Microfluidic devices and methods of using the same in multi-cellular assays of secretions

Abstract

To provide methods of identifying an antibody-secreting cell that produces a virus-neutralizing antibody.SOLUTION: The method comprises: retaining in a plurality of microreactors a plurality of cell populations where individual cell populations comprise one or more antibody-secreting cells: introducing a plurality of accessory particle populations into the plurality of microreactors, where the accessory particle populations comprise a plurality of virus particles operable to infect the one or more readout cells; incubating the individual cell populations, the readout cells and the accessory particle populations within the individual microreactors for a time sufficient to produce a plurality of antibodies; assaying the individual microreactors and determining whether the plurality of antibodies comprises a virus-neutralizing antibody; and identifying the antibody secreting cell that produces the virus-neutralizing antibody.SELECTED DRAWING: Figure 23

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 61 / 806,329, filed on March 28, 2013. The entire disclosure of which is incorporated herein by reference for all purposes. BACKGROUND OF THE INVENTION

[0002] Cells are the basic units of life, and no two cells are the same. For example, differences in genotype, phenotype, and / or morphological characteristics can contribute to cell diversity. In fact, "apparently identical" clonal populations of cells have been shown to exhibit phenotypic differences between cells within the population. Cell differences exist at all levels of life. The range of that level extends from bacterial cells to highly differentiated mammalian cells (e.g., immune cells), and partially differentiated cells (e.g., adult stem cells and progenitor cells). Differences in cell state, function, and response arise from various mechanisms including different histories, different differentiation states, epigenetic diversity, effects on the cell cycle, stochastic variations, differences in genomic sequences, gene expression, protein expression, and interaction effects of different cells.

[0003] Conventional bulk cell - level analysis, including the measurement of expressed proteins or RNAs, has been performed by averaging a fairly large number of cells (generally more than 1000 cells per individual assay). This averaging of cell populations hides the heterogeneous components present within the cell population and obscures the basic biological functions of individual cells within that population. There are many examples where such averaged measurements are inappropriate. For example, measurements in cell processes of a cell population are complicated and asynchronous by the responses of individual cells. Thus, the dynamic characteristics of the process are made unclear. For example, the presence of dominant genes, as well as sub - populations of cells with different phenotypes, result in population measurements that are difficult to reflect the internal states of the majority of cells in the population. See, e.g., Altshuler and Wu. (2010). Cell / / 141, pp 559 - 563.

[0004] Existing methods for isolating a population of unique cell types are often limited in the purity of the population that can be achieved. For example, enriched populations of primary pluripotent stem cells have rarely achieved better than 50% functional purity and are often at a purity of 10% or less. As a result, the molecular signatures of these cells are largely hidden by overwhelming contamination from other cell types. Many cell types interact with each other via both direct contact and secreted factors to promote survival, death, differentiation, or several other functions. And these interactions are difficult to separate and study in a mixture containing the majority of cells. Furthermore, cells differ in genomic sequences and / or cell states that result in different levels or different types of expressed mRNA or protein. When analyzed in bulk populations, specific cells in unique cell states, or specific cells with expressed mRNA or protein of interest, while of high industrial value, are very difficult or impossible to separate from the population.

[0005] To overcome the drawbacks of bulk population cell analysis, single-cell assay platforms have been developed. For example, microfluidic devices have been used in the past to study single cells (Lecault et al. (2012). CURR. OPIN. CHEM. BIOL. 16, pp. 381-390). MA et al. (Nat Med, 17, pp. 738-743 (2011)) applied a single-cell barcode chip to simultaneously measure multiple cytokines (e.g., IL-10, TNF-β, IFN-γ) from human macrophages and cytotoxic T lymphocytes (CTLs) obtained from both healthy donors and metastatic melanoma patients. Micromachined chamber arrays have also been used to screen and select B cells secreting antigen-specific antibodies from both immunized humans and mice (Story et al. (2009). PROC. Natl. Acad. Sci. U.S.A. 105, pp. 17902-17907; Jin et al. (2009). Nat. Med. 15, pp. 1088-1092). In this approach, single B cells were arrayed on a surface containing hundreds of thousands of micromachined wells (~10-100 μm deep) whose well surfaces were functionalized with capture antibodies. After incubation of the cells on the well surface for less than 3 hours, the surface was washed with a fluorescently labeled antigen and scanned to identify antigen-specific B cells. These cells were manually recovered from the array by microcapillary to amplify, sequence, and clone the genes encoding the antibodies from these cells.

[0006] The two-phase microfluidic device has been applied to the analysis of proteins secreted from single immune cells by encapsulating them in sub-nanoliter water droplets separated by an oil flow (Konry et al. (2011). Biosens. Bioelectron. 26. Pp. 2702-2710). These droplets are analyzed in a flow-through format similar to FACS, providing an opportunity for ultra-high-throughput detection of proteins secreted from single cells. The water-in-oil emulsion has also been used to study cell paracrine signaling by co-encapsulated cells in microfluidically generated agarose beads (Tumarkin et al. (2011). Integer. Biol. 3, pp. 653-662). The generation of microfluidic droplets has been used in drug screening and development by allowing the analysis of the viability of single cells exposed to and encapsulated in different compositions (Brouzes et al. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, pp. 14195-14200).

[0007] Antibodies are molecules naturally produced by the human or animal immune system to fight off infections and diseases. This is accomplished by the innate ability of the immune system to generate an enormous diversity of antibodies that have the ability to recognize and bind to specific targets (e.g., proteins, viruses, bacteria). This unparalleled specificity is what makes antibodies very powerful and low-side-effect drugs in clinically approved therapies for a variety of conditions including cancer, autoimmune diseases, inflammation, neurology, and infections. Compared to conventional small molecule drugs, antibodies offer several advantages including excellent pharmacokinetics, fewer side effects, improved tolerability, and a very high success rate in clinical trials (7% versus 27% for small molecules) (Reichert (2009). Mabs 1, pp. 387-389.). This is why antibodies are the fastest growing class of drugs in the global market, which is growing at a rate of 9% per year to $50 billion in 2012 (Nelson et al. (2010). Nat. Rev. Drug Disc. 9(10), pp. 767-774.).

[0008] The discovery of antibodies with optimal therapeutic properties, and in particular antibodies that target surface receptors, remains a major bottleneck in pharmaceutical development. In response to immunization, animals can produce millions of different monoclonal antibodies (mAbs). Each mAb is produced by a single cell called an antibody-secreting cell (ASC), and each ASC produces only one type of mAb. Thus, for example, antibody analysis for drug discovery purposes leads to single cell analysis. However, since ASCs are analyzed individually (not within a bulk population of cells), a single ASC produces only a very small amount of antibody, and when analyzed in the volume of a conventional assay format, the antibody is at a rather low concentration and completely undetectable. Thus, new methods are needed to study individual ASCs and the antibodies they secrete. The present invention addresses this need and other needs. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0009] The present invention provides a microfluidic platform for analyzing extracellular effects resulting from a single effector cell. The effector cell, in one embodiment, is a cell that secretes a biological factor (e.g., an antibody (ASC)). In a further embodiment, the microfluidic analysis of the effector cell is an extracellular effect assay performed on a cell population containing a single effector cell. Means for solving the problems

[0010] In one aspect, a method for identifying a cell population containing effector cells having an extracellular effect is provided. In one embodiment, the method includes the step of holding a cell population containing one or more effector cells in a microreactor. The contents of the microreactor further include a population of readout particles containing one or more readout particles. The method also includes the steps of incubating the cell population with one or more readout cells in the microreactor and assaying the cell population for the presence of an extracellular effect. The population of readout particles or a subpopulation thereof provides a direct or indirect readout of the extracellular effect. The method further includes the step of determining, based on the results of the assay step, whether one or more effector cells within the cell population exert an extracellular effect. In a further embodiment, the microreactor is a microfluidic chamber. In a further embodiment, the microfluidic chamber is part of a microfluidic structure including a membrane valve.

[0011] In this aspect, the effector cell is a cell that secretes a biological factor, such as an antibody. As long as the presence of an extracellular effect is detected within a particular microreactor, it is not necessary to first identify a particular effector cell or a plurality of effector cells having a particular extracellular effect. That is, some or all of the cells in the microreactor in which the effect is measured can be recovered if they are required for further characterization to identify the particular cell providing the extracellular effect.

[0012] In one embodiment, when a cell population containing one or more effector cells is determined to exhibit an extracellular effect, the cell population or a portion thereof is recovered to obtain the recovered cell population. The recovery, in one embodiment, includes the step of perforating a microfluidic chamber containing the cell population containing one or more cells exhibiting the extracellular effect with a microcapillary, and the step of aspirating the contents of the chamber or a portion thereof. Thereby, the recovered aspirated cell population is obtained.

[0013] Also, in one embodiment, when a cell population containing one or more effector cells is determined to exhibit an extracellular effect, the cell population or a portion thereof is recovered to obtain the recovered cell population. The recovered cell population is further analyzed as a cell subpopulation. The method in one embodiment includes the step of holding subpopulations of a plurality of cells derived from the recovered cell population in separate chambers of a microfluidic device. The separate chamber contains a population of readout particles containing one or more readout particles. Also, the method includes the step of incubating the population of readout particles with the individual cell subpopulations in the chamber, and the step of assaying the individual cell subpopulations for the presence of a second extracellular effect. The population of readout particles or a subpopulation thereof provides a readout of the second extracellular effect. The second extracellular effect is the same extracellular effect or a different extracellular effect from the extracellular effect measured in the recovered cell population. When the cell subpopulations are incubated and assayed, the method includes the step of identifying one cell subpopulation from among the plurality of cell subpopulations containing one or more cells exhibiting the second extracellular effect with the population of readout particles or a subpopulation thereof based on the results of the assay step.

[0014] In another aspect, the present invention relates to a method for identifying a cell population that exhibits variability in extracellular effects. In one embodiment, the method comprises the steps of holding a plurality of individual cell populations in separate microfluidic chambers, incubating an individual cell population with a population of readout particles within the microfluidic chamber, and assaying the individual cell population for the presence of an extracellular effect. At least one of the individual cell populations comprises one or more effector cells, and the contents of a separate microfluidic chamber comprise a population of readout particles that includes one or more readout particles. The population of readout particles or a subpopulation thereof provides a readout of the extracellular effect. When the cell population is incubated and assayed, the method comprises identifying, based on the results of the assay, one cell population from among a plurality of cell populations that exhibit variability in extracellular effects as compared to one or more of the remaining cell populations of the plurality of cell populations. In a further embodiment, the one or more effector cells comprise antibody-secreting cells. In another embodiment, the one or more effector cells comprise plasmablasts, B cells, plasmablasts, cells produced through the proliferation of memory B cells, hybridoma cells, T cells, CD8+ T cells and CD4+ T cells, recombinant cells engineered to produce antibodies, recombinant cells engineered to express a T cell receptor, or combinations thereof.

[0015] One or more cell populations that exhibit an extracellular effect or a variation in an extracellular effect are, in one embodiment, recovered to obtain one or more recovered cell populations. The recovery is performed, for example, using a microcapillary. Once one or more individual cell populations are identified and recovered, the one or more individual cell populations are further analyzed to determine the plurality of cells or cells responsible for the observed extracellular effect. In one embodiment, the method includes retaining, in separate chambers of a microfluidic device, a plurality of cell subpopulations derived from one or more recovered cell populations. Each of the separate chambers contains a population of readout particles that includes one or more readout particles. The individual cell subpopulations are incubated in the chamber with the population of readout particles. The individual cell subpopulations are assayed for a variation in a second extracellular effect. The population of readout particles or a subpopulation thereof provides a readout of the second extracellular effect. The second extracellular effect is the same extracellular effect or a different cell effect as the extracellular effect measured in the recovered cell population. Based on the assay of the second extracellular effect, one or more individual cell subpopulations are identified as exhibiting a variation in the second extracellular effect. In one embodiment, the one or more individual cell subpopulations are then recovered for further analysis. Extracellular effect assays are described throughout the specification.

[0016] In one embodiment, cells from the recovered cell population or recovered cell subpopulation are retained in a plurality of containers as a subpopulation or sub-subpopulation of cells, with each cell subpopulation or cell sub-subpopulation being present in an individual container. The individual subpopulations or sub-subpopulations are lysed to provide, and one or more nucleic acids within the lysed cell subpopulation or lysed cell sub-subpopulation are amplified. In a further embodiment, the one or more nucleic acids include antibody genes.

[0017] In one embodiment of the method described herein, the incubating step includes the step of exchanging the medium within each microreactor (e.g., microfluidic chamber) containing an individual cell population or subpopulation. The medium exchange is carried out, for example, to maintain the viability of the cells within the chamber, or to provide reagents for performing an assay of extracellular effects, or to perform a plurality of extracellular effect assays in a continuous format.

[0018] In one embodiment, the incubating step includes the step of incubating a cell population or cell subpopulation with a plurality of accessory particles. The plurality of accessory particles are provided, for example, as additional reagents for an assay of extracellular effects or are provided to maintain cell survival. In one embodiment, the plurality of accessory particles include sphingosine-1-phosphate, lysophosphatidic acid, growth factors, cytokines, chemokines, neurotransmitters, virus particles, secondary antibodies, fluorescent particles, fluorescent substrates, complement pathway inductors, virus particles or accessory cells. Accessory cells are, in one embodiment, fibroblasts, natural killer (NK) cells, killer T cells, antigen presenting cells, dendritic cells, recombinant cells or combinations thereof.

[0019] In one embodiment, the extracellular effects measured by the methods and devices described herein are the binding of effector cells or molecules secreted by effector cells to cell surface proteins, the antagonism or agonism of cell surface receptors in readout cells (type of readout particles). In further embodiments, the cell surface receptors are receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), receptor serine-threonine kinases, receptor tyrosine phosphatases or receptor guanylyl cyclases. The GPCRs are not limited by class or species. For example, the GPCRs are, in one embodiment, the GPCRs provided in Table 3A or 3B herein.

[0020] In another embodiment, the extracellular effects measured by the methods and devices described herein are the binding of effector cells or molecules secreted by effector cells to ion channels, the antagonism of ion channels, or the agonism of ion channels. The ion channel is, in one embodiment, GABA A , glycine (GlyR), serotonin (5-HT), nicotinic acetylcholine (nAChR), zinc-activated ion channel, ionotropic glutamate, AMPA, kainite, NMDA receptor, or ATP-dependent channel.

[0021] The extracellular effect is the agonism, antagonism, or binding of a cell surface receptor or ion channel. The effect in one embodiment is measured by the detection of intracellular cAMP or calcium, the expression of a protein reporter, or the localization of a protein in a readout cell that expresses a cell surface receptor or ion channel.

[0022] In another embodiment, the extracellular effect is the binding interaction between molecules secreted by one or more effector cells or a subset thereof, the regulation of apoptosis, the regulation of cell proliferation, the change in the morphological appearance of readout particles, the change in the localization of proteins within readout particles, the expression of proteins by readout particles, the neutralization of accessory particles operable to affect readout particles, or a combination thereof, to one or more readout particles or one or more accessory particles.

[0023] In some embodiments, the extracellular effect is the effect of a cellular product secreted by an effector cell. The extracellular effect is a binding interaction between a protein produced by an effector cell and either a readout particle or an accessory particle. For example, in one embodiment, the effector cell is an antibody-secreting cell (ASC), and the readout or accessory particle contains an epitope or antigen. The binding interaction is, in one embodiment, to an extent of one or more of antigen-antibody binding specificity, antigen-antibody binding affinity, and antigen-antibody binding reaction rate. In another embodiment, the effector cell is an activated T cell that secretes cytokines, and the readout particle contains one or more antibodies for capturing the secreted cytokines.

[0024] The methods and devices described above may be used to examine or select cells. The cells are rare, for example, less than 1% of the cells in a population, or about 1% to about 10% or about 5% to about 10% of the cells to be examined or selected.

[0025] In another aspect, functional antibodies and receptors found by the methods described herein are provided. In one embodiment of this aspect, the nucleic acid of the effector cell responsible for the extracellular effect is amplified and sequenced. The nucleic acid is a gene encoding a secreted biomolecule (e.g., an antibody or a fragment thereof), or a gene encoding a cell receptor or a fragment thereof (e.g., a T cell receptor). The antibody or a fragment thereof or the cell receptor or a fragment thereof is cloned and / or sequenced by methods known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

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[0181] As used herein, the singular forms "a", "an", and "the" include plural instances unless the context clearly dictates otherwise. Thus, for example, "one" includes one or more such items. The references described herein are not admitted to be prior art to the present invention.

[0182] As used herein, "readout" refers to a method by which an extracellular effect is reported. As described herein, a "readout particle population" can include one or more readout particles.

[0183] As used herein, "extracellular effect" refers to the direct or indirect effect of a readout particle that is extracellular to an effector cell. It includes, but is not particularly limited to, increased cell proliferation, decreased proliferation, apoptosis, lysis, differentiation, infection, binding (e.g., binding to a cell surface receptor or epitope), morphological changes, inhibition of an induction or signaling cascade, enzyme inhibition, virus inhibition, cytokine inhibition, complement activation. As provided herein, in one embodiment, the extracellular effect is the binding of a biomolecule of interest secreted by an effector cell to a readout particle. In another embodiment, the extracellular effect is a response such as apoptosis of a readout cell or accessory cell.

[0184] The methods provided herein are used to identify a cell population containing effector cells that exhibit a variation in extracellular effect or effector cells. The variation in extracellular effect is a variation compared to a control (negative or positive control) or a variation compared to one or more other cell populations.

[0185] Particularly with respect to particles or heterogeneous cell populations, the "heterogeneous population" referred to herein means a population of particles or cells that contains at least two particles or cells having different functions. For example, a characteristic in one embodiment is morphology, size, type of fluorescent reporter, different cell types, phenotype, genotype, cell differentiation type, sequence or functional characteristics of one or more expressed RNA species.

[0186] As used herein, the term "subpopulation" refers to a proportion of a large population of particles (cells). In one embodiment, a population of cells is divided into subpopulations, for example, by isolating individual subpopulations within individual microfluidic chambers. Further, individual subpopulations can be divided into further subpopulations, for example, within a plurality of microfluidic chambers or other reaction vessels. A subpopulation may be only a portion of the particles within a large population located within the same microfluidic chamber. A subpopulation contains one or more particles, and the plurality of particles are present within the subpopulation. The individual particles within the plurality of particles can be uniform or non-uniform with respect to each other.

[0187] In one aspect, a "cell fixture" defines at least one effector zone and at least one readout zone, either continuously or intermittently. The fixture may be a valve, cell fence, external field or field gradient (e.g., gravity, magnetic, electromagnetic, acceleration, etc.) orientation, electrodes or optical components or magnetic probes, localization and / or orientation of a locally generated field by surface modifications (e.g., texturing, coating, etc.) that promote or inhibit cell adhesion, or localization and / or orientation of a locally generated field by the specific gravity of the solution within the chamber, or may be a structural element such as these or a combination of one or more of these.

[0188] As used herein, "coating" may be any addition to the surface of the chamber that promotes or inhibits the ability of effector cells or readout particles to adhere to the surface of the chamber. The coating may be selected from one or more of the following. It may be a cell, a polymer brush, a polymeric hydrogel, a self-assembled monolayer (SAM), a photograft molecule, a protein or protein fragment having cell-binding properties (e.g., a cell-binding domain from actin, fibronectin, integrin, protein A, protein G, etc.). More generally, a peptide sequence motif of arginine-glycine-aspartic acid (serine) (RGD(S)) is used. Poly-L-lysine is also widely used as a polymer coating with PDMS to enhance cell adhesion via electrostatic interactions (phospholipids having cell-binding properties, cholesterol having cell-binding properties, glycoproteins having cell-binding properties, and glycolipids having cell-binding properties). Also, the PDMS surface functionality using biotinylated biomolecules is a simple, very attractive, and flexible approach. It is widely known that bovine serum albumin (BSA) is easily adsorbed by the hydrophobic effect on the PDMS surface due to its hydrophobic domain. Its PDS surface allows for further direct binding of streptavidin-based complexes (proteins, DNA, polymers, fluorophores) in the chamber. Hydrophobic polyethylene glycol-based polymers are also known for their anti-biofouling properties and can be applied on the PDMS surface (adsorption, covalent grafting). Thereby, cell adhesion is prevented. Poly(para-xylylene), e.g., parylene C, can also be deposited on the surface of PDMS using chemical vapor deposition (CVD) to prevent cell attachment.

[0189] As used herein, "isolated" refers to a situation where a particular chamber shows no substantial contamination of effector cells and / or readout particles analyzed using particles or biomolecules from another chamber of the microfluidic device. Such isolation can be achieved, for example, in the case of chambers of a compound, by sealing a set of chambers or a single chamber, by restricting fluid communication between the chambers, or by restricting the flow of fluid between the chambers.

[0190] As used herein, "inlet" or "outlet" includes any opening through which fluid flows into and out of the chamber. The fluid flow may be restricted through the inlet or outlet or both to separate the chamber from its surrounding environment. There may be one or more valves, and the flow may be controlled by restricting the fluid flow path leading to the inlet and outlet having a layer (e.g., a control layer or an insulating layer) that prevents the flow. Alternatively, the flow may be regulated by the speed at which the fluid passes through the device. The inlet or outlet may also provide fluid flow to a device for carrying effector cells or readout particles, or other components carried in a flow as required during the analysis. In some embodiments, the inlet and outlet may be provided by a single opening at the top of the chamber through which fluid flows from the inlet side to the outlet side.

[0191] As used herein, "magnet" includes any ferromagnetic or paramagnetic material. As used herein, "ferromagnetic" means a material composed of iron, nickel, chromium, cobalt or combinations thereof and various alloys. As a result, a magnetic material is attracted to a magnet or is itself a magnet. For example, the magnetic material may be made of ferromagnetic stainless steel, or may be made of stainless steel having magnetism or a rare earth magnet. The magnet may also be made by using a magnetic fluid in a defined shape or orientation. The magnet may also be realized using a coil or other electrically actuated device designed to generate a magnetic field when energized by an electric current.

[0192] As used herein, an "array of wells" means any arrangement of structures within a chamber that restricts the movement of effector cells and / or readout particles by localizing one or the other to a specific readout zone or effector zone. A zone (i.e., effector or readout) is defined by the presence of a particular type of particle therein. For example, one embodiment of an array of wells is shown in FIG. 33. Continuously intersecting cell fences form an array of wells on the surface of the chamber.

[0193] As used herein, a "particle trap" means a structure capable of spatially retaining effector cells or readout particles (beads) at a specific spatial position so as to restrict movement during an assay. A "cell fence" is a type of "particle trap". In one embodiment, when referred to as an "effector cell trap", the particle trap is used to retain effector cells. In some embodiments, when referred to as a "readout particle trap" or "readout cell trap", the particle trap is used to retain readout particles. In one embodiment, a "particle trap" enables particles to be trapped with a fixed position. Having a fixed position simplifies imaging and image analysis. Having a fixed position for readout particles or particles also has the advantage of controlling or restricting the diffusion distance between effector cells and readout particles. Also, having a fixed position can prevent the interaction between effector cells and readout particles.

[0194] As used herein, a "textured surface" may be any type of surface modification that promotes or reduces cell adhesion to the chamber surface. For example, the surface may be textured with one or more of bumps, depressions, roughness, protrusions, hooks, pegs, wells, grooves, ridges, particles, patterns, webs, hydrophobicity, hydrophilicity, etc.

[0195] The human body creates millions to billions of different types of antibodies at all times. These are produced by different single plasma cells called "antibody-secreting cells" or "ASCs". Each ASC has a diameter of about 7μm to about 15μm and, depending on the source, has a diameter of about 1 / 10 the width of a human hair and produces only trace amounts of antibodies. Out of the billions of different ASCs in the human body, a very rare number produce antibodies suitable for use as therapeutic agents. Analyzed in conventional format volumes, this small amount of antibody is very dilute and completely undetectable. For this reason, currently, antibody discovery requires separating each ASC, fusing it with immortal cancer cells to create hybridomas, and "growing" them (see Figure 60). Ultimately, thousands of identical cells that can produce enough antibody to measure are generated. See, for example, McCullough and Spier (1990). Monoclonal Antibodies in Biotechnology: Theory and Practicalization, Chapter 2, Cambridge University Press (which are hereby incorporated by reference in their entirety). This process is not only incredibly inefficient (99.9% of the starting immune cells are lost), but also very slow, expensive, and requires a minimum of three months of labor before the therapeutic function can be tested. As a result, the discovery of antibodies with optimal therapeutic properties is a major unsolved bottleneck in drug development.

