron, 500-7,500 square micron, 500-5,000 square micron, 1,000-25,000 square micron, 1,000-20,000 square micron, 1,000-15,000 square micron, 1,000-10,000 square micron, 1,000-7,500 square micron, 1,000-5,000 square micron, 2,000-20,000 square micron, 2,000-15,000 square micron, 2,000-10,000 square micron, 2,000-7,500 square micron, 2,000-6,000 square micron, 3,000-20,000 micron, 3,000 square micron, 15,000 square micron, 3,000 square, 3,000 micron, 3,000 square micron. The above are examples only, and the cross-sectional area of the passage 122 at the proximal openings 252, 472 can be in other ranges (e.g., a range defined by any of the endpoints described above).

In various embodiments of the growth chamber, the length L of the connecting region 254, 442_conMay be in any of the following ranges: 1-200 microns, 5-150 microns, 10-100 microns, 15-80 microns, 20-60 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, and 100-150 microns. The above are examples only, and the length L of the connecting regions 254, 442_conMay be in a range different from the above examples (e.g., a range defined by any of the endpoints described above).

In various embodiments of the growth chamber, the width W of the connection region 254, 442 at the proximal opening 252_conMay be in any of the following ranges: 20-500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-150 microns, 70-100 microns, and 80-100 microns. The foregoing are examples only, and the width W of the connection regions 254, 442 at the proximal opening 252 _conMay differ from the foregoing examples (e.g., ranges defined by any of the endpoints listed above).

In various embodiments of the growth chamber, the width W of the connecting region 254, 442 at the proximal opening 252, 472_conMay be in any of the following ranges: 2-35 microns, 2-25 microns, 2-20 microns, 2-15 microns, 2-10 microns, 2-7 microns, 2-5 microns, 2-3 microns, 3-25 microns, 3-20 microns, 3-15 microns, 3-10 microns, 3-7 microns, 3-5 microns, 3-4 microns, 4-20 microns, 4-15 microns, 4-10 microns, 4-7 microns, 4-5 microns, 5-15 microns, 5-10 microns, 5-7 microns, 6-15 microns, 6-10 microns, 6-7 microns, 7-15 microns, 7-10 microns, 8-15 microns, and 8-10 microns. The foregoing are examples only, and the width W of the connection region 254, 442 at the proximal opening 252, 472_conMay differ from the foregoing examples (e.g., ranges defined by any of the endpoints listed above).

In various embodiments of the growth chamber, the length L of the connecting region 254, 442_conAnd the width W of the connection region 254, 442 at the proximal opening 252, 472_conThe ratio may be greater than or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. The above are examples only, and the length L of the connection region 254 _conAnd the width W of the connection region 254, 442 at the proximal opening 252, 472_conThe ratio may be different from the above examples.

In various embodiments of the microfluidic devices 100, 200, 240, 290, 400, V_maxMay be set to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 microliters/second. Alternatively, in some other embodiments, V_maxCan be set to about 0.2, 0.3, 0.4, 05, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 microliters/second. In other embodiments, V_maxMay be set to about 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 7.0, 8.0, or about 9.0 microliters/second.

In various embodiments of microfluidic devices having growth chambers, the volume of the separation region 258, 444 of the growth chamber can be, for example, at least 3 x 10³、6×10³、9×10³、1×10⁴、2×10⁴、4×10⁴、8×10⁴、1 ×10⁵、2×10⁵、4×10⁵、8×10⁵、1×10⁶、2×10⁶、4×10⁶、6×10⁶Cubic microns or larger. In various embodiments of microfluidic devices having growth chambers, the volume of the growth chamber may be about 5 x 10³、7 ×10³、1×10⁴、3×10⁴、5×10⁴、8×10⁴、1×10⁵、2×10⁵、4×10⁵、6×10⁵、8×10⁵、 1×10⁶、2×10⁶、4×10⁶、8×10⁶、1×10⁷、3×10⁷、5×10⁷Or about 8X 10⁷Cubic microns or larger. In some embodiments, the microfluidic device has a structure in which no more than 1 × 10 can be maintained ²A growth chamber for the biological cells, and the volume of the growth chamber may not exceed 2X 10⁶Cubic microns. In some embodiments, the microfluidic device has a structure in which no more than 1 × 10 can be maintained²A growth chamber for biological cells, and the growth chamber may not exceed 4 × 10⁵Cubic microns. In other embodiments, the microfluidic device has a growth chamber in which no more than 50 biological cells can be maintained, and the growth chamber can be no more than 4 x 10⁵Cubic microns.

In various embodiments, the microfluidic device has a growth chamber configured as in any of the embodiments described herein, wherein the microfluidic device has from about 100 to about 500 growth chambers, from about 200 to about 1000 growth chambers, from about 500 to about 1500 growth chambers, from about 1000 to about 2000 growth chambers, or from about 1000 to about 3500 growth chambers.

In some other embodiments, the microfluidic device has growth chambers configured as in any of the embodiments described herein, wherein the microfluidic device has from about 1500 to about 3000 growth chambers, from about 2000 to about 3500 growth chambers, from about 2500 to about 4000 growth chambers, from about 3000 to about 4500 growth chambers, from about 3500 to about 5000 growth chambers, from about 4000 to about 5500 growth chambers, from about 4500 to about 6000 growth chambers, from about 5000 to about 6500 growth chambers, from about 5500 to about 7000 growth chambers, from about 6000 to about 7500 growth chambers, from about 6500 to about 8000 growth chambers, from about 7000 to about 8500 growth chambers, from about 7500 to about 9000 growth chambers, from about 8000 to about 9500 growth chambers, from about 8500 to about 10,000 growth chambers, from about 9000 to about 10,500 growth chambers, from about 9500 to about 11,000 growth chambers, from about 10,000 to about 11,500 growth chambers, from about 10,000 to about 12,000 growth chambers, About 11,000 to about 12,500 growth chambers, about 11,500 to about 13,000 growth chambers, about 12,000 to about 13,500 growth chambers, about 12,500 to about 14,000 growth chambers, about 13,000 to about 14,500 growth chambers, about 13,500 to about 15,000 growth chambers, about 14,000 to about 15,500 growth chambers, about 14,500 to about 16,000 growth chambers, about 15,000 to about 16,500 growth chambers, about 15,500 to about 17,000 growth chambers, about 16,000 to about 17,500 growth chambers, about 16,500 to about 18,000 growth chambers, about 17,000 to about 18,500 growth chambers, about 17,500 to about 19,000 growth chambers, about 18,000 to about 19,500 growth chambers, about 18,500 to about 20,000 growth chambers, about 19,000 to about 20,500 growth chambers, about 19,500 to about 21,000 growth chambers, or about 21,000 growth chambers.

Fig. 2F shows a microfluidic device 290 according to one embodiment. The microfluidic device 290 shown in fig. 2F is a stylized schematic diagram of the microfluidic device 100. In practice, the microfluidic device 290 and its constituent tubing elements (e.g., channels 12)2 and growth chamber 128) would have the dimensions discussed herein. The microfluidic circuit 120 shown in fig. 2F has two ports 107, four different channels 122, and four different flow paths 106. The microfluidic device 290 also includes a plurality of growth chambers that open to each channel 122. In the microfluidic device shown in fig. 2F, the growth chamber has a geometry similar to the dock shown in fig. 2E, and thus has both a connection region and a separation region. Thus, microfluidic circuit 120 includes both a swept area (e.g., channel 122 and maximum penetration depth D at secondary flow 262)_pThe portion of the junction region 254 within) also includes non-swept regions (e.g., the separation region 258 and the maximum penetration depth D not within the secondary flow 262)_pThe portion of the inner connecting region 254).

Fig. 3A and 3B illustrate various embodiments of a system 150 that can be used to operate and view microfluidic devices (e.g., 100, 200, 440, 290) according to the present invention. As shown in fig. 3A, the system 150 may include a structure ("nest") 300 configured to hold the microfluidic device 100 (not shown) or any other microfluidic device described herein. Nest 300 can include a socket 302 that can interface with a microfluidic device 360 (e.g., a light-actuated electrokinetic device 100) and provide an electrical connection from power source 192 to microfluidic device 360. Nest 300 may also include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 may be configured to provide a bias voltage to the receptacle 302 such that when the receptacle 302 holds the microfluidic device 360, a bias voltage is applied across a pair of electrodes in the microfluidic device 360. Thus, the electrical signal generation subsystem 304 may be part of the power supply 192. The ability to apply a bias voltage to the microfluidic device 360 does not mean that the bias voltage is always applied when the receptacle 302 holds the microfluidic device 360. In contrast, in most cases, the bias voltage will be applied intermittently, e.g., only when it is desired to facilitate generation of an electrokinetic force (e.g., dielectrophoresis or electrowetting) in the microfluidic device 360.

As shown in fig. 3A, nest 300 may include a Printed Circuit Board Assembly (PCBA) 320. The electrical signal generation subsystem 304 may be mounted on the PCBA 320 and electrically integrated therein. The exemplary nest 300 also includes sockets 302 mounted on the PCBA 320.

Typically, electrical signal generation subsystem 304 will include a waveform generator (not shown). The electrical signal generation subsystem 304 can also include an oscilloscope (not shown) and/or a waveform amplification circuit (not shown) configured to amplify waveforms received from the waveform generator. The oscilloscope (if any) may be configured to measure the waveform supplied to the microfluidic device 360 held by the receptacle 302. In certain embodiments, the oscilloscope measures the waveform at a location proximate to the microfluidic device 360 (and remote from the waveform generator), thereby ensuring a more accurate measurement of the waveform actually applied to the device. Data obtained from oscilloscope measurements may be provided, for example, as feedback to a waveform generator, and the waveform generator may be configured to adjust its output based on such feedback. Red Pitaya^TMIs one example of a suitable combined waveform generator and oscilloscope.

In certain embodiments, nest 300 also includes a controller 308, such as a microprocessor for detecting and/or controlling electrical signal generating subsystem 304. Examples of suitable microprocessors include Arduino ^TMMicroprocessors, e.g. Arduino Nano^TM. The controller 308 may be used to perform functions and analyses or may communicate with the external master controller 154 (shown in FIG. 1) to perform functions and analyses. In the embodiment shown in fig. 3A, the controller 308 communicates with the master controller 154 through an interface 310 (e.g., a plug or connector).

In some embodiments, nest 300 may include an electrical signal generation subsystem 304, which includes Red Pitaya^TMA waveform generator/oscilloscope cell ("Red Pitaya cell") and a waveform amplification circuit, wherein the waveform amplification circuit amplifies the waveform generated by the Red Pitaya cell and transmits the amplified voltage to the microfluidic device 100. In some embodiments, the Red Pitaya cell is configured to measure the amplified voltage at the microfluidic device 360 and then adjust its own output voltage as needed so that the voltage measured at the microfluidic device 360 is a desired value. In some embodiments, the waveform amplification circuit may have a pair of DC-D mounted on the PCBA 320The C-converter generates a +6.5V to-6.5V power supply, thereby generating up to 13Vpp of signal at the microfluidic device 100.

As shown in fig. 3A, nest 300 may also include a thermal control subsystem 306. The thermal control subsystem 306 may be configured to adjust the temperature of the microfluidic device 360 held by the nest 300. For example, the thermal control subsystem 306 may include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device may have a first surface configured to interface with at least one surface of the microfluidic device 360. The cooling unit may be, for example, a cooling block (not shown), such as a liquid-cooled aluminum block. A second surface (e.g., a surface opposite the first surface) of the Peltier thermoelectric device may be configured to interface with a surface of such a cooling block. The cooling block may be connected to a fluid path 330, the fluid path 330 being configured to circulate a cooled fluid through the cooling block. In the embodiment shown in fig. 3A, nest 300 includes an inlet 332 and an outlet 334 to receive cooled fluid from an external reservoir (not shown), introduce the cooled fluid into fluid path 330 and through a cooling block, and then return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, cooling unit, and/or fluid path 330 may be mounted on the housing 340 of the nest 300. In some embodiments, the thermal control subsystem 306 is configured to adjust the temperature of the Peltier thermoelectric device in order to achieve a target temperature for the microfluidic device 360. For example, by such as Pololu ^TMThermoelectric power supplies (Pololu semiconductors and Electronics Corp.) to achieve temperature regulation of Peltier thermoelectric devices. The thermal control subsystem 306 may include feedback circuitry, such as temperature values provided by analog circuitry. Alternatively, the feedback circuit may be provided by a digital circuit.

In some embodiments, nest 300 may include a thermal control subsystem 306 having a feedback circuit, wherein the feedback circuit is an analog voltage divider circuit (not shown) that includes a resistor (e.g., 1k Ω +/-0.1% resistance, temperature coefficient +/-0.02ppm/C0) and an NTC thermistor (e.g., nominal 1k Ω +/-0.01% resistance). In some cases, thermal control subsystem 306 measures the voltage from the feedback circuit and then uses a meterThe calculated temperature value is used as an input to an on-board PID control loop algorithm. The output from the PID control loop algorithm may drive, for example, Pololu^TMDirectional and pulse width modulated signal pins on a motor driver (not shown) to actuate the thermoelectric power supply to control the Peltier thermoelectric device.

Nest 300 may include a serial port 350 that allows the microprocessor of controller 308 to communicate with external master controller 154 via interface 310. Additionally, the microprocessor of the controller 308 may be in communication with the electrical signal generation subsystem 304 and the thermal control subsystem 306 (e.g., via a Plink tool (not shown)). Thus, the electrical signal generation subsystem 308 and the thermal control subsystem 306 may communicate with the external master controller 154 via a combination of the controller 308, the interface 310, and the serial port 350. In this manner, the main controller 154 may assist the electrical signal generation subsystem 308 by performing scaling calculations for output voltage regulation, among other things. A Graphical User Interface (GUI) (not shown) provided by 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 306 and the electrical signal generation subsystem 308, respectively. Alternatively or in addition, the GUI may allow for updating the controller 308, the thermal control subsystem 306, and the electrical signal generation subsystem 304.

As described above, the system 150 may include an imaging device 194. In some embodiments, imaging device 194 includes a light modulation subsystem 422. The light modulation subsystem 422 may include a Digital Mirror Device (DMD) or a micro-shutter array system (MSA), either of which may be configured to receive light from the light source 420 and transmit a portion of the received light into the optical train of the microscope 450. Alternatively, light modulation subsystem 422 may include a device that generates its own light (and thus does not require light source 420), such as an organic light emitting diode display (OLED), a Liquid Crystal On Silicon (LCOS) device, a Ferroelectric Liquid Crystal On Silicon (FLCOS), or a transmissive Liquid Crystal Display (LCD). The light modulation subsystem 422 may be, for example, a projector. Thus, the light modulation subsystem 422 is capable of emitting structured light and unstructured light. One example of a suitable light modulation subsystem 422 is from Andor Technologies^TMMosaic of^TMProvided is a system. In certain embodiments, the imaging module 164 and/or the motion module 162 of the system 150 may control the light modulation subsystem 422.

In certain embodiments, the imaging device 194 further comprises a microscope 450. In such embodiments, the nest 300 and the light modulation subsystem 422 may be individually configured to be mounted on the microscope 450. Microscope 450 may be, for example, a standard research grade optical microscope or a fluorescent microscope. Thus, the nest 300 may be configured to mount on the stage 426 of the microscope 450 and/or the light modulation subsystem 422 may be configured to mount on a port of the microscope 450. In other embodiments, the nest 300 and the light modulation subsystem 422 described herein may be an integrated component of the microscope 450.

In certain embodiments, the microscope 450 may also include one or more detectors 440. In some embodiments, detector 440 is controlled by imaging module 164. The detector 440 may include an eyepiece, a Charge Coupled Device (CCD), a camera (e.g., a digital camera), or any combination thereof. If there are at least two detectors 440, one detector may be, for example, a fast frame rate camera and the other detector may be a high sensitivity camera. Further, the microscope 450 may include an optical train configured to receive light reflected and/or emitted from the microfluidic device 360 and focus at least a portion of the reflected and/or emitted light on the one or more detectors 440. The optical train of the microscope may also include different tube lenses (not shown) for different detectors so that the final magnification on each detector may be different.

In certain embodiments, the imaging device 194 is configured to use at least two light sources. For example, a first light source 420 may be used to generate structured light (e.g., via light modulation subsystem 422), and a second light source 430 may be used to provide unstructured light. The first light source 420 may generate structured light for light-actuated electrical motion and/or fluorescence excitation, and the second light source 430 may be used to provide bright field illumination. In these embodiments, the motion module 164 may be used to control the first light source 420 and the imaging module 164 may be used to control the second light source 430. The optical train of microscope 450 can be configured to (1) receive structured light from light modulation subsystem 422 and focus the structured light onto at least a first area in a microfluidic device (such as a light-actuated electro-kinetic device) when the device is held by nest 300, and (2) receive light reflected and/or emitted from the microfluidic device and focus at least a portion of such reflected and/or emitted light onto detector 440. The optical train can also be configured to receive unstructured light from a second light source and focus the unstructured light on at least a second area of the microfluidic device when the device is held by the nest 300. In certain embodiments, the first and second regions of the microfluidic device may be overlapping regions. For example, the first region may be a portion of the second region.

In fig. 3B, a first light source 420 is shown providing light to a light modulation subsystem 422, which provides structured light to an optical train of a microscope 450 of the system 450. The second light source 430 is shown providing unstructured light to the optical train via the beam splitter 424. The structured light from the light modulation subsystem 422 and the unstructured light from the second light source 430 travel together through an optical train from the beam splitter 424 to the second beam splitter 424 (or dichroic filter 448, depending on the light provided by the light modulation subsystem 422), where the light is reflected down to the sample plane 428 by the objective lens 454. The reflected and/or emitted light from the sample plane 428 then passes through the objective lens 454, through the beam splitter and/or the dichroic filter 448, and back to the dichroic filter 452. Only a portion of the light that reaches the dichroic filter 452 passes through to the detector 440.

In some embodiments, the second light source 430 emits blue light. With an appropriate dichroic filter 452, blue light reflected from the sample plane 428 can pass through the dichroic filter 452 and reach the detector 440. In contrast, structured light from the light modulation subsystem 422 reflects from the sample plane 428, but does not pass through the dichroic filter 452. In this example, the dichroic filter 452 filters out visible light having a wavelength longer than 495 nm. This filtering of light from the light modulation subsystem 422 is accomplished (as shown) only if the light emitted from the light modulation subsystem does not include any wavelengths shorter than 495 nm. In implementation, if the light from the light modulation subsystem 422 includes a wavelength shorter than 495nm (e.g., a blue wavelength), some of the light from the light modulation subsystem may pass through the filter 452 to the detector 440. In such an embodiment, filter 452 acts to change the balance between the amount of light reaching detector 440 from first light source 420 and second light source 430. It is beneficial if the first light source 420 is significantly stronger than the second light source 430. In other embodiments, the second light source 430 may emit red light, and the dichroic filter 452 may filter out visible light other than red light (e.g., visible light having a wavelength shorter than 650 nm).

Additional system components for maintaining cell viability within a growth chamber of a microfluidic device. To promote growth and/or expansion of the cell population, environmental conditions that help maintain functional cells may be provided by additional components of the system. For example, such additional components may provide nutrients, cell growth signaling substances, pH adjustment, gas exchange, temperature control, and removal of waste products from the cells.

A conditioned surface of a microfluidic device. In some embodiments, at least one surface of the microfluidic device is conditioned to support cell growth, viability, portability, or any combination thereof. In some embodiments, substantially all of the interior surface is conditioned. The conditioned surface may be one of the elements that contribute to successful cell incubation within the microfluidic device. Identification of a properly conditioned surface may require balancing a variety of operational requirements. First, the conditioned surface can provide a contact surface for protecting cells from materials that can be used to fabricate such microfluidic devices. Without wishing to be bound by theory, the conditioned surface may be surrounded by hydration water, which provides an aqueous, rather than metallic, contact layer with the cells. Second, the conditioned surface can provide a contact surface by which at least one biological cell can be adequately supported during incubation without substantially inhibiting the ability of the cell to be removed from the growth chamber after incubation is complete. For example, many cells require a contact surface that is hydrophilic to some extent in order to adhere sufficiently to be viable and/or growing. Alternatively, some cells may require a degree of hydrophobicity of the contact surface in order to grow and exhibit a desired level of viability. In addition, some cells may require the presence of selected protein or peptide motifs within the contact surface in order to elicit a viability/growth response. Third, conditioning of at least one surface may allow the motive force used in the microfluidic device to function substantially within the normal operating power range. For example, if light-actuated motive forces are employed, the conditioned surface may substantially allow light to pass through the conditioned surface such that the light-actuated motive forces are not substantially inhibited.

The at least one conditioned surface may comprise a surface of a growth chamber or a surface of a flow region, or a combination thereof. In some embodiments, each of the plurality of growth chambers has at least one conditioned surface. In other embodiments, each of the plurality of fluid flow regions has at least one conditioned surface. In some embodiments, at least one surface of each of the plurality of growth chambers and each of the plurality of fluid flow regions is a conditioned surface.

Including conditioned surfaces of polymers. The at least one conditioned surface may comprise a polymer. The polymer may be covalently or non-covalently attached to at least one surface. The polymers can have a variety of structural motifs, including block polymers (and copolymers); star polymers (star copolymers) and graft or comb polymers (graft copolymers), all of which are suitable for use herein.

The polymer may comprise a polymer comprising alkylene ether moieties. A wide variety of alkylene ether-containing polymers may be suitable for use in the microfluidic devices described herein. One non-limiting exemplary type of alkylene ether containing polymer is a zwitterionic block copolymer comprising blocks of Polyoxyethylene (PEO) and polyoxypropylene (PPO) subunits in different proportions and at different positions in the polymer chain.

The polymers (BASF) are block copolymers of this typeAnd are known in the art to be suitable for use when in contact with living cells. Average molecular weight M of the Polymer_wIn the range of about 2000Da to about 20 KDa. In some embodiments, the PEO-PPO block copolymer may have a hydrophilic-lipophilic balance (HLB) of greater than about 10 (e.g., 12-18). Particularly useful for producing conditioned surfaces

The polymer comprises

L44, L64, P85 and F127 (including F127 NF). Another class of alkylene ether-containing polymers is polyethylene glycol (PEG M)_w<100,000Da) or polyoxyethylene (PEO, M)_w>100,000). In some embodiments, M of PEG_wMay be about 1000Da, 5000Da, 10,000Da or 20,000 Da.

In other embodiments, the polymer conditioned surface may comprise a polymer comprising carboxylic acid moieties. The carboxylic acid subunits may be subunits containing alkyl, alkenyl or aromatic moieties. One non-limiting example is polylactic acid (PLA).

In some other embodiments, the polymer conditioned surface may comprise a polymer comprising urethane moieties, such as, but not limited to, a polyurethane.

In other embodiments, the polymer conditioned surface may comprise a polymer comprising sulfonic acid moieties. The sulfonate subunits may be subunits containing alkyl, alkenyl or aromatic moieties. One non-limiting example is polystyrene sulfonic acid (PSSA) or polyanetholesulfonic acid. These latter exemplary polymers are polyelectrolytes, and may alter the properties of the surface to aid/prevent attachment.

In other embodiments, the polymer conditioned surface may comprise a polymer comprising a phosphate moiety, either at the end of or pendant from the polymer backbone.

In other embodiments, the polymer conditioned surface may comprise a polymer comprising a sugar moiety. In one non-limiting example, polysaccharides (e.g., those derived from seaweed or fungal polysaccharides, such as xanthan or dextran) can be suitable for forming polymer-conditioned surfaces that can aid or prevent cell attachment. For example, dextran polymers having a size of about 3 Kda may be used to provide a conditioned surface within a microfluidic device.

In other embodiments, the polymer conditioned surface may comprise a polymer comprising nucleotide moieties, i.e., nucleic acids, which may have ribonucleic acid moieties or deoxyribonucleic acid moieties. Nucleic acids may contain only natural nucleotide moieties or may contain non-natural nucleotide moieties that comprise nucleobase, ribose, or phosphate moiety analogs, such as, but not limited to, 7-deazaadenine, pentose, methylphosphonate, or phosphorothioate moieties. The nucleic acid-containing polymer can include a polyelectrolyte that can aid or prevent attachment.

In other embodiments, the polymer conditioned surface may comprise a polymer comprising an amino acid moiety. The amino acid moiety-containing polymer can include a natural amino acid-containing polymer or a non-natural amino acid-containing polymer, each of which can include a peptide, polypeptide, or protein. In one non-limiting example, the protein can be Bovine Serum Albumin (BSA). In some embodiments, extracellular matrix (ECM) proteins may be provided on conditioned surfaces for optimal cell attachment to promote cell growth. Cell matrix proteins that may be included in the conditioned surface may include, but are not limited to, collagen, elastin, RGD-containing peptides (such as fibronectin) or laminin. In other embodiments, growth factors, cytokines, hormones, or other cell signaling substances may be provided within at least one conditioned surface of the microfluidic device.

In other embodiments, the polymer conditioned surface can include a polymer comprising amine moieties. The polyamino polymer may comprise a natural polyamino polymer or a synthetic polyamino polymer. Examples of natural polyamines include spermine, spermidine and putrescine.