[0196] ASCs are terminally differentiated cells that cannot be directly grown in culture. As described above, existing methods for overcoming this problem (e.g., the hybridoma method, see Figure 60) are very inefficient and capture only a very small part of antibody diversity (usually <0.1%). These approaches are limited to use in rodents, are very slow, and expensive. They require several months of labor before the therapeutic function can be tested. As described below, the present invention overcomes these limitations by enabling a direct functional assay for antibodies and ASCs, regardless of the source.

[0197] A variety of techniques have advanced to improve the speed and throughput of antibody screening, and these techniques have advanced using information. Specifically, existing techniques are limited in the selection of antibodies based on binding, affinity, and specificity. While sufficient for research purposes, many therapeutic applications require high-affinity antibodies that bind rather than just binding to the target. Rather, therapeutic applications require antibodies that induce the desired biological response (e.g., agonists / antagonists of cell signaling; activation of the immune response; induction of apoptosis; inhibition of cell growth and differentiation). Currently, all high-throughput antibody detection techniques require this functional characterization to be performed downstream after target binding has been evaluated. As a result, a method that is cumbersome, costly, and low-throughput is used, even when compared to the hybridoma method. For this reason, the hybridoma method, developed over 40 years ago, is still the mainstay in therapeutic antibody discovery.

[0198] In one aspect, the present invention utilizes the ability of a microfluidic platform for large-scale parallel assays and small reaction volumes to screen cell populations for a desired property (hereinafter referred to as "extracellular effect"). Each cell population optionally includes one or more effector cells. The extracellular effect is not limited to a particular effect, but rather may be a binding property (specificity, affinity) or a functional property (e.g., antagonism or agonism of a cell surface receptor). In one embodiment, the extracellular effect is an effect exerted by the secretion of a particular effector cell.

[0199] The integrated microfluidic devices and methods provided herein are based in part on the concept that small things are sensitive. Each device includes thousands of nanoliter volume cell analysis chambers. Each chamber is approximately 100,000 times smaller than a conventional plate-based assay. In these small nanoliter chambers, each single effector cell produces high concentrations of biomolecules secreted within minutes. For example, each ASC produces antibodies at high concentrations within minutes. The effect of this concentration is utilized in one embodiment to perform a cell line screening assay. This assay identifies antibodies made by a single first ASC having specific functional characteristics such as the modulation of cell surface receptor activity (e.g., agonism or antagonism). Suitable functional assays for using the methods and devices provided herein are described in detail below. Importantly, in the screening methods provided herein, as long as the presence of an extracellular effect is detected within a specific microfluidic chamber containing a cell population, a specific effector cell or subpopulation of effector cells having specific properties need not be identified. Some or all of the cells within the chamber in which the effect is measured are retrieved for further characterization to identify the cell or cells responsible for the extracellular effect. By completely eliminating the need for cell culture prior to screening, the single cell approach provided herein enables, for the first time, the direct selection of functional antibodies from any species with a throughput of more than 100,000 cells per run in just a few days (Figure 1).

[0200] The microfluidic devices and methods provided herein offer strategic advantages over currently available strategies for evaluating the extracellular effects of single cells (e.g., the extracellular effects of antibodies secreted by a single ASC). For example, the devices described herein are scalable, and reduced reagent consumption and improved throughput can provide a large-scale single cell assay platform for research that would otherwise be unrealistic or prohibitively expensive. Also, currently available single cell assay platforms require handling and processing steps for multiple cells in conventional tubes to generate products required for downstream analysis (e.g., quantitative PCR). Thus, including microfluidic cell handling and processing as described herein provides an important means for improved throughput and cost while improving accuracy and sensitivity through the limitation of volumes.

[0201] Without being bound by theory, the improved concentration and rapid diffusion mixing provided by the nanoliter microfluidic chambers provided herein, in conjunction with accurate cell handling and manipulation (e.g., spatiotemporal control of media conditions), enable single cell analysis of effector cells such as immune cells (e.g., B cells, T cells, and macrophages), whose primary function involves the secretion of different effector proteins such as antibodies and cytokines.

[0202] Embodiments described herein provide a method capable of performing a multicellular assay of products secreted from a cell population comprising one or more effector cells, a microfluidic system, and subsequently provide for the recovery of the cell population for subsequent analysis. In some embodiments, the cell population is a heterogeneous cell population. That is, two or more cells in the population differ in genotype, phenotype, or some other characteristic. Further, the cell populations are assayed in parallel on one device. At least two populations are heterogeneous relative to each other (e.g., different numbers of cells, cell types, etc.). In the assays described herein, the readout particle population comprises one or more readout particles. The readout particles are such that a detection reagent (e.g., a readout cell expressing a cell receptor, a readout bead, a sensor, a soluble enzyme, etc.) is exposed to a cell population comprising one or more effector cells and a product secreted from one or more effector cells at a concentration sufficient (e.g., a fluorescence signal) for the detection of a readout signal. In some embodiments, the readout signal reports a biological response / functional effect (e.g., apoptosis) induced by one or more effector cells in a population of one or more readout particles (e.g., readout cells). For example, for an antibody produced by a given ASC, the cell population comprising one or more ASCs, along with the readout particles and optionally accessory detection reagents, is isolated in a small volume on the device. And the assay is performed (the chamber has a volume of about 100 pL to 50 nL. For example, about 1 nL to about 5 nL). Importantly, since the effector cells in one embodiment are rare cells, not all cell populations assayed by the methods described herein initially contain effector cells. For example, when thousands of cell populations are assayed on a single device, in one embodiment only a portion of the chambers contain effector cells. The methods provided herein enable the identification of effector cells and chambers.

[0203] The present invention employs an approach different from the aforementioned microfluidic methods. The latter takes the approach of loading single cells at a density that maximizes the number of single cells in individual chambers (e.g., droplets or microwells are used). This is achieved by isolating single cells by limiting dilution followed by analysis of a chamber or portion of a volume containing a single cell. Such a strategy sacrifices throughput. Because optimal single cell loading is achieved at about 1 cell per well average density. Similarly, the arrays described for these methods usually cannot accommodate more than a few cells in a chamber and are often designed to physically accommodate only a single cell. In addition to reduced throughput, many technical challenges arise from the approach of isolating and assaying single cells in a single microfluidic chamber. For example, keeping individual cells alive, achieving sufficient cell concentration to realize meaningful readouts from heterogeneous populations, due to nutrient depletion, the need for aeration, unwanted vapor permeation effects, poorly controlled media conditions, and the need for waste removal, pose serious problems for achieving reliable, reproducible single cell microfluidic assays. In many functional assays, it is necessary to maintain both effector cells and readout cells that survive for several days, similar to inhibition of cell proliferation. Such assays are not practical in microwell-based systems and droplets. However, as described herein, the present invention provides a robust platform for assays spanning several days.

[0204] The devices and assays described herein provide single cell assays. Thereby, one or more effector cells are present in individual cell populations within a single microfluidic chamber. The cell populations are assayed for their respective abilities to exert an extracellular effect within each chamber. Thereby, a higher overall throughput is provided than the methods described above. Importantly, the effect of a single effector cell is detected within a larger cell population (e.g., a heterogeneous cell population). By taking a multi-cell assay approach within a single microfluidic chamber, the embodiments described herein operate at a throughput more than 100-fold greater than previously reported throughputs. When a cell population discriminates an extracellular effect of the read-out particles, or a variation in extracellular effect as compared to another population, in one embodiment the cell population is recovered to determine whether effector cells within the population are the cause of the extracellular effect, and further assayed as individual cell sub-populations (e.g., the recovered cell population is assayed by limiting dilution).

[0205] Methods and devices known in the art that are designed to accommodate more than single cells have inappropriate limitations for the types of assays described herein. That is, for example, maintaining cells in a viable state, the inability to selectively recover the effector cells of interest, evaporation within the device, pressure fluctuations, cross-contamination, device architectures that limit imaging capabilities (e.g., providing particles at different focal planes, reduced resolution), and lack of throughput (WO2012 / 072822 and Bocchi et al. (2012), which are hereby incorporated by reference in their entirety).

[0206] The embodiments described herein relate to part of a functional effector cell assay (herein referred to as an extracellular effect assay). The assay enables the detection of a single effector cell of interest present within an individual microfluidic chamber in a heterogeneous cell population. Specifically, when the chamber contains a heterogeneous cell population, each cell of the population secretes an antibody (i.e., a heterogeneous ASC population within a single microfluidic chamber), or only some of the cells in the population secrete an antibody. Thereby, only one effector cell or a subpopulation of effector cells secretes an antibody that brings about the desired extracellular effect on the readout particles. The embodiments described herein provide a method for measuring and detecting a desired extracellular effect. Once the chamber is identified as containing a cell population that exhibits an effect, the population is recovered for downstream analysis, for example, by dividing the cell population into subpopulations by limiting dilution. In one embodiment, as described below, one or more heterogeneous populations of cells presenting an extracellular effect are recovered and further screened by limiting dilution (e.g., from 1 to about 25 cells per assay) to determine that the extracellular effect is cell-derived.

[0207] In one embodiment, the microfluidic assay is performed on a plurality of cell populations present in individual microfluidic chambers to determine whether effector cells in a population secrete an antibody or other biomolecule that inhibits the proliferation of the readout cells. In this embodiment, the proliferation of the readout cells is still equally inhibited even in the presence of a heterogeneous cell population containing a plurality of ASCs that secrete antibodies that do not affect the proliferation of the readout cells. The microfluidic chamber can be identified as containing the desired effector cells and secretions. The contents of the chamber are recovered, for example, by limiting dilution of the effector cells to determine that the effector cells present an effect, for further microfluidic analysis or benchtop analysis. The antibody sequences are also recovered by methods known to those skilled in the art.

[0208] In one embodiment, the novel antibodies are provided by the methods described herein. For example, one or more ASCs can be identified, recovered, and their antibody genes sequenced and cloned by the methods described herein.

[0209] When single cells are loaded into individual chambers at a cell density of about 1 cell per chamber, the devices provided herein enable screening of about 1000 single cells (e.g., ASCs) per experiment. The one or more single cells can be ASCs or different types of effector cells. Screening tens of thousands of cells, or even millions of cells, which is about 10 times higher than the hybridoma method, is often desirable. Examples of this include cases where ASCs are not obtained in high purity (e.g., for species where ASC markers / antibodies are not available, or for cases of poor immune responses), or where the antibodies that bind are frequent, but the antibodies with the desired properties are very rare (such as receptor blockade). However, after identifying a cell population containing one or more effector cells that exhibit the desired extracellular effect, the cell population is, in one embodiment, analyzed again (but by limiting dilution) as single cells, for example, within individual microfluidic chambers, or as small populations within individual chambers (compared to the first screen), in order to determine the identity of the individual effector cells responsible for the extracellular effect. One embodiment of this two-step screening method is shown in FIG. 2. Once the effector cells are identified, their genetic information is amplified and sequenced. In one embodiment, the genetic information includes novel antibody genes.

[0210] In the embodiment shown in FIG. 2, the microfluidic array is loaded at a density of approximately 25 cells per chamber, resulting in a total of approximately 100,000 cells in a single device. The chambers are then isolated and incubated to generate a unique polyclonal mixture of antibodies within each chamber. These antibodies are screened to identify chambers that exhibit a desired extracellular effect, such as antigen binding, high binding affinity, antigen specificity, or one or more functional properties. The contents of each positive chamber are then recovered. In one embodiment, the recovery of each population is done with a single microcapillary, and the contents of the chambers are pooled and reloaded into the microcapillary by limiting dilution in the same device or a different microfluidic device. In the embodiment shown in FIG. 2, cells from any of the arrays are reloaded into different chambers at a density of approximately 1 cell per chamber. The cells from the recovered populations are then rescreened for the same or different extracellular effects. The contents of the positive chambers from the second array are recovered, for example, by next-generation sequencing and / or PCR to identify the antibody sequences of interest. In one embodiment, the antibody sequences are sequenced and cloned. Thus, in one embodiment, the methods provided herein enable the discovery of novel antibody genes.

[0211] In another embodiment, a cell population presenting an extracellular effect is recovered using a system incorporating microfluidic valves that allow the chambers to be addressed individually (e.g., Singhal et al. (2010), Anal. Chem. 82, pp 8671-8679, which is incorporated herein by reference in its entirety). It should be noted that the present invention is not limited to the type of extracellular effect assay performed on the contents of the positive chambers of "Array 1" (FIG. 2). For example, in some embodiments, it may be desirable to further assay the contents of the positive chambers from "Array 1" via bench-top methods rather than from a second microfluidic array. Bench-top methods include, for example, RT-PCR and next-generation sequencing.

[0212] With respect to a single cell and the single and multi - cell microfluidic assays described herein, "chamber" is referred to herein. In that chamber, a cell population optionally containing one or more effector cells is assayed for an extracellular effect (e.g., a functional effect or a binding effect). However, one of ordinary skill in the art will recognize that the devices provided herein provide a large - scale parallel system incorporating hundreds of thousands of chambers. Also, one of ordinary skill in the art will recognize that the assay is performed in parallel on a plurality of individual cell populations optionally containing effector cells or a plurality of effector cells in all or substantially all of the chambers, or all of the chambers within a sub - array of the device. This is because due to the scarcity of some effector cells, not all cell populations will contain effector cells when present within the microfluidic chamber. Fluidic structures (e.g., multiplexers) for handling multiple chambers individually or together are described below.

[0213] Some of the embodiments described herein provide one or more of the following features.

[0214] The ability to concentrate and load a cell population containing one or more effector cells into a microfluidic chamber having a small volume for assaying effector cell products. The ability to co - localize with read - out particles (read - out beads, read - out cells, etc.) used to detect the presence of individual effector cell products (e.g., secreted proteins) having desired properties within the chamber.

[0215] The ability to maintain the growth and / or viability of a cell population assisted by the osmotic baths described herein and the ability to exchange the medium surrounding individual cells at concentrations reported to cause cell viability deficits or growth using conventional culture methods or microfluidic devices.

[0216] The ability to concentrate effector cell products within the chamber for a sufficient time to measure the properties of the desired effector cell products before the effector cells become unhealthy or grow larger than each microfluidic chamber.

[0217] The ability to add detection reagents to cell clusters or selectively exchange media contents while maintaining a population of cells within a microfluidic chamber.

[0218] The ability to recover a specified population of selected cells in a manner that enables their use of one or more microfluidic structures, manual methods, or robotic methods.

[0219] The ability to transfer a population of cells recovered for a second lower throughput screening that enables the analysis of effector cell products from each single cell in a heterogeneous population or multiple clones or one clone generated from each single cell in a heterogeneous population.

[0220] The ability to directly analyze aggregated genetic material from a recovered heterogeneous population of single cells and then use this information or genetic material to identify genes associated with the cells of interest.

[0221] In some embodiments, a method is provided for enriching effector cells that exert an extracellular effect from a starting population of cells. The starting population comprises one or more effector cells that exert an extracellular effect. In one embodiment, the method includes retaining a starting population of cells in a plurality of microfluidic chambers for obtaining a plurality of subpopulations of cells. The average number of effector cells per chamber is greater than Y, the total number of cells in the population is greater than X, and the expected proportion of effector cells in the population is 1 / X. The subpopulations of cells within the microfluidic chambers are subjected to an extracellular effect assay to identify one or more chambers that contain one or more effector cells presenting an extracellular effect. Based on the results of the extracellular effect assay, one or more chambers are then identified as containing one or more effector cells exhibiting an extracellular effect. The contents of the identified chambers are recovered to provide an enriched population of cells. The enriched population of cells enriched for effector cells has a proportion of effector cells of 1 / Y. In further embodiments, 1 / X is less than 0.05, or less than 0.01, or less than 0.001. The extracellular effect can be one or more of the extracellular effects described herein. In one embodiment, the starting population of cells is peripheral blood mononuclear cells (PBMCs) isolated from an immunized or antigen-exposed animal. In another embodiment, the starting population of cells is a population of B cells isolated from an immunized or antigen-exposed animal. In yet another embodiment, the source of the starting population of cells is whole blood from an immunized or antigen-exposed animal.

[0222] As provided herein, in one aspect, the devices and methods of the invention are used to assay a cell population optionally containing one or more effector cells for the presence of an extracellular effect. In another aspect, the devices and methods provided herein enable the identification of a cell population that exhibits variation in extracellular effects as compared to other cell populations. In this aspect, a plurality of individual cell populations are held within separate microfluidic chambers. At least one of the individual cell populations contains one or more effector cells. The separate microfluidic chambers further contain a population of readout particles containing one or more readout particles. The cell population is assayed for the presence of an extracellular effect. Thereby, the population of readout particles or a subpopulation thereof provides a readout of the extracellular effect. One cell population from among the plurality of cell populations can be identified as exhibiting variation in extracellular effects as compared to one or more of the remaining cell populations among the plurality of cell populations. Once a cell population is confirmed to exhibit variation in extracellular effects, the population can be recovered and further assayed by limiting dilution to identify the cell or cells within the population that are responsible for the extracellular effect.

[0223] The cell populations analyzed herein are not limited to a particular type. For example, in one embodiment, the starting population of cells divided into individual cell populations in a microreactor can be peripheral blood mononuclear cells (PBMCs) isolated from an immunized or antigen-exposed animal. In another embodiment, the starting population of cells is B cells isolated from an immunized or antigen-exposed animal. The source of the starting population of cells can be whole blood from an immunized or antigen-exposed animal.

[0224] As used herein, "effector cell" refers to a cell having the ability to exert an extracellular effect. As described in detail below, the cellular effect is a direct or indirect effect of the readout particle. The extracellular effect is due to an effector cell or a molecule secreted by an effector cell (e.g., a signaling molecule, metabolite, antibody, neurotransmitter, hormone, enzyme, cytokine). In one embodiment, the effector cell is a cell that secretes or presents a protein (e.g., a T cell receptor). In the embodiments described herein, the extracellular effect is characterized by using a readout particle (e.g., a readout cell or readout bead, or a readout particle population or subpopulation). For example, in one embodiment described herein, the extracellular effect is agonizing or antagonizing a cell surface receptor, ion channel, or ATP-binding cassette (ABC) transporter present on the readout cell or readout bead. In one embodiment, the effector cell is an antibody-secreting cell (ASC). ASC as used herein refers to any cell type that produces and secretes an antibody. Plasma cells (referred to as "plasma B cells", "plasmacytes" and "effector B cells") are terminally differentiated and are a type of ASC. For the purposes of the present invention, other ASCs considered to be "effector cells" include cells generated by the proliferation of plasmablasts, memory B cells, cell lines expressing recombinant monoclonal antibodies, hybridoma cell lines. In another embodiment, the effector cell is a cell that secretes a protein. Other cell types considered to be effector cells include T cells (e.g., CD8+ T cells and CD4+ T cells), hematopoietic cells, cell lines derived from humans and animals, recombinant cell lines (e.g., recombinant cell lines engineered to produce an antibody, recombinant cell lines engineered to express a T cell receptor).

[0225] Individual cell populations optionally containing one or more effector cells are assayed to determine whether each cell population contains effector cells that exert an extracellular effect or a variation in extracellular effect compared to other individual cell populations or pluralities thereof. As noted above, when cell populations are assayed in parallel on one device, not all cell populations will contain effector cells. The methods described herein enable the identification of cell populations containing one or more effector cells. Also, a cell population containing effector cells need not contain a plurality of effector cells or need not be a population of only effector cells. Rather, in the embodiments described herein, non-effector cells are included in the population. The non-effector cells may be the majority or minority of the population. A heterogeneous population containing effector cells need not contain a plurality of effector cells. Rather, a heterogeneous cell population is heterogeneous as long as two cells are heterogeneous with respect to each other. A cell population may not contain effector cells or may contain one effector cell or pluralities of effector cells. Similarly, a subpopulation of cells may not contain effector cells or may contain one effector cell or pluralities of effector cells.

[0226] An extracellular effect in one embodiment is a binding interaction with an antigen or a functional effect. For example, in one embodiment, the extracellular effect is agonism or antagonism of a cell surface receptor, agonism or antagonism of an ion channel or an ABC transporter, regulation of apoptosis, regulation of cell proliferation, change in the morphological appearance of a readout particle, change in the localization of a protein within a readout particle, protein expression by a readout particle, neutralization of the biological activity of an accessory particle, cell lysis of a readout cell induced by an effector cell, cell apoptosis of a readout cell induced by a cell effector cell, necrosis of a readout cell, internalization of an antibody by a readout cell, internalization of an accessory particle by a readout cell, enzyme neutralization by an effector cell, neutralization of a soluble signaling molecule, or combinations thereof.

[0227] Identifying the presence of effector cells that secrete a biomolecule (e.g., an antibody) that binds to a target of interest (e.g., an antigen) is readily confirmed in embodiments where the effector cells are present in a heterogeneous cell population that includes a plurality of effector cells that secrete antibodies that are not specific for the target of interest. In one embodiment, this is achieved within individual microfluidic chambers by first capturing all or substantially all of the secreted antibodies of the population on a readout particle (e.g., a bead) functionalized to capture antibodies (e.g., functionalized with protein G or protein A) in a chamber, adding a fluorescently labeled antigen into the chamber, and imaging the particles to detect the presence or absence of an increase in fluorescence due to binding of the antigen to the immobilized antibodies. An estimate of the minimum number of antibodies captured on the beads required for reliable detection is obtained by performing experiments that measure antibody secretion from single cells. In one embodiment, antigen-specific antibodies secreted from a single ASC in a heterogeneous population of about 500 cells can be detected. In the case of the present invention, the cell population present within an individual microfluidic chamber can include from about 2 to about 500 cells (e.g., from about 2 to about 250 ASCs). As described above, the cell population can include cells other than effector cells, and not all cell populations include effector cells. This is particularly true when conventional enrichment protocols (e.g., FACS) cannot be used to obtain a substantially pure cell population of the same cell type.

[0228] For a heterogeneous cell population (not all cells are heterogeneous with respect to each other), assume that there are at least two cells in the population that are heterogeneous with respect to each other (effector cells and non-effector cells). The heterogeneous cell population may consist of only two cells. A cell population or cell subpopulation may consist of a single cell. In principle, a heterogeneous cell population may contain any number of cells that are maintained in an executable state for the required period of an extracellular effect assay (e.g., one of the extracellular effect assays provided herein). In one embodiment, the number of cells in the cell population is from 1 cell per chamber to about 500 cells per chamber. In one embodiment, when imaging of individual cells or readout particles is required, the number of cells in the population is selected to be insufficient to cover the floor of the chamber. As a result, the cells to be imaged are arranged in a monolayer. Alternatively, the cell population contains many cells that are insufficient to form a bilayer covering the surface of the chamber.

[0229] In some embodiments, a large population of cells can be present in a single microfluidic chamber or microreactor without inhibiting the detection of effects arising from a single effector cell or a small number of effector cells within the particular population. For example, in one embodiment, the number of cells in the cell population is from 2 to about 900, or from about 10 to about 900, or from about 100 to about 900. In another embodiment, the number of cells in the cell population is from 2 to about 800, or from about 10 to about 800, or from about 100 to about 800. In another embodiment, the number of cells in the cell population is from 2 to about 700, or from about 10 to about 700, or from about 100 to about 700. In another embodiment, the number of cells in the cell population is from 2 to about 600, or from about 10 to about 600, or from about 100 to about 600. In other embodiments, the number of cells in the cell population is from 2 to about 500, or from about 10 to about 500, or from about 100 to about 500. In another embodiment, the number of cells in the cell population is from 2 to about 400, or from about 10 to about 400, or from about 100 to about 400. In another embodiment, the number of cells in the cell population is from 2 to about 300, or from about 10 to about 300, or from about 100 to about 300. In another embodiment, the number of cells in the cell population is from 2 to about 200, or from about 10 to about 200, or from about 100 to about 200. In another embodiment, the number of cells in the cell population is from 2 to about 100, or from about 10 to about 100, from about 50 to about 100. In another embodiment, the number of cells in the cell population is from 2 to about 90, or from about 10 to about 90, or from about 50 to about 900. In yet another embodiment, the number of cells in the cell population is from 2 to about 80, or from about 10 to about 80, or from 2 to about 70, or from about 10 to about 70, or from about 2 to about 60, or from about 10 to about 60, or from about 2 to about 50, or from about 10 to about 50, or from about 2 to about 40, or from about 10 to about 40, or from 2 to about 30, or from about 10 to about 20, or from 2 to about 10. In some embodiments, most of the cells in the cell population are effector cells.