In some embodiments, the polymer conditioned surface may comprise a polymer comprising more than one of an alkylene oxide moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphate moiety, a sugar moiety, a nucleotide moiety, or an amino acid moiety. In other embodiments, a polymer conditioned surface may comprise a mixture of more than one polymer, each having alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, sugar moieties, nucleotide moieties, and/or amino acid moieties, which may be incorporated into the conditioned surface independently or simultaneously.

A covalently linked conditioned surface. In some embodiments, the at least one conditioned surface comprises covalently attached moieties configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device. The covalently linked moiety can include a linking group, wherein the linking group is covalently linked to a surface of the microfluidic device. The linking group is also linked to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device. The surface to which the linking group is attached may comprise a surface of a substrate of a microfluidic device, which may comprise silicon and/or silicon dioxide in embodiments where the microfluidic device comprises a DEP configuration. In some embodiments, the covalently linked conditioned surfaces include all internal surfaces of the microfluidic device.

For microfluidic devices with conditioned surfaces, a schematic representation is shown in fig. 9. As can be seen in fig. 9, the microfluidic device 900 has a first DEP substrate 904 and a second DEP substrate 906 facing an enclosed region 902 of the microfluidic device, which may include at least one growth chamber and/or flow region. The device 900 may be otherwise configured similar to any of the microfluidic devices 100, 200, 240, 290, 400, 500A-E, or 600. Enclosed region 902 may be a region where biological cells are held or input or output. The inner surface 910 (of the second DEP substrate 906) and the inner surface 912 (of the first DEP substrate 904) are modified by a conditioned surface 916, which conditioned surface 916 may be any portion that supports cell growth, viability, portability, or any combination thereof. In this embodiment, the conditioned surface is covalently attached to the oxide functional groups of the inner surface via siloxy linking groups 914.

In some embodiments, the covalently linked moiety configured to support cell growth, viability, portability, or any combination thereof, may comprise an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid.

The covalently linked moiety configured to support cell growth, viability, portability, or any combination thereof of one or more biological cells within the microfluidic device can be any polymer described herein, and can include one or more polymers containing alkylene oxide moieties, carboxylic acid moieties, sugar moieties, sulfonic acid moieties, phosphate moieties, amino acid moieties, nucleic acid moieties, or amino moieties.

In other embodiments, the covalently linked moieties configured to support cell growth, viability, portability, or any combination thereof of one or more biological cells may comprise non-polymeric moieties such as alkyl moieties, fluoroalkyl moieties (including but not limited to perfluoroalkyl), amino acid moieties, alcohol moieties, amino moieties, carboxylic acid moieties, phosphonic acid moieties, sulfonic acid moieties, sulfamic acid moieties, or sugar moieties.

In some embodiments, the covalently attached moiety may be an alkyl group. The alkyl group can include carbon atoms that form a straight chain (e.g., a straight chain of at least 10 carbons or at least 14, 16, 18, 20, 22, or more carbon atoms). Thus, the alkyl group may be an unbranched alkyl group. In some embodiments, an alkyl group may include a substituted alkyl group (e.g., some carbons in the alkyl group may be fluorinated or perfluorinated). The alkyl group can comprise a linear chain of substituted (e.g., fluorinated or perfluorinated) carbons attached to a linear chain of unsubstituted carbons. For example, an alkyl group can include a first segment (which can include a perfluoroalkyl group) bonded to a second segment (which can include an unsubstituted alkyl group). The first and second segments may be joined directly or indirectly (e.g., via an ether linkage). The first segment of the alkyl group may be located distal to the linking group and the second segment of the alkyl group may be located proximal to the linking group. In other embodiments, the alkyl group may include branched alkyl groups, and may further have one or more arylene groups interrupting the alkyl backbone of the alkyl group. In some embodiments, the branched or arylene interrupted portion of the alkyl group or the fluorinated alkyl group is located away from the point of covalent linkage to the surface.

In other embodiments, the covalently linked moiety may comprise at least one amino acid, which may comprise more than one amino acid. The covalently linked moiety may comprise a peptide or a protein. In some embodiments, the covalently linked moiety may include an amino acid, which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof.

The covalently linked moiety may comprise one or more saccharides. The covalently linked saccharide may be a monosaccharide, disaccharide or polysaccharide. Covalently linked saccharides can be modified to introduce reactive partner moieties that allow coupling or tailoring of the linkage to the surface. Exemplary reactive partner moieties may include aldehyde, alkyne, or halo moieties. The polysaccharide may be modified in a random manner, wherein each saccharide monomer or only a fraction of the saccharide monomers in the polysaccharide may be modified to provide reactive partner moieties, which may be coupled directly or indirectly to a surface. One example may include dextran polysaccharides, which may be indirectly coupled to a surface via an unbranched linking moiety.

The covalently linked moiety may comprise one or more amino groups. The amino group can be a substituted amine moiety, guanidine moiety, nitrogen-containing heterocyclic moiety, or heteroaryl moiety. The amino-containing moiety can have a structure that allows for pH modification of the environment within the microfluidic device and optionally within the growth chamber.

The covalently linked moiety may comprise one or more carboxylic, phosphonic, sulfamic or sulfonic acid moieties. In some embodiments, the covalently linked moieties can include one or more nucleic acid moieties, which can have a sequence of individual nucleotides designed to capture nucleic acids from biological cells within the microfluidic device. The capture nucleic acid may have a nucleotide sequence complementary to a nucleic acid from the biological cell and may capture the nucleic acid by hybridization.

The conditioned surface may consist of only one type of moiety or may comprise more than one different type of moiety. For example, a fluoroalkyl-conditioned surface (including perfluoroalkyl) may have a plurality of covalently attached moieties that are identical, e.g., have the same covalent attachment to the surface and have the same number of fluoromethylene units comprising fluoroalkyl moieties that support growth and/or viability and/or portability. Alternatively, the conditioned surface may have more than one moiety attached to the surface. For example, a conditioned surface may include an alkyl or fluoroalkyl group having a specific number of methylene or fluoromethylene units, and may also include another group attached to the surface that has a charged moiety attached to an alkyl or fluoroalkyl chain that has a greater number of methylene or fluoromethylene units. In some embodiments, a conditioned surface with more than one attached moiety can be designed such that a first set of attached ligands (which have a greater number of backbone atoms and thus a greater length from covalent attachment to the surface) can provide the ability to present a larger moiety at the conditioned surface, while a second set of attached ligands (which have different, sterically less demanding ends while having fewer backbone atoms) can help functionalize the entire substrate surface to prevent attachment or contact with the silicon or alumina substrate itself. In another example, the moiety attached to the surface can provide a zwitterionic surface that exhibits alternating charges on the surface in a random manner.

Conditioned surface properties. In some embodiments, the covalently linked moieties can form a monolayer when covalently linked to a surface of a microfluidic device (e.g., a substrate surface of a DEP configuration). In some embodiments, the conditioned surface formed by the covalently attached moieties can have a thickness of less than 10nm (e.g., less than 5nm or about 1.5 to 3.0 nm). In other embodiments, the conditioned surface formed by covalently linked moieties may have a thickness of about 10nm to about 50 nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to function properly in operation within a DEP configuration.

In various embodiments, the conditioned surface of the microfluidic device can provide desired electrical properties. Without wishing to be bound by theory, one factor that affects the durability of the conditioned surface is intrinsic charge trapping. Different surface-conditioned materials can trap electrons, which can lead to decomposition of the material. Defects in the conditioned surface may lead to charge trapping and decomposition of the conditioned surface.

In addition to the composition of the conditioned surface, other factors (e.g., the physical thickness of the hydrophobic material) may affect the DEP force. Various factors may alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid deposition, spin coating, flood coating, and electrostatic coating). The physical thickness and uniformity of the conditioned surface can be measured using an ellipsometer.

In addition to its electrical properties, the conditioned surface may also have properties that are beneficial when used with biomolecules. For example, a conditioned surface containing fluorinated (or perfluorinated) carbon chains may provide benefits in reducing surface fouling relative to alkyl terminated chains. As used herein, surface fouling refers to the amount of any substance deposited on the surface of a microfluidic device, which may include permanent or semi-permanent deposition of biological materials (e.g., proteins and their degradation products, nucleic acids and respective degradation products, and the like).

Various properties of conditioned surfaces that can be used in DEP configurations are included in the table below. It can be seen that for entries 1 to 7, which are all covalently linked conditioned surfaces described herein, the thickness as measured by ellipsometry was thinner than that of entry 8, a CYTOP surface formed by non-covalent spin coating (N/a indicates data not available throughout the table). It was found that fouling is more dependent on the chemistry of the surface than the manner of formation, since fluorinated surfaces are generally less fouling than surfaces conditioned with alkyl (hydrocarbons).

Table 1. properties of various conditioned surfaces prepared by covalent modification of a surface were compared to non-covalently formed surface CYTOP.

CYTOP structure:

spin-coated, non-covalent.

A surface-bound group. The covalently linked moieties forming the conditioned surface are linked to the surface via a linking group. The linking group may be a siloxy linking group formed by reaction of a siloxane-containing reagent with an oxide of the substrate surface (which may be formed from silicon or aluminum oxide). In some other embodiments, the linking group may be a phosphonate ester formed by the reaction of a phosphonic acid-containing reagent with an oxide of the silicon or aluminum substrate surface.

Multi-part conditioned surfaces. The covalently linked conditioned surface can be formed from a surface conditioning agent that is configured to already contain a moiety that provides a conditioned surface (e.g., an alkylsiloxane agent or a fluoro-substituted alkylsiloxane agent, which can include a perfluoroalkylsiloxane agent), as described below. Alternatively, a conditioned surface may be formed by coupling a moiety that supports cell growth, viability, portability, or any combination thereof to a surface modifying ligand that is itself covalently attached to the surface.

Structures and methods of preparation for conditioned surfaces. In some embodiments, the conditioned surface of the oxide covalently attached to the surface of the dielectrophoresis substrate has a structure of formula 1:

The conditioned surface may be covalently attached to an oxide of the surface of the dielectrophoresis substrate. The dielectrophoresis substrate may be silicon or alumina and the oxide may be present as part of the original chemical structure of the substrate or may be incorporated as described below. The conditioned surface may be linked to the oxide via a linking group LG, which may be a siloxy or phosphonate group formed by reaction of a siloxane or phosphonate group with the oxide

The moiety configured to support cell growth, viability, portability, or any combination thereof may comprise an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid. The alkyl or fluoroalkyl moiety can have a backbone chain length equal to or greater than 10 carbons. In some embodiments, the alkyl or fluoroalkyl moiety may have a backbone chain length of about 10, 12, 14, 16, 18, 20, or 22 carbons.

The linking group LG may be directly or indirectly attached to a moiety that provides support for cell growth, viability, portability, or any combination thereof within a microfluidic device. When the linking group LG is directly linked to the moiety, the optional linking moiety L is absent and n is 0. When the linking group LG is indirectly linked to the moiety, the linking moiety L is present and n is 1. The linking moiety L may have a linear moiety, wherein the backbone of the linear moiety may comprise from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemical bonding limitations known in the art. In some non-limiting examples, it may be interrupted by any combination of one or more moieties selected from ether, amino, carbonyl, amido, or phosphonate groups. Furthermore, the linking moiety L may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety. In some embodiments, the backbone of the linking moiety L may comprise 10 to 20 atoms. In other embodiments, the backbone of the linking moiety L can include from about 5 atoms to about 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

A surface conditioning agent. The surface conditioning agent of formula 6 may be used to introduce a conditioned surface when a moiety configured to support cell growth, viability, portability, or any combination thereof and thereby provide a conditioned surface is added to the surface of the substrate in a one-step process.

The surface conditioning agent may have a structure of formula 6:

part- (L)_n-LG

Formula 6

In the surface conditioning agent of formula 6, the surface conditioning agent may include a linking group LG, which may be a siloxane or phosphonic acid group. The linking group LG may be directly or indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. LG may be directly (n ═ 0) attached to a moiety configured to support cell growth, viability, portability, or any combination thereof or indirectly (n ═ 1) attached to the moiety via a connection to the first end of the linking moiety L. The linking moiety L may also comprise a linear moiety, wherein the backbone of the linear moiety may have from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to the limitations of chemical bonding known in the art. The backbone of the linear moiety may also include one or more arylene moieties. The moiety ("moiety") configured to support cell growth, viability, portability, or any combination thereof may include an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid. The moiety configured to support cell growth, viability, portability, or any combination thereof may comprise an alkyl or perfluoroalkyl moiety. The alkyl or perfluoroalkyl moiety may have a backbone chain length of greater than 10 carbons. The portion of the surface conditioning agent configured to support cell growth, viability, portability, or any combination thereof may include a sugar moiety, which may be dextran. In other embodiments, the portion of the surface conditioning agent configured to support cell growth, viability, portability, or any combination thereof may include an alkylene ether moiety. The alkylene ether moiety may be polyethylene glycol. The surface conditioning agent may also include a cleavable moiety, which may be located within the linking moiety L or may be a portion of the surface conditioning agent configured to support cell growth, viability, portability, or any combination thereof. The cleavable moiety can be configured to allow disruption of the conditioned surface, thereby facilitating portability of the cultured one or more biological cells.

In some embodiments, a moiety that supports cell growth, viability, portability, or any combination thereof may be added to the surface of the substrate in a multi-step process. When the moiety is coupled to the surface in a stepwise manner, the linking moiety L may also be a moiety

Including coupling groups as shown in formula 2.

In some embodiments, the coupling group CG is represented by a reactive moiety R_xWith a moiety configured to react therewith, i.e. a reactive partner R_pxThe resulting fraction of the reaction between. For example, a typical CG may include a carboxamido group, which is the result of the reaction of an amino group with a carboxylic acid derivative (e.g., an activated ester, acid chloride, etc.). CG may include triazolylene, carboxamido, thioamido, oxime, mercapto, disulfide, ether or alkenyl groups, or may be comprised of reactive moietiesAny other suitable group formed by reaction of a moiety with its corresponding reactive partner. The coupling group CG may be located at the second end of the linking moiety L to which the moiety is attached. In some other embodiments, the coupling group CG may interrupt the backbone of the linking moiety L. In some embodiments, the coupling group CG is a triazolylene group that is the result of a reaction between an alkynyl group and an azide group, both of which may be reactive moieties or reactive partner moieties known in the art for use in Click coupling reactions. The triazolylene group may also be further substituted. For example, from a compound having a dibenzocyclooctynyl reactive partner R _pxWith the azido-reactive moiety R of a surface-modified ligand_xThe reaction of (a) can result in a dibenzocyclooctynyl fused triazolylene moiety, as described in more detail below. A variety of dibenzocyclooctynyl-modified molecules are known in the art and can be synthesized to incorporate moieties configured to support cell growth, viability, portability, or any combination thereof.

When the conditioned surface is formed in a multi-step process, moieties that support cell growth, viability, portability, or any combination thereof can be introduced by the reaction of a conditioning modifying reagent (formula 5) with a substrate having a structure of formula 3 having a surface modifying ligand covalently attached thereto.

The intermediate modified surface of formula 3 has a surface modifying ligand attached thereto having the formula-LG- (L ") j-R_xWhich is connected to the oxide of the substrate and is formed similarly as described above for the conditioned surface of equation 1. The surface of the DEP substrate is as described above and includes the oxide originally present on or incorporated into the substrate. The linking group LG is as described above. The linking moiety L "may be present (j ═ 1) or absent (j ═ 0). The linking moiety L' may have a linear moiety, wherein the backbone of the linear moiety may comprise from 1 to 100 non-hydrogen atoms, said non-hydrogen atoms Selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, subject to chemical bonding limitations known in the art. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. Furthermore, the linking moiety L "may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety. In some embodiments, the backbone of the linking moiety L "may comprise 10 to 20 atoms. In other embodiments, the backbone of the linking moiety L "can include from about 5 atoms to about 100 atoms; from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

Reactive moiety R_xIs present at the end of the surface-modified ligand distal to the covalent attachment of the surface-modified ligand to the surface. Reactive moiety R_xIs any suitable reactive moiety that can be used in a conjugation reaction to introduce a moiety that supports cell growth, viability, portability, or any combination thereof. In some embodiments, the reactive moiety R _xCan be an azido, amino, bromo, thiol, activated ester, succinimidyl, or alkynyl moiety.

A conditioning modifying agent. The conditioning modification agent (equation 5) is configured to supply a moiety that supports cell growth, viability, portability, or any combination thereof.

Moiety- (L')_m-R_px

Formula 5

By reactive pairing of moieties R_pxWith reactive moieties R_xTo link a portion of the conditioned modifying agent configured to support cell growth, viability, portability, or any combination thereof, to the surface modifying ligand. Reactive partner R_pxIs any suitable reactive moiety R configured to react with the corresponding reactive moiety_xReactive groups of reaction. In a non-limiting example, a suitable reactive partner R_pxCan be an alkyne, and the reactive moiety R_xMay be an azide. Alternatively, the reactive partner R_pxCan be an azide moiety, and the corresponding reactive moiety R_xMay be an alkyne. In other embodiments, the reactive partner R_pxMay be an active ester functional group, and the reactive moiety R_xMay be an amino group. In other embodiments, the reactive partner R_pxMay be an aldehyde, and the reactive moiety R_xMay be an amino group. Other reactive moiety-reactive partner combinations are possible, and these examples are not limiting in any way.

The portion of the conditional modifying agent of formula 5 configured to support cell growth, viability, portability, or any combination thereof can include an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid.

The moiety of the conditional modification agent of formula 5 that provides enhanced cell growth, viability, portability or any combination thereof may be linked directly (L', wherein m ═ 0) or indirectly to the reactive partner moiety R _px. When the reactive partner R is_pxReactive partner moiety R when indirectly linked to a moiety that provides enhanced cell growth, viability, portability or any combination thereof_pxCan be connected to the connecting part L' (m ═ 1). Reactive partner R_pxA moiety that can be attached to a first end of the linking moiety L 'and that provides enhanced cell growth, viability, portability, or any combination thereof can be attached to a second end of the linking moiety L'. The linking moiety L' may have a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to the limitations of chemical bonding known in the art. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. Furthermore, the linking moiety L 'may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety L'. In some embodiments, the backbone of the linking moiety L' may comprise 10 to 20 atoms. In other embodiments, the backbone of the linking moiety L' may comprise from about 5 atoms to about 100 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

When the conditioned modifying reagent (formula 5) reacts with the surface having the surface modifying ligand (formula 3), a substrate having a conditioned surface of formula 2 is formed. Then, the linking moiety L 'and linking moiety L' are the formal parts of the linking moiety L, and the reactive partner R_pxWith reactive moieties R_xTo give the coupling group CG of formula 2.

A surface modification agent. The surface-modifying agent is of the structure LG- (L')_j-R_xA compound of (formula 4). The linking group LG is covalently linked to the oxide of the surface of the dielectrophoresis substrate. The dielectrophoresis substrate may be silicon or alumina and the oxide may be present as part of the original chemical structure of the substrate or may be incorporated as described herein. The linking group LG may be a siloxy or phosphonate group consisting of siloxane or phosphonate groupsClusters form from reaction with the oxide on the substrate surface. Reactive moiety R_xAs described above. Reactive moiety R_xMay be directly (L ", j ═ 0) or indirectly via a linking moiety L" (j ═ 1) to the linking group LG. The linking group LG may be linked to the first end of the linking moiety L' and the reactive moiety R_xMay be attached to the second end of the linking moiety L ", which is remote from the surface of the substrate once the surface modifying ligand has been attached to the surface as shown in formula 3.

The linking moiety L "may have a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. Furthermore, the linking moiety L "may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety L". In some embodiments, the backbone of the linking moiety L "may comprise 10 to 20 atoms. In other embodiments, the backbone of the linking moiety L "can include from about 5 atoms to about 100 atoms; from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

A cleavable moiety. In various embodiments, any of the moiety, linker L', linker L ", or coupling group CG that supports cell growth, viability, portability, or any combination thereof, may further comprise a cleavable moiety, as described below. The cleavable moiety can be configured to allow disruption of the conditioned surface of the microfluidic device, which facilitates portability of the one or more biological cells. In some embodiments, portability of one or more biological cells is desirable so as to enable the cells to be moved after a period of culturing of the cells, and in particular to enable the cells to be output from the microfluidic device.

Composition of a substrate. Accordingly, a composition is provided, comprising a substrate having a Dielectrophoresis (DEP) configuration and a surface; and a conditioned surface of oxide moieties covalently attached to the surface of the substrate. The conditioned surface on the substrate can have a structure of formula 1 or formula 2:

wherein LG is a linking group; l is a linking moiety, which may be present (n ═ 1) or absent (n ═ 0); the portion is a portion that supports cell growth, viability, portability, or any combination thereof within the microfluidic device; and CG is a coupling group, as defined herein.

The conditioned surface may comprise a linking group LG covalently linked to an oxide moiety of the surface. The linking group may be further attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group may be a siloxy linking group. In other embodiments, the linking group may be a phosphonate. The linking group may be directly or indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via attachment to the first end of the linking moiety. The linking moiety may also comprise a linear moiety, wherein the backbone of the linear moiety may have from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, as described above. The backbone of the linear moiety may also include one or more arylene moieties.

The linking moiety may have a coupling group CG as defined above. The coupling group CG may include a triazolylene moiety. The triazolylene moiety may interrupt the linear portion of the linking moiety or may be connected to the linear portion of the linking moiety at the second end. The second end of the connecting portion may be remote from the surface of the substrate. The portion configured to support cell growth, viability, portability, or any combination thereof may include one or more of the following: alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid. In some embodiments, a mixture of different moieties is incorporated in the conditioned surface, such as, but not limited to, a mixture of anionic and cationic functional groups that provide a zwitterionic conditioned surface. The conditioned surface may include alkyl or perfluoroalkyl moieties. The alkyl or perfluoroalkyl moiety may have a backbone chain length of greater than 10 carbons. The conditioned surface may include a sugar moiety and may be dextran. In other embodiments, the conditioned surface may include alkylene ether moieties. The alkylene ether moiety may be polyethylene glycol. The conditioned surface may also include a cleavable moiety. The cleavable moiety may be configured to allow disruption of the conditioned surface, thereby facilitating portability of the one or more biological cells.

Another composition is provided that includes a substrate having a Dielectrophoresis (DEP) configuration and a surface, and a surface modifying ligand covalently attached to an oxide moiety of the substrate surface. The surface with the surface-modified ligand may have the structure of formula 3:

wherein LG is a linking group; l' is an optional linking moiety, and j is 0 or 1. The linking moiety L "is present when j is 1 and absent when j is 0; and R is_xIs a reactive moiety, as described herein.

The reactive moiety of the surface modifying ligand may be an azido, amino, bromo, thiol, activated ester, succinimidyl, or alkynyl moiety. The surface modifying ligand may be covalently linked to the oxide moiety via a linking group. The linking group may be a siloxy moiety. In other embodiments, the linking group may be a phosphonate. The linking group may be indirectly linked to the reactive moiety of the surface modifying ligand via a linking moiety. The linking group can be attached to a first end of the linking moiety and the reactive moiety can be attached to a second end of the linking moiety. The linking moiety L "may comprise a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. The backbone of the linking moiety L "may comprise 10 to 20 atoms. In other embodiments, the backbone of the linking moiety L "can include from about 5 atoms to about 50 atoms. In some embodiments, the backbone of the linking moiety L "may include all carbon atoms. The backbone of the linear moiety may comprise one or more arylene moieties. The linking moiety L "may include a triazolylene moiety. The triazolylene moiety may interrupt the linking moiety L "or may be attached at a terminus of the linking moiety L". The surface modifying ligand may comprise a cleavable moiety. The cleavable moiety can be configured to allow disruption of the conditioned surface of the microfluidic device, thereby facilitating the portability of one or more biological cells.

A preparation method. In some embodiments, the conditioned surface or surface modifying ligand is deposited on the interior surface of the microfluidic device by using chemical vapor deposition. By vapour deposition of molecules, viaThe conditioned surface/surface modifying ligands may achieve a close-packed monolayer, wherein molecules comprising the conditioned surface/surface modifying ligands are covalently linked to molecules of the inner surface of any of the microfluidic devices (100, 200, 240, 290, 400, 500A-E, 600). To achieve a desired bulk density, molecules comprising, for example, alkyl-terminated siloxanes, can be vapor deposited at a temperature of at least 110 ℃ (e.g., at least 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃, etc.) for a period of at least 15 hours (e.g., at least 20, 25, 30, 35, 40, 45, or more hours). Such vapor deposition is typically carried out under vacuum and in a water source (e.g., hydrated sulfate salts (e.g., MgSO)₄·7H₂O)) in the presence of oxygen. Generally, increasing the temperature and duration of vapor deposition results in improved characteristics of the conditioned surface/surface-modified ligands. In some embodiments, the conditioned surface or surface modifying ligand may be introduced by reaction in a liquid phase.

To prepare the microfluidic surface, the cover, microfluidic circuit material, and electrode activated substrate can be treated with oxygen plasma treatment, which can remove various impurities while introducing oxidized surfaces (e.g., oxides at the surface, which can be covalently modified as described herein). The oxygen plasma cleaner may be operated, for example, under vacuum conditions at 100W for 60 seconds. Alternatively, a liquid phase treatment may be used which includes an oxidizing agent such as hydrogen peroxide to oxidize the surface. For example, a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (e.g., a piranha solution, which may have a sulfuric acid to hydrogen peroxide ratio ranging from about 3:1 to about 7: 1).

The vapor deposition process can optionally be modified by, for example, pre-cleaning the lid, the microfluidic circuit material, and the electrode activation substrate. For example, such pre-cleaning may include a solvent bath, such as an acetone bath, an ethanol bath, or a combination thereof. The solvent bath may include sonication.

In some embodiments, vapor deposition is used to coat the interior surfaces of the microfluidic device after the microfluidic device has been assembled to form a housing defining the microfluidic channels.

When the substrate with surface-modified ligands is further reacted with a conditioned modifying reagent to produce a substrate with a conditioned surface, the reaction can be carried out in situ by using any suitable solvent that will dissolve the reagent and will not damage the microfluidic circuit material or the surface with surface-modified ligands. In some embodiments, the solvent is an aqueous solution.

Methods of preparing a conditioned surface or a surface comprising a surface-modified ligand. Accordingly, there is provided a method of preparing a modified surface of a microfluidic device having a Dielectrophoresis (DEP) configuration, comprising the steps of: providing a surface of a substrate of a microfluidic device, wherein the substrate comprises a DEP configuration; the oxide of the surface is reacted with a modifying reagent, thereby converting the surface of the substrate to a modified surface. In some embodiments, the surface of the substrate may be plasma cleaned to provide an oxide on the surface. In some embodiments, the surface may be plasma cleaned prior to assembly of the microfluidic device. In other embodiments, the surface may be plasma cleaned after assembly of the microfluidic device.

A method wherein the step of reacting the oxide of the surface with the modifying agent is performed by exposing the surface to a liquid comprising the modifying agent. In some embodiments, the step of reacting the oxide of the surface may be performed by exposing the surface to a vapor containing the modifying agent under reduced pressure.

In some embodiments, the modifying agent may comprise a surface conditioning agent having: a first portion configured to covalently react with a surface; and a second portion configured to support cell growth, viability, portability, or any combination thereof, thereby modifying the surface to a surface conditioned to support cell growth, viability, portability, or any combination thereof.

The surface conditioning agent may have a structure of formula 6:

part- (L)_n-LG

Formula 6

The first moiety may comprise a linking group LG, which may be a siloxane or phosphonic acid group. The linking group LG may be directly or indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. The first moiety may be directly (n ═ 0) attached to the second moiety or indirectly (n ═ 1) via attachment to the first end of the linking moiety L, which is a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking moiety L may also comprise a linear moiety, wherein the backbone of the linear moiety may have from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. The backbone of the linear moiety may also include one or more arylene moieties. The second portion ("moiety") of the surface conditioning agent may comprise an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid. The second portion of the surface conditioning agent may comprise an alkyl or perfluoroalkyl moiety. The alkyl or perfluoroalkyl moiety may have a backbone chain length of greater than 10 carbons. The second portion of the surface conditioning agent may comprise a sugar moiety and may be dextran. In other embodiments, the second portion of the surface conditioning agent may comprise an alkylene ether moiety. The alkylene ether moiety may be polyethylene glycol. The surface conditioning agent may also include a cleavable moiety, which may be located within the linking moiety L or may be part of the second portion of the surface conditioning agent. The cleavable moiety may be configured to allow disruption of the conditioned surface, thereby facilitating portability of the one or more biological cells.

In various embodiments, the modifying agent may comprise a surface modifying agent having a structure of formula 4 as defined above, wherein the surface modifying agent comprises a first portion LG configured to react with a surface; and a second moiety R_xWhich may or may not be modified to include reactive moieties including azido, amino, bromo, thiol, activated ester, succinimidyl or alkynyl moieties to convert the surface to a surface comprising surface modifying ligands having the structure of formula 3 as described above. In some embodiments, the first portion of the surface modifying agent (which is configured to react with the oxide of the surface) may be a siloxane or a phosphonic acid.

In some embodiments, the method comprises the step of reacting a surface comprising a surface modifying ligand (formula 3) with a conditioned modifying reagent comprising a first moiety configured to support cell growth, viability, portability, or any combination thereof; and a second moiety R_pxA reactive moiety configured to react with a surface modifying ligand; thereby providing a surface conditioned to support cell growth, viability, portability, or any combination thereof, of a biological cell having a structure of formula 2 as described above. The conditional modifying agent may have a structure of formula 5. In some embodiments, the first portion of the conditioning modification agent comprises at least one of an alkylene oxide moiety, an amino acid moiety, a sugar moiety, an anionic moiety, a cationic moiety, and a zwitterionic moiety.

In various embodiments, any of the surface conditioning, surface modifying, or conditioning modifying agents may further comprise a cleavable moiety as described herein.

Conditioned surfaces containing other components. Unlike or in addition to the conditioned surface formed by the polymer or covalently linked moieties, the conditioned surface may additionally include other components, including biocompatible metal ions (e.g., calcium, sodium, potassium, or magnesium), antioxidants, surface activitySex agents and/or essential nutrients. A non-limiting illustrative list includes vitamins such as B7, alpha-tocopherol acetate, vitamin a and its acetate; proteins such as BSA, catalase, insulin, transferrin, superoxide dismutase; small molecules such as corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine dihydrochloride, and triiodothyronine; and salts, including but not limited to sodium selenite, sodium phosphate, potassium phosphate, calcium phosphate, and/or magnesium phosphate. Antioxidants can include, but are not limited to, carotenoids, cinnamic acid and derivatives, ferulic acid, polyphenols such as flavonoids, quinones and derivatives (including mitoxantrone-Q), N-acetyl cysteine, and antioxidant vitamins such as ascorbic acid, vitamin E, and the like. The conditioned surface may include a media supplement, for example

A supplement containing an antioxidant and many of the other components listed above.

Supplements are commercially available in serum-free form from ThermoFisher Scientific (50X) (Cat. No. 17504044).

In some embodiments, the at least one conditioned surface may comprise one or more components of mammalian serum. In some embodiments, the mammalian serum is Fetal Bovine Serum (FBS) or fetal bovine serum (FCS). The conditioned surface may include specific components of mammalian serum, such as specific amounts and types of proteins typically found in serum, which may be provided in defined amounts and types from serum-free media or well-defined media.

In other embodiments, at least one conditioned surface does not comprise mammalian serum. In various embodiments, at least one conditioned surface may not include any titanium, nickel, or iron metal ions. In other embodiments, at least one conditioned surface may not include any significant concentration of titanium, nickel, or iron metal ions. In other embodiments, at least one conditioned surface may not include any gold, aluminum, or tungsten metal ions.

Reagent treatment to reduce sticking. A mixture of reagents. As cells are cultured within the microfluidic device, the cells actively secrete proteins and other biomolecules and passively exude similar biomolecules, which may adhere to surfaces within the microfluidic device. Cells in culture may adhere to each other or to a conditioned surface and become difficult to remove from the growth chamber for output from the microfluidic device. Furthermore, in some cases, it may be desirable to bring additional cells, of the same or different type as the cultured cells, into the microfluidic device. These newly delivered cells may also become attached to the surface, accumulate dirt in the microfluidic environment, and cause difficulty in removal from the device at a later point in time.

With proteases, e.g. trypsin or

(enzyme mixtures with proteolytic and collagenolytic activity, Innovative Cell Technologies) treatments do not necessarily provide sufficient efficiency to allow, for one non-limiting example, the export of attached cells from a microfluidic device. One or more proteins and/or peptides providing anti-attachment properties may be used in a mixture to reduce such attachment in both cases. Biomolecules or small molecules that have activity against one of a variety of cell attachment mechanisms may be used. Some of the cell attachment mechanisms that can be inhibited can be active agonist silk formation and related processes, which can be targeted by the use of compounds such as cytochalasin B (New England Biosciences, Cat.: M0303S, which is a small molecule inhibitor of microfilament extension). Specific receptor-driven attachment processes, such as, but not limited to, inhibition of integrin receptor-mediated attachment to fibronectin (which may be found on fouled surfaces), may be targeted by the use of RGD-containing peptides, for example. Another class of fouling substances, i.e., nucleic acids released by dead cells, It may attract cell binding, which may be targeted by the use of endonucleases that can cleave the fouling nucleic acids. One specific endonuclease, deoxyribonuclease 1 (DNase 1, Sigma Aldrich, catalog number AMPD1-1KT), also binds actin, thereby providing a dual active block to attachment. In some embodiments, a mixture of all three blocking agents may be used to prevent/reduce cell adhesion.

General processing scheme. After culturing: for cells that have been grown within the microfluidic device for 2, 3, 4, or more days, a mixture of three anti-attachment reagents, as described below, or a single anti-attachment reagent, can be flowed into the microfluidic device and allowed to diffuse into the growth chamber for a period of about 20min, 30min, 40min, 50min, or 60min before the cells are exported.

Pretreatment: for cells to be input into the microfluidic device, the cells may be preincubated in a medium containing the mixture or single anti-attachment reagent for about 30min before being input into the microfluidic chip. Inhibition continued for 1, 2, 3, or more hours without further addition of reagents.

The RGD peptide (mw.614.6, Santa Cruz Biotechnology, Cat. sc-201176) may be present in the culture medium or in the incubation medium prior to infusion at a concentration of about 0.1mM to about 20 mM. In some embodiments, the RGD tripeptide may be present at a concentration of about 0.1, 0.5, 0.7, 1.0, 3.0, 5.0, 6.0, 8.0, 10.0mM or any value within this range. Cytochalasin B can be present in the pre-infusion incubation medium at a concentration of about 0.01 μ Μ to about 50 μ Μ or about 0.01, 0.05, 0.1, 2, 4, 6, 8, 10, 20, 30, 50 μ Μ or any value within this range. DNase 1 may be present at a concentration of about 0.001U/μ L to about 10U/μ L or about 0.001, 0.005, 0.01, 0.05, 1.0, 5.0, 10U/μ L or any value within this range.

In some embodiments, a single reagent may be used to reduce adherence before or after culturing cells in the microfluidic device. For example, the RGD tripeptide may be used at a concentration of 5mg/ml, or used for pre-incubation, or may be flowed in as a treatment within the microfluidic device prior to output.

Another inhibitor that may be used is the tetrapeptide fibronectin inhibitor (Arg-Gly-Asp-Ser-OH, mw.433.4, Santa Cruz Biotechnology, Cat. No.: sc-202156)). The fibronectin inhibitor may be used at a concentration of about 1.75 μ g/ml (4 μ M).

Similar to the use of proteins or small molecule reagents to reduce or prevent adherence, output and portability can be achieved within a microfluidic device using antibodies against proteins of interest for extracellular adherence. One non-limiting example is anti-B1 integrin: clone M-106 (Santa Cruz Biotechnology, Cat. No.: sc-8978).

A conditioned surface comprising a cleavable moiety. In some embodiments, the conditioned surface can have a cleavable moiety incorporated into a covalently or non-covalently linked molecule of the conditioned surface. The conditioned surface may include a peptide motif, such as RGD, which has the function as described above; or it may have another peptide motif that promotes cell growth or provides a contact signal for cell proliferation. In other embodiments, the conditioned surface provides non-specific support for the cells and may serve to simply buffer the cells from the silicon or alumina surface of the microfluidic device. After a period of cell culture is complete, it may be desirable to disrupt the conditioned surface to facilitate the export of an expanded cell population within the growth chamber of the microfluidic device. This may be useful when the cells show an adhesive behavior. The conditioned surface can be disrupted, partially or completely removed, by incorporating other peptide motifs that are substrates for proteases that are not highly secreted by the target cell. In one non-limiting example, the ENLYQS peptide motif (Glu-Asn-Leu-Tyr-Gln-Ser) can be incorporated into the conditioned surface at pre-designed intervals. This motif is a substrate for TEV protease (tobacco etch virus cysteine protease, Sigma Aldrich, catalog number T4455), which is highly sequence specific and therefore useful for highly controlled cleavage. After the incubation period is complete, the TEV protease may be flowed into the microfluidic device and allowed to diffuse into the isolated region of the growth chamber. The conditioned surface is then disrupted, which facilitates the export of cells within the microfluidic device. Thus, as can be designed by one skilled in the art, a variety of other proteolytic motifs can be designed and incorporated into a conditioned surface to be cleaved by an appropriate specific protease.

A fluid medium. For the foregoing discussion regarding microfluidic devices having channels and one or more growth chambers, the fluid medium (e.g., the first medium and/or the second medium) can be any fluid capable of maintaining cells in a substantially viable state. The survival state will depend on the biological micro-object and the culture experiment being performed.

The first and/or second fluid media may provide the fluid and dissolved gas components necessary for cell viability, and may also maintain the pH in a desired range by using buffered fluid media or pH monitoring, or both.

If the cell is a mammalian cell, the first fluid medium and/or the second fluid medium may comprise mammalian serum or well-defined serum-free media known in the art that are capable of providing the necessary nutrients, hormones, growth factors or cell growth signals. Similar to the conditioned surface above, the first fluid medium and/or the second fluid medium can comprise Fetal Bovine Serum (FBS) or fetal bovine serum (FCS). Alternatively, the first fluid medium and/or the second fluid medium may not include any animal-derived serum but may include a defined medium that may include any or all of physiologically relevant metal ions (including but not limited to sodium, potassium, calcium, magnesium, and/or zinc), antioxidants, surfactants, and/or essential nutrients. A well-defined medium can be serum-free, but still contain some protein, where the protein is of a defined amount and type. A non-limiting exemplary list of components in a serum-free medium includes vitamins such as B7, alpha-tocopherol acetate, vitamin a, and its acetate; proteins such as BSA, catalase, insulin, transferrin, superoxide dismutase; small molecules such as corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine dihydrochloride, and triiodothyronine; and salts, including but not limited to sodium selenite, sodium phosphate, potassium phosphate, calcium phosphate, and/or magnesium phosphate. The fluid medium may contain any of the antioxidants described above for the conditioned surface.

The fluid medium may be sterile filtered through a 0.22 micron filter unit (VWR, catalog number 73520-.

In some embodiments, a suitable medium may include, or may consist entirely of, any of the following media: dulbecco's Modified Eagle's medium (ThermoFisher Scientific, Cat. No. 11960-; FreeStyle^TMMedia (Invitrogen, ThermoFisher Scientific, Cat. No. 11960-; RPMI-1640(

ThermoFisher Scientific, Cat No. 11875-127); hybridoma-SFM (ThermoFisher Scientific, Cat. No. 12045-076); medium E (Stem Cell, catalog No. 3805); 1X CD CHO medium (ThermoFisher Scientific, Cat. No. 10743-011); iscove's Modified Dulbecco's Medium (ThermoFisher Scientific, Cat. No. 12440-; or CD DG44 medium (ThermoFisher Scientific, Cat. No. 10743-011).

The culture medium may additionally comprise fetal bovine serum (FBS, obtained from

Thermo fisher scientific), heat inactivated fetal bovine serum; or fetal calf serum (FCS, Sigma-Aldrich, Cat. Nos. F2442, F6176, F4135, and others). FBS can be present at a concentration of about 1% to about 20% v/v; about 1% to about 15% v/v, about 1% to about 10% v/v, or about 1% to about 5% v/v, or any value within any range. The culture medium may additionally comprise human AB serum (Sigma-Aldrich, cat # S2146), and it may be present at a concentration of about 1% to about 20% v/v; about 1% to about 15% v/v, about 1% to about 10% v/v or about 1% to about 5% v/v- v, or any value within any range.

The medium may additionally include penicillin-streptomycin (ThermoFisher Scientific, Cat. No. 15140-163). Penicillin-streptomycin may be present at a concentration of about 0.01% to about 10% v/v; about 0.1% to about 10% v/v; about 0.01% to about 5% v/v; about 0.1% to about 5% v/v; about 0.1% to about 3% v/v; about 0.1% to about 2% v/v; about 0.1% to about 1% v/v; or any value within any range. In other embodiments, the culture medium may comprise geneticin (ThermoFisher Scientific, Cat. No. 101310-. Geneticin can be present at a concentration of about 0.5 μ g/ml; about 1.0 μ g/ml; about 5.0 μ g/ml; about 10.0 μ g/ml; about 15 μ g/ml; about 20 μ g/ml; about 30 μ g/ml; about 50 μ g/ml; about 70 μ g/ml; about 100 μ g/ml; or any value within these ranges.

The culture medium may include a buffer. The buffer may be one of Good's buffers. The buffer may be, but is not limited to, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) (ThermoFisher Scientific, Cat. No. 15630-080). The buffer may be present at a concentration of about 1 mM; about 3 mM; about 5 mM; about 7 mM; about 9 mM; about 10 mM; about 12 mM; about 15 mM; about 20 mM; about 40 mM; about 60 mM; about 100 mM; or any value within these ranges.

The medium may additionally comprise GlutaMAX, a dipeptide substitute for glutamine^TM(

ThermoFisher Scientfic, Cat. No. 35050-. The substitute for glutamine can be present at a concentration of about 0.2 mM; about 0.5 mM; about 0.7 mM; about 1.0 mM; about 1.2 mM; about 1.5 mM; about 1.7 mM; about 2.0 mM; about 2.5 mM; about 3.0 mM; about 4.0 mM; about 7.0mM or about 10.0mM, or any value within these ranges. The medium may include MEM non-essential amino acids (ThermoFisher Scientific, Cat. No. 10370-088). MEM non-essential amino acids may be present in an amount of about 0.2 mM; about 0.5 mM; about 0.7 mM; about 1.0 mM; about 1.2 mM; about 1.5 mM; about 1.7 mM; about 2.0 mM; about 2.5 mM; about 3.0 mM; about 4.0 mM; about 7.0mM or about 10.0mM, or any value within these ranges.

The culture medium may additionally comprise glucose (ThermoFisher Scientific, Cat. No. 15023-. Glucose may be present at a concentration of about 0.1 g/L; about 0.1 g/L; about 0.1 g/L; about 0.3 g/L; about 0.5 g/L; about 0.8 g/L; about 1.0 g/L; about 1.5 g/L; about 2.0 g/L; about 2.5 g/L; about 3.0 g/L; about 3.5 g/L; about 4.0 g/L; about 5.0 g/L; about 7.0 g/L; about 10.0 g/L; or any value within these ranges.

The medium may additionally include mercaptoethanol (ThermoFisher Scientific, Cat. No. 31350-010). Mercaptoethanol may be present at a concentration of from about 0.001% to about 1.5% v/v; about 0.005% to about 1.0% v/v; about 0.01% to about 1.0% v/v; about 0.15% to about 1.0% v/v; about 0.2% to about 1% v/v; or any value within these ranges.

The media may include OPI media supplements including oxaloacetate, pyruvate, and insulin (Sigma-Aldrich, Cat. No. O-5003). The OPI medium supplement may be present at a concentration of about 0.001% to about 1.5% v/v; about 0.005% to about 1.0% v/v; about 0.01% to about 1.0% v/v; about 0.15% to about 1.0% v/v; about 0.2% to about 1% v/v; or any value within these ranges. The medium may contain a B-27 supplement (50X), which is serum-free (ThermoFisher Scientific, Cat. No. 17504-. The B-27 supplement may be present at a concentration of about 0.01% to about 10.5% v/v; about 0.05% to about 5.0% v/v; about 0.1% to about 5.0% v/v; about 0.5% to about 5% v/v; or any value within these ranges.

As described herein, the culture medium or additives for the culture medium may include one or more substances useful for obtaining a conditioned surface

A polymer, and may comprise

L44, L64, P85, F68 and F127 (including F127 NF).

The polymer may be present in the medium at a concentration of about 0.001% v/v to about 10%v/v; about 0.01% v/v to about 5% v/v; about 0.01% v/v to about 1% v/v or about 0.05% to about 1% v/v. For media supplements that can be provided in the form of a kit, the concentration can be 1X, 5X, 10X, 100X, or about 100X of the final media concentration.

The culture medium may include IL 6(Sigma-Aldrich, catalog number SRP3096-20 UG). IL 6 may be present at a concentration of about 1 nM; about 5nM, about 10nM, about 15nM, about 20nM, about 25nM, about 30nM, about 40nM, about 50nM, or any value within these ranges.

The medium may additionally include sodium pyruvate (ThermoFisher Scientific, Cat. No. 11360-. The substitute for glutamine can be present at a concentration of about 0.1 mM; about 0.02 mM; about 0.04 mM; about 0.06 mM; about 0.08 mM; about 0.1 mM; about 0.5 mM; about 0.7 mM; about 1.0 mM; about 1.2 mM; about 1.5 mM; about 1.7 mM; about 2.0 mM; about 2.5 mM; about 3.0 mM; about 4.0 mM; about 7.0mM or about 10.0mM, or any value within these ranges.

A gaseous environment. The system provides a gas mixture for cell viability including, but not limited to, oxygen and carbon dioxide. Both gases are dissolved in the fluid medium and can be used by the cells to change the gas content of the fluid medium in the separation region of the growth chamber over time. In particular, the carbon dioxide content may vary over time, which affects the pH of the fluid medium in the microfluidic device. In some experimental conditions, a non-optimal oxygen partial pressure may be used.

And (4) controlling the temperature. In some embodiments, at least one conditioned surface of the growth chamber and/or the flow region is conditioned by controlling the temperature of the at least one conditioned surface. The system may include components that can control and adjust the temperature of at least one conditioned surface of a growth chamber and/or a flow region of a microfluidic device. The system may include Peltier heating, resistive heating, or any other suitable method for providing temperature adjustment to the microfluidic device. The system may also include sensors and/or feedback components to control the heat input within a predetermined range. In some embodiments, the temperature of at least one conditioned surface is at least about 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, or about 40 ℃ and is stable at that temperature. In some embodiments, the temperature of at least one surface is greater than about 25 ℃. In other embodiments, the temperature of at least one surface is between about 30 ° and 40 ℃; from about 35 ℃ to about 38 ℃; or in the range of about 36 ℃ to about 37 ℃. In some embodiments, the temperature of the at least one conditioned surface is at least about 30 ℃.

A flow controller to provide perfusion during incubation. The flow controller can perfuse the first fluid medium in the flow region during incubation, as described above, to provide nutrients to the cells in the growth chamber and carry waste products away from the growth chamber, wherein exchange of nutrients and removal of waste products occurs substantially via perfusion. The controller may be a separate component from the microfluidic device or may be incorporated as part of the microfluidic device. The flow controller may be configured to non-continuously perfuse the medium in the flow region. The flow controller may be configured to perfuse the medium in a periodic manner or an irregular manner.

In some other embodiments, the controller can be configured to perfuse the fluid culture medium in the fluid region about every 4h, 3h, 2h, 60min, 57min, 55min, 53min, 50min, 47min, 45min, 43min, 40min, 37min, 35min, 33min, 30min, 27min, 25min, 23min, 20min, 17min, 15min, 13min, 10min, 7min, or 5 min. In some embodiments, the controller may be configured to perfuse the fluid medium about every 5min to about every 20 min. In other embodiments, the controller may be configured to perfuse the fluid medium about every 15min to about every 45 min. In other embodiments, the controller may be configured to perfuse the fluid medium every 30min to about every 60 min. In other embodiments, the controller may be configured to perfuse the fluid medium every 45min to about every 90 min. In some other embodiments, the controller may be configured to perfuse the fluid medium every 60min to about 120 min. Alternatively, the controller may be configured to perfuse the fluid medium every 2h to every 6 h.

In some embodiments, the controller 226 may be configured to perfuse the medium for a period of time, which may be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 seconds. In other embodiments, the controller can be configured to perfuse the medium for about 1min, 1.2min, 1.4min, 1.5min, 1.6 min, 1.8min, 2.0min, 2.2min, 2.4min, 2.5min, 2.6min, 2.8min, 3.0min, 3.2min, 3.4min, 3.5min, 3.6min, 3.8min, or 4.0 min.

In various embodiments, the controller may be configured to perfuse the medium for about 5 seconds to about 4min, about 10 seconds to about 3.5min, about 15 seconds to about 3min, about 15 seconds to about 2min, about 25 seconds to about 90 seconds about 30 seconds to about 75 seconds, about 40 seconds to about 2.0min, about 60 seconds to about 2.5min, about 90 seconds to about 3.0min, or 1.8min to about 4 min.

The flow controller (not shown) can be configured to perfuse the first fluid medium in the flow region at a rate that is much higher than the average rate of perfusion of the component from the separation region of the growth chamber to the flow channel. For example, the rate of fluid flow in the flow region can be about 0.009, 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 7.0, 8.0, or 9.0 μ L/sec, any rate being the rate that sweeps across the connected region of the growth chamber (but not across the isolated region of the growth chamber). The controller may be capable of providing a first fluid medium rate that is a non-sweeping rate of fluid medium velocity, i.e., any suitable rate below V _max(i.e., the maximum speed of the microfluidic device that avoids the microfluidic device from rupturing due to excessive pressure and limits the movement of components to diffuse between the second fluid medium in the growth chamber and the first fluid medium in the flow region). In some embodiments, the controller may be configured to control the flow rate of the fluid at about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 0.15, 0.20, 0.30, 0.40, 1.50, 1.60, or 0.10,1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 microliters/second perfuse the first fluid medium through the flow region. In some embodiments, the controller can be configured to perfuse the first fluid medium through each of the plurality of fluid zones at about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, or about 0.11 μ Ι _ per sec.