[0230] In one aspect of the invention, the cells or cell populations analyzed by the methods provided herein comprise one or more effector cells (e.g., antibody-secreting cells (ASCs) or multiple ASCs). In one embodiment, the cells are separated into multiple cell populations in thousands of microfluidic chambers. And individual cell populations (i.e., within a single microfluidic chamber) containing one or more effector cells are assayed for extracellular effects. When the effector cells within one or more populations exert a variation in extracellular or extracellular effects, one or more individual cell populations are identified and recovered. The extracellular effect is determined by the user and, in one embodiment, is a binding interaction with an antigen, cell surface receptor, ABC transporter, or ion channel.

[0231] The methods provided herein are used to identify single effector cells (alone or within a heterogeneous population) based on binding interactions (e.g., antigen affinity and specificity), but the invention is not limited thereto. Rather, in one embodiment, the identification of cell populations is performed directly through the implementation of functional assays. Accordingly, one aspect of the invention includes methods and devices that enable the direct detection of ASCs within cell populations that secrete "functional antibodies" without the need to first screen for "functional antibodies" for binding properties (e.g., affinity and selectivity for antigen targets).

[0232] Along these directions, in one aspect, receptors and functional antibodies that can be discovered by the methods herein are provided. In one embodiment of this aspect, the nucleic acids of effector cells that cause extracellular effects are amplified and sequenced. The nucleic acids are genes encoding secreted biomolecules (e.g., antibodies or fragments thereof), or genes encoding cell receptors or fragments thereof (e.g., T cell receptors). The antibody or fragment thereof or cell receptor or fragment thereof is cloned and / or sequenced by methods known in the art. For example, in one embodiment, an ASC that secretes a functional antibody that can be discovered by the methods and devices provided herein modulates cell signaling by binding to a target cell surface protein (e.g., an ion channel receptor, an ABC transporter, a G-protein coupled receptor (GPCR), a receptor tyrosine kinase (RTK), or a receptor having an intrinsic enzymatic activity such as an intrinsic guanylate cyclase activity).

[0233] In one aspect of the invention, a cell population comprising one or more effector cells is identified in a microreactor (e.g., a microfluidic chamber) based on the results of an extracellular effect assay performed in a chamber. When an extracellular effect or variation in an extracellular effect is measured in the microreactor, the cell population is recovered and analyzed (e.g., FIG. 2) to determine the effector cell or effector cells within the population that are the cause of the effect. In embodiments where the effector cell secretes an antibody, the DNA sequence encoding the antibody produced by the ASC or ASCs is determined and subsequently cloned. In one embodiment, the DNA sequence of the antibody is expressed and cloned in a cell line to provide an immortal source of monoclonal antibodies for further validation and preclinical testing.

[0234] As described herein, a heterogeneous cell population typically comprises a population of cells having a number in the range of from 2 to about 1000, or from 2 to about 500, or from about 2 to about 250, or from about 2 to about 100. A heterogeneous cell population characterizes a population of cells. The population of cells includes at least two cells having fundamental differences in genotype, protein expression, mRNA expression or differentiation state (where at least one cell is an effector cell). In particular, a heterogeneous cell population, in one embodiment, includes two or more effector cells (e.g., from about 2 to about 250 cells). The two or more effector cells express or contain different immunoglobulin genes, express or contain different genes derived from immunoglobulin genes, express or contain different genes derived from T cell receptor genes, secrete different immunoglobulin proteins, secrete different proteins derived from immunoglobulin proteins, secrete different proteins, or express different proteins derived from T cell receptor proteins.

[0235] In one aspect, the cell population includes cells genetically engineered to express a library of molecules capable of binding to a target epitope, cells genetically engineered to express a fragment of a gene or a gene from a cDNA library of interest, cells genetically engineered with reporters for various biological functions, and cells from immortalized cell lines or primary sources. Notably, clones derived from a single cell are, in one embodiment, heterogeneous with respect to each other due to, for example, gene silencing, differentiation, changes in gene expression, morphological changes, etc. In addition, cells from immortalized cell lines or primary sources are considered to be heterogeneous with respect to each other, rather than being the same clone of a single cell. Rather, a clonal population of cells is derived from a single cell and is not genetically modified, not transduced with RNA, not transduced with DNA, not infected with a virus, not differentiated, or not otherwise manipulated by important functional and molecular means to create different cells. Cells derived from a single cell (but which undergo somatic hypermutation naturally or are engineered to undergo somatic hypermutation (e.g., by inducing the expression of activation-induced cytidine deaminase, etc.)) are not considered clones. Thus, these cells, when present together, are considered a heterogeneous cell population.

[0236] Throughout, in one aspect, methods and devices are provided for the assay of a plurality of individual cell populations optionally including one or more effector cells to identify one or more cell populations. The cell population includes at least one effector cell having a cell effect (e.g., secretion of a biomolecule having a desired property) with a population of readout particles or a subpopulation thereof. When a cell population is identified, in one embodiment, it is selectively recovered to obtain the recovered cell population. When a plurality of cell populations are identified, in one embodiment, they are recovered and pooled to obtain the recovered cell populations. The recovered cell populations are enriched for effector cells and compared to the starting population of cells initially loaded onto the device. In this regard, the former has a higher proportion of effector cells compared to the latter.

[0237] Subpopulations of the recovered cell population are assayed for the presence of a second extracellular effect of the readout particle population. The readout particle population or subpopulation provides a readout of that second extracellular effect. The extracellular effect can be the same effect as the effect assayed in the identified cell population, or a different extracellular effect. In further embodiments, subpopulations of the identified population each contain from about 1 to about 10 cells. In even further embodiments, subpopulations of the identified population each contain on average 1 cell. One or more subpopulations presenting the extracellular effect are then identified and recovered to obtain the recovered subpopulations. It is, in one embodiment, enriched for effector cells. When multiple cell subpopulations are identified, in one embodiment they are recovered and pooled to obtain the recovered cell subpopulations. The genetic information from the recovered cell subpopulations is then isolated, amplified, and / or sequenced.

[0238] The present invention is not limited by the type of effector cell or cell population that can be assayed according to the methods of the present invention. Examples of the types of effector cells used in the present invention include, for example, the above, primary antibody-secreting cells from any species (e.g., human, mouse, rabbit, etc.), primary memory cells (e.g., which can assay for IgG, IgM, IgD, or other immunoglobulin antibodies presented on the surface of the cell, or be grown / differentiated into plasma cells), T cells, hybridoma fusions immediately after selection or fusion, cell lines (either stable or transient) into which nucleic acids have been introduced with one or more libraries of monoclonal antibodies (mAbs) (e.g., for affinity maturation of mAbs identified using a library of mutants in the fab region, or for optimization of effector function using identified mAbs having mutations in the Fc region), combinations of heavy chains (HC) and light chains (LC) from amplified HC / LC variable regions obtained from human / animal / libraries, or cell lines into which nucleic acids have been introduced with combinations of cell-expressed mAbs (either characterized or uncharacterized for exploring synergistic effects).

[0239] Plasma cells (also called "plasmablasts", "plasmocytes" and "effector B cells") are terminally differentiated and are a type of effector cell (ASC) that can be assayed with the devices and methods of the present invention. Other ASCs considered as "effector cells" for the purposes of the present invention include plasmablasts, cells generated by the proliferation of memory B cells, cell lines expressing recombinant monoclonal antibodies, primary hematopoietic cells secreting cytokines, T cells (e.g., CD4+ and CD8+ T cells), dendritic cells presenting proteins or peptides on their surface, recombinant cell lines secreting proteins, hybridoma cell lines, recombinant cells engineered to produce antibodies, and recombinant cells engineered to express T cell receptors.

[0240] Also, it is understood that the cell populations for use in the present invention are not limited by their source, rather, they may be from human or other mammalian or any animal including other sources, from in vitro tissue cultures. Cells may be directly analyzed after enrichment of a population having desired characteristics (e.g., secretion of an antibody that binds to a specific antigen) by using various protocols known in the art (e.g., flow cytometry), or may be analyzed directly after collection from the source, for example. In one embodiment, the animal is immunized with one or more immunizations prior to collection from the animal source. In one embodiment, flow cytometry is used to enrich effector cells prior to loading onto one of the devices provided herein. And the flow cytometry is fluorescence-activated cell sorting (FACS). When the starting cell population is enriched for effector cells (e.g., ASCs) and held as individual cell populations within individual microfluidic chambers, the individual cell populations need not be composed entirely of effector cells. Rather, other cell types may be present as the majority or minority. Further, one or more individual cell populations may not contain effector cells.

[0241] There are several methods known to those skilled in the art for concentrating animal-derived ASCs. It is used to concentrate the starting population of cells for analysis by the methods and devices provided herein. For example, in one embodiment, FACS uses surface markers CD19+CD20 low CD27 hi CD38 hi to concentrate human ASCs (Smith et al. (2009). Nature Protocols, 4. pp. 372-384, which is incorporated herein by reference in its entirety). In another embodiment, the cell population is concentrated by magnetic immunocapture system positive or negative selection of cells presenting surface markers. In another embodiment, plaque assay (Jerne et al. (1963). Science 140, p. 405, which is incorporated herein by reference in its entirety), ELISPOT assay (Czerkinsky et al. (1983). J. Immunol. Methods 65, pp.109-121, which is incorporated herein by reference in its entirety), droplet assay (Powel et al. (1990). Bio / Technology 8, pp. 333-337, which is incorporated herein by reference in its entirety), cell surface fluorescence-conjugated immunosorbent assay (Yoshimoto et al. (2013). Scientific Reports, 3, 1191, which is incorporated herein by reference in its entirety) or cell surface affinity matrix assay (Manz et al. (1995). Proc. Natl. Acad. Sci. U.S.A. 92, pp. 1921-1925, which is incorporated herein by reference in its entirety) is used to concentrate ASCs before performing one of the methods provided herein or loading the starting cell population into one of the devices provided herein.

[0242] In various embodiments, two or more effector cells within a cell population produce and secrete a cellular product (e.g., an antibody that has a direct or indirect effect on a population of readout particles or a subpopulation thereof). With respect to the devices provided herein, note that not all chambers on the device necessarily contain a cell population and / or a population of readout particles. For example, there may be empty chambers or partially filled chambers. Also, as provided overall, it may be only a subset of the cells within the cell population, or individual cells within a population that produce and secrete an antibody, within an individual chamber. In some embodiments, the cell population within the microfluidic chamber does not contain effector cells. These chambers can be identified by performing one or more extracellular effect assays on each of the cell populations.

[0243] In some aspects, it is desirable to have one or more accessory particles. The accessory particles can include one or more accessory cells present in a microreactor (e.g., a microfluidic chamber) to support the viability and / or function of one or more cells within the cell population or to perform an extracellular effect assay. For example, in one embodiment, the accessory cell or accessory cells include fibroblasts, natural killer (NK) cells, killer T cells, antigen-presenting cells, dendritic cells, recombinant cells, or combinations thereof.

[0244] In one embodiment, an accessory particle or cell, or a population containing the same, is carried to a microreactor (e.g., a microfluidic chamber) together with the cell population. In other words, the accessory cells in one embodiment are part of the cell population that is transported to the microfluidic chamber. Alternatively or additionally, the accessory particle or accessory cell is sent to the chamber before and after loading a heterogeneous population of cells containing effector cells or a plurality of effector cells into a microreactor or a plurality of microreactors (e.g., a microfluidic chamber or a plurality of microfluidic chambers).

[0245] As used herein, the term "accessory particle" refers to any particle including, but not limited to, proteins, protein fragments, and cells. Such particles (i) support the viability and / or function of effector cells, (ii) facilitate extracellular effects, (iii) facilitate the measurement of extracellular effects, or (iv) detect extracellular effects of effector cells.

[0246] Accessory particles include, but are not limited to, proteins, peptides, growth factors, cytokines, neurotransmitters, lipids, phospholipids, carbohydrates, metabolites, signaling molecules, amino acids, monoamines, glycoproteins, hormones, virus particles, or combinations thereof. In one embodiment, one or more accessory particles include sphingosine-1-phosphate, lysophosphatidic acid, or combinations thereof.

[0247] In one embodiment, as an example of accessory cells, a population of fibroblasts (which do not secrete antibodies) is included in a cell population enriched for effector cells (e.g., ASCs) to enhance the survival rate of effector cells (e.g., ASCs) within the population. In another embodiment, a population of NK cells may be added as accessory particles to perform an antibody-dependent cell-mediated cytotoxicity assay. NK cells attack and lyse target cells upon binding of antibodies to their surface. In embodiments where a functional cell assay is performed on one or more cell populations, it is understood that effector cells within the one or more cell populations need to maintain viability for an extended period of time while within the chamber of the microfluidic device. For this purpose, accessory particles and / or accessory cells are used in one embodiment to maintain the viability of a cell population optionally containing one or more effector cells. As described below, accessory particles, e.g., accessory cells, are also used to improve or maintain the viability of a readout cell population or a subpopulation thereof. Either may be a single readout cell.

[0248] One advantage of the embodiments described herein is that the analysis of more than one effector cell within a single microreactor (e.g., a microfluidic chamber), and / or the analysis of single or few effector cells in the presence of other cells, enables a much greater assay throughput and thus enables the identification and selection of desired effector cells. Otherwise, the desired effector cells are too rare to be efficiently detected. This is advantageous in many cases, such as when methods for enriching for the desired cell type are limited or when such enrichment has a detrimental effect of reducing the viability of the assayed cells. One embodiment of the invention constructed features an array of 3500 cell analysis chambers. This device, when operated with an average of 30 cells per chamber, has an overall assay throughput of 105,000 cells per experiment. This throughput is used, for example, for the selection of dozens or hundreds of effector cells present at less than 1% of the total cell population.

[0249] For example, antibody-secreting cells can be identified and isolated without the need for enrichment based on surface markers. In B cells isolated from peripheral blood mononuclear cells (PBMCs) after immunization, the frequency of ASCs may be between 0.01% and 1%. With a throughput of 105,000 cells per device operation, hundreds of ASCs can be directly selected without further purification. This is particularly important since FACS purification of ASCs can reduce cell viability. This is also important since appropriate reagents for ASC enrichment may not be available for the host species of interest. Indeed, after immunization, the frequency of antibody-secreting cells in peripheral blood mononuclear cells (PBMCs) may be between 0.01% and 1% and can be detected using the microfluidic arrays provided herein (e.g., a microfluidic array of 3500 chambers loaded at an average density of 30 cells per chamber). Thus, since isolation of peripheral blood mononuclear cells (PBMCs) is performed for any species without specific capture reagents, some of the methods of the invention provide a rapid and economical selection of cells secreting the antibody of interest from any species.

[0250] In one embodiment, the ASC from the human basal level is identified by the methods and devices provided herein. Animals are immunized to generate new antibodies against many antigens. On the other hand, except for approved vaccines, the same procedure cannot be widely performed on humans. However, humans who are naturally exposed to antigens or vaccinated at some point in their lives generally have a low basal level of antibody-secreting cells against the antigens. The present invention can be used to identify and isolate very rare effector cells that secrete specific antibodies from a large number of cells (e.g., more than 100,000 per device operation). Such methods are used herein, for example, for the detection of functional antibodies as therapeutic agents for autoimmune diseases and cancers where autoantibodies are present.

[0251] As provided throughout, the present invention relates in part to extracellular effects that are performed in large-scale parallel processing within a single microfluidic chamber. Assays are performed to measure and detect extracellular effects exerted by effector cells present in a cell population, or a plurality thereof. A population of readout particles, or a subpopulation thereof, provides a readout of the extracellular effect. For example, the methods described herein enable the identification of heterogeneous cell populations containing effector cells that exert an extracellular effect (e.g., secretion of an antibody specific for a desired light source) against a background of up to about 250 cells that do not exert the extracellular effect.

[0252] "Readout particle" as used herein means any particle, including beads or cells (e.g., functionalized beads or cells that report a function or property), or is used in an assay to determine an extracellular effect (e.g., a function or property) of an effector cell. As described herein, a "readout particle" can exist as a single readout particle or can be present within a homogeneous or heterogeneous population of readout particles within a single microfluidic chamber. In one embodiment, the readout particle is a functionalized bead for binding to one or more biomolecules secreted by an effector cell (e.g., one or more antibodies), or is a bead released by an effector or accessory cell upon lysis. A single readout particle may be functionalized to capture one or more different types of biomolecules (e.g., proteins and / or nucleic acids) or one or more different monoclonal antibodies. In one embodiment, the readout particle is a cell or bead that can bind to an antibody produced by an effector cell that produces and / or secretes the antibody. In some embodiments, the effector cell may also be a readout particle. For example, the secretion of one effector cell within a population has an effect on a larger or different subpopulation of effector cells. Alternatively, the secretion of one effector cell is taken up by the same cell for complementary readout.

[0253] "Readout cell" as used herein is a type of readout particle that exhibits a response in the presence of a single effector cell or a population of effector cells, including one or more effector cells (e.g., one or more effector cells that secrete antibodies). In various embodiments, the readout cell is a cell that presents a surface antigen or receptor specific for a secreted molecule (e.g., a GPCR or RTK). In one embodiment, the binding of the secreted molecule to the readout cell is an extracellular effect. The readout cell is fluorescently labeled and / or has a fluorescent reporter that is activated upon binding.

[0254] As described above, in some embodiments, the cell population subjected to the methods described herein comprises ASCs or a plurality of ASCs. And the readout particle population or a subpopulation thereof presents a plurality of target epitopes or one target epitope. The readout particle population in one embodiment is a population of beads functionalized to capture antibodies by a particular epitope or epitopes. Alternatively or additionally, the readout particle population is specific for the Fc region of the antibody. Thus, the readout particle population does not distinguish between antibodies having different epitopes. In one embodiment, the readout particle population or a subpopulation thereof is labeled with a fluorescent conjugate molecule containing a target epitope, for example, to perform an ELISA assay. Fluorescent antibody and cytokine bead assays are known in the art (e.g., Singhal et al. (2010). Anal. Chem. 82, pp. 8671-8679, Luminex® Assays (Life Terchnologies), BD™ Cytometric Bead Array, the disclosures of which are incorporated by reference in their entirety). These methods are used herein to determine whether effector cells have extracellular effects on the readout particles.

[0255] Also, as described herein, individual microreactors (e.g., microfluidic chambers) are configured such that their reagent exchange is possible within the individual chambers, thereby removing or substantially removing cross-contamination between the chambers. This enables the detection of multiple extracellular effects (e.g., functional effects and / or multiple antigen-binding effects within a single chamber) within a single chamber, for example, by exchanging the antigen and secondary antibody to label each binding complex, followed by imaging. In these embodiments of a series of detections, each reaction is performed continuously after a washing step, so that the assay can be performed using the same fluorophore. Alternatively, different fluorophores are used to detect different extracellular effects in a single microreactor (e.g., a microfluidic chamber) in a sequential or parallel manner.

[0256] In another embodiment, the readout particle population is a readout cell population. At least a portion of the readout cells present a target epitope on their surface. In one embodiment, the readout cell population or a subpopulation thereof is viable and capable of survival. In another embodiment, the readout cell population or subpopulation is fixed. As will be appreciated from the above description, when antibody binding is assayed, "antibody binding" is considered to be an extracellular effect of one effector cell or a plurality of effector cells. Antibody binding can be detected, for example, by staining the cells with one or more fluorescently labeled secondary antibodies. In another embodiment, as described herein, the binding of an antibody to a target epitope on a readout particle or readout cell causes death of the readout cell or some other readout cell response (e.g., secretion of biomolecules, activation or inhibition of a cell signaling pathway).

[0257] The readout cells in the population are distinguished by characteristics such as, for example, morphology, size, surface attachment, motility, fluorescence response, etc. For example, in one embodiment, the population of readout cells is labeled on its surface or intracellularly to determine whether the readout cells exhibit a response as selected by the user of the assay. For example, calcein, carboxyfluorescein succinimidyl ester reporter (CFSE), GFP / YFP / RFP reporters are used to label one or more reporter cells including extracellular receptors and intracellular proteins and other biomolecules.

[0258] In some embodiments, the readout particle population is a heterogeneous readout particle population and can be a heterogeneous readout cell population. Here, for example, one or more ASCs are present in the cell population. Individual readout particles within the population may present different target epitopes, or two different cell receptors (e.g., GPCRs or RTKs or combinations thereof). Thus, the specificity of the extracellular effect (e.g., the specificity of an antibody for a target epitope or the inhibition of a specific cell surface receptor) is evaluated. In another embodiment, the effector cells within the cell population are ASCs and the readout particle population comprises a heterogeneous bead population. The population non-selectively captures all antibodies (e.g., the Fc specific region) and a bead population specific for the native target epitope.

[0259] In one embodiment, accessory particles are provided to facilitate the measurement of an extracellular effect or to facilitate the readout of an extracellular effect. As described throughout, the extracellular effect includes the effect exerted by the secreted product (e.g., an antibody) of an effector cell. For example, in one embodiment, natural killer (NK) cells are provided as accessory particles to facilitate the measurement of the lysis of readout cells. In this embodiment, the extracellular effect includes the lysis of readout cells that bind to a specific epitope or cell receptor by natural killer (NK) cells when an antibody secreted by the effector cell binds to the readout cell.

[0260] In some embodiments, the accessory particles include a protein, protein fragment, peptide, growth factor, cytokine, neurotransmitter (e.g., neuromodulator or neuropeptide), lipid, phospholipid, amino acid, monoamine, glycoprotein, hormone, virus particle, or factors necessary to activate the complement pathway when binding effector cell secretions to the readout cells, or combinations thereof. In one embodiment, the one or more accessory particles include sphingosine-1-phosphate, lysophosphatidic acid, or combinations thereof. The various cell effects measurable with the devices and methods provided herein (including lysis of readout cells that bind antibodies) are described in detail below.

[0261] For example, cytokines that can be used as accessory particles include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. In some embodiments, the accessory particles are produced by the readout cells. In some embodiments, the cytokine is used as an accessory particle and is one or more cytokines provided in Table 1 below. In another embodiment, one or more of the following cytokines are used as accessory particles. For example, interleukin (IL)-1α, IL-1β, IL-1RA, IL18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL17, IL-18, IL-19, IL-20, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor, oncostatin M, interferon (IFN)-α, IFN-β, IFN-γ, CD154, lymphotoxin β (LTB), tumor necrosis factor (TNF)-α, TNF-β, transforming growth factor (TGF)-β, erythropoietin, megakaryocyte growth and development factor (MGDF), FMS-related tyrosine kinase 3 ligand (FLT-3L), stem cell factor, colony-stimulating factor-1 (CSF-1), macrophage-stimulating factor, 4-1BB ligand, proliferation-inducing ligand (APRIL), cluster of differentiation 70 (CD70), cluster of differentiation 153 (CD153), cluster of differentiation 178 (CD17)8, glucocorticoid-induced TNF receptor ligand (GITRL), LIGHT (TNF ligand superfamily member 14, also known as HVEM ligand, CD258), OX40L (also designated as CD252 and is a ligand for CD134), TALL-1, TNF-related apoptosis-inducing ligand (TRAIL), tumor necrosis factor weak inducer of apoptosis (TWEAK), TNF-related activation-induced cytokine (TRANCE), or combinations thereof.

Table 1

[0262] In one embodiment, the accessory particles are cytokines or other factors operable to stimulate a response of the readout cells. For example, the readout cells are incubated with one effector cell or a plurality thereof and pulsed with cytokines operable to affect the readout cells. Alternatively or additionally, cytokine-secreting cells operable to affect the readout particles are supplied into the chamber as accessory cells. In one embodiment, neutralization of cytokines secreted by the secretions of effector cells is detected by the lack of the predicted effect of the cytokines on the readout cells. In another embodiment, the accessory particles are viruses operable to infect one or more readout cells. And neutralization of the virus is detected as a decrease in infection of the readout cells by the virus.