In various embodiments, the flow rate and duration of perfusion provide at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, 25, 30, 35, 50, 75, 100, 200, 300, or more times the volume of the total volume of the first fluid culture medium in the flow channel.

In various embodiments, perfusion may be achieved using varying durations, varying flow rates, and perfusion stop durations, as shown in the methods of fig. 7 and 8 and described below.

Reservoir, media conditioning and introduction assembly. The system can also include a reservoir configured to contain a fluidic medium that can be introduced at the inlet 124 of the microfluidic device and can be perfused by the flow controller. The reservoir may be in fluid connection with any of the microfluidic devices described above (non-limiting examples include 100, 200, 240, 290, or 400) at an upstream location (fig. 5A-E). The fluid medium may be conditioned in the reservoir to contain the desired balance of gases, i.e., for one non-limiting example, a mixture containing 5% carbon dioxide, which provides optimal growth for the cells being cultured, and may also adjust the pH in the microfluidic device.

In some embodiments, the reservoir may further comprise a population of cells different from the cells being studied in the microfluidic device. The cell population may be feeder cells that produce soluble signaling or growth factors necessary for the growth and/or viability of the cells in the microfluidic device. In this manner, the fluid medium may be conditioned to optimize growth and/or viability prior to introduction to the microfluidic device. The use of a reservoir to contain a population of feeder cells can prevent contamination of the cultured cell population in the microfluidic device; soluble secretions from feeder cells may be incorporated into the fluid medium delivered to the microfluidic device, but feeder cells may not be pumped away with the fluid medium.

One embodiment of the reservoir, conditioning and introduction components of the system is shown in fig. 5A. The reservoir in this embodiment may be another microfluidic device 502 that contains a fluid medium 202 (not shown) that is conditioned within the microfluidic device 502. Microfluidic device 502 has a housing 510 and a base 512, at least one of which is gas permeable. The microfluidic device 502 may also contain a population of feeder cells maintained such that the feeder cells produce soluble growth factors or other cell signaling components necessary for the growth and/or viability of the cells in the microfluidic device 500A. The reservoir 502 may be housed within a chamber 516 that provides a 5% carbon dioxide gas environment (one non-limiting example for a gas environment). The fluid medium 202 in the reservoir 502 absorbs the gas mixture (e.g., 5% carbon dioxide in air) through the gas permeable walls of the reservoir and also absorbs soluble secretions from the feeder cells. The culture medium 202 is perfused by the pump 514 through the air-tight connecting line 506 from the reservoir 502 into the microfluidic device 500A via the inlet port 124 and forms a stream 212 in the flow channel 134 of the microfluidic device 500A. In this embodiment, neither the pump connection lines 504 (not labeled), the transfer connection lines 506, the base 104, or the housing 102 are air permeable. Fluid medium flow 212 sweeps across the growth chamber of microfluidic device 500A and allows the waste components of fluid medium 204 to diffuse out of the growth chamber (not shown), while allowing the components to diffuse into the growth chamber from fluid medium 202 of flow channel 134. Eventually, spent fluid media 202 '(not shown) exits microfluidic device 500A via output port 124' in output connecting line 508.

In another embodiment, fluid medium 202 is transferred to microfluidic device 500B through pump connection line 504 and through gas permeable block 518, as shown in fig. 5B. A breathable block 518 is incorporated into and forms a part of the upper surface of the housing 102. The portion of the upper surface of the housing 102 formed by the gas permeable block 518 may be upstream of the growth chamber of the microfluidic device 500B. The microfluidic device 500B is housed within a chamber 516 that provides a gaseous environment (e.g., 5% carbon dioxide) that is exchanged into a fluid medium in the microfluidic device 500B. The chamber 516 may additionally provide conditioning in temperature and/or humidity to the microfluidic device 500B. Neither the pump connection tubing 504, the housing 102, or the base 104 are gas permeable, and exchange through the gas permeable block 518 may function as a "lung" of the microfluidic device 500B and appropriately condition the media within the microfluidic device 500B. In this embodiment, the fluid medium 202 may be additionally conditioned in another element prior to loading into the pump 514, and thus may also contain secreted substances, for example, from feeder cell cultures.

In another embodiment, a gas permeable block is integrated into the upper surface of the housing 102 of the microfluidic device 500C, forming a gas permeable section 518', as shown in fig. 5C. The fluid medium may be conditioned and introduced as described above for the embodiment of fig. 5B, and may also include substances secreted from the feeder cell population. The microfluidic device 500C may be housed in a chamber 516 containing a gaseous environment, such as 5% carbon dioxide in air. The gaseous environment may be exchanged across a gas permeable portion 518' (which may be one or more portions of the upper surface of the housing 102). The chamber 516 may further condition the device 500C to an appropriate temperature and humidity. In this embodiment, the pump connection lines 504, the housing 102 (other than the gas permeable block 518'), and the base 104 may be gas impermeable. In some embodiments, at least one gas-permeable portion 518' is located above the growth chamber of microfluidic device 500C. In another embodiment, at least one gas-permeable portion 518' is located above the flow stream region 134 of the microfluidic device 500C. In other embodiments, gas-permeable portion 518' may be located above both the at least one growth chamber and the at least one flow field 134.

In other embodiments, a gas permeable line 504' may be used to condition (e.g., equilibrate) the fluid medium prior to introducing the medium into the microfluidic device 500D, as shown in fig. 5D. Pipeline capable of selecting air permeability504' are of a length to provide sufficient surface area to allow for efficient gas exchange and equilibration within enclosure 516, which may contain a gaseous environment, such as, in a non-limiting example, 5% carbon dioxide in air. 516 may further condition the temperature and/or humidity of the medium within the gas permeable pump connecting line 504'. One non-limiting example of a breathable material that may be used for breathable connecting lines is

And (5) AF. Prior to introduction into the pump assembly 514, the fluid medium may be conditioned by contact with a population of feeder cells, and as a result may contain secreted substances that may optimize the growth and/or viability of the cells being cultured in the microfluidic device 500D. Prior conditioning with feeder cell populations may occur within chamber 516 or may be performed in another culture assembly having its own environmental controls for any of temperature, humidity, pH, and/or gas environments. In an embodiment, the housing 102 and the base 104 of the microfluidic device 500D may be gas impermeable.

In another embodiment of the reservoir, media conditioning and introduction assembly of the system, the media can be conditioned in a reservoir 502' that can be placed in an appropriate gas environment, as shown in fig. 5E. The reservoir 502' need not be a microfluidic device or any particular type of culture assembly. The reservoir 502' is placed in a suitable gaseous environment (e.g., 5% carbon dioxide in air) by providing a connecting feed 526 from a gaseous environment source 524. The fluid medium within reservoir 502' has a gas exchange with the gaseous environment provided by source 524 and is thus conditioned. The fluid medium of the reservoir 502' may also contain a culture of feeder cells to provide secreted substances that may optimize the growth and/or viability of the cells being cultured in the microfluidic device 500E. Conditioned fluid medium may be transferred from reservoir 502' via transfer connecting line 522 (which is connected to valve 520 on pump 514) and may be injected by pump 514 into channel 134 of microfluidic device 500E via connecting line 504. The fluid medium injected into microfluidic device 500E forms fluid stream 212. After passing through the flow channel 134, the spent fluidic media 202' exits the microfluidic device 500E via the outlet tubing 508. In this embodiment, the transfer connecting line 522, the connecting line 504, the valve 520, the pump 514, the housing 102, and the base 104 may all be gas impermeable. In some embodiments, the connection line 526 connecting the source 524 to the reservoir 502' may be substantially gas impermeable. In other embodiments, the connecting line 526 need not be substantially air impermeable, but may be relatively air impermeable.

In some embodiments of the system shown in fig. 5E, the gas (not shown) may be continuously flowing or may be pulsed, e.g., periodically replaced (not shown), input from source 524, which may be 5% carbon dioxide in air. In other embodiments, the gas input from source 524 may be 100% carbon dioxide. When 100% carbon dioxide gas is used, a small amount of carbon dioxide gas may be injected into the headspace (not shown) of the reservoir 502' to maintain the headspace as a 5% carbon dioxide mixture. In some embodiments, when injecting gas into the headspace of the reservoir 502 ', the reservoir 502' may also include a fan (not shown) to mix the injected air with other gas components (not shown) already present in the headspace (not shown). In some embodiments, where the input of gas is pulsed, the lid 102 of the microfluidic device 500E may have a carbon dioxide sensor (not shown) incorporated or connected thereto. In some embodiments, 100% carbon dioxide gas may be input from source 524 to save costs compared to commercially available gas mixtures of 5% carbon dioxide in air. In other embodiments, 100% carbon dioxide gas may be introduced into source 524 and mixed therein with air to produce a 5% carbon dioxide mixture.

In any of the above embodiments, the chamber 516 may be further humidified such that the gaseous environment of the chamber does not change the osmotic pressure of the fluidic medium in the microfluidic device and/or reservoir.

In another embodiment, another method of providing appropriate gas exchange to the cells being cultured in the growth chamber may provide a gas flow through the fluid flow region of the microfluidic device (not shown). A suitable gas (e.g., 5% carbon dioxide) may be pumped or pulsed directly through the flow channel. Because the separation region of the growth chamber is designed to be a largely unswept volume, the cells located therein are not disturbed by air or air bubbles moving through the flow channel (swept area). This provides a very rapid gas exchange between the gas in the flow channel and the fluid medium inside the growth chamber, because the diffusion distance is very small compared to that in, for example, a 50mL conical tube. The gas may then be replaced by the fluid medium after any selected amount of time. The gas flow may be repeated at any desired frequency to maintain the dissolved gas components at a stable concentration, which also has an effect on the pH of the fluid medium. Alternatively, less than optimal gas compositions or repetitions may be used to perturb the environment of the cells.

In summary, there are a variety of components and configurations that can be used to provide conditioned media to cells in the growth chamber of the microfluidic devices described herein. Any of the microfluidic devices 100, 200, 240, 290, or 400 may be used with any of the embodiments of fig. 5A-5E. The systems and kits may include connecting tubing configured to connect to an inlet and/or an outlet of a microfluidic device. The connecting line may also be configured to connect to a reservoir and/or a pump assembly.

Accordingly, there is provided a microfluidic device for culturing one or more biological cells, comprising: a flow region configured to contain a flow of a first fluid medium; and at least one growth chamber comprising at least one surface conditioned to support cell growth, viability, portability, or any combination thereof within the microfluidic device, wherein the at least one growth chamber comprises a separation region and a connection region, the separation region is in fluidic connection with the connection region, and the connection region comprises a proximal opening to the fluid flow region. In various embodiments, the separation region of the microfluidic device can be configured to contain a second fluid medium. When the flow region and the at least one growth chamber are substantially filled with the first and second fluid media, respectively, a component of the second fluid media may diffuse into the first fluid media and/or a component of the first fluid media may diffuse into the second fluid media; and the first culture medium does not substantially flow into the separation region. In various embodiments, the at least one conditioned surface may be conditioned to support portability of one or more biological cells within the microfluidic device. In some embodiments, portions of the conditioned surface may be configured to support portability of biological cells within the microfluidic device.

In some embodiments, at least one conditioned surface of a microfluidic device can include a polymer comprising an alkylene ether moiety. In other embodiments, at least one conditioned surface of a microfluidic device can include a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety. In other embodiments, at least one conditioned surface of the microfluidic device can comprise a polymer comprising a sugar moiety. In some embodiments, the polymer comprising a saccharide moiety can be dextran. In some other embodiments, at least one conditioned surface of a microfluidic device can comprise a polymer comprising an amino acid moiety.

Alternatively, at least one conditioned surface of the microfluidic device may comprise one or more components of mammalian serum. The components of the mammalian serum may be supplements for the culture medium. In some embodiments, the mammalian serum can be Fetal Bovine Serum (FBS) or fetal bovine serum (FCS).

In various embodiments of the microfluidic device, the at least one conditioned surface may comprise a sugar moiety. In some embodiments, at least one conditioned surface may comprise alkylene ether moieties. In other embodiments, at least one conditioned surface may comprise an amino acid moiety. In some other embodiments, at least one conditioned surface may comprise alkyl or perfluoroalkyl moieties. In some embodiments, the alkyl or perfluoroalkyl moiety may have a backbone chain length of greater than 10 carbons. In some embodiments, the at least one conditioned surface may comprise a moiety that: which may be an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid.

In various embodiments of the microfluidic device, the at least one conditioned surface may comprise a linking group covalently attached to a surface of the microfluidic device, and the linking group may be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof within the microfluidic device. In some embodiments, the linking group may be a siloxy linking group. In other embodiments, the linking group may be a phosphonate linking group. In some embodiments, the linking group can be indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In some embodiments, portions of the conditioned surface may be configured to support portability of biological cells within the microfluidic device. In other embodiments, the linking group may be directly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In other embodiments, the linking group may be indirectly attached via a linking moiety to a moiety configured to support cell growth, viability, portability, or any combination thereof. In various embodiments, the linking moiety may include a triazolylene moiety.

In various embodiments of the microfluidic device, at least one conditioned surface may comprise a zwitterion. In other embodiments, at least one conditioned surface may comprise a phosphonic acid moiety or a carboxylic acid moiety. In other embodiments, the conditioned surface may comprise anions. In some other embodiments, at least one conditioned surface may comprise amino or guanidine moieties. In other embodiments, at least one conditioned surface may comprise cations.

In various embodiments of the microfluidic device, the at least one conditioned surface may comprise at least one cell adhesion blocking molecule. At least one cell adhesion blocking molecule may disrupt the formation of an agonist filament, block an integrin receptor, or reduce binding of cells to a DNA-contaminated surface. The at least one cell adhesion blocking molecule may be cytochalasin B, an RGD-containing peptide, or a DNase 1 protein. In other embodiments, the at least one cell adhesion blocking molecule may comprise a combination of more than one type of cell adhesion blocking molecule.

In various embodiments of the microfluidic device, the at least one conditioned surface is configured to be heated to a temperature of at least about 30 ℃. The at least one conditioned surface may be configured to be stable at a temperature of at least about 30 ℃.

In various embodiments of the microfluidic device, the microfluidic device can further comprise a microfluidic channel comprising at least a portion of the flow region. In some embodiments, at least one growth chamber connection region may open directly into the microfluidic channel. In some embodiments, the separation region of at least one growth chamber of the microfluidic device may have a size sufficient to support cell expansion to a range of about 100 cells. In some embodiments, no more than 1 x 10 can be maintained in at least one growth chamber²And (4) biological cells. In some embodiments, the volume of the at least one growth chamber may be less than or equal to about 2 x 10⁶Cubic microns. In other embodiments, no more than 1 × 10 may be maintained in at least one growth chamber²Biological cells, and the volume of at least one growth chamber may be less than or equal to about 1X 10⁷Cubic microns.

In various embodiments of the microfluidic device, the microfluidic device can further comprise at least one inlet port configured to input the first or second fluid medium into the flow region; and at least one outlet port configured to receive the first medium as it exits the flow region.

In various embodiments of the microfluidic device, the microfluidic device may further include a substrate having a Dielectrophoresis (DEP) configuration configured to introduce one or more biological cells into the growth chamber or move one or more biological cells away from the growth chamber. The DEP configuration may be light-actuated.

In various embodiments of the microfluidic device, the microfluidic device may further comprise a deformable cover region over the at least one growth chamber or separation region thereof, such that depressing the deformable cover region applies a force sufficient to output the biological cells from the separation region into the fluid region. In some embodiments, the microfluidic device may include a cover, wherein at least a portion of the cover is gas permeable, thereby providing a source of gas molecules to a fluidic medium located in the microfluidic device. In some embodiments, the gas permeable portion of the lid may be positioned above the at least one growth chamber. In some embodiments, the gas permeable portion of the lid may be located above the liquid flow region. In some embodiments, the microfluidic device may further comprise a deformable cap region over the at least one growth chamber or separation region thereof, such that depressing the deformable cap region applies a force sufficient to output the biological cells from the separation region into the flow region.

In various embodiments of the microfluidic device, the conditioned surface can include a cleavable moiety. The cleavable moiety can be configured to allow disruption of the conditioned surface, thereby facilitating portability of the cultured one or more biological cells.

In various embodiments of the microfluidic device, the at least one growth chamber may comprise a plurality of growth chambers.

In various embodiments of the microfluidic device, the one or more biological cells can include a plurality of biological cells. In some embodiments, the one or more biological cells may comprise one or more mammalian cells. In some embodiments, the one or more biological cells can include one or more hybridoma cells. In some embodiments, the one or more biological cells may include one or more lymphocytes or leukocytes. In other embodiments, the one or more biological cells may include B cells, T cells, NK cells, macrophages, or a combination thereof. In various embodiments, the one or more biological cells can comprise one or more adherent cells. In some embodiments, the one or more biological cells in the growth chamber may be a single cell or the colony may be a clonal colony of biological cells.

And a pH sensor. The system may further comprise at least one sensor connected to at least one inlet port 124 and/or at least one outlet port 124' of the microfluidic device 600, as shown in fig. 6. The apparatus 600 may alternatively be any of the apparatuses 100, 200, 240, 290, 400, or 500A-E. The sensor may be configured to detect the pH of the first fluidic medium as it enters the microfluidic device 600. Alternatively, the sensor may be configured to detect the pH of the first fluidic medium as it exits the microfluidic device 600. The sensor may be incorporated into the microfluidic device, or it may be a separate component that can be connected to or in series with the inlet port 124 and/or the outlet port 124' of the microfluidic device.

In some embodiments, the pH sensor is a light sensor. Light sensors may provide advantages over electrode-based bench-top devices because electrode-based devices may include large volumes of probes, making it difficult or impossible to measure the pH of small (μ Ι _ L) amounts of fluids. Similarly, serial flow-through solutions may have very long settling times (5 to 15 minutes) due to the nature of the microelectrodes, and may require extensive calibration operations before each use. Furthermore, the electrodes may deteriorate rapidly, thus requiring more maintenance.

The light sensor may be an integrated electrodeless device including an LED for illumination and an integrated colorimeter sensor for visible color detection. The colorimeter sensor may be a phototransistor that is sensitive to color. The colorimeter sensor may detect within the visible wavelength range (e.g., about 390nm to about 700 nm). Media stained with a pH-dependent dye (such as, but not limited to, phenol red) can provide an immediate and contactless light signal. The optical electrodeless measurement method using such a light sensor requires neither contact with the culture medium nor calibration by the user. The optical measurements may be corrected to remove the temperature dependence. Furthermore, the use of light sensors minimizes the risk of sensor fouling, thereby reducing maintenance or replacement. The miniaturization of Light Sources (LEDs) and color sensors also makes them suitable for testing very small volumes of liquid (<1 μ L) and integration into portable or handheld instruments. The system may include drive electronics controlled by a control/monitoring device 180 for the LED and phototransistor sensors, and may further provide an alarm component controlled by the control module 172 if the detection of pH determines that the pH is outside of a desired range. Furthermore, since the stabilization time of the color detection is fast (sub-second scale), it is possible to insert the sensor into a feedback loop to adjust the pH of the medium by adjusting the carbon dioxide content in the gaseous environment surrounding the medium. Alternatively, the control module 172 or the control/detection device 180 may further provide components to adjust the pH of the incoming fluid medium to correct the pH back to the desired range by adding buffers and/or acidic or basic medium components.

In some embodiments, the sensor 610 is connected to the fluid media inlet line 606 adjacent to the at least one inlet 124 of the microfluidic device. The line 606 may be transparent, substantially transparent, or translucent. LED 614 illuminates line 606 and stained fluid media 202 a' within line 606. The integrated colorimeter sensor 612 may monitor the pH of the incoming fluid media; determining that the pH value is within a desired range for a particular culture experiment; and alarms if the pH is outside the desired range.

In some embodiments, the sensor 610 'is connected to the fluid media outlet line 608 adjacent to the at least one outlet 124' of the microfluidic device. Line 608 may be transparent, substantially transparent, or translucent. LED 614' illuminates the stained outflow of fluid medium 202a "in line 608 and line 606. The integrated colorimeter sensor 612' may monitor the pH of the incoming fluid media; determining that the pH value is within a desired range for a particular culture experiment; and alarms if the pH is outside the desired range.

A cell. The cells that can be used in the systems and methods of the invention can be any type of cell. For example, the cell may be an embryo, oocyte or sperm, a stem cell, progenitor cell, or cell isolated from a tissue, a blood cell, a hybridoma, a cultured cell, a cell from a cell line, a cancer cell, an infected cell, a transfected and/or transformed cell (cell line including, but not limited to, a Chinese Hamster Ovary (CHO) cell), a reporter cell, and the like.

In some embodiments, the cells may be from a population of cells actively growing in culture or obtained from a fresh tissue sample (e.g., by isolating a solid tissue sample, such as a biopsy or fine needle puncture), blood, saliva, urine, or other bodily fluid. Alternatively, one or more biological cells may be from a culture of other samples that have been previously frozen.

In some embodiments, the one or more biological cells can include one or more hybridoma cells. In other embodiments, the one or more biological cells may include one or more lymphocytes or leukocytes. In some embodiments, the cell is a B cell, T cell, NK cell, dendritic cell, macrophage or other immune cell type, or a precursor thereof, such as a progenitor cell or hematopoietic stem cell.

In various embodiments, the one or more biological cells are one or more adherent cells. When one or more adherent cells are introduced into the microfluidic device, additional conditioning treatments may be provided to provide the adherent cells with appropriate soluble or non-soluble environmental factors (e.g., one or more extracellular matrix components) that allow for sustained viability and/or cell proliferation.

Depending on the particular objective of the experiment, only one cell or a plurality of cells may be introduced into the microfluidic device for culture or/and cloning. When only one cell is introduced into the growth chamber of the system and incubated according to the methods described herein, the resulting expanded population is a clonal colony of cells initially introduced into the growth chamber.

A method. A method is provided for culturing at least one biological cell in a system comprising a microfluidic device having at least one growth chamber and a flow region. Culturing one (or more cells) in a growth chamber of a microfluidic device that also has a flow stream region may allow for the specific introduction of nutrients, growth factors, or other cell signaling substances for a selected period of time to achieve control of cell growth, viability, or portability parameters. Introducing at least one biological cell into at least one growth chamber having at least one conditioned surface, wherein the conditioned surface supports cell growth, viability, portability, or any combination thereof. In some embodiments, the conditioned surface supports cell portability within the microfluidic device. In some embodiments, portability includes preventing cell adhesion to the microfluidic device. In other embodiments, transplantability includes providing a conditioned surface to adherent cells that will support cell growth, viability, transplantability, or any combination thereof, while also allowing the cells to be moved after a period of culture within the microfluidic device. The at least one conditioned surface may be any conditioned surface described herein. The introduction of at least one biological cell can be accomplished using a variety of different motives described herein, some of which can allow precise control of the placement of a particular biological cell at a particular location on the microfluidic device, e.g., in a preselected growth chamber. The precise control of cell placement/removal and nutrient/signaling/environmental stimulation that can be achieved by the methods described herein is difficult or impossible to achieve with large-scale culture or other microfluidic culture methods.

After placement, the at least one biological cell is then incubated for at least a period of time sufficient to expand the at least one biological cell, thereby producing a colony of biological cells. When the biological cells are entered into the isolated growth chamber, the resulting expanded colonies can be accurately identified for further use as a population of separable biological cells. When only one biological cell is introduced into the growth chamber and allowed to expand, the resulting colony is a clonal population of biological cells. Any suitable cell may be used in the method, including but not limited to cells as described above.

The microfluidic device may be any of the microfluidic devices 100, 300, 400, 500A-E, or 600 described herein, and the microfluidic device may be part of a system having any of the components described herein. The at least one growth chamber may include a plurality of growth chambers, and any suitable number of growth chambers described herein may be used. In some embodiments of the method, the microfluidic device may have from about 500 to about 1500 growth chambers, from about 1000 to about 2000 growth chambers, from about 1000 to about 3500 growth chambers, from about 2000 to about 5000 growth chambers, from about 3000 to about 7000 growth chambers, from about 5000 to about 10000 growth chambers, from about 7500 to about 15000 growth chambers, from about 10000 to about 17500 growth chambers, or from about 12500 to about 20000 growth chambers.

In the method of culturing one or more biological cells, the at least one conditioned surface can be any conditioned surface described herein. The conditioned surface can be covalently attached to a microfluidic device. In some embodiments, the conditioned surface may include a linking group covalently attached to the surface, and the linking group may also be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device. In some embodiments, a microfluidic device having a conditioned surface may be provided prior to inputting one or more biological cells.

Introducing at least one biological cell. In some embodiments, introducing the at least one biological cell into the at least one growth chamber may comprise using a Dielectrophoresis (DEP) force of sufficient strength to move the at least one biological cell. Electronic tweezers, such as optoelectronic tweezers (OET), can be used to generate DEP forces. In some other embodiments, introducing one or more biological cells into at least one growth chamber may include using fluid flow and/or gravity (e.g., by tilting the microfluidic device such that the cells drip into the growth chamber located beneath the cells).

In some embodiments, at least one biological cell is introduced into the microfluidic device through an inlet port 124 into a flow region (e.g., a flow channel) of the microfluidic device. The flow of the culture medium in the flow channel can carry the cells to a location adjacent to the opening to the growth chamber. After being positioned adjacent the opening to the growth chamber, the biological cells can then be moved into the growth chamber using any of the motive forces described herein, including dielectrophoresis or gravity. Dielectrophoretic forces may include electrical actuation or photodynamic forces, and DEP forces may also be provided by optoelectronic tweezers (OET). The at least one biological cell may be moved through the flow channel to the proximal opening of the connection region of the at least one growth chamber, wherein the connection region opens directly to and is in fluid connection with the flow channel/region. The connection region of the at least one growth chamber is also in fluid connection with the separation region of the at least one growth chamber. The at least one biological cell may be further moved through the connection region and into the separation region of the at least one growth chamber. The separation region of the at least one growth chamber may be of a size sufficient to support cell expansion. However, typically the size of the growth chamber limits such expansion to no more than about 1X 10 in culture ³、5×10²、4×10²、3×10²、2×10²、1×10²50, 25, 15 or even as few as 10 filamentsAnd (4) cells. In some embodiments, the size of the isolation region may be sufficient to support expansion of cells into culture by no more than about 1 x 10²50, 25, 15 or 10 cells. It has surprisingly been found that cells are incubated and/or expanded to about 1X 10²Cells can be successfully cultured in volumes of no more than about 1.0X 10⁷Cubic micron, 6 x 10⁶Cubic micron, 2 x 10⁶Cubic micron, 1.5X 10⁶Cubic micron or 1.0X 10⁶In a separate area of cubic microns. In some other embodiments, the cells are incubated and/or expanded to about 1 × 10²Cells can be successfully cultured in volumes of no more than about 4X 10⁵In a separate area of cubic microns. Depending on the cell type, the size of the biological cells can vary widely, from bacteria about 1 micron in diameter and about 1 cubic micron in volume, small human cells (e.g., red blood cells) about 7-8 microns in diameter and about 100 cubic microns in volume, an immortalized cell line (e.g., HeLa) about 40 microns in diameter (non-confluent) and about 2000 cubic microns in volume, megakaryocytes about 25 microns up to about 60 microns in diameter and about 4700 cubic microns to about 100,000 cubic microns in volume, or human oocytes about 120 microns in diameter and about 900,000 cubic microns in volume. Thus, the volume is about 4X 10 ⁵A cubic micron growth chamber may allow for very few megakaryocytes of large variation (about 1X 10 in volume)⁵Cubic microns) up to less than 5 cells in total. Alternatively, the same small growth chamber (volume about 4X 10)⁵Cubic microns) can allow for the expansion of bacterial cells (having a volume of about 1 cubic micron) up to about 400,000 bacterial cells.

The method can further include introducing a first fluidic medium into a microfluidic channel of a flow region of the microfluidic device. In some embodiments, the introduction of the first fluid medium is performed prior to the introduction of the at least one biological cell. When the first fluid medium is introduced prior to the introduction of the at least one biological cell, the flow rate may be selected such that the first fluid medium flows from the flow channel of the microfluidic device into the growth chamber at, for example, any suitable flow rate. Alternatively, if the microfluidic device has been loaded with a medium containing an excess of one or more conditioning agents, the first fluidic medium flows into the microfluidic channel at a flow rate such that: the flow rate is such that the first fluid medium displaces any remaining medium in the flow field containing excess conditioning agent.

When introducing the flow of the first fluid medium after introducing the at least one biological cell into the growth chamber, the flow rate of the first fluid medium may be selected to not sweep across the separation region, which does not displace the at least one biological cell from the separation region. The fluid medium of the at least one biological cell in the separation region surrounding the at least one growth chamber is a second fluid medium, which may be the same or different from the first fluid medium. In some embodiments, the second fluid medium may be the same as the first fluid medium, but during the incubation step, the cell waste product and depleted media components may be such that the second fluid medium is different from the first fluid medium.

The cells were incubated. In the methods described herein, at least one biological cell is incubated for a period of time at least long enough to expand the cells to produce colonies of biological cells. The period of time may be selected to be about 1 day to about 10 days. In other embodiments, the incubation period may be extended for more than 10 days, and may last for any desired period of time. Since the cells in the separation region of the growth chamber are provided with nutrients and waste is removed by perfusion of the fluid medium, the cells can grow indefinitely. As the separation region is filled with the expanded cell population, any additional expansion will result in the expanded biological cells occupying the connecting region of the growth chamber (which is the swept area of the growth chamber). The perfused medium may sweep the expanded biological cells out of the connection region of the growth chamber and then out of the microfluidic device. Thus, depending on the size of the biological cells and the size of the separation region of the growth chamber, the number of cells present in the separation region of the growth chamber can be stabilized at a maximum number. The ability to stabilize the maximum number of cells in a cell population provides an advantage over other currently available cell culture methods because lengthy cell population spotting (split) can be eliminated.

In some embodiments, the incubation can be performed for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or longer. The incubation period can range from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, or from about 1 day to about 2 days. In other embodiments, the incubation may be performed for less than about 5 days, less than about 4 days, less than about 3 days, or less than about 2 days. In some embodiments, the incubation may be performed for less than about 3 days or less than about 2 days. In other embodiments, the incubation can be performed for about 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10 h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, or about 23 h.

During the culturing step, images of at least one growth chamber and any cells contained therein may be monitored at one or more time points throughout the culturing step. The image may be stored in a memory of a processing component of the system.

Cells were perfused. During the incubation step, the second fluid medium present in the separated region of the growth chamber may become depleted of nutrients, growth factors, or other growth stimuli. The second fluid medium may accumulate cellular waste products. Furthermore, as the at least one biological cell continues to grow during the incubation period, it may be desirable to change the nutrient, growth factor or other growth stimulus to be different from the nutrient, growth factor or other growth stimulus of the first or second medium at the beginning of the incubation. Culturing in the growth chamber of a microfluidic device as described herein may obtain the ability to be specific and selective to introduce and alter the chemical gradient perceived by at least one biological cell, which may be much closer to in vivo conditions. Alternatively, altering the chemical gradient perceived by at least one biological cell to a purposefully non-optimized set of conditions may allow the cell to expand under the conditions designed to study a disease or therapeutic pathway. Thus, the method may comprise perfusing the first fluid medium during the incubating step, wherein the first fluid medium is introduced via the at least one inlet 124 of the microfluidic device, and wherein the first fluid medium optionally containing components from the second fluid medium is output via the at least one outlet of the microfluidic device.

Exchange of components of the first fluid medium (thereby providing fresh nutrients, soluble growth factors, etc.) and/or exchange of waste components of the medium surrounding the cells within the separation region occurs substantially under diffusion conditions at the interface of the swept and non-swept areas of the growth chamber. It has surprisingly been found that an efficient exchange is obtained under substantially no flow conditions. Thus, it has surprisingly been found that a successful incubation does not require constant perfusion. As a result, the perfusion may be discontinuous. In some embodiments, the perfusion is periodic, and in some embodiments, the perfusion is irregular. The interruption between perfusion periods may be of sufficient duration to allow diffusion of components of the second fluid medium in the separation region into the first fluid medium in the flow channel/region, and/or diffusion of components of the first fluid medium into the second fluid medium, neither of which significantly flows into the separation region.

In another embodiment, a low perfusion rate may also be employed to obtain efficient exchange of components of the fluid medium inside and outside the non-swept area of the growth chamber.

Thus, one method of perfusing at least one biological cell in at least one growth chamber of a microfluidic device is shown in fig. 7 and includes a perfusion step 7002, wherein at a first perfusion rate R ₁For a first perfusion time D₁The first fluid medium is flowed through a flow region of the microfluidic device into a flow region fluidly connected to the growth chamber. R₁May be selected to be a non-sweeping flow rate as described herein. Method 700 further includes step 7004, wherein the flow of the fluid medium is stopped for a first perfusion stop time S₁. Steps 7002 and 7004 are repeated W times, where W may be an integer selected from 1 to about 1000, after which the perfusion process 700 is complete. In some embodiments, W may be an integer from 2 to about 1000.

Another method 800 of perfusing at least one biological cell in at least one growth chamber of a microfluidic device is shown in FIG. 8, which includes a first perfusionAn infusion cycle comprising a step 8002, wherein at a first infusion rate R₁For a first perfusion time D₁The fluid medium is flowed through a flow region of the microfluidic device into a flow region that is fluidly connected to the growth chamber. R₁May be selected to be a non-sweeping flow rate as described herein. The first perfusion cycle includes step 8004, where the flow of the fluid medium is stopped for a first perfusion stop time S₁. The first perfusion cycle may be repeated W times, wherein W is an integer selected from 1 to about 1000. After completion of the W-th iteration of the first perfusion cycle, method 800 further includes a second perfusion cycle, including step 8006, wherein at a second perfusion rate R ₂For a second perfusion time D₂Flowing a first fluid medium, wherein R₂Is selected as the non-swept flow rate of the flow. The second perfusion cycle of method 800 further includes step 8008, where the flow of the fluid medium is stopped for a second perfusion stop time S₂. Thereafter, the method returns to steps 8002 and 8004 of the first perfusion cycle, and the combined two cycle perfusion process is repeated V times, where V is an integer from 1 to about 5000. The combination of W and V may be selected to meet the desired endpoint during incubation.

In various embodiments of methods 700 or 800, perfusion rate R₁Can be any non-sweeping flow rate of the fluid medium as described above for the flow controller configuration. In some embodiments, R₁Can be about 0.009, 0.010, 0.020, 0.030, 0.040, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00.2.10, 2.20, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 μ L/sec.

In various embodiments of method 800, the second perfusion rate R ₂Can be any non-sweeping flow rate of the fluid medium as described above for the flow controller configuration. In some embodiments, R₂Can be 0.009, 0.010, 0.020, 0.030, 0.040, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10. 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, or 3.00 μ L/sec. The flow rate R may be selected in any combination₁And/or R₂. Generally, the perfusion rate R₂Can be greater than the perfusion rate R₁And may be R₁About 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, or higher. In some embodiments, R₂At least the ratio of R₁Ten times faster. In other embodiments, R₂At least the ratio of R₁Twenty times faster. In another embodiment, R₂At least R₁100x of the rate of (a).

In various embodiments of methods 700 or 800, the first perfusion time D₁May be any suitable perfusion duration as described above for the flow controller configuration. In various embodiments, D ₁Can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 seconds. In other embodiments, D₁May be in the range of time, for example, about 10 to about 40 seconds, as described above. In some embodiments, D₁And may be about 30 seconds to about 75 seconds. In other embodiments, D₁And may be about 100 seconds. In other embodiments, D₁And may range from about 60 seconds to about 150 seconds. In other embodiments, D₁Can be about 20min, 30min, 40min, 50min, 60min, 80min, 90min, 110min, 120min, 140min, 160min, 180min, 200min, 220min, 240min, 250min, 260min, 270min, 290min, or 300 min. In some embodiments, D₁From about 40min to about 180 min.

In various embodiments of methods 700 or 800, the second perfusion time D₂May be any suitable perfusion duration as described above for the flow controller configuration. In various embodiments, D₂May be about 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds,30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 60 seconds, 65 seconds, 70 seconds, 80 seconds, 90 seconds, or about 100 seconds. In other embodiments, D ₂May be in the range of time, for example, from about 5 seconds to about 20 seconds, as described above. In other embodiments, D₂And may be about 30 seconds to about 70 seconds. In other embodiments, D₂And may be about 60 seconds.

In various embodiments of methods 700 or 800, the first perfusion time D₁Can be associated with a second perfusion time D₂The same or different. D may be selected in any combination₁And D₂. In some embodiments, the duration of perfusion is D₁And/or D₂May be selected to be shorter than the stop period S₁And/or S₂。

In various embodiments of methods 700 or 800, the first perfusion stop time S₁May be selected as any suitable time period as described above for the interval time between perfusion time periods of the flow controller configuration. In some embodiments, S₁Can be about 0min, 5min, about 10min, about 15min, about 20min, about 25min, about 30min, about 35min, about 40min, about 45min, about 60min, about 65min, about 80min, about 90 min, about 100min, about 120min, about 150min, about 180min, about 210min, about 240min, about 270min, or about 300 min. In various embodiments, S₁Any suitable time range for the interval between infusions as described above for the flow controller configuration may be used, for example, about 20 to about 60 min. In some embodiments, S ₁Can be from about 10min to about 30 min. In other embodiments, S₁May be about 15 min. In other embodiments, S₁Can be about 0 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, or about 90 seconds. In some embodiments, S₁Is about 0 seconds.

In various embodiments of methods 700 or 800, the second perfusion stop time S₂May be selected as any suitable time period as described above for the interval time between perfusion time periods of the flow controller configuration. In some embodiments, S₂Can be about 0min, 5min, about 6min, about 7min, about 8min, about 9min, about 10min, about 20min, about 30min, about 45min, about 50min, about 60, about 90min, about 120min, about 180min, about 240min, about 270min, or about 300 min. In various embodiments, S₂Any suitable time range for the interval between infusions as described above for the flow controller configuration may be used, for example, about 15 to about 45 min. In some embodiments, S₂Can be from about 10min to about 30 min. In other embodiments, S₂It may be about 8min or 9 min. In other embodiments, S₂Is about 0 min.

In various embodiments of methods 700 or 800, the first perfusion stop time S may be independently selected from any suitable value ₁And a second perfusion stop time S₂。S₁Can be reacted with S₂The same or different.

In various embodiments of method 800 or 900, the number of W repeats may be selected to be the same as or different from the number of V repeats.

In various embodiments of method 700 or 800, W may be about 1, about 4, about 5, about 6, about 8, about 10, about 12, about 15, about 18, about 20, about 24, about 30, about 36, about 40, about 45, or about 50. In some embodiments, W may be selected from about 1 to about 20. In some embodiments, W may be 1.

In various embodiments of method 800, V may be about 5, about 10, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 80, about 100, about 120, about 240, about 300, about 350, about 400, about 450, about 500, about 600, about 750, about 900, or about 1000. In some embodiments, V may be selected to be from about 10 to about 120. In other embodiments, V may be from about 5 to about 24. In some embodiments, V may be from about 30 to about 50 or may be from about 400 to about 500.

In various embodiments of method 800, the number of W repeats may be selected to be the same as or different from the number of V repeats.

In various embodiments of methods 700 or 800, the total time for the first perfusion step (represented by steps 7002/7004 or 8002/8004) is about 1h to about 10h, and W is an integer of 1. In various embodiments, the total time for the first perfusion step is from about 9min to about 15 min.

In various embodiments of method 800, the total time of the second perfusion cycle step (represented by step 8006/8008) is from about 1min to about 15min or from about 1min to about 20 min.

In either method 700 or 800, the perfusion method can be continued throughout the incubation period of the biological cells, for example for about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 days, or longer.

In another non-limiting embodiment of the method 800 of fig. 8, the controller may be configured to prime during the priming step 8002 for a longer priming period D₁The fluid medium is perfused in the fluid flow region. The controller may perfuse the fluid medium at the first rate for about 45min, about 60min, about 75min, about 90min, about 105min, about 120 min, about 2.25h, about 2.5h, about 2.45h, about 3.0h, about 3.25h, about 3.5h, about 3.75h, about 4.0 h, about 4.25h, about 4.5h, about 4.75h, about 5h, or about 6 h. During the first perfusion period D₁At the end, the flow of the fluid medium may be stopped for a period S₁It can be about 0 seconds, 15 seconds, 30 seconds, about 45 seconds, about 1min, about 1.25min, about 1.5min, about 2.0min, about 3.0min, about 4min, about 5min, or about 6 min. In some embodiments, the first flow rate R ₁Can be selected to be about 0.009, 0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.4, or about 0.5 mul/sec. The flow of the fluid medium may be stopped, during which the perfusion is stopped S₁Is less than about 1 minute, or S₁May be 0 seconds. Or, S₁Can be about 30 seconds, about 1.5min, about 2.0min, about 2.5min, or about 3 min. Followed by a second perfusion period D₂Using different perfusion rates. In some embodiments, the second perfusion rate may be higher than the first perfusion rate. In some embodiments, the second perfusion rate R₂May be selected from about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.7, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 6.0, 7.0, 8.0 or about9.0. mu.L/sec. Second perfusion period D₂Can be about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 10 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 65 seconds, about 75 seconds, about 80 seconds, or about 90 seconds. Then a second perfusion stop period S may be provided₂Stopping the filling, the second filling stopping period S₂Can be about 0 seconds, 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 1.5min, about 1.75min, about 2.0min, about 2.5min, about 2.75min, about 3.0min, or about 4.0 min. In some embodiments, D ₁Can be about 2 h, about 3h, or about 4 h. In various embodiments, D₁May be about 4 hours. In various embodiments, S₁And may be 0 seconds or less than about one minute. Second perfusion period D₂And may be about 1 second to about 6 seconds. In some embodiments, the second perfusion-stop period S₂And may be about 40 seconds to about 1.5 minutes.

Accordingly, there is provided a method for perfusing at least one biological cell in at least one growth chamber of a microfluidic device, comprising the steps of: perfusing at least one biological cell using a first perfusion step, the first perfusion step comprising: at a first perfusion rate R₁For a first perfusion time D₁Flowing a first fluid medium through a flow region of the microfluidic device, wherein the flow region is in fluidic connection with the growth chamber, wherein R₁Is selected to be a non-sweeping flow rate; stopping the flow of the first fluid medium for a first perfusion stop time S₁(ii) a And repeating the first perfusion step W times, wherein W is an integer selected from 1 to 1000. The method may further comprise the step of perfusing the at least one biological cell with a second perfusion step comprising: at a second perfusion rate R₂For a second perfusion time D ₂Flowing a first fluid medium, wherein R₂Is selected as a non-sweep rate; stopping the flow of the first fluid medium for a second perfusion stop time S₂(ii) a And repeating the first and then the second perfusion steps V times, wherein V is an integer from 1 to 1000.

Second perfusion rate R₂May be greater than the first perfusionRate R₁. First perfusion time D₁Can be associated with a second perfusion time D₂The same or different. First perfusion stop time S₁May be associated with a second perfusion stop time S₂The same or different. When the second perfusion step is performed, the number of W repeats may be the same as or different from the number of V repeats. R₂Can be compared with R₁At least ten times faster. Or, R₂Can be compared with R₁At least twenty times faster. R₂Can be reacted with R₁At least 100 times as fast. D₁And may be about 30 seconds to about 75 seconds. In other embodiments, D₁Can be from about 40min to about 180min or from about 180min to about 300 min. In some other embodiments, D₁And may be about 60 seconds to about 150 seconds. S₁Can be from about 10min to about 30 min. In other embodiments, S₁Can be from about 5min to about 10 min. In other embodiments, S₁May be zero. In some embodiments, D₁Can be from about 40min to about 180min, and S ₁May be zero. In other embodiments, D₁Can be about 60 seconds to about 150 seconds, and S₁Can be from about 5min to about 10 min. In other embodiments, D₁Can be about 180min to about 300min, and S₁May be zero. The total time for the first perfusion step may be about 1h to about 10 h. In other embodiments, the total time for the first perfusion step may be about 2h to about 4 h. In some embodiments, W may be an integer greater than 2. In some embodiments, W may be from about 1 to about 20. In some embodiments, D₂And may be about 10 seconds to about 25 seconds. In other embodiments, D₂And may be about 10 seconds to about 90 seconds. In some embodiments, S₂Can be from about 10min to about 30 min. In other embodiments, S₂May be about 15 min. In some embodiments, V may be from about 10 to about 120. In some embodiments, V may be from about 30 to about 50 or may be from about 400 to about 500. In some embodiments, D₂Can be about 1 second to about 6 seconds, and S₂May be 0 seconds. In some embodiments, D₂Can be about 10 seconds to about 90 seconds and S₂And may be about 40 seconds to about 1.5 minutes. In some embodiments, the total time for one repetition of the second perfusion step may be about 1min to about 15 min.

The medium was conditioned. To provide a culture medium (e.g., the first or second culture medium) that maintains and enhances the growth and/or viability of the at least one biological cell, the first fluid culture medium can contain both liquid and gaseous components (e.g., the gaseous component can be dissolved in the liquid component). In addition, the fluid medium may include other components, such as biomolecules, vitamins and minerals dissolved in the liquid component. Any suitable component may be used in the fluid medium, as known to those skilled in the art. Some non-limiting examples are discussed above, but many other media compositions may be used without departing from the methods described herein. The culture medium may or may not contain serum of animal origin. In some embodiments, the fluid medium may comprise a chemically defined medium (at least prior to contact with the cells or the fluid containing the cells), and may also be a protein-free or peptide-free chemically defined medium. In some embodiments, the fluid medium may comprise a serum-reduced medium.

The first fluid medium may be prepared by saturating the initial fluid medium with dissolved gas molecules prior to introduction into the microfluidic device. Furthermore, the initial saturation of the fluid medium with dissolved gas molecules may continue until the point in time at which the first fluid medium is introduced into the microfluidic device. Saturating the initial fluidic medium may include contacting the microfluidic device with a gaseous environment capable of saturating the initial fluidic medium with dissolved gaseous molecules. Gas molecules that may saturate the initial fluid medium include, but are not limited to, oxygen, carbon dioxide, and nitrogen.

The first fluid medium may further comprise adjusting the pH of the first fluid medium. Adjusting the pH of the first fluid medium may occur, for example, before and/or during introduction of dissolved gas molecules. This adjustment can be achieved by adding a buffer substance. One non-limiting example of a suitable buffer substance is HEPES. Other buffer substances may be present in the culture medium and may be dependent on and independent of the presence of carbon dioxide (e.g., a carbonate buffer system) and may be selected by one skilled in the art. Salts, proteins, carbohydrates, lipids, vitamins and other small molecules necessary for cell growth may also form part of the first fluid medium composition.

In some embodiments, saturating the first fluid medium with the gas component prior to introduction via the inlet port can be performed in the reservoir. In other embodiments, saturating the first fluid medium with the gas component may be performed in a gas-permeable connecting line between the reservoir and the inlet. In other embodiments, saturating the first fluidic medium with the gas component can be performed through a gas-permeable portion of a lid of the microfluidic device. In some embodiments, gas saturation of the fluidic medium further comprises maintaining humidity in the gas exchange environment such that the fluidic medium within the microfluidic device does not change its osmotic pressure during incubation.

The composition of the first fluid medium may also include at least one component secreted by the feeder cell culture. Secreted feeder cell components may include growth factors, hormones, cytokines, small molecules, proteoglycans, and the like. The introduction of the at least one component secreted by the feeder cell culture may be performed in the same reservoir as the saturation of the first fluid medium with the gas component is performed, or the introduction of the at least one component secreted by the feeder cell culture to the first fluid medium may be performed before the saturation step.

In some other embodiments, the composition of the first medium may further include an additive designed to provide an altered fluid medium to test the response of the cells to the additive. The supplement may, for example, increase or decrease cell viability or growth.

In some embodiments, the method may include detecting the pH of the first fluid medium as it is introduced via the at least one inlet. The pH measurement may be performed at a location directly adjacent to the inlet. In some embodiments, the method can include detecting a pH of the first fluid medium as the first fluid medium is output via the outlet. The pH measurement may be performed at a location directly adjacent to the outlet. Either or both detectors for detecting pH may be optical sensors. In some embodiments, the detector may be capable of providing an alarm if the pH deviates from an acceptable range. In some other embodiments, the composition of the first fluid medium may be changed when the pH value measured by the detector deviates from an acceptable range.

During the incubation step, an image of the at least one growth chamber and any cells contained therein may be monitored.

Outputting at least one biological cell. After the incubation step is complete, the at least one biological cell or cell colony can be output from the growth chamber or the isolation region thereof. The outputting may comprise using a sufficiently strong Dielectrophoresis (DEP) force to move one or more biological cells/colonies of cells. DEP forces can be optically or electrically actuated. For example, the microfluidic device may comprise a substrate having a DEP configuration, such as an opto-electronic tweezers (OET) configuration. In other embodiments, at least one biological cell or colony of cells can be output from the growth chamber or separation region using fluid flow and/or gravity. In other embodiments, at least one biological cell or colony of cells can be output from the growth chamber or separation region using pressure on a region of the deformable cover above the growth chamber or separation region thereof, thereby causing a localized flow of fluid exiting the growth chamber or separation region.

After outputting at least one biological cell or colony of cells from the growth chamber or separation region, the cells can then be output from the flow region (e.g., channel) out of the microfluidic device. In some embodiments, outputting the cells from the flow region comprises using a DEP force strong enough to move one or more colonies of biological cells/cells. DEP forces can be generated as described above. In some other embodiments, outputting the cell from the flow region out of the microfluidic device comprises using fluid flow and/or gravity to move the cell.

During the outputting step, an image of the at least one growth chamber and any cells contained therein may be monitored.