[0263] As is apparent from the discussion above with respect to accessory particles, the extracellular effects measurable and detectable by the devices and methods provided herein are not limited to the binding of antibodies to target epitopes. Rather, in one embodiment, the extracellular effects as described herein are functional effects. Functional effects in one embodiment include apoptosis, regulation of cell proliferation, changes in the morphological appearance of readout particles, changes in the aggregation of multiple readout particles, changes in the localization of proteins within readout particles, protein expression by readout particles, protein secretion by readout particles, induction of cell signaling cascades, readout cell internalization of molecules secreted by effector cells, neutralization of accessory particles operable to affect readout particles, and the like.

[0264] When an extracellular effect is identified within a microreactor (e.g., a microfluidic chamber) containing a cell population, the population is recovered. A downstream assay is performed on a subpopulation of the recovered cell population to determine whether the effector cells were the cause of the extracellular effect that was measured. The downstream assay in one embodiment is a microfluidic assay. In a further embodiment, the downstream assay is performed on the same device as the first extracellular effect assay. However, in another embodiment, the downstream assay is performed on a different microfluidic device or via a non-microfluidic method (e.g., a benchtop single cell reverse transcriptase (RT)-PCR reaction). The antibody gene sequences of the effector cells identified and recovered in one embodiment are isolated, cloned, and expressed to provide novel functional antibodies.

[0265] The functional effects of a single ASC can be measured by the methods and devices provided herein, but the affinity, binding, and specificity can also be measured as an "effect" of the effector cells (e.g., the effect of effector cell secretions). For example, the binding assay provided by Dierks et al. (2009). Anal. Biochem. 386, pp.30-35 (which is hereby incorporated by reference in its entirety) is used in the devices provided herein to determine whether the ASC secretes an antibody that binds to a specific target.

[0266] In another embodiment, the extracellular effect is the affinity for an antigen or cell receptor. And the method described by Singhal et al. (2020). Anal. Chem. 82, pp. 8671-8679 (which is hereby incorporated by reference for all purposes) is used to assay the extracellular effect.

[0267] In one embodiment, the parallel analysis of multiple extracellular effects is performed in a single microreactor (e.g., a microfluidic chamber) by using multiple types of readout particles. Alternatively or additionally, the parallel analysis of multiple functional effects is performed on a single microfluidic device by using different readout particles in at least two different chambers.

[0268] In one embodiment, the readout particle is an enzyme that exists as a soluble molecule. Or it is an enzyme that is immobilized on the surface of the microfluidic chamber or on another physical support within the readout zone of the chamber. In this case, in one embodiment, the binding of an antibody that inhibits the enzymatic activity of the readout particle is detected by a reduced signal that reports on the enzymatic activity (including a fluorescence signal or a colorimetric signal or a precipitation reaction).

[0269] In various embodiments, determining whether one effector cell or multiple effector cells, a population of readout particles or a subpopulation thereof in a cell population has an extracellular effect involves optical and / or fluorescence microscopy of the microfluidic chamber containing the cell population. Accordingly, one embodiment of the present invention includes the step of maintaining a population of readout particles in a single plane so as to facilitate imaging of the particles by microscopy. In one embodiment, the population of readout particles within the chamber is maintained in a single plane. That single plane is imaged through the device material or a portion thereof (e.g., glass or PDMS) to generate one or more high-resolution images of the chamber. In one embodiment, the high-resolution image is an image equivalent to that achieved using standard microscopy with equivalent optical equipment (such as lenses, objectives, illumination, contrast mechanisms, etc.).

[0270] According to one aspect of the present invention, a method is provided for identifying a cell population comprising one or more effector cells that exhibit variability in extracellular effects. In one embodiment, the method includes the step of holding a plurality of individual cell populations within separate microreactors (e.g., microfluidic chambers). At least one of the individual cell populations includes the contents of a separate microfluidic chamber and one or more readout particles, and further includes a readout particle population comprising one or more populations of readout particles. The cell population and the readout particle population are incubated within the microreactor, and the cell population is assayed for the presence of extracellular effects. The readout particle population or a subpopulation thereof provides a direct or indirect readout of the extracellular effect. Based on the results of the assay, it is determined whether one of the plurality of cell populations contains one or more effector cells that exhibit an extracellular effect.

[0271] In some embodiments, one or more of the individual cell populations and the readout particle population are disposed within an "effector zone" and a "readout zone" of the microreactor, respectively. However, the present invention is not limited thereto. When effector and readout zones are used, in one embodiment, they are substantially defined by the nature of the cells or particles disposed therein. That is, one or more effector cells are separated into the effector zone and one or more readout particles are separated into the readout zone. The effector zone is in fluid communication with the readout zone. Thus, in some embodiments, when the cell population and the readout particles are provided within the chamber at low density (e.g., less than two effector cells and readout particles per chamber), physical separation of the readout particles from the effector cells is achieved. However, it is recognized that the present invention need not be practiced in discrete zones within the chamber. As will be apparent from the description of the present invention, such separation is not necessarily required to perform the extracellular effect assay described herein.

[0272] The cell population and the readout particle population are loaded into the microreactor, for example, via different inlet ports or simultaneously into one mixture via a single inlet port. Alternatively, the effector cells and the readout particles are continuously loaded into the microreactor (e.g., a microfluidic chamber). One skilled in the art will understand that the cell population is provided to the microreactor before (or after) loading of the readout particles into the chamber. However, the readout particle population and the cell population can be provided simultaneously as a mixture.

[0273] The devices and methods disclosed herein provide a robust platform for performing an assay of one or more extracellular effects on a plurality of cell populations, for example, to identify one cell population from a plurality of cell populations presenting variations in extracellular effects. The variations are due to one or more effector cells present in the cell population. Each cell population is limited to a single microfluidic chamber, and the effector cell assays performed in each chamber of the device are the same (e.g., all cytotoxicity assays, all binding assays, etc.) or different (e.g., one binding assay, one apoptosis assay). The ability of a particular chamber on the device (e.g., having reagents for performing an assay of effector cells and different readout particles) to accommodate different assays enables a variety of analytical methods.

[0274] The microfluidic device described herein includes a plurality of chambers. Each of the plurality of chambers has the ability to accommodate a cell population and a readout particle population, and determines whether any effector cell in the cell population presents an extracellular effect on the readout particle population or a subpopulation thereof. The readout particle population may be composed of a single readout particle. As provided below, the devices provided herein are designed and assembled to assay multiple cell populations on one device. For example, hundreds to thousands of cell populations are assayed on one device to identify one or more cell populations that exhibit variations in extracellular effects. At least a portion of the cell populations are heterogeneous with respect to each other. The variations are, for example, variations compared to extracellular effects presented by a plurality of other populations. For example, in one embodiment, the method is provided for identifying one cell population from among a plurality of cell populations. The selected population has variations in extracellular effects compared to the remaining cell populations. The variations in extracellular effects are detectable, for example, by differences in the fluorescence intensity of one of the chambers compared to the remaining plurality of populations or subpopulations thereof.

[0275] According to one aspect of the invention, this method is provided for identifying one cell population from among a plurality of cell populations that exhibit variations in extracellular effects. The extracellular effects are induced by the binding of one or more soluble factors secreted from effector cells to a readout particle population or a subpopulation thereof. The readout particle population is a homogeneous or heterogeneous population. In one embodiment, the method provided herein includes the step of retaining individual readout particle populations within different chambers of a microfluidic device. The number of readout particles analyzed varies and is determined according to considerations similar to those for cell populations as described above.

[0276] The individual readout particle populations and cell populations are held within a single chamber of one of the microfluidic devices provided herein. Optionally, the chamber is substantially separated from other chambers of the microfluidic device (including individual cell populations and readout particle populations), for example, to minimize contamination between chambers. However, such separation is not necessary to practice the methods provided herein. In one embodiment, when separation is desired, the separation includes separation of fluids. Separation of the chamber fluids is accomplished by physically sealing the chamber (e.g., by using valves surrounding the chamber). However, in another embodiment, separation is accomplished without physically closing the chamber by restricting fluid communication between one chamber and other chambers of the microfluidic device so as to eliminate contamination between chambers.

[0277] When a chamber or chambers containing a cell population optionally contain one or more effector cells and the readout particle population is separated, the cell population in that chamber or chambers, specifically, one chamber (or chambers), is incubated. The first incubation step can occur prior to the addition of the readout particles and / or after the readout particles have been added to the chamber containing the cell population.

[0278] For example, the incubation step includes medium exchange or a cell washing step to maintain a healthy cell population. Incubation also includes the addition of accessory particles used to perform an assay for extracellular effects.

[0279] In one embodiment, the incubation process includes controlling one or more properties of the chamber (e.g., humidity, temperature, and / or pH) to maintain the viability of the cells (effector cells, accessory cells, or readout cells) and / or to maintain one or more functional characteristics of the cells in the chamber (secretion, surface marker expression, gene expression, signaling mechanisms, etc.). In one embodiment, the incubation process includes flowing a perfusion fluid through the chamber. The perfusion fluid is selected according to the type of effector cells and / or the readout cells are in a specific chamber. For example, the perfusion fluid in one embodiment is selected to maintain the viability of the cells (e.g., to replenish depleted oxygen or remove waste production), or to maintain the state of the cells (e.g., to replenish essential cytokines), or to assist in assaying for a desired effect (e.g., to add a fluorescence detection reagent). Perfusion is used to exchange reagents and, for example, to assay multiple extracellular effects in a continuous manner.

[0280] In another embodiment, the process of incubating a cell population includes flowing a perfusion fluid through the chamber to induce a cellular response of the readout particles (e.g., readout cells). For example, the incubation process in one embodiment includes adding a fluid containing a signaling cytokine to the chamber containing the cell population. The incubation process can be continuous, periodic, or a combination thereof. For example, the process of flowing a perfusion fluid through a microfluidic chamber or multiple chambers is periodic, continuous, or a combination thereof. In one embodiment, the flow rate of the incubation fluid (e.g., perfusion fluid) is controlled by an integrated microfluidic microvalve, micropump. In another embodiment, the flow of the incubation fluid is pressure-driven by using, for example, compressed air, a syringe pump, or gravity to regulate the flow of pressure.

[0281] Once individual chambers within the device are provided with a cell population and a population of readout particles, the method is performed to determine whether the cells within the population exert an extracellular effect on the population of readout particles or a subpopulation thereof. The cell population and the population of readout particles and / or a subpopulation thereof are examined, if desired, to determine whether the cells within the population exert an extracellular effect or, when compared to other cell populations, exert a variation in extracellular effect. As long as the presence and / or variation is detected within the chamber, it is not necessary that the specific cells or cells that exert the extracellular effect or its variation be identified within the chamber. In one embodiment, once the cell population is identified as one that exerts a variation in extracellular effect or an extracellular effect, the cell population is recovered for further characterization to identify the specific effector cells responsible for the extracellular effect or its variation. In another embodiment, once the cell population is identified as one that exerts an extracellular effect or a variation in extracellular effect, the cell population is recovered, nucleic acid from the cell population is amplified, and sequenced.

[0282] The extracellular effect in one embodiment is a binding interaction between a protein produced by an effector cell and a readout particle (e.g., a bead or a cell). In one embodiment, one or more effector cells in the population are antibody-producing cells and the readout particle contains an antigen having a target epitope. The extracellular effect in one embodiment is the binding and variation of an antibody to an antigen, e.g., a greater binding compared to one control chamber or other populations among a plurality. Also, the variation in the cellular effect is the presence of effector cells that secrete antibodies having a modulated affinity for a particular antigen. That is, the binding interaction is one or more measurements of antigen-antibody binding specificity, antigen-antibody binding affinity, and antigen-antibody binding reaction rate. Alternatively or additionally, the extracellular effect is the regulation of apoptosis, the regulation of cell proliferation, a change in the morphological appearance of the readout particle, a change in the localization of a protein within the readout particle, protein expression by the readout particle, neutralization of the biological activity of accessory particles, cell lysis of readout cells induced by effector cells, apoptosis of readout cells induced by effector cells, readout cell necrosis, internalization of antibodies, internalization of accessory particles, enzyme neutralization by effector cells, neutralization of soluble signaling molecules, or combinations thereof. In some embodiments, at least two different types of readout particles are provided in a chamber where one type of readout particle does not contain the target epitope.

[0283] The different types of readout particles may be distinguished by one or more characteristics (e.g., changing levels of fluorescent labeling, fluorescence intensity, morphology, size, surface staining, and location within the chamber).

[0284] When incubated with a cell population containing effector cells, a readout particle population or a subpopulation thereof is examined to determine whether one or more cells within the cell population exert an extracellular effect (whether direct or indirect), or a variation in the extracellular effect, on one or more readout particles. The cell population is confirmed to have a variation in the extracellular effect assayed and is then recovered for downstream analysis. Importantly, as provided throughout, it is not necessary that a particular effector cell having a particular extracellular effect on one or more readout particles be identified so long as the presence of the extracellular effect is detected within a particular microreactor (e.g., a microfluidic chamber).

[0285] In some embodiments, one or more effector cells within the cell population secrete a defined biomolecule (e.g., an antibody). The extracellular effect of these factors is then evaluated with one readout particle or a plurality of readout particles (e.g., readout cells) to detect a cell population exhibiting the extracellular effect. However, the extracellular effect is not limited to the effect of the secreted biomolecule. For example, in one embodiment, the extracellular effect is the effect of a T cell receptor, e.g., binding to an antigen.

[0286] In one embodiment, the readout particle population is a heterogeneous population of readout cells and includes readout cells engineered to express a cDNA library. Thereby, the cDNA library encodes cell surface proteins. Binding of an antibody to these cells is used to recover cells that secrete an antibody that binds to the target epitope and, optionally, to analyze the cells.

[0287] In some embodiments, a method for measuring an extracellular effect in a readout particle population or a subpopulation thereof includes adding one or more accessory particles to the chamber in which the effect is being measured. For example, at least one factor required to activate complement by binding of an antibody to a readout cell may be provided as an accessory particle. As described above, in one embodiment, natural killer cells or a plurality thereof are added to the chamber as accessory cells when cytolysis is being measured. One of ordinary skill in the art can determine whether accessory particles are needed based on the assay being used.

[0288] In some embodiments, when one or more readout particles include readout cells that express or present a target antigen, natural killer cells or a plurality thereof are provided to the chamber as "accessory cells" that facilitate the functional effect (lysis) being measured. The accessory cells are provided to the cell population, the readout particles, and the chamber either before the readout particles are loaded or after the readout particles are loaded into the chamber. In embodiments where natural killer cells are used, the natural killer cells target one or more readout cells to which an antibody produced by an effector cell has bound. The extracellular effect thus includes lysis of one or more readout cells by the natural killer cells. Lysis can be measured by, for example, viability dyes, membrane integrity dyes, release of fluorescent dyes, enzyme assays, and the like.

[0289] In some embodiments, the extracellular effect is the neutralization of accessory particles (or accessory reagents) operable to affect the readout particles (e.g., cytokines (accessory particles) operable to stimulate the response of at least one readout cell). For example, cytokine-secreting cells operable to affect the readout particle cells may further be provided to the chamber. The neutralization of cytokines secreted by effector cells may be detected as the absence of the expected effect (e.g., proliferation) of the cytokine in the readout cells. In another embodiment, the accessory particle is a virus operable to infect the readout cells, and the neutralization of the virus is detected as a decrease in the infection of the readout cells by the virus.

[0290] In some embodiments, the extracellular effect of one effector cell type induces the activation of a different type of effector cell (e.g., secretion of antibodies or cytokines). It can induce at least one intracellular response in the readout cells.

[0291] As provided throughout, the methods and devices provided herein are used to identify effector cells that exhibit a change in extracellular effect with readout particles. The effector cells are present as single effector cells within a microfluidic chamber or within a population of cells in a single chamber. The extracellular effect can be, for example, the extracellular effect of the secretions of the effector cells. When the effector cells are present within a larger population of cells, the extracellular effect is first attributed to the cell population. The population is then isolated and sub-populations of the isolated population are analyzed to determine the cytological basis of the extracellular effect. Sub-populations that exhibit the extracellular effect are isolated and can be further analyzed, for example, by limiting dilution to single cells, or subjected to nucleic acid analysis. In one embodiment, the sub-population of the isolated cell population comprises single cells.

[0292] In one aspect, the cell population comprises ASCs that secrete monoclonal antibodies. In one embodiment, the read bead-based assay is used in a method for detecting the presence of effector cells that secrete antibodies in the background of one or more additional cells that do not secrete antibodies. For example, the bead-based assay is used in one embodiment in a method for detecting ASCs within a cell population in the presence of one or more additional ASCs that secrete antibodies that do not bind to the target epitope of interest (the antibody binds to the target epitope of interest).

[0293] In another embodiment, the ability of an antibody to specifically bind to a target cell is evaluated. Referring to FIG. 3, this assay includes at least two read particles (e.g., read cells 181 and 186) in addition to at least one effector cell 182 (ASC). The read cell 181 expresses (naturally or via genetic engineering) a known target epitope of interest (i.e., target epitope 183) on its surface. In contrast, the read cell 186 does not. The two types of read cells 181 and 186 are distinguishable from themselves and the effector cell 182 by distinguishable fluorescent markers, other stains, or morphology. The effector cell 182 secretes an antibody 184 in the same chamber as the read cells 181 and 186. The antibody 184 secreted by the effector cell 182 binds to the read cell 181 via the target epitope 183, but does not bind to the read cell 186. A secondary antibody is used to detect the selective binding of the antibody 184 to the read cell 181. The microfluidic chamber is then imaged to determine whether antibody 184 that binds to the read cell 181 and / or the read cell 186 has been generated.

[0294] Such assays are also used to evaluate the position of antibodies that bind to the interior or on the surface of the readout cells using a high-resolution microscope. In this embodiment, the readout particles include particles / cells prepared with different particle types (e.g., cell types), or different methods (e.g., permeabilization and fixation) for evaluating binding specificity and / or localization. For example, such assays are used to identify antibodies that bind to the native structure of a target on live cells and the organized structure on fixed cells. Such assays may alternatively be used to determine the position of epitopes on a target molecule by first blocking other portions of the molecule having antibodies to known epitopes with different populations of readout particles having different blocked epitopes.

[0295] In another embodiment, individual heterogeneous readout particle populations (e.g., a readout cell population containing malignant and normal cells) and individual cell populations are provided to one or more microfluidic chambers (e.g., more than 1000 chambers) of the devices provided herein. At least one of the individual cell populations includes effector cells. For example, referring to FIG. 4, the absence of binding to healthy readout cells 426 and binding to one or more malignant readout cells 425 in a population of readout cells is used to identify a population of target cells that includes one or more effector cells that produce an antibody of interest (i.e., effector cells 427 that produce an antibody 428 specific for one or more malignant cells in the population). The two types of readout cells 425 and 426 within the chamber are distinguished by at least one characteristic (e.g., fluorescent labeling, position within the microfluidic chamber, surface staining, size, changing the level of morphological fluorescence intensity). The cells are then imaged and incubated within individual chambers to determine whether one or more of the chambers contain a cell population that exhibits an extracellular effect (i.e., an ASC that secretes an antibody that binds to malignant readout cells but not healthy readout cells).

[0296] If present within the chamber, a cell population comprising one or more ASCs that secrete antibodies that bind to malignant readout cells 425 rather than healthy readout cells 426 is then recovered to obtain the sequence of the antibody within the chamber or to perform other downstream assays on individual cells within the population (e.g., an assay to determine which effector cells of the population have the desired binding characteristics). Accordingly, novel functional antibodies are provided that can be discovered by one or more of the methods described herein. The epitope on the malignant readout cell 425 is either known or unknown. In the latter case, the epitope for the antibody can be identified by the following method.

[0297] In one embodiment, a single cell type can function as both an effector cell and a readout cell. Referring to FIG. 5, this assay is performed using a heterogeneous cell subpopulation of a single cell type (i.e., effector cells 430 and readout cells 431, both of which are functionalized to capture the molecule of interest 432 on their surfaces). For example, a tetrameric antibody 433 directed against a surface marker and the molecule of interest 432, or an affinity matrix on the cell is used to bind a biotinylated antibody. Referring to FIG. 6, the tetrameric antibody complex consists of an antibody (A) 435 that binds to the cell and an antibody (B) 436 that binds to the antibody secreted from the cell. Antibodies A and B are connected by two antibodies 437 that bind the Fc proteins of antibodies A and B. Such tetrameric antibody complexes are described in the art (Lansdorp et al. (1986). European Journal of Immunology 16, pp. 679-683, which is hereby incorporated by reference in its entirety for all purposes) and are commercially available (Stemcell Technologies, Vancouver, Canada). Using these tetramers, the secreted antibody is captured and bound to the surface of the cell. Thus, the effector cell also functions as a readout particle. Once bound to the surface of the cell, these antibodies are assayed for binding, for example, by adding a fluorescently labeled antigen. For example, when attempting to identify a chamber containing cells that secrete a monoclonal antibody that binds to a particular target, the antibody secreted from the effector cells is captured on the surface of these effector cells and other effector cells in the chamber using an appropriate capture agent. Referring again to FIG. 5, it is understood that effector cell 430 can also function as a readout cell. That is, effector cells that secrete the molecule of interest 432 can capture the molecule of interest more efficiently than readout cells 431.

[0298] In one embodiment, the assay of the extracellular effect is performed in parallel in a plurality of microfluidic chambers with a substantially uniform population of cells in each chamber and a non-uniform population of readout particles (e.g., a non-uniform readout cell population). Individual effector cells within the substantially uniform population each produce the same antibody. In a further embodiment, the readout particles are readout cells genetically engineered to express a library of proteins or protein fragments to determine the target epitopes of the antibodies secreted by the effector cells. Referring to FIG. 7, one embodiment of the assay includes a plurality of effector cells 190 that secrete antibody 191. The assay further includes a non-uniform readout cell population including readout cells 192, 193, 194, 195 that present epitopes 196, 197, 198, and 199. Effector cells 190 secrete antibody 191 that diffuses towards readout cells 192, 193, 194, and 195. Antibody 191 binds to readout cell 194 via target epitope 198, but does not bind to readout cells 192, 193, or 195. A secondary antibody may be used to detect the selective binding of antibody 191 to readout cell 194.

[0299] The cell population containing antibody 191 that binds to readout cell 194 (or other epitopes) is then recovered from the device and subjected to further assays.

[0300] In one aspect, a functional assay is provided to determine whether individual ASCs within a cell population activate lysis of target cells (i.e., whether they activate antibody-dependent cell-mediated cytotoxicity (ADCC)). ADCC is a cell-mediated immune defense mechanism. Effector cells of the immune system lyse target cells. Their membrane surface antigens are bound by specific antibodies (i.e., antibodies secreted by ASCs within the specific microfluidic chambers provided herein). Classical ADCC is mediated by natural killer (NK) cells. However, macrophages, neutrophils, and eosinophils can also mediate ADCC and are provided herein as accessory cells to be used in ADCC extracellular effect assays.

[0301] One embodiment of the ADCC assay provided herein includes a cell population comprising one or more effector cells, a readout cell population (having the epitope of interest on their surfaces), and NK cells as accessory cells. The assay is performed to determine whether ASCs from the cell population induce NK cells that attack and lyse the target cells. Referring to FIG. 8, the illustrated embodiments each include a cell population comprising ASCs 200 and 201 that secrete antibodies 202 and 203, respectively. The illustrated embodiments further include a heterogeneous readout cell population comprising readout cells 204 and 205 that present respective epitopes 206 and 207. ASCs 200 and 201 secrete antibodies 202 and 203 that diffuse towards readout cells 204 and 205. Antibody 202 binds to readout cell 205 via the target epitope 207 but does not bind to readout cell 204. Antibody 203 does not bind to either readout cell 204 or 205. NK cell 208 detects readout cell 205 to which antibody 202 is bound while leaving unbound readout cell 204 intact and proceeds to kill readout cell 205.