Conditioning at least one surface. In some embodiments, a microfluidic device is provided having at least one surface of at least one growth chamber in a conditioned state. In other embodiments, the surface of the at least one growth chamber is conditioned prior to introducing the at least one biological cell, and may be performed as part of a method of culturing one or more biological cells. Conditioning the surface may include treating the surface with a conditioning agent, such as a polymer.

In some embodiments, a method for processing at least one surface of at least one growth chamber of a microfluidic device (100, 300, 400, 500A-E, and 600) is provided, comprising the steps of: flowing a fluid medium comprising an excess of a conditioning agent into the flow channel (FIGS. 1, 2A-2F, 3A-3B, 4A-4C); incubating the microfluidic device for a selected period of time; and displacing the medium in the channel. In other embodiments, a method for priming a microfluidic device comprises the steps of: flowing a loading solution containing a conditioning agent into the flow channel; incubating the device for a selected period of time, thereby conditioning at least one surface of the growth chamber; and displacing the solution in the channel with a fluid medium. The loading solution may contain any of the fluid media as described herein. The fluid medium replacing the conditioned solution or fluid medium with excess conditioning agent may be any medium described herein and may additionally contain cells.

In some embodiments, at least one surface may be treated with a polymeric conditioning agent comprising an alkylene ether moiety. The polymer conditioning agent having an alkylene ether moiety can comprise any suitable alkylene ether-containing polymer, including, but not limited to, any of the alkylene ether-containing polymers described above. In one embodiment, the surface of the growth chamber may be treated with amphiphilic nonionic block copolymers comprising blocks of polyethylene oxide (PEO) and polypropylene oxide (PPO) subunits at different ratios and locations within the polymer chain (e.g.,

a polymer). Details useful for obtaining conditioned surfaces

The polymer comprises

L44, L64, P85, F68 and F127 (including F127 NF).

In other embodiments, the surface may be treated with a polymeric conditioning agent comprising carboxylic acid moieties. Non-limiting examples of suitable carboxylic acid-containing polymeric conditioning agents are discussed above, and any suitable carboxylic acid-containing polymeric conditioning agent can be used to treat a surface.

In other embodiments, the surface may be treated with a polymeric conditioning agent comprising a sugar moiety. Non-limiting examples of suitable sugar-containing polymeric conditioning agents are discussed above, and any suitable sugar-containing polymeric conditioning agent can be used to treat a surface.

In other embodiments, the surface may be treated with a polymeric conditioning agent containing sulfonic acid moieties. Non-limiting examples of suitable sulfonic acid-containing polymeric conditioning agents are discussed above, and any suitable sulfonic acid-containing polymeric conditioning agent can be used to treat a surface.

In other embodiments, the surface can be treated with a polymeric conditioning agent comprising an amino acid moiety. Non-limiting examples of suitable amino acid-containing polymeric conditioning agents are discussed above, and any suitable amino acid-containing polymeric conditioning agent can be used to treat a surface. The amino acid-containing polymer conditioning agent may comprise a protein. In some embodiments, the surface is treated with a protein, where the protein may include components present in or part of mammalian serum. In other embodiments, the surface is treated with a component of mammalian serum. In some embodiments, mayTreating surfaces with cell culture medium supplements, e.g.

The supplement ((50X), serum-free, available from ThermoFisher Scientific, Cat. No. 17504044). The mammalian serum can be Fetal Bovine Serum (FBS). Alternatively, the mammalian serum may be Fetal Calf Serum (FCS).

In other embodiments, the surface may be treated with a polymeric conditioning agent containing a nucleic acid moiety. Non-limiting examples of suitable nucleic acid-containing polymeric conditioning agents are discussed above, and any suitable nucleic acid-containing polymeric conditioning agent can be used to treat a surface.

In some embodiments, a mixture of more than one polymeric conditioning agent may be used to treat the surface of the growth chamber.

In some other embodiments, the conditioning step may comprise treating at least one surface of at least one growth chamber with at least one cell adhesion blocking molecule. In some embodiments, the step of treating at least one surface of at least one growth chamber with at least one cell adhesion blocking molecule may be performed prior to outputting the cells from the microfluidic device. In some embodiments, the conditioning step may comprise pre-incubating the cells with at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule may act to disrupt the formation of an agonist filament. In some embodiments, the cell adhesion blocking molecule may be cytochalasin B. In other embodiments, the at least one cell adhesion blocking molecule may block an integrin receptor. In some embodiments, the cell adhesion blocking molecule may comprise a peptide comprising the RGD motif. In some other embodiments, the at least one cell adhesion blocking molecule may reduce binding of the cell to the DNA-contaminated surface. Cell adhesion blocking molecules that can reduce the binding of cells to DNA-contaminated surfaces may include DNase 1 protein. In other embodiments, the at least one cell adhesion blocking molecule may comprise a small molecule fibronectin inhibitor. In other embodiments, the at least one cell adhesion blocking molecule may be an antibody, such as an anti-B1 integrin antibody. In some embodiments, the at least one cell adhesion blocking molecule may comprise a combination of more than one type of cell adhesion blocking molecule.

In other embodiments, conditioning comprises heating the surface of the growth chamber to a temperature of about 30 ℃. In some embodiments, the method includes heating the surface to a temperature of at least about 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, or about 40 ℃. In some embodiments, the method comprises heating the surface to a temperature greater than about 25 ℃. In other embodiments, the method comprises heating the surface to about 30 ° to 40 ℃; from about 35 ℃ to about 40 ℃; or a temperature in the range of about 36 ℃ to about 38 ℃. In some embodiments, the method comprises heating the surface to a temperature of at least about 30 ℃. In some embodiments, the heated surface comprises at least one surface conditioned by treatment with a polymer.

A clonal population. The methods described herein also include methods in which only one biological cell is introduced into at least one growth chamber. Methods are provided for cloning biological cells in a system comprising a microfluidic device having a fluid flow region configured to contain a flow of a first fluid medium; and at least one growth chamber comprising a separation region and a connection region, the separation region being in fluid connection with the connection region and the connection region comprising a proximal opening to the flow region, the method comprising the steps of: introducing biological cells into at least one growth chamber, wherein the at least one growth chamber is configured with at least one conditioned surface to support cell growth, viability, portability, or any combination thereof; and incubating the biological cells for at least a sufficient period of time to expand the biological cells to produce a clonal population of biological cells. In some embodiments, the system can be any system described herein. The microfluidic device may be any of the microfluidic devices described herein.

In some embodiments of the methods for cloning biological cells, the at least one conditioned surface may comprise a linking group covalently attached to the surface, and the linking group may be attached to a moiety configured to support cell growth, viability, or portability of one or more biological cells within the microfluidic device. In some embodiments, the linking group may comprise a siloxy linking group. In other embodiments, the linking group may comprise a phosphonate linking group. In some embodiments, the linking group can be indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. In other embodiments, the linking group may be directly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly attached to the moiety configured to support cell growth, viability, or mobility via a connection to the linking moiety. In some embodiments, the linking group can be indirectly attached to the moiety configured to support cell growth, viability, or mobility via a connection to the first end of the linking moiety. In some embodiments, the linking moiety may also include a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In some embodiments, the backbone of the linear moiety may include one or more arylene moieties. In other embodiments, the linking moiety may include a triazolylene moiety. In some embodiments, the triazolylene moiety may interrupt the linear portion of the linking moiety or may be linked to the linear portion of the linking moiety at the second end. In various embodiments, the moiety configured to support cell growth and/or viability and/or portability may comprise an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid. In some embodiments, at least one of the conditioned surfaces comprises an alkyl or perfluoroalkyl moiety. In other embodiments, at least one of the conditioned surfaces comprises an alkylene ether moiety or a dextran moiety.

In various embodiments, the method may further comprise the step of conditioning at least one surface of at least one growth chamber. In some embodiments, conditioning comprises treating at least one surface with one or more agents that support cell portability within the microfluidic device. In some embodiments, conditioning may comprise treating at least one surface of at least one growth chamber with a conditioning reagent comprising a polymer. In some embodiments, the polymer may include alkylene ether moieties. In some embodiments, the polymer may include carboxylic acid moieties. In some embodiments, the polymer may include a sugar moiety. In other embodiments, the polymer may include sulfonic acid moieties. In other embodiments, the polymer may include an amino acid moiety. In other embodiments, the polymer may include a nucleic acid moiety. In some embodiments, conditioning may comprise treating at least one surface of at least one growth chamber with one or more components of mammalian serum. In some embodiments, the mammalian serum can be Fetal Bovine Serum (FBS) or fetal bovine serum (FCS). In various embodiments, conditioning may comprise treating at least one surface of at least one growth chamber with at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule may comprise an RGD-containing peptide. In other embodiments, the at least one cell adhesion blocking molecule may be cytochalasin B, an anti-integrin antibody, a fibronectin inhibitor, or a DNase 1 protein. In various embodiments, conditioning may include treating at least one surface of at least one growth chamber with a combination of more than one type of cell adhesion blocking molecule.

In various embodiments, conditioning may include heating at least one surface of at least one growth chamber to a temperature of about 30 ℃.

In various embodiments, the method can further comprise the step of introducing a first fluidic medium into a microfluidic channel of a flow region of the microfluidic device. In some embodiments, introducing the first fluid medium may be performed prior to introducing the biological cells. In some embodiments, introducing the biological cells into the at least one growth chamber may comprise using a Dielectrophoresis (DEP) force having an intensity sufficient to move the biological cells. In some embodiments, the DEP force may be light actuated. In some embodiments, DEP forces can be generated by optical tweezers (OET). In some other embodiments, introducing the biological cells into the at least one growth chamber may comprise using fluid flow and/or gravity.

In some embodiments, introducing biological cells into the at least one growth chamber may further comprise introducing biological cells into a separation region of the at least one growth chamber. In some embodiments, the size of the separation region of at least one growth chamber may be sufficient to support cell expansion to no more than 1 x 10 ²And (4) cells. In some embodiments, the separation region can be at least substantially filled with a second fluid medium. In some embodiments, the liquid flow region may be fluidly connected to the proximal opening of the connection region of the at least one growth chamber, and further wherein the connection region may also be fluidly connected to the separation region of the growth chamber.

In various embodiments, the method may further comprise the step of perfusing the first fluid medium during the incubating step, wherein the first fluid medium may be introduced via at least one inlet port of the microfluidic device, and wherein the first fluid medium, optionally comprising components from the second fluid medium, may be exported via at least one outlet port of the microfluidic device. In some embodiments, the perfusion may be discontinuous. In some other embodiments, the perfusion may be periodic. In other embodiments, the perfusion may be random. In some embodiments, perfusion of the first fluid culture medium can be performed at a rate sufficient to allow diffusion of a component of the second fluid culture medium in the separation region into the first fluid culture medium in the flow region and/or diffusion of a component of the first fluid culture medium into the second fluid culture medium in the separation region; and the first culture medium may not substantially flow into the separation region. In some embodiments, perfusing the first fluid medium may be performed for a duration of about 45 seconds to about 90 seconds, about every 10min to about every 30 min. In some embodiments, perfusing the first fluid medium may be performed for a duration of about 2h to about 4 h. In some embodiments, the period of incubation of the at least one biological cell may be from about 1 day to about 10 days.

In some embodiments, the composition of the first fluid medium may include both liquid and gaseous components. In various embodiments, the method may further comprise the step of saturating the first fluid medium with dissolved gas molecules prior to introducing the first fluid medium into the microfluidic device. In various embodiments, the method may further comprise the step of contacting the microfluidic device with a gaseous environment capable of saturating the first fluidic medium or the second fluidic medium with dissolved gas molecules. In various embodiments, the method may further comprise the step of adjusting the pH of the first fluid medium after introducing the dissolved gas molecules. In some embodiments, saturating the first fluidic medium with the gaseous component can be performed in the reservoir prior to introduction via the inlet port, in a gas-permeable connector between the reservoir and the inlet port, or via a gas-permeable portion of a lid of the microfluidic device. In some embodiments, the composition of the first fluid medium may include at least one component secreted by the feeder cell culture.

In various embodiments, the method can further include the step of detecting the pH of the first fluid medium as it is output via the at least one outlet port. In some embodiments, the detecting step may be performed at a location directly proximate to the at least one outlet. In various embodiments, the method may further comprise the step of detecting the pH of the first fluid medium as it is introduced via the at least one inlet port. In some embodiments, the sensor may be a light sensor. In various embodiments, the method may further comprise the step of altering the composition of the first fluid medium.

In various embodiments, the method may further comprise the step of monitoring an image of the at least one growth chamber and any cells contained therein.

In various embodiments, the biological cell can be a mammalian cell. In some embodiments, the biological cell may be an immune cell. In some embodiments, the biological cell may be a lymphocyte or a leukocyte. In some embodiments, the biological cell may be a B cell, a T cell, an NK cell, a macrophage, or a dendritic cell. In some embodiments, the biological cells can be adherent cells. In some embodiments, the biological cell can be a hybridoma cell.

In some embodiments, the biological cell may be a plurality of biological cells and the at least one growth chamber is a plurality of growth chambers. In various embodiments, the method can further comprise the step of moving no more than one of the plurality of biological cells into each of the plurality of growth chambers.

In some embodiments of the method of cloning a biological cell, the conditioned surface can further comprise a cleavable moiety. The method may comprise the step of lysing the cleavable moiety before one or more biological cells of the output clonal population leave the growth chamber or the separation region thereof.

In various embodiments, the method may further comprise the step of outputting the one or more biological cells of the clonal population out of the growth chamber or an isolated region thereof. In some embodiments, outputting may include using a sufficiently strong Dielectrophoresis (DEP) force to move the one or more biological cells. In some embodiments, the DEP force is optically actuated. In some embodiments, DEP forces can be generated by optical tweezers (OET). In some embodiments, outputting may include using fluid flow and/or gravity. In some embodiments, the outputting may comprise using pressure on the deformable cover region above the growth chamber or the separation region thereof. In various embodiments, the method can further comprise the step of outputting the one or more biological cells of the clonal population from the fluid flow region away from the microfluidic device. In some embodiments, outputting may include using a sufficiently strong DEP force to move one or more biological cells. In some embodiments, the DEP force is optically actuated. In some embodiments, DEP forces can be generated by optical tweezers (OET). In some embodiments, outputting may include using fluid flow and/or gravity.

A kit. A kit can be provided for culturing biological cells, wherein the kit comprises: a microfluidic device having a fluid flow region configured to contain a flow of a first fluidic medium; and at least one growth chamber; and a surface conditioning agent. In this embodiment, the at least one growth chamber is not pre-treated to condition at least one surface of the at least one growth chamber, and the conditioned surface is generated by treatment with a surface conditioning reagent prior to introduction of the cells. Other kits for culturing biological cells are also provided, wherein the kit includes a microfluidic device having a fluid flow region configured to contain a flow of a first fluid medium; and at least one growth chamber comprising a separation region and a connection region, wherein the separation region is in fluid connection with the connection region and the connection region comprises a proximal opening to the flow region; and further wherein at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof. Other kits for culturing biological cells are also provided, including a microfluidic device comprising a flow region configured to contain a flow of a first fluid medium; and at least one growth chamber comprising a separation region and a connection region, wherein the separation region is in fluid connection with the connection region and the connection region has a proximal opening to the flow region; wherein at least one growth chamber has at least one surface with a surface-modified ligand. Alternatively, a kit for culturing biological cells may be provided, wherein the kit comprises: a microfluidic device having a fluid flow region configured to contain a flow of a first fluidic medium; and at least one growth chamber having at least one conditioned surface that can support cell growth, viability, portability, or any combination thereof; and a surface conditioning agent. The microfluidic device of any of the kits can be any of the microfluidic devices 100, 200, 240, 290, 400, 500A-E, or 600, and have any of the features described above.

The microfluidic device of any of the kits may further comprise a microfluidic channel comprising at least a portion of the flow region, and the device may further comprise a growth chamber having a connection region opening directly into the microfluidic channel. The growth chamber may further comprise a separation region. The separation region may be fluidically connected to the connection region and may be configured to contain a second fluid medium, wherein when the flow region and the at least one growth chamber are substantially filled with the first and second fluid media, respectively, a component of the second fluid medium diffuses into the first fluid medium and/or a component of the first fluid medium diffuses into the second fluid medium; and the first culture medium does not substantially flow into the separation region.

In various embodiments of the kit, the growth chamber can be configured similar to the growth chambers 124, 126, 128, 130, 244, 246, 248, or 436 of FIGS. 1, 2A-2F, 3A-3B, and 4A-4C, wherein the volume of the separation region of the growth chamber can be configured to support no more than about 1 x 10 in culture³、5×10²、4×10²、3×10²、2× 10²、1×10²50, 25, 15 or 10 cells. In other embodiments, the separation region of the growth chamber can have a volume that supports up to about 10, 50, or 1 x 10 ²And (4) cells. Any of the configurations of growth chambers described above may be used in the growth chambers of the microfluidic devices of the kits.

In various embodiments of any of the kits, the growth chamber can be sized to hold no more than 1 x 10²Personal lifeThe cells may be maintained, wherein the volume of the growth chamber may be no more than 1 x 10⁷Cubic microns. In other embodiments, wherein no more than 1 × 10 can remain²The volume of the growth chamber can be not more than 5 × 10⁶Cubic microns. In other embodiments, no more than 50 biological cells may be maintained, and the volume of the growth chamber may be no more than 1 x 10⁶Cubic micron or not more than 5 x 10⁵Cubic microns. In the kit, the microfluidic device may have any number of growth chambers as described above.

The microfluidic device of any of the kits can further comprise at least one inlet port configured to input a fluidic medium (e.g., a first or second fluidic medium) into the flow region; and at least one outlet configured to receive the fluid media (e.g., spent first fluid media) as it exits the flow region.

The microfluidic device of any of the kits can further comprise a substrate having a plurality of DEP electrodes, wherein a surface of the substrate forms a surface of the growth chamber and the flow region. The plurality of DEP electrodes can be configured to generate a sufficiently strong Dielectrophoretic (DEP) force to move one or more biological cells (e.g., clonal populations) into the growth chamber or isolated region thereof or to move one or more cells of the biological cell culture out of the growth chamber or isolated region thereof. The DEP electrodes, and thus the DEP force, may be optically actuated. Such optically actuated DEP electrodes can be dummy electrodes (e.g., regions of an amorphous silicon substrate having increased conductivity due to incident light), phototransistors, or electrodes switched by corresponding phototransistors. Alternatively, the DEP electrodes, and thus the DEP force, may be electrically actuated. In some other embodiments, the microfluidic device may further comprise a substrate having a plurality of transistors, wherein a surface of the substrate forms a surface of the growth chamber and the flow region. The plurality of transistors may be capable of generating a sufficiently strong Dielectrophoresis (DEP) force to introduce the biological cells or move one or more cells of the biological cell culture away from the growth chamber or the separation region thereof. Each of the plurality of transistors may be optically actuated, and the DEP force may be generated by an optoelectronic tweezer.

The microfluidic device of any of the kits can further comprise a deformable cover region over the at least one growth chamber or the isolation region thereof, such that depressing the deformable cover region applies a force to transport one or more biological cells (e.g., clonal population) from the growth region to the flow region.

The microfluidic device of either kit may be configured with a lid that is substantially gas impermeable. Alternatively, all portions of the cover may be configured to be breathable. The gas permeable portion of the lid may be permeable to at least one of carbon dioxide, oxygen, and nitrogen. In some embodiments, the lid (or a portion thereof) may be permeable to a combination of more than one of carbon dioxide, oxygen, or nitrogen.

Any of the kits may further include a reservoir configured to contain a fluid medium. The reservoir may be fluidically connected to any of the microfluidic devices described herein. The reservoir may be configured such that the fluidic medium present in the reservoir is contacted by a gaseous environment capable of saturating the fluidic medium with dissolved gas molecules. The reservoir may also be configured to contain a population of feeder cells in fluidic contact with the fluid medium.

Any of the kits can include at least one connecting line configured to connect to an inlet port and/or an outlet port of a microfluidic device. The connecting line may also be configured to connect to a reservoir or flow controller, such as a pump assembly. The connecting line may be gas permeable. The gas permeable connecting line may be permeable to at least one of carbon dioxide, oxygen and nitrogen. In some embodiments, the gas permeable tubing may be permeable to a combination of more than one of carbon dioxide, oxygen, or nitrogen.

Any of the kits may further include a sensor configured to detect a pH of the first fluid medium. The sensor may be connected (or connectable) to an inlet port of the microfluidic device or a connecting line connected thereto. Alternatively, the sensor may be integrated into the microfluidic device. The sensor may be attached near the point where the fluid medium enters the microfluidic device. The kit may include a sensor configured to detect a pH of the fluid medium at an outlet of the microfluidic device. The sensor may be connected (or connectable) to an outlet port of the microfluidic device or a connecting line connected thereto. Alternatively, the sensor may be integrated into the microfluidic device. The sensor may be attached near the point where the fluid medium exits the microfluidic device. The sensor, whether connected to the inlet and/or outlet of the microfluidic device, may be an optical sensor. The light sensor may include an LED and an integrated colorimeter sensor, which may optionally be a phototransistor sensitive to color. The kit may also include drive electronics to control the pH sensor and receive output therefrom. The kit may also include a pH detection reagent. The pH detecting reagent may be a pH-sensitive dye that can detect under visible light.

Either kit may also include a culture medium having components capable of enhancing viability of biological cells on the microfluidic device. These components may be any suitable media components known in the art, including any of the components described above for the fluid media components.

Any of the kits may further comprise at least one reagent for detecting the status of a biological cell or cell population. Reagents configured to detect status are well known in the art and can be used, for example, to detect whether a cell is viable or dead; whether a target substance, such as an antibody, cytokine, or growth factor, is secreted; or whether a target cell surface marker is present. Such reagents may be used in the kits and methods described herein without limitation.

For any of the kits provided herein, the components of the kit can be in separate containers. For any component of the kit provided in solution form, the component may be present at a concentration of about 1X, 5X, 10X, 100X, or about 1000X of the concentration used in the methods of the invention.

For such kits: wherein at least one growth chamber of the microfluidic device is not pretreated to condition at least one surface of the at least one growth chamber, and wherein treatment with a surface conditioning agent produces a conditioned A surface; or for such kits: comprising a microfluidic device having a flow region configured to contain a flow of a first fluidic medium; and at least one growth chamber having at least one conditioned surface that can support cell growth, viability, portability, or any combination thereof; and a surface conditioning reagent, the surface of the growth chamber being pre-conditioned with the surface conditioning reagent. The surface conditioning agent may comprise a polymer, which may be any one or more of the polymers described above for use as surface conditioning agents. In some embodiments, the surface conditioning agent may comprise a polymer having an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, an amino acid moiety, a nucleic acid moiety, a sugar moiety, or any combination thereof. The surface conditioning agent may comprise a PEO-PPO block copolymer, for example

A polymer (e.g., L44, L64, P85, or F127). In some embodiments, the surface conditioning agent may comprise one or more components of mammalian serum. The mammalian serum can be Fetal Bovine Serum (FBS) or fetal bovine serum (FCS).

Alternatively, the surface conditioning reagents for conditioning the surface of the growth chamber may be included in the kit separately from the microfluidic device. In other embodiments of the kit, a pre-conditioned microfluidic device is included with a surface conditioning reagent (which is different from the surface conditioning reagent used to condition the surface of the growth chamber). The different surface conditioning agent may be any of the surface conditioning agents described above. In some embodiments, more than one surface conditioning agent is included in the kit.

In various embodiments of kits having a microfluidic device in which at least one growth chamber of the microfluidic device is not pretreated to condition at least one surface, the kit may further comprise a culture medium suitable for culturing one or more biological cells. In some embodiments, the kit may further comprise a media supplement comprising an agent capable of replenishing the conditioning of the surface of the growth chamber. The media supplement may include a conditioning agent as described above or another chemical that enhances the ability of at least one surface of at least one growth chamber to support cell growth, viability, portability, or any combination thereof. This may include growth factors, hormones, antioxidants or vitamins, and the like.

The kit may also include a flow controller configured to perfuse at least the first fluidic medium, which may be a separate component from the microfluidic device, or may be incorporated as part of the microfluidic device. The controller may be configured to non-continuously perfuse the fluid medium. Thus, the controller may be configured to perfuse the fluid medium in a periodic manner or an irregular manner.

In another aspect, a kit for culturing biological cells is provided, comprising a microfluidic device having a fluid flow region configured to contain a flow of a first fluid medium; and at least one growth chamber comprising a separation region and a connection region, wherein the separation region is in fluid connection with the connection region and the connection region comprises a proximal opening to the flow region; and further wherein at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof. The microfluidic device may be any of the microfluidic devices described herein, and may have any of the growth chambers described herein. The microfluidic device may have a substrate with any type of DEP configuration described herein. The DEP configuration may be light-actuated. The substrate of the microfluidic device can have a surface that includes a substrate composition of formula 1 or formula 2 described herein, and has all of the features described above.

The at least one conditioned surface of the microfluidic device of the kit may comprise a sugar moiety, an alkylene ether moiety, an amino acid moiety, an alkyl moiety, a fluoroalkyl moiety (which may comprise a perfluoroalkyl moiety), an anionic moiety, a cationic moiety, and/or a zwitterionic moiety. In some embodiments, the conditioned surface of the microfluidic device can include a sugar moiety, an alkylene ether moiety, an alkyl moiety, a fluoroalkyl moiety, or an amino acid moiety. The alkyl or perfluoroalkyl moiety may have a backbone chain length of greater than 10 carbons. In some embodiments, the moiety that supports cell growth, viability, portability, or any combination thereof may comprise an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid.

In some embodiments of the kit, the conditioned surface may include a linking group covalently attached to a surface of the microfluidic device, and the linking group may be attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device. The linking group may be a siloxy linking group. Alternatively, the linking group may be a phosphonate linking group. In some embodiments of the kit, the linking group of the conditioned surface may be directly linked to a moiety configured to support cell growth, viability, portability, or any combination thereof.

In other embodiments, the linking group may be indirectly attached via a linking moiety to a moiety configured to support cell growth, viability, portability, or any combination thereof. The linking group can be indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via attachment to the first end of the linking moiety. The linking moiety may also include a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms. In some embodiments of the kit, the linking moiety of the conditioned surface may further comprise a triazolylene moiety. The cleavable moiety is configured to allow disruption of the conditioned surface, thereby facilitating the portability of the biological cell. The kit may further comprise a reagent configured to cleave a cleavable moiety of the conditioned surface.

In various embodiments of the kit, the kit can further comprise a surface conditioning agent. In some embodiments, the surface conditioning agent can include a polymer comprising at least one of an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or a sugar moiety. In some other embodiments, the surface conditioning agent comprises a polymer comprising at least one of an alkylene ether moiety, an amino acid moiety, or a sugar moiety. In some other embodiments, the conditioned surface can include a cleavable moiety.

In other embodiments of the kit, the surface conditioning reagent comprises at least one cell adhesion blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule may disrupt the formation of an agonist silk, block an integrin receptor, or reduce binding of cells to a DNA-contaminated surface. In some embodiments, the at least one cell adhesion blocking molecule may be cytochalasin B, an RGD-containing peptide, a DNase 1 protein, a fibronectin inhibitor, or an anti-integrin antibody. In some embodiments, the at least one cell adhesion blocking molecule may comprise a combination of more than one type of cell adhesion blocking molecule.

In various embodiments of the kit, the surface conditioning agent may comprise one or more components of mammalian serum. The mammalian serum can be Fetal Bovine Serum (FBS) or fetal bovine serum (FCS). In various embodiments of the kit, the kit can further comprise a culture medium suitable for culturing one or more biological cells. In some embodiments, the kit may include a media supplement comprising a reagent configured to supplement conditioning of at least one surface of the growth chamber. The media supplement may include a conditioning agent as described above or another chemical that enhances the ability of at least one surface of at least one growth chamber to support cell growth, viability, portability, or any combination thereof. This may include growth factors, hormones, antioxidants or vitamins, and the like.

In various embodiments of the kit, the kit can include at least one reagent that detects the status of one or more biological cells.

In another aspect, a kit for culturing biological cells is provided, comprising a microfluidic device for culturing one or more biological cells, the microfluidic device comprising a flow region configured to contain a flow of a first fluid medium; and at least one growth chamber comprising a separation region and a connection region, wherein the separation region is in fluid connection with the connection region and the connection region has a proximal opening to the flow region; and at least one growth chamber has at least one surface with a surface modifying ligand. The microfluidic device may be any of the microfluidic devices described herein. The surface may include a substrate having a Dielectrophoresis (DEP) configuration. The DEP configuration can be any DEP configuration described herein. The DEP configuration may be light-actuated. The substrate is any substrate having a surface modifying ligand described herein, and may have the structure of formula 3, and may include all of the features described above:

In various embodiments of kits comprising a microfluidic device having at least one surface comprising a surface modifying ligand, the surface modifying ligand can be covalently attached to an oxide moiety on the surface of the substrate. The surface modifying ligand may include a reactive moiety. The reactive moiety of the surface modifying ligand may be an azido, amino, bromo, thiol, activated ester, succinimidyl, or alkynyl moiety. The surface modifying ligand may be covalently linked to the oxide moiety via a linking group. In some embodiments, the linking group may be a siloxy moiety. In other embodiments, the linking group may be a phosphonate moiety. The linking group may be indirectly linked to the reactive moiety of the surface modifying ligand via a linking moiety. The linking moiety may comprise a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some embodiments, the surface modifying ligand may comprise one or more cleavable moieties. The one or more cleavable moieties can be configured to allow disruption of the conditioned surface of the microfluidic device once formed, thereby facilitating portability of the one or more biological cells after culture.

In some embodiments of kits comprising a microfluidic device having at least one surface comprising a surface modifying ligand, the kit may further comprise a conditioning modifying agent comprising a first portion configured to support cell growth, viability, portability, or any combination thereof; and a second moiety configured to react with the reactive moiety of the surface modifying ligand, which may have the structure of formula 5, and having any of the features described herein:

moiety- (L')_m-R_px

Formula 5

The second portion may be configured to convert the surface modifying ligand to a conditioned surface configured to support cell growth, viability, portability, or any combination thereof of one or more biological cells within the growth chamber after reaction with the reactive portion of the surface modifying ligand of the microfluidic device of the kit. The first portion may include an alkylene oxide portion, a sugar portion; an alkyl moiety, a perfluoroalkyl moiety, an amino acid moiety, an anionic moiety, a cationic moiety, or a zwitterionic moiety. In some embodiments, the first moiety may comprise an alkyl or fluoroalkyl (which includes perfluoroalkyl) moiety; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, 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 (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxylic acid betaine; a sulfobetaine; sulfamic acid; or an amino acid. The second moiety may be an amino, carboxylic acid, alkyne, azide, aldehyde, bromo, or thiol moiety. In some embodiments, the first portion or linking moiety L' (as described above for formula 5) of the conditional modification reagent may comprise a cleavable moiety. The cleavable moiety may be configured to allow disruption of the conditioned surface, thereby promoting portability of the biological cell. In some embodiments, the kit may further comprise a reagent configured to cleave a cleavable moiety of the conditioned surface.

In some embodiments of kits comprising a microfluidic device having at least one surface comprising a surface modifying ligand, the kit may further comprise a surface conditioning reagent.

In some embodiments of kits comprising a microfluidic device having at least one surface comprising a surface modifying ligand, the surface conditioning agent can comprise a polymer comprising at least one of an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or a sugar moiety. In some other embodiments, the surface conditioning agent comprises a polymer comprising at least one of an alkylene ether moiety, an amino acid moiety, or a sugar moiety. In some other embodiments, the conditioned surface can include a cleavable moiety.

In some embodiments of kits comprising a microfluidic device having at least one surface comprising a surface-modified ligand, the surface conditioning reagent comprises at least one cell attachment blocking molecule. In some embodiments, the at least one cell adhesion blocking molecule may disrupt the formation of an agonist silk, block an integrin receptor, or reduce binding of cells to a DNA-contaminated surface. In some embodiments, the at least one cell adhesion blocking molecule may be cytochalasin B, an RGD-containing peptide, a DNase 1 protein, a fibronectin inhibitor, or an anti-integrin antibody. In some embodiments, the at least one cell adhesion blocking molecule may comprise a combination of more than one type of cell adhesion blocking molecule.

In some embodiments of kits comprising a microfluidic device having at least one surface comprising a surface modifying ligand, the surface conditioning reagent may comprise one or more components of mammalian serum. The mammalian serum can be Fetal Bovine Serum (FBS) or fetal bovine serum (FCS).

In some embodiments of kits comprising a microfluidic device having at least one surface comprising a surface-modified ligand, the kit can further comprise a culture medium suitable for culturing one or more biological cells. In some embodiments, the kit may further comprise a media supplement comprising a reagent configured to supplement conditioning of at least one surface of the growth chamber. The media supplement may include a conditioning agent as described above or another chemical that enhances the ability of at least one surface of at least one growth chamber to support cell growth, viability, portability, or any combination thereof. This may include growth factors, hormones, antioxidants or vitamins, and the like.

In some embodiments of kits comprising a microfluidic device having at least one surface comprising a surface-modified ligand, the kit can further comprise at least one reagent that detects a state of one or more biological cells.

In some aspects of the present application, the present application further relates to the following:

a microfluidic device for culturing one or more biological cells, comprising:

a flow region configured to contain a flow of a first fluid medium; and

at least one growth chamber comprising a separation region and a connection region, the separation region being in fluidic connection with the connection region and the connection region comprising a proximal opening to the flow region,

wherein the at least one growth chamber further comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof within the microfluidic device.

The microfluidic device of item 1, wherein at least one conditioned surface is conditioned with one or more reagents that support cell portability within the microfluidic device.

Item 3. the microfluidic device of item 1 or 2, wherein at least one conditioned surface is conditioned with a polymer comprising alkylene ether moieties.

The microfluidic device of any preceding claim, wherein at least one conditioned surface is conditioned with a polymer comprising a sugar moiety.

The microfluidic device of any preceding claim, wherein at least one conditioned surface is conditioned with a polymer comprising an amino acid moiety.

The microfluidic device of any preceding claim, wherein at least one conditioned surface of the microfluidic device is conditioned with a polymer comprising a carboxylic acid moiety, a sulfonic acid moiety, a nucleic acid moiety, or a phosphonic acid moiety.

The microfluidic device of any preceding claim, wherein at least one conditioned surface comprises a linking group covalently attached to a surface of the microfluidic device, and wherein the linking group is attached to a moiety configured to support cell growth, viability, portability, or any combination thereof within the microfluidic device.

The microfluidic device of item 7, wherein the linker is a siloxy linker.

Item 9 the microfluidic device of item 7 or 8, wherein at least one conditioned surface comprises an alkyl or fluoroalkyl moiety.

The microfluidic device of item 9, wherein the alkyl or fluoroalkyl moiety has a backbone chain length greater than 10 carbons.

The microfluidic device of any one of claims 7 to 10, wherein the linking group is indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via a linking moiety.

The microfluidic device of item 11, wherein the linking moiety comprises a triazolylene moiety.

The microfluidic device of any preceding claim, wherein at least one conditioned surface comprises a sugar moiety.

The microfluidic device of any preceding claim, wherein at least one conditioned surface comprises an alkylene ether moiety.

The microfluidic device of any preceding claim, wherein at least one conditioned surface comprises an amino acid moiety.

The microfluidic device of any one of claims 7 to 15, wherein at least one conditioned surface comprises a zwitterion.

The microfluidic device of any preceding claim, wherein at least one conditioned surface comprises at least one cell adhesion blocking molecule.

The microfluidic device of item 17, wherein the at least one cell adhesion blocking molecule is an RGD-containing peptide.

The microfluidic device of item 17 or 18, wherein the at least one cell adhesion blocking molecule is a combination of more than one cell adhesion blocking molecule.

The microfluidic device of any preceding claim, wherein the conditioned surface comprises a cleavable moiety.

The microfluidic device of any preceding claim, wherein at least one conditioned surface of the microfluidic device comprises one or more components of mammalian serum.

The microfluidic device of any preceding claim, wherein the microfluidic device further comprises a substrate having a Dielectrophoresis (DEP) configuration.

The microfluidic device of item 22, wherein the DEP configuration is optically actuated.

The microfluidic device of any preceding claim, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of mammalian cells.

The microfluidic device of any preceding claim, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of immune cells.

The microfluidic device of item 25, wherein the immune cell is a lymphocyte or a leukocyte.

The microfluidic device of item 25, wherein the immune cell is a B cell, a T cell, an NK cell, a macrophage, or a dendritic cell.

The microfluidic device of any one of items 1 to 24, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof of adherent cells.

The microfluidic device of any one of claims 1 to 24, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of hybridoma cells.

The microfluidic device of any preceding claim, wherein the at least one growth chamber comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof, of a clonal colony of individual cells and corresponding biological cells.

An item 31. a system for culturing one or more biological cells on a microfluidic device, the system comprising:

a microfluidic device, comprising:

a flow region configured to contain a flow of a first fluid medium; and

at least one growth chamber, wherein the growth chamber has at least one surface conditioned to support cell growth, viability, portability, or any combination thereof within the microfluidic device.

The system of item 32, item 31, wherein the microfluidic device is the microfluidic device of any one of items 1 to 30.

The system of item 33, item 31 or 32, further comprising a flow controller configured to perfuse at least the first fluid medium.

The system of item 33, wherein the controller is configured to non-continuously perfuse the at least first fluid medium.

The system of any of items 31-34, wherein the microfluidic device further comprises a substrate having a Dielectrophoresis (DEP) configuration, the substrate configured to introduce or remove one or more biological cells into or from the growth chamber.

Item 36 the system of item 35, wherein the DEP configuration is optically actuated.

The system of any one of items 31 to 36, further comprising a reservoir configured to contain the first fluidic medium, wherein the reservoir is fluidically connected to the microfluidic device.

Item 38. the system of item 37, wherein the reservoir is configured to be contacted by a gaseous environment capable of saturating the first fluidic medium with dissolved gas molecules.

The system of any one of items 31 to 38, further comprising a sensor connected to at least one inlet port of the microfluidic device, wherein the sensor is configured to detect a pH of the first fluidic medium.

The system of any one of items 31 to 39, further comprising a sensor connected to at least one outlet, wherein the sensor is configured to detect the pH of the first fluid medium as it exits the microfluidic device.

Item 41 the system of item 39 or 40, wherein the sensor is a light sensor.

The system of any of items 31 to 41, further comprising a detector configured to capture an image of the at least one growth chamber and any biological cells contained therein.

A composition, according to item 43, comprising:

a substrate having a Dielectrophoresis (DEP) configuration and a surface; and

a conditioned surface of an oxide portion covalently attached to the surface of the substrate.

The composition of item 44, item 43, wherein the conditioned surface comprises a linking group covalently attached to an oxide moiety of the surface, and wherein the linking group is attached to a moiety configured to support cell growth, viability, portability, or any combination thereof.

Item 45 the composition of item 44, wherein the linking group is a siloxy linking group.

The composition of clause 44 or 45, wherein the linking group is indirectly attached to a moiety configured to support cell growth, viability, portability, or any combination thereof.

The composition of any one of items 44 to 46, wherein the linking group is indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via a connection to a first end of a linking moiety.

The composition of item 48, wherein the linking moiety further comprises a linear moiety, wherein the backbone of the linear moiety comprises from 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms.

The composition of any one of items 49, 47 to 48, wherein the linking moiety further comprises a triazolylene moiety.

The composition of any of items 43 to 49, wherein the moiety configured to support cell growth, viability, portability, or any combination thereof comprises an alkyl, fluoroalkyl, mono-or polysaccharide, alcohol, polyol, alkylene ether, polyelectrolyte, amino group, carboxylic acid, phosphonic acid, sulfonate anion, carboxylic betaine, sulfobetaine, sulfamic acid, or amino acid.

The composition of any of items 43 to 50, wherein at least one conditioned surface comprises amino acids, alkyl moieties, perfluoroalkyl moieties, dextran moieties, and/or alkylene ether moieties.

The composition of any one of claims 43 to 51, wherein the conditioned surface further comprises one or more cleavable moieties.

The composition of item 52, wherein the cleavable moiety is configured to allow disruption of the conditioned surface, thereby promoting transplantability of the cultured one or more biological cells.

An item 54. a kit for culturing biological cells, comprising:

a microfluidic device, comprising:

a flow region configured to contain a flow of a first fluid medium; and

wherein at least one growth chamber further comprises at least one surface conditioned to support cell growth, viability, portability, or any combination thereof within the microfluidic device.

The kit of item 54, item 55, wherein the microfluidic device is the microfluidic device of any one of items 1-30.

Item 56 the kit of item 54 or 55, wherein at least one conditioned surface of the microfluidic device comprises an alkyl moiety, a fluoroalkyl moiety, a mono-or polysaccharide moiety, an alcohol moiety, a polyol moiety, an alkylene ether moiety, a polyelectrolyte moiety, an amino moiety, a carboxylic acid moiety, a phosphonic acid moiety, a sulfonate moiety, a carboxylic acid betaine moiety, a sulfobetaine moiety, a sulfamic acid moiety, or an amino acid moiety.

The kit of any one of items 54 to 56, wherein at least one conditioned surface of the microfluidic device comprises at least one of a sugar moiety, an alkylene ether moiety, an alkyl moiety, a fluoroalkyl moiety, or an amino acid moiety.

The kit of item 58. item 57, wherein the alkyl or fluoroalkyl moiety has a backbone chain length of more than 10 carbons.

The kit of any one of items 54 to 58, wherein at least one conditioned surface comprises a linking group covalently attached to a surface of the microfluidic device, and wherein the linking group is attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of one or more biological cells within the microfluidic device.

The kit of item 59, wherein the linker is a siloxy linker.

The kit of item 59 or 60, wherein the linking group is indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof.

The kit of item 61, item 62, wherein the linking group is indirectly attached to the moiety configured to support cell growth, viability, portability, or any combination thereof via a linking moiety.

The kit of any one of items 54 to 62, further comprising a surface conditioning agent.

Item 64. the kit of item 63, wherein the surface conditioning reagent comprises a polymer comprising at least one of an alkylene ether moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphonic acid moiety, an amino acid moiety, a nucleic acid moiety, or a sugar moiety.

Item 65 the kit of item 63, wherein the surface conditioning reagent comprises at least one cell adhesion blocking molecule.

The kit of item 63, wherein the surface conditioning agent comprises one or more components of mammalian serum.

The kit of any one of items 54 to 66, further comprising a media supplement comprising a reagent configured to supplement the conditioning of at least one surface of the growth chamber.

The kit of any one of items 54 to 67, wherein the conditioned surface comprises a cleavable moiety.

The kit of item 68, wherein the kit further comprises a reagent configured to cleave the cleavable moiety of the conditioned surface.

Item 70. a method of culturing at least one biological cell in a microfluidic device having a flow region configured to contain a flow of a first fluidic medium; and at least one growth chamber, the method comprising the steps of:

Introducing the at least one biological cell into the at least one growth chamber, wherein the at least one growth chamber is configured with at least one surface conditioned to support cell growth, viability, portability, or any combination thereof; and

incubating the at least one biological cell for at least a period of time sufficient to expand the at least one biological cell to produce a colony of biological cells.

The method of item 70, item 71, wherein the microfluidic device is the microfluidic device of any one of items 1-30.

The method of any one of items 70 to 71, wherein at least one conditioned surface comprises a linking group covalently attached to the surface, and wherein the linking group is attached to a moiety configured to support cell growth, viability, portability, or any combination thereof, of the one or more biological cells within the microfluidic device.

The method of any one of items 70 to 72, wherein at least one conditioned surface comprises alkyl or perfluoroalkyl moieties.

The method of any of clauses 70 to 72, wherein at least one conditioned surface comprises an alkylene ether moiety or a dextran moiety.

The method of any one of items 70 to 74, further comprising conditioning at least one surface of the at least one growth chamber.

The method of item 75, wherein conditioning comprises treating at least one surface of the at least one growth chamber with a conditioning agent comprising a polymer.

The method of any one of items 75 to 76, wherein conditioning comprises treating at least one surface of the at least one growth chamber with one or more components of mammalian serum.

The method of any one of items 75 to 77, wherein conditioning comprises treating at least one surface of the at least one growth chamber with at least one cell adhesion blocking molecule.

The method of any of clauses 70 to 78, wherein introducing at least one biological cell into the at least one growth chamber comprises moving the at least one biological cell using a Dielectrophoresis (DEP) force of sufficient strength.

Item 80 the method of item 79, wherein the DEP force is optically actuated.

The method of any one of items 70 to 80, further comprising the step of perfusing the first fluidic medium during the incubating step, wherein the first fluidic medium is introduced via at least one inlet port of the microfluidic device and output via at least one outlet port of the microfluidic device, wherein upon output, the first fluidic medium optionally comprises components from a second fluidic medium.

The method of any one of items 70 to 81, further comprising a step of lysing the one or more cleavable moieties of the conditioned surface after the incubating step, thereby facilitating export of one or more biological cells from the growth chamber or the separation region thereof and into the liquid flow region.

The method of any one of items 70 to 82, further comprising the step of outputting one or more biological cells from the growth chamber or the separation region thereof into the flow region.

The method of any one of items 70 to 83, wherein the at least one biological cell is a mammalian cell.

The method of any one of items 70 to 84, wherein the at least one biological cell is an immune cell.

The method of item 85, wherein the immune cell is a lymphocyte or a leukocyte.

The method of item 85, wherein the immune cell is a B cell, T cell, NK cell, macrophage, or dendritic cell.

The method of any one of items 70 to 85, wherein the at least one biological cell is an adherent cell.

The method of any one of items 70 to 85, wherein the at least one biological cell is a hybridoma cell.

Item 90. the method of any one of items 70 to 89, wherein introducing the at least one biological cell into the at least one growth chamber comprises introducing a single cell into the growth chamber, and wherein the colonies of biological cells produced by the incubating step are clonal colonies.

Examples

Example 1 culturing and growth of K562 erythroleukemia cells

Materials: k562 cells, a human immortalized myeloid leukemia cell line, obtained from the American Type Culture Collection (ATCC) (Cat. No.)

CCl-243^TM) And is provided in the form of a suspension cell line. By inoculation of 1X 10³The culture was maintained at viable cells/mL and incubated at 37 ℃ using a 5% carbon dioxide gas environment. The cells were cultured at 1X 10⁶One cell/mL or every 2-3 days. Cells were frozen in 5% dimethyl sulfoxide (DMSO)/95% complete growth medium.

Culture medium:modified Iskoff's medium (A)

Catalog No. 30-2005) plus 10% fetal bovine serum (Hyclone catalog No. SH30071.2) to prepare a complete growth medium. When perfused during incubation, the complete growth medium was continuously conditioned with 5% carbon dioxide in air prior to introduction into the microfluidic device.

Solution filling: contains 0.1 percent of

F127(Life

Catalog No. P6866).

Systems and microfluidic devices: manufactured by Berkeley Lights, inc. The system includes at least a flow controller, a temperature controller, a fluid medium conditioning and pumping assembly, a light source for light activated DEP configuration, a microfluidic device, a mounting stage, and a camera. The volume of the growth chamber of the microfluidic device used in this experiment was about 1.4X 10⁵Cubic microns. The cross-sectional area of the flow channel is about 4X 10³Square micron. The microfluidic device has 8 channels.

Preparation of culture: the microfluidic device was loaded onto the system and purged (purge) with 100% carbon dioxide at 15psi for 5 min. Immediately after the carbon dioxide purge, the loading solution was perfused through the microfluidic device at 5 μ L/sec for 8 min. The complete growth medium was then flowed through the microfluidic device at 5 μ L/sec for 5 min.

The culture conditions are as follows: the temperature of the microfluidic device was maintained at 37 ℃. The medium was perfused at a constant flow rate of 0.001. mu.L/sec throughout the culture experiment.

Gravity was used to load single K562 cells into one growth chamber of the microfluidic device. A photograph of the growth chamber at t 0h after loading the cells is shown (see fig. 10A). Arrow 1002 points to the location of the individual cells in the growth chamber.

After the completion of the 16h culture, the cells were expanded to a population of 2 cells as shown in the photographs taken at this time point (see fig. 10B). Arrow 1004 points to the location of the two cells in the growth chamber.

After the completion of the 34h culture, the cell population increased to a total of 4 cells as shown in the photograph of fig. 10C. Arrows 1006 and 1008 point to two populations of two cells, each, located within the growth chamber.

After the 54-hour culture was completed, the population of K562 cells increased to a total of 8 cells, as shown in the photograph of fig. 10D. Arrows 1010 and 1012 point to cells on either side of the cell population within the growth chamber.

After the completion of the 70-hour culture, the population of K562 cells increased to a total of 16 cells, as shown in the photograph of fig. 10E. Arrows 1014, 1016, and 1018 point to the cells of the population. A K562 clonal amplification population is provided in a growth chamber of a microfluidic device.

Example 2 culture and growth of OKT3 hybridoma cells.

Materials: OKT3 cell, murine myeloma hybridoma cell line, obtained from ATCC (ATCC: (Novo)

Directory number CRL-8001^TM). The cells are provided in the form of a suspension cell line. About 1X 10 by inoculation⁵To about 2X 10⁵The culture was maintained at viable cells/mL and incubated at 37 ℃ using 5% carbon dioxide in air as the gaseous environment. Cells were bottled every 2-3 days. OKT3 cell number and viability were counted and cell density was adjusted to 5X 10 ⁵Ml for loading into a microfluidic device.

Culture medium: 500ml of an improved Ersikov medium (c)

Cat No. 30-2005), 200ml fetal bovine serum (

Catalog No. 30-2020) and 1ml penicillin-streptomycin (Life)

Catalog number 15140-122) to prepare the media. The complete medium was filtered through a 0.22 μm filter and stored at 4 ℃ protected from light until use.

When perfused during incubation, the medium was continuously conditioned with 5% carbon dioxide in air prior to introduction into the microfluidic device.

Solution filling: contains 0.1 percent of

F127(Life

Directory number P6866). The medium of (1).