[0302] One of ordinary skill in the art will understand that NK cells may be added to the chamber during or after incubation of the readout cells and effector cells, provided that the NK cells are added to the chamber such that they facilitate access to the readout cells. The NK cells may be from a heterogeneous population of accessory cells (e.g., peripheral blood mononuclear cells). The NK cells may be from an animal or human cell line and may be engineered to increase ADCC activity. One of ordinary skill in the art will further understand that this assay can be performed using other hematopoietic cell types capable of mediating ADCC, such as macrophages, eosinophils, and neutrophils. In this case, macrophages, eosinophils, and neutrophils are accessory cells in the assay. The cell types capable of mediating ADCC can also be animal or human cell lines engineered to increase ADCC activity or engineered to report a signal for antibody binding on the target cell. In the latter case, the cells mediating ADCC are the readout particles, while the target cells are the accessory particles.

[0303] The ADCC extracellular effect assay can be performed on a single effector cell, a homogeneous cell population, or a heterogeneous cell population as shown in FIG. 8. Similarly, the ADCC assay can be performed using a single readout cell, a homogeneous readout cell population, or a heterogeneous readout cell population as shown in FIG. 8. However, in many cases, it is desirable to perform the ADCC assay with multiple readout cells to avoid false positive detection due to random death of the readout cells.

[0304] Cytolysis, in one embodiment, is quantified by a clonogenic assay, by adding a membrane integrity dye, by loss of fluorescent molecules within the cell, or by release of intracellular molecules into solution. The released biomolecules can be measured directly in solution or captured by readout particles for measurement. In some cases, additional accessory molecules (substrates for redox assays or substrates for enzyme assays) are added. Referring to FIG. 9, for example, a cell population comprising effector cell 500 that secretes a first biomolecule 502 and a second effector cell 501 that does not secrete the first biomolecule 502 is incubated in the presence of a heterogeneous readout particle population (comprising readout cells 503 and readout particles 504) and accessory particles (e.g., natural killer cells 505). Binding of the first biomolecule 502 to the readout cells 503 induces the recruitment of natural killer cells 505 that lyse the readout cells 503. Upon cytolysis, a second biomolecule 506 is released from the readout cells 503 and captured onto readout particles 504 (e.g., different types of readout particles functionalized to capture the second biomolecule 506 via molecule 507). Molecule 507 is, in one embodiment, a protein, antibody, enzyme, reactive group, and / or nucleic acid. The captured second biomolecule 506 can be any molecule present in the readout cells 503 such as a protein, enzyme, carbohydrate, or nucleic acid. In one embodiment, binding of the second biomolecule 506 to the readout particles 504 is quantified using a fluorescence assay, a colorimetric assay, a bioluminescence assay, or a chemiluminescence assay. If the captured biomolecule 506 is an enzyme that converts a substrate to a product with different optical properties, the assay is performed, for example, directly on the readout particles 504 or indirectly in the surrounding solution. The assay is performed in one of a plurality of chambers of a device provided herein to determine whether any of the chambers contain effector cells that secrete a biomolecule that induces cytolysis.

[0305] ADCC assays are known in the art and components are commercially available. For example, the Guava Cytotoxicity Kit for flow cytometry (Millipore), ADCC Reporter Bioassay Core Kit (Promega), ADCC Assay (GenScript), LIVE / DEAD Cell-Mediated Cytotoxicity Kit (Life Technologies), and DELFIA Cytotoxicity Assay are utilized in the devices provided herein.

[0306] In another embodiment, the assay of the extracellular effect is a complement-dependent cytotoxicity (CDC) assay. In one embodiment of CDC, the method is provided to identify the presence of ASC (or secreted antibody of ASC) within a cell population. That cell population binds to the readout cells in the presence of soluble factors sufficient and / or necessary to induce lysis of the readout cells via the classical complement pathway. Thus, this assay is to determine whether the antibody secreted by ASC promotes lysis of one or more target cells via the classical complement pathway.

[0307] The CDC assay includes at least one effector cell and at least one readout cell. One embodiment of the CDC is shown in FIG. 10. The embodiment includes a cell population comprising effector cells 210 and 211 that secrete antibodies 212 and 213, respectively. The further illustrated embodiment includes a heterogeneous readout cell population comprising readout cells 214 and 215 that present epitopes 216 and 217, respectively. Effector cells 210 and 211 secrete antibodies 212, 213 that diffuse towards readout cells 214 and 215. Antibody 212 binds to readout cell 215 via target epitope 217, but does not bind to readout cell 214. Antibody 213 does not bind to either readout cell 214 or 215. Enzyme C1 218, accessory particles, and one of the soluble factors necessary to induce cell lysis via the classical complement pathway bind to the complex of antibody 212 and readout cell 215, leaving unbound readout cell 214 intact. Binding enzyme C1 208 to the complex of antibody 212 and readout cell 215 operates the classical complement pathway involving the necessary additional soluble factors and induces cell lysis via the classical complement pathway (not shown), thereby resulting in the rupture and death of readout cell 215.

[0308] The soluble factors necessary to induce lysis of the readout cells (i.e., the accessory particles necessary for the assay) are added to the chamber during or after incubation of the readout and effector cells if it is added to the chamber in a manner that facilitates access to the readout cells. The CDC assay provided herein can be performed on a single effector cell, a homogeneous effector cell population, or a heterogeneous cell population as shown in FIG. 8. Similarly, the CDC assay can be performed on a single readout cell, a homogeneous readout cell population, or a heterogeneous readout cell population as shown in FIG. 8. However, it is often preferred to perform the CDC assay on a readout cell population to avoid false positive detection due to random death of the readout cells.

[0309] Cell lysis via the complement pathway is quantified according to methods known to those skilled in the art. For example, cell lysis can be quantified by a colony formation assay, by adding a membrane integrity dye, by loss of fluorescent molecules within the cell, or by release of intracellular molecules into solution. The released biomolecules are measured directly in solution or captured on readout particles. In some cases, additional accessory molecules (substrates for redox assays or enzyme assays) may be added. Referring to Figure 11, for example, a cell population comprising effector cell 510 that secretes a first biomolecule 512 and a second effector cell 511 that does not secrete the first biomolecule 512 is incubated in the presence of accessory particles 515 (e.g., complement proteins) and in the presence of one or more heterogeneous readout particles (e.g., readout cells 513 and readout particles 514). In the presence of accessory particles 515, binding of the biomolecule 512 to the readout cell 513 causes the readout cell 513 to lyse. Upon cell lysis, a second biomolecule 516 is released and captured by readout particles 514 (e.g., a second type of readout particle functionalized to capture the biomolecule 516 via molecule 517). Molecule 517 may be one or more types of molecules such as protein molecules, antibodies, enzymes, reactive groups, and / or nucleic acids. The supplemented biomolecule 516 is not limited to a type. Rather, the captured biomolecule 516 can be any molecule present in the readout cell 513 such as a protein, enzyme, dye, carbohydrate, or nucleic acid. Binding of the second biomolecule 516 to the readout particles 514 is quantified using a fluorescence assay, a colorimetric assay, a bioluminescence assay, or a chemiluminescence assay. For example, it is understood that if the captured biomolecule 516 is an enzyme that converts a substrate to a product with different optical properties, the assay may be performed directly on the readout particles 514 or indirectly in the surrounding solution.

[0310] In another embodiment, the assay is provided to determine whether effector cells alone, or effector cells within a cell population, regulate growth cells. Specifically, the assay is used to determine whether effector cells secrete a biomolecule (e.g., a cytokine or an antibody) that regulates the growth rate of the readout cells. Referring to FIG. 12, the illustrated embodiment includes a cell population including effector cell 220 and effector cell 221 that secrete biomolecules 222 and 223, respectively. The further illustrated embodiment includes a homogeneous readout cell population including readout cell 224. Effector cells 220 and 221 secrete biomolecules 222 and 223 that diffuse toward readout cell 224. Biomolecule 222 binds to readout cell 224 (shown by the dashed line) to induce the growth of readout cell 224. Biomolecule 223 does not bind to readout particle 224. A microscopic image of the chamber is used to evaluate the growth of readout cell 224 associated with cells in other chambers not exposed to the biomolecule.

[0311] The cell growth modulation assay is performed using a cell population optionally including one or more effector cells. As described above, in some embodiments, not all cell populations include effector cells due to their rarity and / or the difficulty of enrichment in the starting population. The starting population is first loaded into one of the devices provided herein. The present invention enables the identification of these rare cells by identifying a cell population including one or more effector cells.

[0312] The cell growth modulation assay is performed using a single readout cell, or a heterogeneous readout cell population within a single chamber. However, in many cases, it is desirable to perform the cell growth regulation assay with a homogeneous readout cell population to enable a more accurate measurement of the growth rate.

[0313] The cell growth modulation assay is adapted, in one embodiment, to screen for cells that produce a biomolecule that inhibits cell growth. In another embodiment, the method is adapted to screen for cells that produce a molecule that modulates (i.e., increases or decreases) the growth rate of the readout cells. The growth rate is measured, in one embodiment, by manual or automated cell counting from an optical microscope image, the total fluorescence intensity of cells expressing fluorescence, the average fluorescence intensity of cells labeled with a dilution dye (e.g., CFSE), nuclear staining, or any other method known to those of skill in the art.

[0314] Commercially available assays for measuring proliferation include the alamarBlue® cell viability assay, the CellTrace® CFSE Cell Proliferation Kit and the CellTrace® Violet Cell Proliferation Kit (all from Life Technologies). They can be used with the methods and devices described herein.

[0315] In another embodiment, a functional assay of apoptosis is provided for selecting a cell population that includes one or more effector cells (i.e., readout cells or accessory cells) that induce apoptosis in other cells. In one embodiment, the method is used to identify the presence of effector cells that secrete a biomolecule (e.g., a cytokine or an antibody) that induces apoptosis in the readout cells or accessory cells. Referring to FIG. 13, the illustrated embodiment includes a cell population that includes effector cell 230 and effector cell 231 that secrete biomolecule 232 and biomolecule 233, respectively. In a further illustrated embodiment, a homogeneous readout cell population that includes readout cell 234 is included. Effector cell 230 and effector cell 231 each secrete biomolecules 232 and 233 that diffuse towards readout cell 234. Biomolecule 232 binds to readout cell 234 and induces apoptosis of readout cell 234. Biomolecule 233 does not bind to the readout cells. A microscopic image of the chamber is used in one embodiment to evaluate apoptosis by potentially using other markers and stains for apoptosis known in the art (e.g., annexin 5, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling, disruption of mitochondrial membrane potential, etc.). In one embodiment, cell death rather than apoptosis is measured using a commercially available dye or kit, e.g., propidium iodide (PI), LIVE / DEAD® viability / cytotoxicity kit (Life Technologies) or LIVE / DEAD® cell-mediated cytotoxicity kit (Life Technologies).

[0316] The apoptosis assay is performed, in one embodiment, on a cell population containing a single effector cell, a cell population optionally containing one or more effector cells, or a cell population containing one or more effector cells. In one embodiment, the apoptosis assay is performed using a single readout cell, or a heterogeneous population of readout cells. However, in many cases, it is desirable to perform the apoptosis assay on a homogeneous population of readout cells to enable a more accurate assessment of apoptosis.

[0317] In another embodiment, the microfluidic device provided herein is used to select effector cells that secrete biomolecules, such as cytokines or antibodies that induce autophagy in readout cells. One embodiment of this method is shown in FIG. 14. Referring to FIG. 14, the illustrated embodiment includes a cell population containing effector cell 441 and effector cell 442. The illustrated embodiment further includes a heterogeneous population of readout cells including a first type of readout cell 444 presenting a target epitope 449 and a second type of readout cell 445 lacking the target epitope. Effector cell 441 secretes biomolecule 443. It diffuses towards the first type of readout cell 444 and the second type of readout cell 445. Biomolecule 443 binds to the first type of readout cell 444 and induces autophagy in the first type of readout cell 444. The body molecule 443 does not bind to the second type of readout cell 445. The microscopic image of the chamber is used, in one embodiment, to evaluate autophagy using a cell line engineered with an autophagy reporter. Such autophagy reporters are known in the art (e.g., FlowCellect® GFP-LC3 Reporter Autophagy Assay Kit (20S) (EMD Millipore), Premo® Autophagtan tandem sensor RFP-GFP-LC3B Kit (Life Technologies)).

[0318] In one embodiment, the autophagy assay is performed on a cell population containing a single effector cell, a cell population optionally containing one or more effector cells, or a cell population containing one or more effector cells. In one embodiment, the autophagy assay is performed using a single readout cell, or a heterogeneous population of readout cells, or a homogeneous population of readout cells. This assay is, in one embodiment, performed using a homogeneous population of readout cells.

[0319] In another embodiment, the method is provided to identify the presence of effector cells or to select effector cells that secrete a biomolecule (e.g., an antibody). The biomolecule interferes with the function of a known biomolecule (e.g., a cytokine) that induces a readout cell to respond. The response is not limited by type. For example, the response in one embodiment is selected from cell death, cell proliferation, reporter expression, morphological changes, or some other reaction selected by the user of the method. One embodiment of the method is provided in FIG. 15. Referring to FIG. 15, the illustrated embodiment includes a cell population containing effector cells 240 and 241 that secrete biomolecules 242 and 243, respectively, and effector cell 241. The illustrated embodiment further includes a homogeneous population of readout cells containing readout cell 244. Effector cells 240, 241 secrete antibodies 242 and 243. It diffuses into the medium in the chamber. The chamber is pulsed with cytokine 245, which normally has a known effect on readout cell 244. Antibody 242 binds to cytokine 245, thereby preventing them from binding to readout cell 244. Accordingly, the expected response is not observed. This includes that one of effector cells 240 and 241 secretes an antibody that can neutralize the ability of cytokine 245 to stimulate readout cell 244 that responds.

[0320] In one embodiment, the cytokine neutralization assay is used to identify the presence of effector cells. These effector cells produce biomolecules that target receptors for cytokines present on the readout cells. In this case, the binding of an antibody (e.g., antibody 242) to the receptor 246 for cytokine 245 on the readout cell 244 blocks the interaction between the cytokine and the receptor so that the response is not stimulated. In other embodiments, the cytokine receptor is “solubilized” or “stabilized,” e.g., a cytokine receptor engineered via the HeptaresSTAR® platform.

[0321] The response to a cytokine is confirmed, in one embodiment, by microscopic measurement of associated signaling as known in the art, but is not limited to cell death, cell proliferation, expression of a fluorescent reporter protein, localization of cell components, changes in cell morphology, motility, chemotaxis, cell aggregation, etc. In one embodiment, the response of the effector cells and the chamber is compared to a chamber lacking effector cells to determine whether the response is inhibited. If the response is inhibited, the effector cells within the chamber are harvested for further analysis.

[0322] In one embodiment, the cytokine assay is performed within individual microreactors of a cell population containing a single effector cell, a cell population optionally containing one or more effector cells, or a cell population containing one or more effector cells. Of course, the method is performed in parallel in multiple microchambers of multiple cell populations. In one embodiment, the cytokine assay is performed using a single readout cell, or a heterogeneous population of readout cells. In one embodiment, the method is performed using a heterogeneous population of readout cells that allows for a more accurate assessment by stimulation (or rather the lack thereof) of the readout cells.

[0323] Examples of commercially available cytokine-dependent or cytokine-sensitive cell lines for such assays include, but are not limited to, TF-1, NR6R-3T3, CTLL-2, L929 cells, A549, HUVEC (human umbilical vein endothelial cells), BaF3, BW5147.G.1.4.OUAR.1, (all available from ATCC), PathHunter® CHO cells (DiscoveRx) and TANGO cells (Life Technologies). Those skilled in the art will understand that primary cells (e.g., lymphocytes, monocytes) can also be used as readout cells for cytokine assays.

[0324] In one embodiment, the signaling assay is used to identify a cell population comprising one or more effector cells that secrete a molecule (e.g., an antibody or a cytokine). The molecule has agonist activity for the receptor of the readout cells. When bound to the receptor, the effect on the readout cell population includes activation of the visualized signaling pathway, such as by expression of a fluorescent reporter, translocation of an intracellular fluorescent reporter, change in growth rate, cell death, change in morphological differentiation, change in a protein expressed on the surface of the readout cells, etc.

[0325] Some engineered reporter cell lines are commercially available and are used to perform such assays. Examples include PathHunte cells® (DiscoverRx), TANGO™ cells (Life Technologies), EGFP reporter cells (ThermoScientific).

[0326] In one embodiment, a virus neutralization assay is performed to identify and / or select a cell population comprising one or more effector cells that secrete a biomolecule (e.g., an antibody). The biomolecule interferes with the ability of a virus to infect a target readout cell or a target accessory cell. One embodiment of this method is shown in FIG. 16. Referring to FIG. 16, in the illustrated embodiment, a cell population comprising effector cell 250 and effector cell 251 that each secrete a biomolecule (e.g., antibodies 252 and 253) is included. The illustrated embodiment further includes a homogeneous population of readout cells including readout cell 254. Effector cells 250, 251 secrete a biomolecule, e.g., antibodies 252 and 253. It diffuses into the medium within the chamber. The chamber is then pulsed with virus 255 (accessory particles). It typically does not infect readout cell 254. Antibody 252 or 253 binds to virus 255, thereby preventing the virus from binding to readout cell 254. Thus, no expected infection is observed. This includes that one of effector cells 250 or 251 secretes an antibody that can neutralize virus 255.

[0327] The virus neutralization assay is also suitable for identifying the presence of effector cells that produce a biomolecule that binds to the receptor of the virus on the readout cell. In this case, binding of an antibody (e.g., antibody 252) to the receptor of virus 255 on readout cell 254 blocks the receptor-virus interaction so that no infection is observed.

[0328] The evaluation of virus infection can be carried out using methods known in the art. For example, the virus can be engineered to contain a fluorescent protein. This fluorescent protein is expressed by the cells that are the readout after infection, the expression of the fluorescent protein in the readout cells that are upregulated during virus infection, the secretion of proteins from the readout cells or accessory cells (which are measured and supplemented on the particles that increase during virus infection), the death of the readout cells or accessory cells, the morphological changes of the readout cells or accessory cells, and / or the aggregation of the readout cells.

[0329] In one embodiment, the virus neutralization assay within individual microreactors is performed with a cell population containing a single effector cell, a cell population optionally containing one or more effector cells, or a cell population containing one or more effector cells. In one embodiment, the virus neutralization assay is performed using a single readout cell, or a heterogeneous population of readout cells. In one embodiment, the method is performed using a heterogeneous population of readout cells that allows for a more accurate assessment by the stimulation (or rather the lack thereof) of the readout cells that respond. Of course, this method can be performed in parallel in multiple microchambers of multiple cell populations.

[0330] For example, commercially available cell lines for virus neutralization assays are MDCK cells (ATCC) and CEM-NKR-CCR5 cells (NIH AIDS Reagent Program), which can be used with the methods and devices described herein.

[0331] In another embodiment, the enzyme neutralization assay is performed to determine whether effector cells display or secrete a biomolecule that inhibits a target enzyme. One embodiment of the method is provided in FIG. 17. Referring to FIG. 17, the illustrated embodiment includes a cell population including effector cells 280 and effector cells 281 that secrete biomolecules (e.g., proteins 282 and 283), respectively. Further, the illustrated embodiment includes a homogeneous population of readout particles (e.g., beads 284) to which the target enzyme 285 is conjugated. However, in another embodiment, the target enzyme 285 is bound to or dissolved on the surface of the device. Proteins 282 and 283 diffuse in the medium, and protein 282 binds to the target enzyme 285, thereby inhibiting its activity. Protein 283 does not bind to the target enzyme. In one embodiment, the detection (or rather the lack thereof) of the enzymatic activity of the substrate present in the chamber is evaluated by methods known in the art, including, but not limited to, fluorescence readout, colorimetric readout, precipitation, etc.

[0332] In another embodiment, the enzyme neutralization assay is performed on a cell population containing a single effector cell, a cell population optionally containing one or more effector cells, or a cell population containing one or more effector cells per individual chamber. In one embodiment, the enzyme neutralization assay is performed using a single readout particle within an individual chamber. In one embodiment, the enzyme neutralization assay is performed on a plurality of cell populations to identify a cell population having a change in the response of the assay.

[0333] In another embodiment, the assay method is provided to identify the presence of effector cells that present or secrete a molecule that induces activation of a second type of effector particle. The effector cells sequentially secrete molecules that have an effect on the readout particles. Thus, in this embodiment, the cell population is provided to individual microfluidic chambers. One embodiment of this method is provided in FIG. 18. Referring to FIG. 18, the illustrated embodiment includes a cell population that includes one effector cell 460 presenting a molecule 461 (e.g., an antibody, a surface receptor, a major histocompatibility complex molecule, etc.) on its surface. The cell 460 activates adjacent effector cells of a different type (in this case, effector cell 462) and induces the secretion of another type of molecule 463 (e.g., a cytokine, an antibody) captured by the readout particle 464. In this example, the readout particle 464 is functionalized with a receptor or antibody 465 specific for the secreted molecule 463.

[0334] In another embodiment, effector cells activated by accessory particles exhibit phenotypic changes such as proliferation, viability, morphology, motility, and differentiation. In this case, the effector cells are also the readout particles. This effect can be caused by autocrine secretion of proteins by the activated effector cells and / or by accessory particles.

[0335] Referring to FIG. 19, the illustrated embodiment includes a cell population that includes an effector cell 470 that secretes a molecule 471 (e.g., an antibody, a cytokine, etc.). The molecule 471 activates a second type of effector cell of a different type (in this case, effector cell 472). Once activated, the effector cell 472 secretes another type of molecule 473 (e.g., a cytokine, an antibody) captured by the readout particle 474. In this example, the readout cell 474 is functionalized with a receptor or antibody 475 specific for the secreted molecule 473.

[0336] As provided herein, monoclonal antibodies having a low off-rate are detectable in the presence of a large background of monoclonal antibodies having a fast off-rate (in the same chamber) that are specific for the same antigen. However, affinity can also be measured with the devices and methods provided herein. Thus, on-rates can also be measured. These measurements depend on not only the sensitivity of the optical system but also the binding ability of the capture reagent (e.g., beads). To assay specificity, the capture reagent (readout particle) may be designed to be present with the epitope of interest such that it binds only antibodies having the desired specificity for the epitope of interest.

[0337] Referring to FIG. 20, the illustrated embodiment includes a homogeneous population of cells that secrete antibodies specific for the same antigen but have different antibodies. This assay is used to identify effector cells within a population containing at least one effector cell that produces a high-affinity antibody. Effector cells 450, 451 secrete antibodies 453 and 454 that have a low affinity for a target epitope (not shown). In contrast, effector cell 452 secretes antibody 455 that has a higher affinity for the target epitope. Antibodies 453, 454 are captured by a homogeneous population of readout particles that includes readout beads 456. The readout beads are incubated with a fluorescently labeled antigen (not shown) that binds to all antibodies. Upon washing with unlabeled antigen (not shown), the fluorescently labeled antigen remains only if the readout beads present high-affinity antibody 455 on their surface.

[0338] Referring to FIG. 21, another illustrated embodiment includes effector cells 260 that secrete a biomolecule (e.g., antibody 261). The illustrated embodiment further includes a heterogeneous population of readout particles. The population is optically distinguishable readout particles, e.g., beads 262 and 263 that each present different target epitopes 264, 265. Antibody 261 diffuses into the chamber. Antibody 261 binds to antibody 264 but does not bind to antibody 265. In one embodiment, the preferential binding of antibody 261 to epitope 264 was observed on the fluorescent surface of bead 262 but not on bead 263.

[0339] In the illustrated embodiment, beads 262 and 263 are optically distinguishable by shape for evaluating cross-reactivity. However, the readout particles can also be distinguishable by other means. Other means include one or more characteristics such as fluorescent labels (including different fluorescence wavelengths), changes in levels of fluorescence intensity (e.g., Starfire™ beads having different fluorescence intensities, morphologies, sizes, surface stains, and locations within a microfluidic chamber).

[0340] In one embodiment, beads 262 and 263 are optically distinguishable when separated into separate readout zones, e.g., by a cell fence. Alternatively, different colored fluorophores are used to optically distinguish the readout beads.

[0341] Alternatively, specificity can be measured by including another antibody that competes with the secreted antibody that binds to the target epitope. For example, in one embodiment, the presence of the secreted antibody bound to the readout particles presenting the antigen is identified using a fluorescently labeled secondary antibody. The unlabeled competing antibody is generated from a different host and is known to bind to a known target epitope of the antigen. Subsequent addition of the unlabeled competing antibody will result in a decrease in fluorescence due to displacement of the secreted antibody only if the secreted antibody binds to the same target epitope as the competing antibody. Alternatively, specificity is measured by adding a mixture of various antigens. The antigens compete with the binding of the secreted antibody to the target epitope when the secreted antibody has low specificity. Alternatively, specificity is measured using differentially labeled antigens to evaluate the binding properties of the secreted antibody after capturing the secreted antibody on beads.