Systems and microfluidic devices: manufactured by Berkeley Lights, inc. The system includes at least a flow controller, a temperature controller, a fluid medium conditioning and pump assembly, a light source and projector for light activated DEP configuration, a microfluidic device, a mounting stage, and a camera. The volume of the growth chamber of the microfluidic device used in this experiment was about 1.5X 10⁶Cubic microns. The cross-sectional area of the flow channel is about 8X 10³Square microns and there are a total of six channels on the microfluidic device.

Preparation of culture: the microfluidic device was loaded onto the system and purged with 100% carbon dioxide at 15psi for 5 min. Immediately after the carbon dioxide purge, the loading solution was perfused through the microfluidic device at 8 μ Ι/sec until a total volume of 2.5ml was perfused through the microfluidic device. The media was then flowed through the microfluidic device at 8 μ L/sec until a total of 1ml of media was perfused through the microfluidic device. The prepared microfluidic device is shown in the photograph of fig. 11A before introduction of the cells. A row of four growth chambers extends along the bottom of the photograph.

The culture conditions are as follows: the temperature of the microfluidic device was maintained at 37 ℃. The culture medium was perfused throughout the culture experiment using a varied perfusion method that included an initial 4h period of perfusion at 0.01 μ L/sec, followed by a short high speed perfusion at 8 μ L/sec for about 3 seconds, followed by a short perfusion stop period of approximately less than 1 minute. This cycle, which includes alternating perfusion rates and stops, continues throughout the culture experiment.

A single OKT3 cell was introduced into the growth chamber using gravity. A photograph of a growth chamber with one cell at t-0 is shown in fig. 11B, where arrow 1102 points from the left to the second chamber, in particular to a single cell within the chamber, where the region where the cell resides is further surrounded by a circle.

FIGS. 12A-12C show photographs of the microfluidic device at a later time point in a culture experiment and demonstrate that the cells expanded to form clonal populations. The photograph of fig. 12A was taken at the completion of a day of culture, and arrow 1202 points from the left to the point of introduction of a single OKT3 cell to a population of about four cells in the second chamber. Fig. 12B is a photograph taken after 2 days of culture were completed, and arrow 1204 points from the left to a further increased cell population in the second chamber. Fig. 12C is a photograph taken after completing 3 days of culture, and arrow 1206 shows numerous expanded OKT3 cells obtained from culturing a single OKT3 cell.

Fig. 13A-13C show photographs of the microfluidic device after three days of culture were completed (i.e., after the 12C time point) and demonstrate that selected expanded OKT 3 cells were exported using the dielectrophoretic force generated by the optoelectronic tweezers. In fig. 13A, the pattern of light that induces the dielectrophoretic force (i.e., the optical trap indicated by arrow 1302) is shown as a white box around the cell. The cells are moved from the bottom of the growth chamber to the flow channel by the force of light-actuated dielectrophoresis. The photograph of fig. 13B shows that the expanded OKT 3 cells move further into the flow stream region. The cell is still trapped in the optical trap and is forced to move with the optical trap (arrow 1304). The photograph of fig. 13C shows the release (arrow 1306) of the expanded cells once they are moved completely into the flow field. By using light-actuated DEP forces, gravity or fluid flow, these cells are exported out of the microfluidic device for further study or expansion.

This experiment demonstrates the selectivity, accuracy and flexibility provided by using the apparatus and methods described herein.

Example 3. surfaces of microfluidic devices were conditioned using serum-free media to remove adherent cells.

Systems and microfluidic devices: the volume of the growth chamber was about 7X 10 as in example 1 ⁵Cubic microns.

The filling scheme is as follows: 250 microliters of 100% carbon dioxide was flowed at a flow rate of 12 μ L/sec. Subsequently 250. mu.l were made to contain 0.1%

F27(Life

Catalog No. P6866) was flowed in at 12 μ L/sec. The final step of filling included an inflow of 250 microliters of PBS at 12 μ L/sec. This is followed by the introduction of the culture medium.

Perfusion protocol: the perfusion method is one of the following two methods:

1. perfusing at 0.01 μ L/sec for 2 h; perfused at 2 μ L/sec for 64 seconds; and repeated.

2. Perfused at 0.02 μ L/sec for 100 seconds; stop the flow for 500 seconds; perfused at 2 μ L/sec for 64 seconds; and repeated.

And (4) a culture medium. Serum-free medium (ThermoFisher Scientific, Cat. No. 12045-096).

Systems and microfluidic devices. By conditioning the media supplement at 36 deg.C

Supplements (2% v/v) adherent cells (which may be, for example, JIMT1 cells, which are commercially available from addex bio, catalog No. C000605) were preincubated for 30min in serum-free medium to demonstrate the ability to remove adherent cells from the flow channels of the microfluidic device after culture. Following pre-incubation, adherent cells are introduced into the flow channel, flow is stopped, and the adherent cells are cultured for a period of 2h to about 24 h. After the end of the assay, a flow of serum-free medium was introduced at a flow rate of 5. mu.L/sec. Approximately 750 microliters of flow passes through the microfluidic device, representing approximately 150X the microfluidic device volume, and all attached JIMT1 cells are exported out of the flow channel and out of the microfluidic device. The experiment shows that the medicine can contain Serum-free media with supplement components such as commercially available B27 can prevent adherence during the assay process incorporating adherence reporter cells and allow for export of adherent cells from the microfluidic device.

Example 4. removal of adherent cells using a conditioning mixture to condition the surface of a microfluidic device.

Adherent cells: as described above for example 3.

And (4) a culture medium. Serum-free medium (ThermoFisher Scientific, Cat. No. 12045-076) with added components, including but not limited to FBS (commercially available from ThermoFisher Scientific, Cat. No. 16000-036) and penicillin-streptomycin (ThermoFisher Scientific, Cat. No. 15140-163).

Conditioning the mixture: cytochalasin B (Sigma Aldrich, Cat. No. C2743-200 UL); DNaseI (New England Biosciences, Cat. No: M0303S); and RGD tripeptide (Santa Cruz Biotechnology, Cat. No.: sc-201176).

Preparation of adherent cells: the medium was adjusted with the conditioning mixture such that the final concentration was: 4 μ M cytochalasin B; 0.1 unit/. mu.L DNaseI; and 1mM RGD tripeptide. Adherent cells were incubated at 36 ℃ for 30min prior to input into the microfluidic device.

Systems and microfluidic devices. As described above, wherein the volume of the growth chamber is about 7 x 10 ⁵Cubic microns.

The ability to remove adherent cells (e.g., JIMT1 cells) from the flow channel of a microfluidic device after culture was demonstrated by pre-incubating populations of adherent cells pre-incubated with the conditioning mixture. Notably, the use of a conditioning mixture allows for the use of serum-containing media, such as the media used in this example, in a microfluidic environment while still providing for the removal of adherent cells.

Introducing pre-incubated adherent cells into a flow channel of a microfluidic device and incubating the adherent cells for a period of time from 2h to about 24 h. After the end of the measurement, a flow of medium was introduced at a flow rate of 5. mu.L/sec. Approximately 750 microliters of flow passes through the microfluidic device, representing approximately 150X microfluidic device volume, and all adherent cells are exported out of the flow channel and out of the microfluidic device. This experiment shows that the conditioning mixture can prevent adherence and allow export of adherent cells.

Example 5. preparation of a microfluidic device with a conditioned surface.

For all preparations: a microfluidic device: produced by Berkeley Lights, inc, and used as received, as described above for example 1. In all cases, the silicon substrate with the patterned polysiloxane and the ITO/glass substrate (PPS) were subjected to an oxygen plasma clean in a Nordson Asymtek plasma cleaner (100W power, 50s) prior to synthesis of the conditioned surface.

A. Perfluoroalkyl siloxy conditioned surfaces.

Materials: heptadecafluoro-1, 1,2, 2-tetrahydrodecyltrimethoxysilane was obtained from Gelest (cat. No. SIH5841.5) and used in the form obtained. MgSO (MgSO)₄·7H₂O (Acros) is used in the form obtained.

A preparation method. The assembled microfluidic device was chemically modified by exposing it to heptadecafluoro-1, 1,2, 2-tetrahydrodecyltrimethoxysilane and water vapor at elevated temperature and reduced pressure. 300 microliters of heptadecafluoro-1, 1,2, 2-tetrahydrodecyltrimethoxysilane and 0.5g MgSO₄·7H₂O (water source) was added to a separate aluminum evaporation dish at the bottom of a clean, dry 6 "glass vacuum dryer. The microfluidic device was supported on a multi-well plate above the silane reagent and the hydrate salt (water source). The desiccator was pumped to 750mTorr at room temperature and sealed. The desiccator was then placed in an oven at 110 ℃ for 24 h. The microfluidic device with perfluoroalkyl-conditioned surface was then removed from the desiccator and used.

In some experiments, microfluidic devices are chemically modified prior to mounting them to a printed wiring board.

B. Dextran conditioned surface.

A material. 11-azidoundecyltrimethoxysilane (Gelest) was synthesized from 11-bromoundecyltrimethoxysilane by replacing the bromide moiety with sodium azide. In a typical reaction, 4.00g of 11-bromoundecyltrimethoxysilane (Gelest) was added to a solution containing 2.00g of sodium azide (Sigma-Aldrich) in 60mL of dry Dimethylformamide (DMF) (Acros). The solution was stirred at room temperature under nitrogen for 24 h. The solution was then filtered and the filtrate was extracted with dry pentane (Acros). The crude 11-azidoundecyltrimethoxysilane was concentrated by rotary evaporation and purified by two successive vacuum distillations.

Dibenzocyclooctyne (DBCO) -modified dextran (MW-3000 Da) was purchased from Nanocs and used as obtained.

A preparation method. And introducing a surface modification ligand. The surface of the assembled microfluidic device was chemically modified by exposing it to 11-azidoundecyltrimethoxysilane and water vapor at elevated temperature and reduced pressure. 300 μ l of 11-azidoundecyltrimethoxysilane and 0.5g of MgSO₄·7H₂O (water source) was added to a separate aluminum evaporation dish at the bottom of a clean, dry 6 "glass vacuum dryer. The microfluidic device was supported on a multi-well plate above the silane and hydrate salt (water source). The desiccator was pumped to 750mTorr at room temperature and sealed. The desiccator was then placed in an oven at 110 ℃ for 24 h. The microfluidic chip with the surface-modified ligand 11-azidoundecylsiloxy moiety was then removed from the desiccator. In some experiments, microfluidic devices are chemically modified prior to mounting them to a printed wiring board.

Incorporation of dextran-conditioned surfaces. After vapor deposition, the azide-terminated microfluidic device surface is reacted with DBCO-dextran by flowing at least 250 microliters of an aqueous solution containing 166 μ Μ DBCO-dextran through the microfluidic device having the surface-modified azide ligand. The reaction was allowed to proceed at room temperature for at least 1 h. The chips were then rinsed by flowing at least 250 microliters of DI water over the chips.

C. Polyethylene glycol (PEG) conditioned surfaces.

A material. 11-azidoundecyltrimethoxysilane was synthesized as described above. Alkyne-modified PEG (MW-5000 Da) was purchased from JenKem and used as received. Sodium ascorbate and copper sulfate pentahydrate were purchased from Sigma-Aldrich and used as received. (3[ tris (3-hydroxypropyl-triazolylmethyl) amine) THPTA copper catalyzed click reagent (Glen Research).

A preparation method. And introducing a surface modification ligand. A microfluidic chip with 11-azidoundecylsiloxy surface-modified ligands was prepared as described above.

PEG-conditioned surfaces were introduced. The azide-terminated surface of the microfluidic device is reacted with alkyne-modified PEG by flowing at least 250 microliters of an aqueous solution containing 333 μ M alkyne-modified PEG, 500 μ M copper sulfate, 500 μ M THPTA ligand, and 5mM sodium ascorbate through the microfluidic device having the 11-azidoundecylsiloxy surface-modified ligand. The reaction was allowed to proceed at room temperature for at least 1 hour. The microfluidic device with the PEG conditioned surface was then rinsed by flowing at least 250 microliters of deionized water through the device.

D. An alkyl modified surface having a phosphonate linking group to the surface.

A material. Octadecylphosphonic acid was purchased from Sigma Aldrich and used as received. Acetone and ethanol were purchased from Sigma Aldrich.

A preparation method. The surface of the microfluidic device was exposed to a 10 mM solution of octadecylphosphonic acid in dry ethanol for 48 hours at 35 ℃. After deposition, the resulting microfluidic device with the alkyl-conditioned surface attached via phosphonate linking groups was rinsed thoroughly with ethanol and DI water.

Example 6:t lymphocytes are cultured and exported on conditioned microfluidic surfaces.

A material. CD3+ cells were obtained from AllCells Inc. and magnetic beads (anti-CD 3/anti-CD 28

Thermofeisher Scientific, Cat. No. 11453D) was mixed at a ratio of 1 bead/1 cell. The mixture was heated at 37 ℃ in 5% CO₂Incubate for 5 hours in the same medium as the culture experiment itself. After incubation, the T cell/bead mixture was incubatedAnd (5) resuspending for later use.

And (4) a culture medium. RPMI-1640(

ThermoFisher Scientific, Cat # 11875-127), 10% FBS, 2% human AB serum (50U/ml IL 2; r&D System)。

And (3) filling operation: as described above for example 3.

Perfusion protocol: as described above for example 3.

Systems and microfluidic devices: as described above for example 3. The volume of the growth chamber is about 7X 10 ⁵Cubic microns.

A conditioned surface. The microfluidic device had a covalently linked dextran conditioned surface prepared as described above.

The T cell beaded suspension is introduced into the microfluidic device by flowing the heavy suspension through the fluid inlet and into the microfluidic channel. Flow was stopped and T cells/beads were randomly loaded into the growth chamber by tilting the chip and allowing gravity to pull the T cells/beads into the growth chamber.

After loading the T cells/beads into the growth chamber, the media was perfused through the microfluidic channels of the nanofluidic chip for a period of 4 days. Fig. 14A shows the growth of T cells on a dextran-conditioned surface of a growth chamber of a microfluidic device. T cell growth on dextran conditioned surfaces was improved relative to non-conditioned surfaces of similar microfluidic devices (data not shown).

The T cells are then removed from the growth chamber by gravity (e.g., tilting the microfluidic device). Fig. 14B shows the extent of removal from the growth chamber at the end of the 20 minute period, indicating an excellent ability to export expanded T cells into the flow channel, which is improved relative to removing T cells from the non-conditioned surface of a similar microfluidic device. The T cells are then exported from the microfluidic device (not shown).

The embodiments illustrated herein are exemplary and do not in any way limit the scope of the methods and apparatuses described throughout the specification.

Claims (30)

1. A kit for preparing a microfluidic device having covalently attached thereto a monolayer and comprising:

an inner surface facing an interior of at least one growth chamber, the inner surface having the monolayer covalently attached thereto.

2. The kit of claim 1, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the one or more independent microfluidic circuits including the at least one growth chamber, the microfluidic circuits defined by a housing, the housing including:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

a cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber,

wherein the monolayer comprises a reactive moiety Rx and a linking group configured to covalently attach to a conditioning modifier for supporting cell growth, viability, portability, or any combination thereof within the at least one growth chamber, and further wherein each linking group of the monolayer towards the interior surface of the interior of the growth chamber is the same linking group, and

Wherein the kit comprises a reactive partner portion Rpx comprising a reactive partner configured to react with the reactive portion Rx of the monolayer and a portion for supporting cell growth, viability, portability or any combination thereof.

3. A method of making a microfluidic device having a monolayer covalently attached thereto, the method comprising:

introducing a conditioning modifier to the microfluidic device, wherein the microfluidic device comprises an inner surface facing the interior of at least one growth chamber, the inner surface having a monolayer covalently attached thereto.

4. The method of claim 3, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the one or more independent microfluidic circuits including the at least one growth chamber, the microfluidic circuits defined by a housing, the housing including:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

a cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber,

Wherein the monolayer comprises a linking group and a reactive moiety Rx,

wherein the conditioning modifier comprises a reactive partner portion Rpx and a portion configured to support cell growth, viability, portability, or any combination thereof,

wherein the reactive partner moiety Rpx of the conditioning modifier reacts with the reactive partner moiety Rx of the monolayer to form a modified conditioned surface configured to support cell growth, viability, portability, or any combination thereof.

5. A method of culturing at least one biological cell in a microfluidic device, the method comprising:

introducing the at least one biological cell into a growth chamber of the microfluidic device;

incubating the at least one biological cell in the microfluidic device for a period of time sufficient to expand the at least one biological cell to generate a colony of biological cells; and

discontinuously perfusing a first fluid medium through a fluid flow region of the microfluidic device during the incubating step.

6. The method of claim 5, wherein the microfluidic device comprises a separate microfluidic circuit configured to contain a flow of the first fluidic medium, the separate microfluidic circuit comprising a fluid inlet port, at least one growth chamber, and a flow region connecting the fluid inlet port and the at least one growth chamber, wherein the growth chamber has a surface conditioned to support cell growth, viability, portability, or any combination thereof.

7. A microfluidic device comprising a conditioned surface having a thickness of between 10nm and 50nm, the microfluidic device comprising:

an inner surface facing an interior of the at least one growth chamber, the inner surface having a conditioned surface covalently attached thereto.

8. The microfluidic device of claim 7, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the microfluidic circuits defined by a housing, the housing comprising:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

a cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber,

wherein the conditioned surface comprises a linking group configured to covalently attach to a conditioning modifier for supporting cell growth, viability, portability, or any combination thereof within the at least one growth chamber and a reactive moiety Rx.

9. A kit for preparing a microfluidic device having a modified conditioned surface configured to support cell growth, viability, portability, or any combination thereof, the kit comprising:

The microfluidic device of claim 7 or 8; and

a conditioning modifier comprising a reactive partner portion Rpx configured to react with the reactive moiety Rx and a moiety for supporting cell growth, viability, portability, or any combination thereof.

10. A method of making a microfluidic device for providing a modified conditioned surface configured to support cell growth, viability, portability, or any combination thereof, the method comprising:

introducing a conditioning modifier into the microfluidic device of any one of claims 7 to 9, wherein the microfluidic device comprises an interior surface facing the interior of at least one growth chamber, the interior surface having a conditioned surface covalently attached thereto.

11. The method of claim 10, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the one or more independent microfluidic circuits including the at least one growth chamber, the microfluidic circuits defined by a housing, the housing including:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

A cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber,

wherein the conditioned surface comprises a linking group and a reactive moiety Rx,

wherein the conditioning modifier comprises a reactive partner portion Rpx and a portion configured to support cell growth, viability, portability, or any combination thereof,

wherein the reactive partner moiety Rpx of the conditioning modifier reacts with the reactive moiety Rx of the conditioned surface to form a modified conditioned surface configured to support cell growth, viability, portability, or any combination thereof.

12. A method of culturing at least one biological cell in a microfluidic device having the modified conditioned surface of claim 10 or 11, the method comprising:

introducing the at least one biological cell into at least one growth chamber of a microfluidic device, wherein each of the one or more independent microfluidic circuits further comprises a flow region connecting a fluid inlet port and the at least one growth chamber; and

Incubating the at least one biological cell in the microfluidic device for a period of time sufficient to expand the at least one biological cell to generate a biological cell colony.

13. A microfluidic device having a conditioned surface with a thickness of less than 10nm, the microfluidic device comprising:

an inner surface facing an interior of at least one growth chamber, the inner surface having the conditioned surface covalently attached thereto.

14. The microfluidic device of claim 13, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the microfluidic circuits defined by a housing, the housing comprising:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

a cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber,

wherein the conditioned surface comprises a linking group configured to covalently attach to a conditioning modifier for supporting cell growth, viability, portability, or any combination thereof within the at least one growth chamber and a reactive moiety Rx.

15. A kit for preparing a microfluidic device having a modified conditioned surface configured to support cell growth, viability, portability, or any combination thereof, the kit comprising:

the microfluidic device of claim 13 or 14; and

a conditioning modifier comprising a reactive partner portion Rpx configured to react with the reactive moiety Rx and a moiety for supporting cell growth, viability, portability, or any combination thereof.

16. A method of making a microfluidic device for providing a modified conditioned surface configured to support cell growth, viability, portability, or any combination thereof, the method comprising:

introducing a conditioning modifier to the microfluidic device of any one of claims 13 to 15, wherein the microfluidic device comprises an interior surface facing the interior of at least one growth chamber, the interior surface having a conditioned surface covalently attached thereto.

17. The method of claim 16, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the one or more independent microfluidic circuits including the at least one growth chamber, the microfluidic circuits defined by a housing, the housing including:

A base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

a cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber,

wherein the conditioned surface comprises a linking group and a reactive moiety Rx,

wherein the conditioning modifier comprises a reactive partner portion Rpx and a portion configured to support cell growth, viability, portability, or any combination thereof,

wherein the reactive partner moiety Rpx of the conditioning modifier reacts with the reactive moiety Rx of the conditioned surface to form a modified conditioned surface configured to support cell growth, viability, portability, or any combination thereof.

18. A method of culturing at least one biological cell in a microfluidic device having the modified conditioned surface of claim 16 or 17, the method comprising:

introducing the at least one biological cell into at least one growth chamber of a microfluidic device, wherein each of the one or more independent microfluidic circuits further comprises a flow region connecting a fluid inlet port and the at least one growth chamber; and

Incubating the at least one biological cell in the microfluidic device for a period of time sufficient to expand the at least one biological cell to generate a biological cell colony.

19. A microfluidic device comprising a conditioned surface having a cleavable moiety, the microfluidic device comprising:

an interior surface facing an interior of at least one growth chamber, the interior surface having a conditioned surface covalently attached thereto, wherein the cleavable moiety is configured to allow disruption of the at least one conditioned surface to promote portability of one or more biological cells.

20. The microfluidic device of claim 19, wherein the at least one conditioned surface is configured to support cell growth, viability, portability, or any combination thereof within the microfluidic device.

21. The microfluidic device of claim 19 or 20, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the microfluidic circuits defined by a housing, the housing comprising:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

A cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber.

22. A kit for preparing the microfluidic device of any one of claims 19-21, wherein the kit comprises a reactive partner Rpx comprising a reactive moiety Rx configured to covalently link to a conditioned surface, thereby forming a conditioned surface having a cleavable moiety and configured to support cell growth, viability, portability, or any combination thereof.

23. A method of making the microfluidic device of any one of claims 19-21, the method comprising:

introducing into the microfluidic device a conditional modifier comprising a moiety for supporting cell growth, viability, portability, or any combination thereof and a reactive partner Rpx, wherein the reactive partner Rpx is covalently attached to the reactive moiety Rx of the conditioned surface.

24. A method of culturing at least one biological cell in the microfluidic device of any one of claims 19-21, the method comprising:

Introducing the at least one biological cell into at least one growth chamber;

incubating the at least one biological cell for a period of time at least long enough to expand the at least one biological cell to produce a biological cell colony; and (c).

25. A system for culturing one or more biological cells, comprising:

a microfluidic device comprising at least one growth chamber, wherein an interior surface facing an interior of the at least one growth chamber comprises a conditioned surface covalently attached thereto and configured to support cell growth, viability, portability, or any combination thereof within the microfluidic device; and

temperature control configured to adjust the temperature of the at least one conditioned surface within a predetermined temperature range that helps maintain functional cells.

26. The system of claim 25, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the one or more independent microfluidic circuits including the at least one growth chamber, the microfluidic circuits defined by a housing, the housing including:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

A cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber.

27. A method of culturing at least one biological cell within the microfluidic device of claim 25 or 26, the method comprising:

introducing the at least one biological cell into at least one growth chamber; and

incubating the at least one biological cell for a period of time at least long enough to expand the at least one biological cell to produce a biological cell colony, wherein incubating the at least one biological cell comprises using temperature control to control the temperature of the at least one conditioned surface within a predetermined temperature range that facilitates maintaining functional cells.

28. A system for culturing one or more biological cells in a microfluidic device, the system comprising:

a microfluidic device, comprising:

a flow region configured to contain a flow of a first fluid medium;

at least one growth chamber configured to support cell growth, viability, portability, or any combination thereof; and

a reservoir fluidically coupled to the microfluidic device and configured to contain the first fluidic medium, wherein the reservoir is configured to be contacted by a gaseous environment capable of providing a desired level of dissolved gas molecules to the first fluidic medium to facilitate supporting cell growth, viability, portability, or any combination thereof within the microfluidic device.

29. The system of claim 28, wherein an interior surface facing the at least one growth chamber comprises a conditioned surface configured to support cell growth, viability, portability, or any combination thereof.

30. The system of claim 29, wherein the microfluidic device comprises:

one or more independent microfluidic circuits each configured to hold a fluid, the one or more independent microfluidic circuits including the at least one growth chamber, the microfluidic circuits defined by a housing, the housing including:

a base comprising a first material and having an inner surface;

a microfluidic conduit structure comprising a second material and having an inner surface; and

a cover comprising a third material and having an inner surface,

wherein an inner surface of the base, an inner surface of the microfluidic conduit structure, and an inner surface of the lid form an inner surface facing an interior of the at least one growth chamber.

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