[0342] The measurement of specificity described herein is essentially a polyclonal measurement when performed on a cell population containing two or more effector cells that secrete antibodies that bind to one of the targets.

[0343] In various embodiments of the present invention, methods for identifying the presence of effector cells that secrete a biomolecule are paired with the analysis of the presence or absence of one or more intracellular compounds of the effector cells. Referring to FIG. 22, a cell population includes at least one effector cell type 520 and another effector cell type 521. Effector cell type 520 secretes a biomolecule 522 of interest (e.g., an antibody, or a cytokine). Effector cell type 521 does not secrete the biomolecule of interest. This cell population is incubated in the presence of a population of readout particles 523 functionalized to capture the biomolecule of interest. After the incubation period, the cell population including effector cell types 520 and 521 is lysed to release the intracellular contents of the cells in the population. Readout particles 523 are also functionalized to capture an intracellular biomolecule 524 of interest (e.g., a nucleic acid, a protein, an antibody, etc.) in the cells. Cell lysis can be achieved by various methods known to those skilled in the art.

[0344] In one embodiment, the method is provided for identifying a polyclonal mixture of secreted biomolecules having desired binding properties. The assay may be performed with a heterogeneous mixture of effector cells that produce antibodies having a known affinity for a target epitope, a target molecule, or a target cell type. Binding of the target in the context of the mixture is compared to binding of the target in the context of individual effector cells alone, for example, to determine whether the mixture provides an enhanced effect.

[0345] Multifunctional assays combining the binding and / or functional assays described herein may be performed by having multiple readout regions, multiple effector regions, multiple readout particle types, or combinations thereof. For example, a perfusion step can be performed during an extracellular effect assay to exchange reagents for different functional experiments.

[0346] One embodiment of the multifunctional assay is provided in FIG. 23. Referring to FIG. 23, a microfluidic chamber 410 is generally shown for simultaneously evaluating the extracellular effects of effector cells on three subsets of readout particles. The microfluidic chamber 410 includes cell fences 420 and 421 that divide the chamber into four zones. The cell fences 420 and 421 are shown to be perpendicular to each other in this example, but one of ordinary skill in the art will understand that the exact positioning of the fences can be varied as long as four zones are created. Further, one of ordinary skill in the art will understand that the cell fences are not necessarily required to perform a multifunctional assay (in series or in parallel). In one embodiment, configurations other than cell fences are included to create zones as long as the different zones are addressable in terms of the transport of cell populations and readout particles. In another embodiment, the multifunctional assay is performed without cell fences or structures. Rather, each assay is performed within the chamber, for example, simultaneously or sequentially, with fluorescent molecules that emit light of different wavelengths.

[0347] In the embodiment shown in FIG. 23, effector cells 411 and 422 that secrete antibodies 419 and 423, respectively, are supplied to the upper left region of chamber 410. Thereby, an effector zone 415 is defined. Readout particles 412, 413, and 414 are supplied to the remaining regions. Thereby, readout zones 416, 417, and 418 are defined, respectively. The readout particles 412, 413, and 414 constitute, in one embodiment, a heterogeneous population of readout particles. For example, in one embodiment, readout particles 412 and 414 are beads of different sizes presenting different epitopes of the same antigen, and readout particle 413 is a cell presenting an antigen in the presence of natural killer cells 424. Thus, the presence or absence of effector cells that can selectively bind to a given epitope and induce the killing of readout cells by natural killer cells can be simultaneously evaluated within a single chamber.

[0348] Alternatively, the readout particles 412, 413, and 414 are identical and enable multiple independent measurements of extracellular effects provided by a single set of effector cells within a single chamber. Also, particles 412 and 414 are distinguishable and the assays are performed sequentially.

[0349] In one embodiment, the presence of one or more extracellular effects is analyzed. Depending on the effect, one of ordinary skill in the art will recognize that the presence or absence of a conjugated extracellular effect may be desired. Similarly, the desired characteristics can include the presence of one or more types of extracellular effects and the presence of different types of extracellular effects. For example, in one embodiment, a multifunctional assay is used to identify effector cells that secrete an antibody that binds to a receptor epitope on the readout cells but does not induce activation of the corresponding signaling pathway.

[0350] In one embodiment, a multifunctional assay is performed by introducing, for example, different effector cells or combinations of effector cells into a chamber containing multiple effector regions in different areas. For example, in one embodiment, different cell populations that produce antibodies with known affinity for a target antigen are introduced into different effector zones of the chamber. The binding of the target in the context of the mixture is then compared to the binding of the target in the context of individual effector cells alone. Thus, such use of multiple effector zones enables screening of multiple combinations within a single chamber.

[0351] In one aspect of the assays provided herein, after the readout particles are incubated with a cell population in a microfluidic chamber, fluorescence measurements are taken to determine whether the cells in the population exhibit an extracellular effect. In this embodiment, the readout particle population is fluorescently labeled and the change in fluorescence correlates with the presence and / or magnitude of the extracellular effect. The readout particle population is labeled directly or indirectly with a fluorescent label. In some embodiments provided throughout, accessory particles (e.g., accessory cells) are supplied to the chamber to help facilitate a fluorescent readout. As will be appreciated by those skilled in the art, attention has been paid to designing an assay that provides readout particles and effector cells in a focal plane to enable accurate imaging and fluorescence measurements.

[0352] In one embodiment, the response of the readout particles is monitored using automated high-resolution microscopy. For example, imaging can be monitored by using a 20x (0.4 NA) objective lens on an Axiovert 200 (Zeiss) or DMIRE2 (Leica) motorized inverted microscope. Using the automated microscopy system provided herein, complete imaging of a 4000-chamber array containing one brightfield and three fluorescent channels can be achieved in about 30 minutes. This platform is adaptable to various chip designs as described by Lecault et al. (2011). Nature Methods 8, pp. 581-586 is incorporated herein by reference in its entirety for all purposes. Importantly, the imaging methods used herein obtain sufficient signal in the effect positive chambers while minimizing photodamage to the cells.

[0353] In one embodiment, the effector cell assays provided herein benefit from long-term cell cultures. Thus, the effector cell assays require that the effector cells maintained within the device are viable and healthy cells. In embodiments where readout cells or accessory cells are used in the effector cell assay, it is understood that they are maintained in a sufficiently healthy state to be viable and healthy. The fluidic structures provided herein enable precise and accurate control of the media conditions to maintain effector readout cell viability such that a functional assay can be performed. For example, some cell types require autocrine or paracrine factors that rely on the accumulation of secreted products. For example, the growth rate of CHO cells depends strongly on seeding density. Confining a single CHO cell within a 4 nL chamber corresponds to a seeding density of 250,000 cells / ml, comparable to conventional macroscale cultures. As shown in FIG. 73, single CHO cells have a higher growth rate in the microfluidic device than when seeded in a multiwell plate. Since they propagate at high seeding densities, CHO cells may not require perfusion for several days. However, other cells, particularly those that are cytokine-dependent (e.g., ND13 cells, BaF3 cells, hematopoietic stem cells), generally do not reach high concentrations in macroscale cultures. Also, frequent feeding within the microfluidic device may be required to prevent cytokine depletion. Cytokines can be added to the media or produced by feeder cells. For example, bone marrow-derived stromal cells and eosinophils have been shown to support the survival of plasma cells due to their production of IL-6 and other factors (Wols et al., (2002), Journal of Immunology 169, pp. 4213-21; Chu et al. (2001), Nature Immunology, 2, pp. 151-159, which are incorporated herein by reference in their entirety). In this case, the perfusion frequency is adjusted to allow sufficient accumulation of paracrine factors while preventing nutrient depletion.

[0354] In one aspect, the present invention provides a method for determining whether a cell population optionally containing one or more effector cells exerts an extracellular effect of a readout particle (e.g., a cell including a cell surface receptor). The effector cells can be present in a heterogeneous cell population, a homogeneous population, or a single cell. In one embodiment, the effector cells are antibody-secreting cells. In one embodiment, the one or more extracellular properties include an extracellular effect of the readout particle, e.g., inhibition (antagonism) or activation (agonism) of a cell surface receptor on the readout cell (e.g., agonist and / or antagonist properties of an antibody secreted by an antibody-secreting cell). In a further embodiment, the extracellular effect is an agonist or antagonist effect of a transmembrane protein. It is, in a further embodiment, a G protein-coupled receptor (GPCR), a receptor tyrosine kinase (RTK), an ion channel or an ABC transporter. In a further embodiment, the receptor is a cytokine receptor. The extracellular effects of metabotropic receptors other than RTKs with GPCRs can be evaluated. For example, the extracellular effect of a guanylate cyclase receptor is evaluated by incubating a cell population having a readout cell population that expresses the guanylyl cyclase receptor.

[0355] In embodiments where readout cells are used, the readout cells can be alive or fixed. For fixed readout cells, the extracellular effect in one embodiment is the effect of an intracellular protein of the fixed readout cell. The extracellular effect can also be measured on extracellular proteins of live or fixed readout cells, or secreted proteins of live readout cells.

[0356] In another embodiment, the readout cells express one of the following types of cell receptors, and the extracellular effect assay measures binding, agonism or antagonism of the cell receptor (receptor serine / threonine kinase, histidine kinase-related receptor).

[0357] Certain receptors, such as receptor serine / threonine kinases, histidine kinase-related receptors or GPCRs, are orphan receptors. That is, in embodiments where no ligand is known to activate a particular receptor, the methods provided herein enable the discovery of ligands for a particular orphan receptor by performing an extracellular assay in reporter cells that express the particular orphan receptor and identifying a cell population or subpopulation. The cell population or subpopulation comprises effector cells that have variability in the extracellular effects of the reporter cells that express the orphan receptor.

[0358] In one embodiment, the cell surface protein is a transmembrane ion channel. In a further embodiment, the ion channel is a ligand-gated ion channel. Also, the extracellular effect measured in the microfluidic assay is the regulation of the opening and closing of the ion channel, for example, the opening of the ion channel by agonist binding, or the closing / blocking of the ion channel by antagonist binding. The antagonist or agonist is, for example, a biomolecule (e.g., an antibody) secreted by one or more effector cells in a heterogeneous cell population comprising one or more effector cells. The extracellular assay described herein is used to measure the extracellular effect of effector cells on cells that express ligand-gated ion channels in the cysteine loop superfamily, ionotropic glutamate receptors and / or ATP-gated ion channels. Specific examples of anionic cysteine loop ion-dependent channels include GABA AIt includes a receptor and a glycine receptor (GlyR). Specific examples of cationic cysteine loop ion-dependent channels include serotonin (5-HT) receptors, nicotinic acetylcholine (nAChR) and zinc-activated ion channels. The above one or more channels are expressed by the readout cells to determine whether the effector cells have an extracellular effect on the respective cells by agonizing or antagonizing the ion channels. Measurement of ion flux generally occurs over a short time (i.e., seconds) and requires precise fluid control for their implementation. Examples of commercially available ion channel assays include the Fluo-4 Direct Calcium Assay Kit (Life Technologies) and the FLIPR Membrane Potential Assay Kit (Molecular Devices). Cell lines expressing ion channels (e.g., PrecisION™ cell lines, EMD Millipore) are also commercially available.

[0359] In one embodiment, the cell surface protein is an ATP-binding cassette (ABC) transporter and the measured extracellular effect is the transport of a substrate across the membrane. The readout particles are membrane vesicles derived from cells expressing a protein that can be immobilized on beads (e.g., GenoMembrane ABC Transporter Vesicles (Life Technologies)). For example, the ABC transporter can be a permeable glycoprotein (multidrug resistance protein). The effect can be measured by the fluorescence intensity of calcein in the readout cells. The Vybrant™ Multidrug Resistance Assay Kit (Molecular Probes) is commercially available for performing such assays.

[0360] The extracellular effects are also evaluated in readout cells that express ionotropic glutamate receptors such as AMPA receptors (class GluA), kainate receptors (class GluK), or NMDA receptors (class GluN). Similarly, the extracellular effects are evaluated in readout cells that express ATP-dependent channels or phosphatidyl 4,5-bisphosphate (PIP2)-dependent channels.

[0361] As provided throughout, the present invention provides a method for identifying a cell population that exhibits a variation in an extracellular effect. In one embodiment, the method includes the step of holding a plurality of individual cell populations in separate microfluidic chambers. At least one of the individual cell populations includes one or more effector cells, and the contents of another microfluidic chamber further include a readout particle population that includes one or more readout particles. The method also includes the steps of incubating the individual cell populations and the readout particle population within the microfluidic chambers, and assaying the individual cell populations for the presence of the extracellular effect. The readout particle population or a subpopulation thereof provides a readout of the extracellular effect. In one embodiment, the extracellular effect is, for example, the effect of a receptor tyrosine kinase (RTK) that binds to an RTK, an antagonism of an RTK, or an agonism of an RTK. RTKs are high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. To date, there are approximately 60 receptor kinase proteins identified in the human genome (Robinson et al. (2000). Oncogene 19, pp. 5548-5557, which is hereby incorporated by reference in its entirety for all purposes). RTKs have been shown to regulate cell processes and have roles in the progression and development of many types of cancer (Zwick et al. (2001). Endocr. Relat. Cancer 8, pp. 161-173, which is hereby incorporated by reference in its entirety for all purposes).

[0362] When the extracellular effect is that of an RTK, the invention is not limited to a particular RTK class or member. Approximately 20 different RTK classes have been identified, and the extracellular effects of members of any of these classes are screened by the methods and devices provided herein. Table 2 provides the different RTK classes and representative members of each class. Each is suitable for use herein when expressed on a readout particle (e.g., a readout cell or vesicle). In one embodiment, the method provides a step of screening a plurality of cell populations in parallel to identify one or more populations herein. The one or more populations include effector cells having the extracellular effect of one RTK of the subclasses provided in Table 2. In one embodiment, the method further includes a step of isolating one or more cell populations comprising ASCs having an extracellular effect to provide an isolated cell population. Further, the method includes a step of subjecting the isolated subpopulation to one or more additional extracellular effect assays by limiting dilution to identify ASCs having an extracellular effect. The additional extracellular effect assays can be performed via the microfluidic methods or benchtop assays provided herein. Alternatively, if a cell population is confirmed to have cells that exhibit the extracellular effect of an RTK, the cell population is recovered, lysed, and the nucleic acid is amplified. In a further embodiment, the nucleic acid is one or more antibody genes.

[0363] In one aspect, the invention relates to the identification of a cell population comprising effector cells that antagonize or agonize an RTK (i.e., an extracellular effect), for example, via a secretion (e.g., a monoclonal antibody). Effector cells are present in isolated effector cells or in a cell population comprising one or more effector cells.

[0364] [Table 2] JPEG0007699639000007.jpg195138JPEG0007699639000008.jpg195138JPEG0007699639000009.jpg195138JPEG0007699639000010.jpg194136JPEG0007699639000011.jpg137137

[0365] In one embodiment, the RTK is platelet-derived growth factor receptor (PDGFR), e.g., PDGFRα. PDGFs are a family of soluble growth factors (A, B, C, and D) that bind to form various homo- and hetero-dimers. These dimers are recognized by two closely related receptors (PDGFRa and PDGFRb) with different specificities. Specifically, PDGF-α selectively binds to PDGFRα and has been shown to drive the pathological mesenchymal response in fibrotic diseases including pulmonary fibrosis, liver cirrhosis, scleroderma, glomerulosclerosis, and cardiac fibrosis (Andrae et al. (2008). Genes Dev. 22, pp. 1276-1312, which is incorporated herein by reference in its entirety). It has also demonstrated that constitutive activation of PDGFRα in mice leads to progressive fibrosis in multiple organs (Olson et al. (2009). Dev. Cell 16, pp. 303-313, which is incorporated herein by reference in its entirety). Therefore, therapies that inhibit PDGFRα hold great potential for the treatment of fibrosis and for conditions that exacerbate up to 40% of the disease and represent a major medical problem inappropriate for the aging population. Antibodies (imatinib and nilotinib) have been investigated as inhibitors of PDGFRa but have significant off-target effects on other important RTKs including c-KIT and FLT-3, resulting in many side effects. Thus, while imatinib and nilotinib can effectively inhibit PDGFRα and PDGFRβ, their side effects are unacceptable for the treatment of fibrotic diseases. This highlights the potential for highly specific antibody inhibitors. The present invention overcomes this problem by providing, in one embodiment, an antibody with higher PDGFRα specificity compared to imatinib and nilotinib.

[0366] PDGFRα has been previously established as a target for the treatment of fibrosis. Two anti-human PDGFRα monoclonal antibody antagonists are in development for early clinical trials in cancer treatment (e.g., Shah et al. (2010). Cancer 116, pp. 1018-1026, which is incorporated herein by reference in its entirety). The methods provided herein facilitate the identification of the secretions of effector cells that bind to PDGFRα. In further embodiments, the secretions block the activity of both human and mouse PDGFRa in both cancer and fibrosis models.

[0367] One embodiment of an effector cell assay for determining whether the secretions of effector cells bind to PDGFRα is based on the use of suspension cell lines (e.g., 32D and Ba / F3). Those suspension cell lines are strictly dependent on the cytokine IL-3 for survival and proliferation. However, this “IL-3 dependence” is treated through the expression and activation of almost any tyrosine kinase. This approach was first used by Daley and Baltimore to evaluate the BCR-ABL fusion oncogene and has been widely used for high-throughput screening of small molecule tyrosine kinase inhibitors (e.g., Warmuth et al. (2007). Curr. Opom. Oncology 19, pp. 55-60; Daley and Boltimore (19898). Proc. Natl. Acad. Sci. U.S.A. 85, pp. 9312-9316, each incorporated by reference in its entirety for all purposes). To monitor signaling, PDGFRα and PDGFRβ (both human and mouse forms) are expressed in 32D cells (readout cells), a mouse hematopoietic cell line that does not naturally express either receptor. This allows for the separation of each pathway (otherwise it would be somewhat difficult as both receptors are co-expressed). Expression of human PDGFRα / β in 32D cells has previously been confirmed to give a functional PDGF-induced mitogenic response (Matsui ete al. (1989). Proc. Natl. Acad Sc. U.S.A. 86, pp. 8314-8318, incorporated by reference in its entirety). In the absence of IL-3, 32D cells do not divide at all, but PDGF stimulation of cells expressing an RTK alleviates the requirement for IL-3 and provides a rapid mitogenic response that is detectable by microscopy. Detectable responses are, in one embodiment, cell proliferation, morphological changes, increased motility / chemotaxis or cell death / apoptosis in the presence of an antagonist. The light multiplexing method is, in one embodiment, used in one of the devices provided herein to simultaneously measure the inhibition / activation of the responses of both PDGFRα and PDGFRβ.In another embodiment, in one of the devices provided herein, inhibition / activation of the responses of both PDGFRα and PDGFRβ is measured by two extracellular assays that are performed sequentially within the same microfluidic chamber.

[0368] Full-length cDNAs of human / mouse PDGFRα and PDGFRβ (Sino Biological) are expressed in 32D cells (ATCC; CRL-11346) in one embodiment. The 32D cells use a modified pCMV expression vector containing an IRES sequence with either GFP or RFP. Thereby, two types of "readout cells" that can be identified by fluorescence imaging are created. The readout cells are characterized by optimizing the medium and feeding conditions, determining the dose response to the PDGF ligand, and characterizing the morphology and response kinetics. The use of suspension cells (e.g., 32D or Ba / F3) offers the advantages that a single cell can be easily identified by image analysis and that it is physically smaller (in terms of projected area) than adherent cells. As a result, a single chamber can accommodate more than 100 readout cells before reaching confluence. In another embodiment, instead of 32D cells or Ba / F3 cells, another IL-3-dependent mouse cell line having properties similar to 32D is used as the readout cell. Both 32D cells and Ba / F3 cells are of bone marrow origin, grow well in media optimized for ASCs, and secrete IL-6, a growth factor important for the maintenance of ASCs (e.g., Cassese et al. (2003). J. Immunol. 171, pp. 1684-1690, which is incorporated herein by reference in its entirety).

[0369] Preclinical models have been developed to evaluate the role of PDGFRa in fibrosis. Specifically, two models of cardiac fibrosis are provided here. The first is based on ischemic injury (isoproterenol-induced cardiac injury; ICD), and the second is based on myocardial infarction (MI) induced by coronary artery ligation. At the time of injury, the fibrotic response is initiated by the rapid expansion of PDGFRα+ / Sca1+ positive progenitor cells. Thereby, it accounts for more than 50% of the cells that proliferate in response to injury. Subsequently, the differentiation of these progeny occurs in the matrix that produces PDGFRα low / Sca1 low myofibroblasts. Gene expression by RT-qPCR shows the expression of multiple markers associated with fibrotic matrix deposition, including α-smooth muscle actin (αSMA) and type I collagen (Col1). They are detectable in (Sca1+) progenitor cells but are substantially upregulated in the differentiated population. In one embodiment, the cell population has been identified to contain effector cells that secrete monoclonal antibodies that attenuate the progenitor expansion leading to decreased fibrosis. This extracellular effect assay is performed by monitoring two independent markers (Sca1+ / PDGFRα+ progenitor cells and the initial proliferation of E. coli-driven GFP). Specifically, after MI, the fibrotic response is characterized by the initial GFP expression of PDGFRα+ / Sca1+ progenitor cells in the emerging population of myofibroblasts and later at increased intensity.

[0370] The present invention provides a method and device for screening a plurality of cell populations in parallel to identify one or more cell populations having an extracellular effect, or a variation in an extracellular effect, that is compared to other populations. The identified cell populations include one or more effector cells that are the cause of the extracellular effect. The extracellular effect is, for example, an extracellular effect of a GPCR (e.g., GPCR binding), an agonist or an antagonist. As described herein, the extracellular effect need not be attributable to each cell, or a plurality of cells, in the population. Rather, the methods provided herein enable the detection of the extracellular effect of a single effector cell when the effector cell is present in a heterogeneous population containing tens to hundreds of cells (e.g., from about 10 to about 500 cells, or from about 10 to about 100 cells) or from about 2 to about 10 cells (e.g., from about 2 to about 10 cells).

[0371] GPCRs are a superfamily of seven-transmembrane receptors that include over 800 members in the human genome. Each GPCR has a C-terminal tail facing the cytosol and an amino terminus on the outer surface of the cell. Inside the cell, the GPCR binds to a heterotrimeric G protein. When an agonist binds, the GPCR undergoes a conformational change that results in the activation of the associated G protein. Approximately half of these are olfactory receptors that have remaining portions that respond to the full range of different ligands. The full range is from calcium and metabolites to cytokines and neurotransmitters. The present invention, in one embodiment, provides a method for selecting one or more ASCs having an extracellular effect of a GPCR. The GPCR is not particularly limited herein. Rather, screening methods for any GPCR are suitable for use in the present invention.

[0372] The type of G protein that is naturally associated with a particular GPCR determines the transfected cell signaling cascade. For Gq-coupled receptors, the signal resulting from receptor activation is an increase in intracellular calcium levels. For Gs-coupled receptors, an increase in intracellular cAMP is observed. For Gi-coupled receptors, which constitute 50% of GPCRs, activation leads to inhibition of cAMP production. For embodiments where the effector cell property is the activity of a Gi-coupled GPCR, it may be necessary to stimulate the readout cells with a non-specific activator of adenylyl cyclase. In one embodiment, the activator of adenylyl cyclase is forskolin. Thus, activation of a Gi-coupled receptor by one or more effector cells prevents the induced forskolin from increasing cAMP. Accordingly, forskolin is used as an accessory agent in the extracellular effect assay of one or more GPCRs provided herein.

[0373] In one embodiment, the present invention provides a means for determining whether effector cells (e.g., ASCs) within a cell population have an extracellular effect of a GPCR. The GPCR is present on one or more readout particles within a microfluidic chamber. And the extracellular effect in one embodiment is binding to the GPCR, demonstrated affinity or specificity, inhibition or activity. The GPCR may be a stabilized GPCR such as one of the GPCRs made by the method of Heptares Therapeutics (stabilized receptor, STAR™ technology). The effector cells (e.g., ASCs) are, in one embodiment, present as a single cell. Or present in a homogeneous or heterogeneous cell population within the microfluidic chamber. In one embodiment, the methods and devices provided herein are used to identify one or more ASCs or a plurality of cell populations. The one or more ASCs secrete one or more antibodies that exhibit an extracellular effect of one of the GPCRs described in Table 3A and / or the table, or one of the GPCRs disclosed in PCT International Publication WO 2004 / 040000 (which is incorporated by reference in its entirety). For example, in one embodiment, the GPCR belongs to one of the following classes (class A, class B, class C, adhesion, frizzled).

[0374] In another embodiment, the extracellular effect has an effect on endothelial differentiation, G protein-coupled (EDG) receptors. The EDG receptor family includes 11 GPCRs (S1P1-5 and LPA1-6) involved in lipid signaling and binds lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Signaling through LPA and S1P regulates many functions in disease and health, including cell proliferation, activation of immune cells, migration, invasion, inflammation, and angiogenesis. There has been little success in generating potent and specific small molecule inhibitors for this family. Therefore, monoclonal antibodies are made very attractive alternatives. In one embodiment, the EDG receptors are S1P3 (EDG3), S1PR1 (EDG1). The latter has been shown to activate NF-κB and STAT3 in several types of cancer, including breast cancer, lymphoma, ovarian cancer, and melanoma, and plays an important role in immune cell trafficking and cancer metastasis (Milstien and Spiegel (2006). Cancer Cell 9, pp. 148-15, which is incorporated herein by reference in its entirety). A monoclonal antibody that neutralizes the S1P ligand (Sonepcizumab) recently entered a Phase II trial for the treatment of advanced solid tumors (NCT00661414). In one embodiment, the methods and devices provided herein are used to identify and isolate ASCs that secrete antibodies with higher affinity than Sonepcizumab or antibodies that inhibit S1P more potently than Sonepcizumab. In another embodiment, the extracellular effect is the effect of the LPA2 (EDG4) receptor. LPA2 is overexpressed in thyroid, colon, gastric and thoracic carcinomas, as well as in many ovarian tumors. Therefore, LPA2 is the first contributor to the detrimental and sensitivity to LPA.

[0375] In one aspect, the cell population is assayed for its ability to exert an extracellular effect on the chemokine receptor present on the readout particles. In a further embodiment, the chemokine receptor is also the CXC chemokine receptor type 4 (CXCR-4), also known as fusin or CD184. CXCR4 binds the potent chemotactic SDF1α (CXCL12) for immune cell recruitment, known as CXC motif chemokine 12 (CXCL12). DNA immunization is used to generate 92 hybridomas against this target. 75 of those hybridomas exhibited different chain usage and epitope recognition (Genetic Eng and Biotech news, Aug 2013). The selection of hybridomas indicates that it captures only a fraction of the diverse antibodies available. Signaling through the CXCR4 / CXCL12 axis has been shown to play a central role in tumor cell proliferation, angiogenesis, cell survival, and has been shown to be involved in mediating the growth of secondary metastases in CXCL12-producing organs such as the liver and bone marrow (Teicher and Fricker (2010). Clin. Cncer Res. 16, pp. 2927-2931, which is hereby incorporated by reference in its entirety).

[0376] In another embodiment, the cell population is screened for its ability to exert an effect on the chemokine receptor CXCR7, which has recently been found to bind SDF1α. Unlike CXCR4, which signals through canonical G-protein coupling, CXCR7 signals independently through the β-arrestin pathway.

[0377] In another embodiment, the GPCR is a protease-activated receptor (PAR1, PAR3, and PAR4). The GPCR is a class of GPCRs activated by thrombin-mediated cleavage of the exposed N-terminus. Protease-activated receptors are involved in fibrosis. In yet another embodiment, the GPCR is one of the GPCRs in Table 3A or Table 3B below.

[0378] In embodiments where the extracellular effect is that of a GPCR, the invention is not limited to specific GPCRs. For example, cell lines expressing specific GPCRs designed to provide a readout of binding, activity or inhibition are commercially available from, for example, Life Thechnologies (GeneBLAzer® and Tango™ cell lines), DiscoveRx, Cisbio, Perkin Elmer, etc., and are suitable for use as the readout cells described herein.

[0379] In one embodiment, a GPCR from one of the following receptor families is expressed on one or more of the readout cells herein. The extracellular effect is measured in relation to one or more of the following GPCRs. Namely, acetylcholine receptor, adenosine receptor, adrenergic receptor, angiotensin receptor, bradykinin receptor, calcitonin receptor, calcium sensing receptor, cannabinoid receptor, chemokine receptor, cholecystokinin receptor, complement component (C5AR1), corticotropin releasing factor receptor, dopamine receptor, endothelial differentiation gene receptor, endothelin receptor, formyl peptide-like receptor, galanin receptor, gastrin releasing peptide receptor, ghrelin receptor of the receptor, gastric inhibitory polypeptide receptor, glucagon receptor, gonadotropin releasing hormone receptor, histamine receptor, kisspeptin (KISS1) receptor, leukotriene receptor, melanin concentrating hormone receptor, melanocortin receptor, melatonin receptor, motilin receptor, neuropeptide receptor, nicotinic acid, opioid receptor, orexin receptor, orphan receptor, platelet factor receptor, prokineticin receptor, prolactin releasing peptide, prostanoid receptor, protease activated receptor, P2Y (purinergic) receptor, relaxin receptor, secretin receptor, serotonin receptor, somatostatin receptor, tachykinin receptor, vasopressin receptor, oxytocin receptor, vasoactive intestinal peptide (VIP) receptor or pituitary adenylate cyclase activating polypeptide (PACAP) receptor. JPEG0007699639000012.jpg148133JPEG0007699639000013.jpg194136JPEG0007699639000014.jpg77131JPEG0007699639000015.jpg110131JPEG0007699639000016.jpg194136JPEG0007699639000017.jpg195136JPEG0007699639000018.jpg196136JPEG0007699639000019.jpg196136JPEG0007699639000020.jpg196136JPEG0007699639000021.jpg196136JPEG0007699639000022.jpg141136

[0380] In one embodiment, effector cells are assayed for the extracellular effects of readout cells expressing GPCRs by one or more assays provided in Table 4 below. In another embodiment, the readout particle population comprises beads or vesicles (available from Integral Molecular) functionalized with membrane extracts, or stabilized solubilized GPCRs (e.g., from Heptares).

[0381] GPCRs are phosphorylated and interact with a protein called arrestin. There are three main methods for measuring arrestin activation. (i) Microscopy using fluorescently labeled arrestin (e.g., GFP or YFP), (ii) microscopy utilizing enzyme complementation, and (iii) microscopy using the TANGO™ reporter system (β-lactamase) (Promega). In one embodiment, the TANGO™ reporter system is used in one or more readout cells. This technique uses a GPCR linked to a transcription factor via a cleavable linker. Arrestin is fused to an inactive protease. When arrestin binds to the GPCR, the high local concentration of the linker and protease leads to cleavage of the linker, releasing a nuclear transcription factor that activates transcription. The β-lactamase assay does not require cell lysis, can be performed on live cells, and can be imaged within just 6 hours of agonist incubation.

[0382] In one embodiment, the β-arrestin GPCR assay, which is commonly used for the detection of antagonists and agonists of GPCR signaling, is used in the methods and devices provided herein to identify effector cells that secrete biomolecules that bind to the GPCR (Rossi et al. (1997). Proc. Natl. Acad. Sci. U.S.A. 94, pp. 8405-8410, which is incorporated by reference in its entirety for all purposes). This assay is based on the β-galactosidase (β-Gal) enzyme complementation technology currently commercialized by DiscoveRx. The GPCR target is fused in frame with a small N-terminal fragment of the β-Gal enzyme. Upon activation of the GPCR, a second fusion protein containing β-arrestin linked to the N-terminal sequence of β-Gal binds to the GPCR, resulting in the formation of a functional β-Gal enzyme. The β-Gal enzyme then rapidly converts the non-fluorescent substrate di-β-D-galactopyranoside (FDG) to fluorescein, providing great amplification and excellent sensitivity. In this embodiment, the readout cells (which have GPCRs) are pre-loaded (off-chip) with a cell-permeable pro-substrate (acetylated FDG) that is converted to cell-impermeable FDG by esterase cleavage of the acetate group. Fluorescein is actively transported out of the live cells by performing this assay within a microfluidic chamber, while the fluorescent product is concentrated. Thereby, it provides a significantly enhanced sensitivity over plate-based assays. DiscoveRx has validated this assay strategy used in a micro-well format across a large panel of GPCRs.

[0383] In one embodiment, activation of a GPCR by an effector cell is determined in a microfluidic format by detecting an increase in cytoplasmic calcium in one or more readout cells. In a further embodiment, the increase in cytoplasmic calcium is detected using one or more calcium-sensitive dyes. Calcium-sensitive dyes have low fluorescence levels in the absence of calcium and undergo an increase in fluorescence properties once bound by calcium. The peak of the fluorescence signal at about 1 minute is detectable in the 5- to 10-minute time range. Thus, for detecting activity using fluorescent calcium, the addition and detection of the agonist are closely related. To achieve this relationship, the effector cell is simultaneously exposed to a population of readout cells and one or more calcium-sensitive dyes. In one embodiment, one or more calcium-sensitive dyes are one provided in the FLIPR™ Calcium Assay (Molecular Devices).

[0384] In one embodiment, the recombinant expressed jellyfish luminescent protein, aequorin, is used in a functional GPCR screen (i.e., an extracellular effect assay). The extracellular effect is the modulation of a GPCR. Aequorin is a calcium-sensitive reporter protein that generates a luminescence signal when a coelenterazine derivative is added. Cell lines engineered with GPCRs expressed in a mitochondrial-targeted version of apoaequorin are commercially available (Euroscreen). In one embodiment, one or more cell lines available from Euroscreen are used as a population of readout cells in a method for assessing the extracellular effect, or variation in the extracellular effect, of an effector cell.

[0385] In one embodiment, the extracellular effect of a GPCR is measured using, as a population of readout cells, one of the ACTOne cell lines (Codex Biosolutions) that express the GPCR and cyclic nucleotide-gated (CNG) channels. In this embodiment, the extracellular effect assay is coupled with a cell line that contains an exogenous cyclic nucleotide-gated (CNG) channel. The channel is activated by elevated intracellular levels of cAMP. This results in ion flux (often detectable by a calcium-responsive dye) and depolarization of the cell membrane detected using a fluorescent membrane potential (MP) dye. The ACTOne cAMP assay enables both kinetic and endpoint measurements of the fluctuations of intracellular cAMP using a fluorescence microplate reader.

[0386] In one embodiment, the reporter gene assay is used to determine whether an effector cell regulates a specific GPCR. In this embodiment, the regulation of the GPCR is the extracellular effect being evaluated. The reporter gene assay is based, in one embodiment, on GPCR second messengers such as calcium (AP1 or NFAT response elements) or cAMP (CRE response element) to activate or inhibit response elements placed upstream of a minimal promoter. This in turn regulates the expression of a reporter protein selected by the user. The expression of the reporter is, in one embodiment, bound to the response element of a transcription factor activated by signal transduction via the GPCR. For example, the expression of the reporter gene can be bound to the response element of one of the following transcription factors: ATF2 / ATF3 / AFT4, CREB, ELK1 / SRF, FOS / JUN, MEF2, GLI, FOXO, STAT3, NFAT, NFκB, etc. In a further embodiment, the transcription factor is NFAT. The reporter gene assay is commercially available, for example, from SA Biosciences.

[0387] Reporter proteins are known in the art and include, for example, β-galactosidase, luciferase (e.g., Paguio. et al. (2006). "Use of luciferase reporter assay for screening of GPCR modulators", Cell Notes Issue 16, pp. 22-25; Dual-Glo™ luciferase assay system technical manual #TM058; pGL4 luciferase reporter vector technical manual #TM259, all of which are incorporated by reference in their entirety for all purposes), GFP, YFP, CFP, β-lactamase. Reporter gene assays for measuring GPCR signaling are commercially available and can be used in the methods and devices described herein. For example, the GeneBLAzer® assay from Life Technologies is suitable for use in the present invention.

[0388] In one embodiment, overexpression of G protein in reporter cells is engineered to constitutively signal through calcium to cAMP-coupled GPCRs. This is referred to as coupling strength.

[0389] In one embodiment, a Gq-coupled cell line is used as a readout cell line by the methods described herein. In one embodiment, a Gq-coupled cell line reports a GPCR that signals through β-lactamase. For example, there is one of the cell line GPCR reporter cell lines (GeneBLAzer®, Life Technologies). The reporter cell line can be a dividing cell or can contain normal dividing cells.

[0390] Cyclic AMP response element-binding protein (CREB) is a transcription factor as described above and is used in one embodiment for Gs and / or Gi coupled GPCRs. In a further embodiment, forskolin is utilized as an accessory particle. CRE reporter is available in the form of a plasmid or lentivirus that drives GFP expression from SA Biosciences and is suitable for use in the methods and devices described herein. For example, in one embodiment, an assay system available from SA Biosciences is used herein to generate readout cells (http: / / www.sabiosciences.com / reporter_assay_product / HTML / CCS-002G.html). Life Technologies has CRE-responsive cell lines that express specific GPCRs and these can be used as readout cells, as well as in the methods described herein.

[0391] In one aspect, one or more effector cells present in a cell population are assayed for their ability to activate or antagonize GPCRs present in one or more readout cells by detecting an increase or decrease in cAMP levels inside the one or more readout cells. ELISA-based assays, homogeneous time-resolved fluorescence (HTRF) (Dogorce et al. (2009). Current Chemical Genomics 3, pp. 22-32, the disclosure of which is incorporated herein by reference in its entirety), and complementary enzymes are all used in the microfluidic devices and assays provided herein to determine cAMP levels in readout cells. Each of these cAMP detection methods requires cell lysis to release cAMP for detection, such as the actually measured cyclic AMP.

[0392] Assays for measuring cAMP in whole cells and assays for measuring adenylate cyclase activity in membranes are commercially available (e.g., Gabriel et al. (2003). Assay Drug Dev. Technol. 1, pp. 291-303; Williams (2004). Nat. Rev. Drug Discov. 3, pp. 125-135, which are incorporated by reference in their entirety) and are suitable for use in the devices and methods provided herein. That is, a cell population within one or more microfluidic chambers is assayed according to these methods.

[0393] Cisbio International (Codolet, France) has developed a sensitive high-throughput homogeneous cAMP assay (HTRF) based on time-resolved fluorescence resonance energy transfer technology (Degorce et al. (2009). Current Chemical Genomics 3, pp. 22-32, the disclosure of which is incorporated by reference in its entirety). It is used herein to screen effector cells that exhibit the effects of GPCRs. This method is a competitive immunoassay between native cAMP produced by cells and a cAMP-labeled dye (cAMP-D2). cAMP-D2 binding is visualized by a MaB anti-cAMP labeled with cryptate. The specific signal (i.e., energy transfer) is inversely proportional to the concentration of cAMP in the sample, in this case, the amount of cAMP activated in the readout cells by effector cells or the secretions of effector cells. The readout cells are first lysed to release the cAMP for detection so that cAMP can be measured. This assay has been confirmed for both Gs-coupled (β2-adrenergic, histamine H2, melanocortin MC4, CGRP and dopamine D1) receptors and G-i / o-coupled (histamine H3) receptors.

[0394] cAMP assay kits based on fluorescence polarization are commercially available, for example, from Perkin Elmer, Molecular Devices, and GE Healthcare, each of which is suitable for use as an effector cell assay in the methods and devices provided herein. Accordingly, one embodiment of the invention includes the step of selecting a cell population comprising one or more effector cells based on the results of a fluorescence polarization assay of the effector cells and / or cAMP. This method is used in one embodiment to determine whether an effector cell activates (agonizes) or inhibits (antagonizes) a particular GPCR.

[0395] In one embodiment, the Perkin Elmer AlphaScreen™ cAMP assay, a sensitive bead-based chemiluminescent assay that requires laser activation, is used in the devices provided herein to screen for effector cells that have an effect (specifically, activation or inhibition of a GPCR) on the readout cells.

[0396] DiscoveRx (http: / / www.discoverx.com) offers a homogeneous high-throughput cAMP assay kit called HitHunter™ based on patented enzyme (β-galactosidase) complementation technology that uses either a fluorescent or luminescent substrate (Eglen and Singh (2003). Comb Chem. High Throughput Screen 6, pp. 381-387; Weber et al. (2004). Assay Drug Dev. Technol. 2, pp. 39-49; Englen (2005). Comb. Chem. High Throughput Screen 8, pp. 311-3188, each of which is incorporated by reference in its entirety). This assay can be performed herein to detect variations in the extracellular effects of readout cells expressing a GPCR or effector cells having extracellular effects.

[0397] Cell events resulting from the activation or inhibition of GPCR receptors can also be detected to determine the characteristics of effector cells (e.g., antibodies that produce the ability to activate or antagonize cells) in the readout cells. For example, in the case of Gq-coupled receptors, when the GPCR is activated, the Gq protein is activated. This leads to the cleavage of phospholipase C of membrane lipids. This cleavage leads to the production of inositol trisphosphate 3 (IP3). Free IP3 binds to its target on the surface of the endoplasmic reticulum, which causes the release of calcium. Calcium activates specific calcium-responsive transcription vectors such as the nuclear factor of activated T cells (NFAT). Thus, by monitoring the activity or expression of NFAT, an indirect readout of GPCR in the readout cells is established. See, for example, Crabtree and Olson (2002). Cell 109, pp. S67-S79 (which is hereby incorporated by reference in its entirety).

[0398] Once activated, more than 60% of all GPCRs are internalized. Using tagged GPCRs (usually made with a C-terminal GFP tag), the distribution of the receptor in one embodiment is imaged in the presence and absence of the ligand. Upon ligand stimulation, receptors that are normally evenly distributed are often displayed as endocytosis sites. JPEG0007699639000023.jpg202124JPEG0007699639000024.jpg203126JPEG0007699639000025.jpg20383

[0399] One embodiment of a workflow for single cell antibody secreting cell (ASC) / antibody selection pipeline is shown in FIG. 1. In this embodiment, the host animal is immunized with the target antigen and cells are obtained from the spleen, blood, lymph nodes and / or bone marrow one week after the final immune boost. These samples are then optionally enriched for ASCs by flow cytometry (e.g., FACS) or magnetic bead purification using established surface markers (if available) or microfluidic enrichment. Thereafter, the enriched ASC population is loaded into a microfluidic array of nanoliter volume chambers at a loading concentration selected to achieve about 1 to about 500 cells per chamber or about 1 to about 2050 cells per chamber. Depending on the pre-enrichment step, individual chambers contain multiple ASCs, a single ASC, or no ASCs. The chambers are then isolated by closing the microvalves and incubated to allow antibodies to be secreted into the small chamber volume. ASCs generally secrete antibodies at a rate of 1000 antibody molecules per second. And since the volume of the individual chambers provided herein is on the order of 2 nL, a concentration of about 10 nM of the secreted monoclonal antibody is provided in about 3 hours (each ASC secretes a unique monoclonal antibody). In a further embodiment, integrated microfluidic control is used for reagent delivery and exchange to perform an effector cell assay on an image system read using automated microscopy and real-time image processing. A cell population or individual ASCs containing one or more ASCs that secrete antibodies with desired properties (e.g., binding, specificity, affinity, function) are then recovered from the individual chambers. Further analysis of the cell population recovered by limiting dilution is then performed. Further analysis of individual ASCs can also be performed if individual ASCs are supplied to and recovered from the chambers. For example, in one embodiment, further analysis includes single cell RT-PCR to amplify pairs of HV and LV for cloning and sequence analysis in cell lines.

[0400] In another embodiment, after immunizing an animal and obtaining cells from the spleen, blood, lymph nodes, and / or bone marrow, the cells form a starting population. That starting population, i.e., as a plurality of cell populations, is directly loaded into the individual chambers of the microfluidic device provided herein. The individual cell populations are present within each microfluidic chamber. An extracellular effect assay is performed on the individual cell populations within the individual chambers to determine whether any of the individual cell populations contain one or more effector cells that are the cause of the extracellular effect.

[0401] The host animal is immunized with the target antigen prior to microfluidic analysis, although the invention is not limited thereto. For example, in one embodiment, the cells are obtained from the spleen, blood, lymph nodes, or bone marrow from a host (including humans). This is followed by enrichment for ASCs. Alternatively, no enrichment step is performed and the cells are directly loaded (i.e., as a plurality of cell populations) into the chambers of the device provided herein. The individual cell populations are present within each chamber.

[0402] The methods provided herein enable the selection of antibodies from any host species. This provides two important advantages for the discovery of therapeutic antibodies. First, the ability to work against species other than mice and rats enables the selection of mAbs against targets with high homology to mouse proteins and mAbs against human proteins. The mAbs against those human proteins cross-react with mice and are used in readily accessible preclinical mouse models. Second, immunization of mice leads to responses that are immunodominant to some epitopes. This results in low diversity of the generated antibodies. As a result, expanding to other species significantly improves the diversity of antibodies that recognize different epitopes. Thus, in the embodiments described herein, mice, rats, and rabbits are used for immunization. Subsequently, ASCs from these immunized animals are selected. In one embodiment, rabbits are immunized with an antigen and ASCs from the immunized rabbits are selected using the methods and devices provided herein. As will be appreciated by those skilled in the art, rabbits offer the advantage of a well-defined mechanism of affinity maturation using gene conversion to obtain greater antibody diversity, greater physical size (diversity of multiple antibodies), and greater evolutionary distance from humans (more recognized epitopes).

[0403] The immunization strategy, in one embodiment, is immunization with the protein, cells, and / or DNA. For example, for PDGFRα, the extracellular domain obtained from expression in mammalian cell lines or purchased from a commercial source (Calixar) is used to immunize animals. For CXCR4, in one embodiment, preparations of virus-like particles (VLPs) from a commercial source (Integral Molecular), nanoparticles with high expression of the GPCR in an unfolded conformation are used. Cell-based immunization is performed by overexpressing the full-length protein in cell lines (e.g., 32D-PDGFRα cells for mice / rats) and rabbit fibroblast cell lines (SIRC cells for rabbits). It includes protocols for using the new cell lines as the final boost for concentrating specific mAbs. Various established DNA immunization protocols are also suitable for use in the present invention. DNA immunization is a method of choice for complex membrane proteins.This is because it has been demonstrated to 1) eliminate the need for protein expression and purification, 2) ensure the native conformation of the antigen, 3) reduce the potential for non-specific immune responses to other cell membrane antigens, and 4) be effective for challenging targets (Bates et al. (2006). Biotechniques 40, pp. 199-208; Chambers and Johnston (2003). Nat. Biotechnol. 21, pp. 1088-1092; Nagata et al. (2003). J. Immunol. Methods 280, pp. 59-72; Chowdhury et al. (2001). J. Immunol Methods 249, pp. 147-154; Surman et al. (1998). J. Immunol. Methods 214, pp. 51-62; Leinonen et al. (2004). J. Immunol. Methods 289, pp. 157-167; Takatasuka et al. (2011). J. Pharmacol. and Toxicol. Methods 63, pp. 250-257, each of which is hereby incorporated by reference in its entirety for all purposes). All immunizations are performed in accordance with animal care requirements and established protocols.

[0404] Anti-PDGFRα antibodies have been previously produced in rats, mice, and rabbits, and comparison of the extracellular domains of PDGFRα shows several sites of substantial variation (Figure 24). Thus, a good immune response is expected to be obtained from this antigen. Anti-CXCR4 mAbs have been previously generated using both lipid particles and DNA immunization. As a result, this target has the potential to generate a good immune response. Optionally, we use co-expression of GroEl or GM-CSF (either co-expressed or as a fusion) as a molecular adjuvant and test and investigate different adjuvants and immunization schedules (Takatsuka et al. (2011). J. Pharmacol. and Toxicol. Methods 63, pp. 250-257, Fujimoto et al. (2012). J. Immunol. Methods 375, pp. 243-251, which are hereby incorporated by reference in their entirety for all purposes).

[0405] The devices provided herein are based on multilayer soft lithography (MSL) microfluidics (Unger et al. (2000). Science 7, pp. 113-116, which is hereby incorporated by reference in its entirety). MSL is a manufacturing method that provides increased sensitivity through small-volume reactions, high scalability and parallelization, robust cell culture, fluid handling control required for complex assays, and significant reduction in cost and reagent consumption.

[0406] The number of effector cells isolated per run of the device (i.e., the number of cells within each chamber of the device) is a function of the concentration of cells in the cell suspension loaded onto the device, the frequency of the particular effector cells in the cell suspension, and the total number of chambers of the device. Devices having arrays of 40,000 or more effector cell assay chambers are contemplated.

[0407] Among all microfluidic technologies, MSL is unique in the rapid and inexpensive prototyping of devices with thousands of integrated microvalves (Thorsen et al. (2002). Science 298, pp. 58 - 584, which is incorporated herein by reference in its entirety). These valves are used to construct high - level fluid components including mixers, peristaltic pumps (Unger et al. (2000). Science 7, pp. 113 - 116), and fluid multiplexers (Thorsen et al. (2002). Science 298, pp. 58 - 584; Hansen and Quake (2003). Curr. Opin. Struc. Biol. 13, pp. 538 - 544, which are incorporated herein by reference in their entirety). Thus, it enables high - level integration and on - chip liquid handling (Hansen et al. (2004). Proc. Natl. Acad. Sci. U.S.A. 101, pp. 14431 - 1436; Maerkl and Quake (2007). Science 315, pp. 233 - 237, which are incorporated herein by reference in their entirety) (FIG. 25).

[0408] FIG. 25A shows an optical micrograph of a valve fabricated by MSL. Two intersecting microfabricated channels (one "flow channel" (vertical) for the active fluid and the other "control channel" (horizontal) for valve actuation) create the valve structure. The flow channel is separated from the control channel by a thin elastomeric membrane to create a "pinch valve". Pressurization of the control channel deflects the membrane to close the flow channel. FIG. 25B shows a cross - section of an MSL device integrating multiple valves (filled with green and blue food dyes). FIG. 25C is a cross - section of a device having a total of 16,000 valves, 4000 chambers, and more than 3000 layer - layer interconnects (arrows). FIG. 25D shows a penny for scale and an example of a microfluidic device. The device shown is for illustrative purposes of MSL manufacturing technology.

[0409] In one embodiment, the assay chamber of the device has an average volume of about 100 pL to about 100 nL. For example, in one embodiment, one or more characteristics of effector cells are assayed within a microfluidic chamber containing a cell population. The volume of the microfluidic chamber is about 100 pL, about 200 pL, about 300 pL, about 400 pL, about 500 pL, about 60 pL, about 700 pL, about 800 pL, about 900 pL or about 1 nL. In another embodiment, the volume of the microfluidic chamber is about 2 nL. In another embodiment, the volume of the microfluidic chamber for assaying the characteristics of effector cells within a cell population is from about 100 pL to about 100 nL, from about 100 pL to about 50 nL, from about 100 pL to about 10 nL, from about 100 nL to about 1 nL, from about 50 pL to about 100 nL, from about 50 pL to about 50 nL, from about 50 pL to about 10 nL, or from about 50 pL to about 1 nL. In yet another embodiment, the volume of the microfluidic chamber for assaying the characteristics of effector cells within a cell population is about 10 nL, about 20 nL, about 30 nL, about 40 nL, about 50 nL, about 60 nL, about 70, about 80 nL, about 90 nL, or about 100 nL.

[0410] The MSL manufacturing process utilizes well-established photolithography techniques and advances in microelectronics manufacturing technology. The first step in MSL is to draw the design of the flow channels and control channels using computer-aided design software printed on a high-resolution mask. A silicon (Si) wafer covered with photoresist is exposed to ultraviolet light. The ultraviolet light is filtered by the mask in specific regions. Depending on whether the photoresist is negative or positive (either the exposed area (negative) or the non-exposed area (positive)), cross-linking and polymerization of the resist occur. The non-polymerized resist is soluble in the developer and is subsequently washed away. By combining spin coating at different speeds with different photoresists, the silicon wafer is patterned with various different shapes and heights that define various channels and chambers. The wafer is then used as a mold for transferring the pattern to polydimethylsiloxane (PDMS). In one embodiment, before molding with PDMS and after defining the photoresist layer, the mold is coated with parylene (chemical vapor deposited poly(p-xylylene) polymer barrier) to reduce the adhesion of the PDMS during molding, to improve the durability of the mold, and to enable the replication of small features.

[0411] In MSL, stacking different layers of PDMS casts from different molds on top of each other is used to create channels for overlaying a "flow" layer and a "control" layer. Two (or more) layers are bonded by mixing a curing agent component and a potting prepolymer component in complementary stoichiometric ratios to achieve vulcanization. To create a simple microfluidic chip, the "thick" layer (e.g., about 200 - 2000 μm) is cast from a mold containing the flow layer. Also, the "thin" layer (e.g., about 25 - 300 μm) is cast from a mold containing the control layer. After partial vulcanization of both layers, the flow layer is peeled from its mold and aligned with the control layer (which is still on its mold). The control layer and the flow layer are bonded, for example, at 80 °C for about 15 - 60 minutes. The double slab is peeled from the control mold, inlet and outlet holes are punched, and the double slab is adhered to a blank layer of PDMS (i.e., a flat layer of PDMS without structural features). After spending more time for bonding, the completed device is mounted on a glass slide. The flow of fluid within the device is controlled using an off-chip computer-programmable solenoid that actuates the pressure applied to the fluid within the channels of the control layer. When pressure is applied to these control channels, a flexible membrane between the overlapping orthogonal controls and the flow line is deflected within the flow path, effectively valve-regulating the flow. Different combinations of these valves are used to create peristaltic pumps, multiplexer controls, and separate different regions of the chip.

[0412] Regarding the fluidized bed, the assay chamber and the channels that control the flow of fluid into and out of the assay chamber are defined by a photoresist layer. As will be understood by those skilled in the art, the thickness of the photoresist layer is partially controlled by the spin coating speed and the specific photoresist selected for use. In one embodiment, most of the assay chamber is defined by the function of SU-8 100 directly on a Si wafer. As is known to those skilled in the art, SU-8 is a commonly used epoxy-based negative photoresist. Alternatively, other photoresists known to those skilled in the art are used to define the assay chamber having the height described above. In some embodiments, the assay chamber has a width of 50 - 500 μM and a height of 50 - 500 μM, respectively, as defined by the characteristics of SU-8.

[0413] The MSL manufacturing technology enables a wide range of chamber volumes and device densities to be manufactured. In one embodiment, for the devices provided herein, from about 2000 to about 10,000 effector cell analysis chambers are provided in a single integrated device. The effector cell analysis chamber, in one embodiment, has an average volume of from about 1 nL to about 4 nL, for example, from about 1 nL to about 3 nL, or from about 2 nL to about 4 nL. The effector cell analysis chambers, in one embodiment, are connected in a continuous format as shown in FIG. 26. As an example, a device having 4032 individual analysis chambers (2.25 nL average volume) connected in a continuous format achieves a screening throughput of about 100,000 cells per run (FIG. 8). The integrated microfluidic valves utilized in the devices provided herein enable chamber separation and programmable washing with reagents selected from multiple inlets (e.g., from 2 to about 32 inlets, from 2 to about 20 inlets, from 2 to about 15 inlets, from 2 to about 10 inlets, or from 2 to about 9 inlets, or from 2 to about 8 inlets, or from 2 to about 7 inlets or from 2 to about 6 inlets). An additional inlet is provided to control the pressure of the valve (FIG. 26).

[0414] The devices provided herein utilize gravity-based immobilization of cells and / or particles. Gravity-based immobilization enables perfusion of non-adherent cell types and generally allows for the exchange of buffers and reagents within the chamber. Each chamber has an access channel passing through the top and is cubic in shape (Figure 26). For example, in one embodiment, the chambers provided herein have the following dimensions. Dimensions of 50 - 250 μm × 50 - 250 μm × 50 - 250 μm, l × w × h (e.g., 150 μm × 100 μm × 150 μm, l × w × h). During loading, the particles (e.g., cells or beads) follow the streamlines and pass over the top of the chamber, but when the flow stops, they fall to the bottom of the chamber. Due to the laminar flow profile, the flow rate is negligible near the bottom of the chamber. This enables perfusion of the chamber array and exchange of reagents by combined convection / diffusion without disturbing the position of non-adherent cells (or beads) within the chamber.

[0415] Importantly, the devices provided herein enable long-term culture and maintenance of cells (regardless of changes in effector cells, accessory cells or readout cells). The microfluidic array of chambers is fabricated within a thick membrane of PDMS elastomer (e.g., thickness from about 150 μm to about 500 μm, thickness of about 200 μm, thickness of about 300 μm, thickness of about 400 μm, or thickness of about 500 μm) covered by a reservoir of medium (e.g., the 1 mL of medium described above) (Lecault et al. (2011). Nature Methods 8, pp. 581 - 586, which is hereby incorporated by reference in its entirety for all purposes). The proximity of the medium reservoir (osmotic bath) to the cell chambers effectively blocks evaporation (through the gas-permeable PDMS material) and ensures robust cell viability (cells are not fully differentiated and are grown over several days). Also important for achieving long-term culture in nL volumes while having cell responses and growth rates identical to the μL volume format. Figure 27 shows a schematic of the layers of the device used herein.

[0416] The membrane design of the devices provided herein also enables the selective retrieval of cells from any chamber by means of microcapillaries that penetrate the upper membrane.

[0417] The device structures provided herein are designed such that the soluble secretions of effector cells are not washed away from the chamber when additional components (e.g., accessory particles or cell signaling ligands) are added to the chamber. Further, the devices provided herein allow for the addition of components to the chamber without introducing cross-contamination of secretions (e.g., antibodies) between the individual chambers of the device. In embodiments where the chambers are connected in series, medium exchange requires flushing the entire array. Flushing the entire array results in the loss of antibodies from each chamber and introduces cross-contamination into downstream chambers in one embodiment. However, if the secretions bind to the surface of the chamber or the readout particles bind to the surface, the secretions are immobilized, so cross-contamination and / or loss of secretions is not a significant problem.

[0418] In one embodiment, cross - contamination and / or loss of secretions is minimized by using an “expandable chamber” design as shown in FIGS. 28A - D. Each chamber has a single inlet and is connected to a common channel via a short “access channel” controlled by a microvalve. The top of each chamber is covered by a recess and separated from the chamber by a thin (~10 micron) membrane. This allows for a significant volume expansion when the chamber is pressurized. In one embodiment, the chamber can be inflated to twice its volume (e.g., from 2 nL to 4 nL, from 1 nL to 2 nL, or from 2.5 nL to 5 nL). In another embodiment, the chamber can be inflated to 1.5 times its volume (e.g., from 2 nL to about 3.5 nL) depending on the application. Particles (cells or beads) are loaded into the expandable chamber in one embodiment by inflating the chamber, allowing the particles to sediment under gravity, and then contracting the chamber. Similarly, the continuous process of inflation, diffusive mixing, and contraction allows for washing of the chamber contents and / or exchange of media. This approach allows for the addition of soluble ligands without loss of secretions because they are only diluted to less than 50%. Since the chambers are not connected in series, the expandable chambers also eliminate the possibility of cross - contamination. The simplicity of this structure allows for high - density array integration and is applicable in one embodiment to a device having 10,000 chambers in an area of only 1 square inch.

[0419] In one aspect, the microfluidic devices provided herein are operated in a flow regime that suppresses inertial effects and is influenced by viscosity. This flow regime is characterized by a low Reynolds number Re = ρdu / η, where ρ is the density of the fluid, d is the characteristic length scale of the channel, u is the characteristic velocity of the flow, and η is the viscosity. At low Reynolds numbers, the streamlines of the flow are predictable and can be designed for a desired effect. Referring to FIGS. 29 and 30, for example, if the goal is to load effector cells 40 only on the left side of chamber 41, the device of one embodiment is designed with an upstream restriction of the chamber that preferentially directs effector cells to the left side of the inlet flow path 43 (e.g., formed by deflector 42 in FIG. 30). Since effector cells 40 enter the downstream chamber 41, effector particles follow the streamlines of the flow and continue to the left side of the chamber. The transit time is selected so that effector cells do not diffuse substantially across the streamline. Similarly, in one embodiment, readout particles are directed to the right side of the chamber by using an auxiliary channel connected to the inlet channel. Its inlet flow path provides for the positioning of readout particles on the right side of the inlet flow path. In another embodiment, readout particles are introduced via a different channel that accesses the chamber containing the outlet channel. It will be appreciated by those skilled in the art that the design of the laminar flow profile provides great flexibility in the direction of particles with respect to specific regions of the chamber when a specific arrangement within the chamber is desired.

[0420] Separation of effector cells from readout particles within a particular chamber can also be achieved by using structural elements by manipulating the flow within the flow channel or channels of the device, stochastic loading, magnetic, electric or dielectric fields, gravitational fields, deformation of the surface of the microfluidic chamber affecting adhesion, and the relative buoyancy of effector cells and readout particles, or combinations thereof.

[0421] In one embodiment, effector cells and / or readout cells are contained within or are part of a chamber (e.g., an effector zone or a readout zone) that uses components within the chamber.

[0422] As used herein, an "effector zone" is a region of a microfluidic chamber in which effector cells, a population of effector cells (or a subpopulation thereof) are maintained.

[0423] As used herein, a "readout zone" is a region of a microfluidic chamber in which readout particles are separated and maintained from effector cells and the functionality of the effector cells is detected. For example, when the "effector zone" and the "readout zone" are three-dimensional regions of a microfluidic device, they may be individual chambers (e.g., compound chambers). The chambers are in fluid communication with each other.

[0424] As described herein, in one embodiment, one or more components are used for the distribution of effector cells and / or readout particles within one or more chambers. In a further embodiment, one or more components are used for the retention of effector cells and / or readout particles within a defined region of the chamber (e.g., an effector zone and / or a readout zone). In some embodiments, the use of such components is associated with the use of a field (e.g., gravity, dielectric, magnetic, etc.) to effect a retention function. For example, in one embodiment, a cell fence is utilized to trap cells (effector or readout) at a particular chamber location. Cell fences are described in PCT Publication No. WO 2012 / 162779, the disclosure of which is incorporated herein by reference in its entirety for all purposes. As used herein, a "cell fence" refers to any structure that functions to restrict the movement of cells and readout particles, but can allow the movement of other cell products within the microfluidic chamber.

[0425] In an embodiment using cell fences, each fence is defined by the function of the thin SU-8 2010 on top of the SU-8 100 fence. Such a structure is schematically shown in FIG. 31. The manufacture of cell fences is described in PCT Application Publication No. 2012 / 162779, which is hereby incorporated by reference in its entirety. In one embodiment where the cell fence is used to capture a cell population optionally containing one or more effector cells or effector cells, the microfluidic chamber is a standard protocol except that the final step of development is omitted. It is defined using an SU-8 100 negative photoresist (generally 160 μm in height). Specifically, the spinning of the Si wafer with the SU-8 100 photoresist is cooled after the post-exposure bake. Then, instead of developing the photoresist, SU-8 2010 is spun on top of the undeveloped SU-8 100 (typically 10 - 20 μm in height). The thickness of the SU-8 2010 determines the height of the fence. The depth of each chamber was the combined height of both photoresist layers.

[0426] In an alternative cell fence embodiment, the SU-8 100 layer is fully developed and the wafer is coated with an additional layer of SU-8 100 higher than the first layer. The difference is the height of the fence.

[0427] The structural elements described herein can utilize gravity to provide retention of effector cells and readout particles on different sides of the chamber. In this case, gravity is directed towards the floor of the chamber and provides a force that prevents cells from rising over the fence. It should be noted that the microparticles may rely on Brownian motion in which they "diffuse" over the fence stochastically. However, the probability that the particles spontaneously rise over the fence by Brownian motion is determined by the Boltzmann distribution, and the Boltzmann factor e -E / KTis proportional to. Here, E is the potential energy that raises the particles to a height equal to the height of the fence (E = volume of the particles × difference between the density of the particles and the density of the liquid × gravity × height of the fence), K is the Boltzmann constant, and T is the temperature of the liquid. Therefore, one skilled in the art can design and manufacture the height of the fence such that the probability of spontaneous "diffusion" of the particles over the fence becomes negligibly small. Also, depending on the potential energy required to pass over the fence, it is understood by those skilled in the art that a fence of a given height represents a barrier to some particles (e.g., beads or cells). However, it does not represent a barrier to particles with an appropriately low apparent mass in the liquid. For example, a fence having a height of about 20 μm allows cells to pass through naturally by diffusion but basically does not present a barrier to protein diffusion. In one embodiment, the component for retaining the cells is used in combination with gravity. However, other forces such as magnetic gradient force, dielectric force, centrifugal force, flow force, or optical force can equally be used.

[0428] The structural element is also effective in retaining effector cells or readout particles by providing a mechanical barrier to the passageway. The use of a fence reaching into the gap "d" in the roof of the chamber acts as a barrier without the need for another application of force. Here, "d" is smaller than the diameter of all effector cells or readout particles. Such a structure may be designed to selectively separate effector cells or particles on different sides of the chamber by designing a gap d through which one type of particle (but not others) can pass. Referring to FIG. 32, for example, in one embodiment, effector cells 120 have a diameter of 10 microns, and a mixture of effector cells and readout particles 121 (having a diameter of 1 micron) is loaded on one side of the chamber. And one side of the chamber is separated from the opposite side by a fence 122 having a gap of 5 microns (as measured in the roof of the chamber). By properly tilting the device, the 1-micron readout particles 121 move to the other side of the chamber while leaving the effector cells 120 on one side. In this way, the effector cells are placed in the effector zone of the chamber. On the other hand, the readout particles are placed in the readout zone.

[0429] The wells within the chamber are also used for capture assay reagents, effector cells and / or readout cells. For example, representative device shapes are shown in FIGS. 33 and 34. In one embodiment, the chamber is designed using an array of wells. Thereby, small wells are defined at the bottom of the chamber (FIG. 33). The wells can be of any shape and they are designed based on effector cells or readout particles designed to be associated. Both square and circular wells are suitable for use in the devices described herein. And generally, polygonal wells are suitable for use within the chamber. Various prototypes were made with square and circular wells as well as square and circular "posts". Effector cells and readout particles are loaded into the device at a concentration such that each well within the chamber contains a subpopulation of cells or readout particles. For example, in one embodiment, each well within the chamber is made to contain one effector cell or not contain an effector cell. Such a design can spatially restrict the particles / cells. In embodiments where separate "effector zones" and "readout zones" are utilized, effector cells define the wells that exist as the "effector zone" and readout particles define the wells that exist as the "readout zone".

[0430] FIG. 34 shows an embodiment of a "bead trap" that can be used with the device of the present invention. This design spatially confines a plurality of readout particles (e.g., a plurality of beads or readout cells) or cell populations to a specific spatial location within the chamber. Such a design overcomes problems associated with moving beads or particles throughout the assay, thereby simplifying downstream imaging and image analysis. Having a fixed position for the readout particles is an advantage due to the well-controlled diffusion distance between effector cells and readout particles.

[0431] In one embodiment, the operation of the bead trap (shown in FIG. 34) occurs as follows. The readout particles are loaded into the chamber and then sediment to the bottom of the chamber by gravity. Meanwhile, the chamber is tilted towards the upper right corner (i.e., the zone surrounded by the vertical cell fence and the substantially circular particle trap opening towards the upper right quadrant). After the readout particles or particles have settled to the bottom of the chamber, the device is tilted in the opposite direction (along the same axis). As a result, the readout particles or particles slide or roll along the bottom of the chamber and into the circular "trap" feature at the center of the chamber. As described above, the same method and device can be used to isolate effector cells.

[0432] In one embodiment, effector cells and readout particles are placed within the chamber in an effector zone and a readout zone using a microfabricated structure designed to hold one or more types of particles (e.g., cells). For example, in one embodiment, the flow of effector cells or readout particles is designed to intersect a microfabricated cup structure. The microfabricated cup structure is designed such that the flow can pass through but is designed to hold particles. Such a structure is designed to be able to adjust only a fixed number of particles or cells, or a defined size range of particles or cells. Thereby, the structure is made selective for different particle types. Such a trap may be placed substantially in the chamber, or at the inlet and outlet of the chamber.

[0433] In another embodiment, referring to FIG. 35, effector cells 81 are provided to the bottom of the chamber using gravity. Meanwhile, readout particles 82 are placed at the outlet 83 of the chamber and a trap structure 84 fabricated at the outlet.

[0434] According to one embodiment of the present invention, one or more recesses (hereinafter referred to as "cups") are provided at the bottom of the chamber to separate effector cells from the readout particles. Referring to FIG. 36, for example, the dead-end cup 361 is provided at the bottom of the chamber 362. This dead-end cup 361 has a width that is smaller than the average diameter of the effector cells 363 but larger than the diameter of the readout particles 364. In this configuration, the readout particles 364 sink to the bottom of the cup 361, while the effector cells 363 are held above the entrance of the cup. In some embodiments, the bottom of the cup 361 is accessed downward through a porous membrane and a channel structure in a dead-end cup covered with effector cells to enhance fluid access to the readout particles.

[0435] In one embodiment, a component is disposed within a flow path or chamber to hold one or more particles or cells. Referring to FIG. 37, the structural element 371 (or a functionalized surface patch, etc.) is disposed within a flow path (not necessarily a microwell) to hold one or more effector cells 372 and / or one or more readout particles 373. For example, the trap structure may be designed to hold cells or particles having specific physical properties (e.g., size), or may be non-specific (random distribution of effector cells and readout particles). The valve 374 is used to separate adjacent chambers.

[0436] As an extension of the above-described design (e.g., FIG. 37), the components in the flow path are designed to hold effector cells and readout particles in close proximity to each other. Referring to FIG. 38, in one embodiment, one unit includes an effector cell trap 381 that traps effector cells 382 and a microstructure 383 that holds one or more readout particles 384. Readout particles having a diameter smaller than the gap of the cell trap pass through the cell trap and are held in the microstructure 383 located downstream of the effector cell trap 331. Effector cells having a diameter larger than the gap 385 of the effector cell trap 381 are held as described above. In another embodiment, the microstructure is designed such that the different types of distances and positions of the readout particles (or effector cells) are clearly defined.

[0437] In addition to the fence-like barrier between the effector cells and the readout particles, the porous membrane also functions as a barrier and is thus suitable for use in the present invention. For example, referring to FIG. 39, in one embodiment, a porous membrane 391 (also referred to as a diffusion channel) is fabricated between an effector cell chamber 392 that holds effector cells 395 and a readout particle chamber 393 that holds readout particles 394. Thereby, a horizontal arrangement is provided. In such a horizontal arrangement, the porous membrane 391 is replaced by a flow path or a sieve valve having a cross-section smaller than the diameter of the effector cells or the readout particles in one embodiment. The sieve valve is described in U.S. Patent Application Publication No. 2008 / 0264863, the entire disclosure of which is incorporated by reference for all purposes.

[0438] In another embodiment, the porous membrane is fabricated between PDMS layers to provide a vertical arrangement. In this embodiment, the effector zone is provided in one PDMS layer and the readout zone is provided in a second PDMS layer. One advantage of the vertical arrangement is that the spacing between the effector zone and the readout zone is clearly defined.

[0439] The porous membrane incorporated within the PDMS device has been reported, for example, by Aran et al. (2010). Lab Chip 10, pp. 548-552; Cheuh et al. (2007). Anal. Chem. 79, pp. 3504-350 (the disclosures of which are incorporated herein by reference in their entireties for all purposes). In one embodiment, the porous membrane is manufactured according to any of the following embodiments. In another embodiment, the PDMS layer is made porous by adding an immiscible fluid to the uncured components of the PDMS. During the firing process, the immiscible fluid evaporates. In yet another embodiment, the PDMS membrane is perforated after curing using laser ablation or other removal techniques. In yet another embodiment, the PDMS membrane is molded onto one or more microstructures having a height exceeding the thickness of the membrane to make the PDMS membrane porous.

[0440] In one embodiment, the structure that holds one or more effector cells and / or readout particles is a temporary structure and / or is removable. In one embodiment, as shown in FIG. 40, the readout particles 401 (e.g., beads) are stacked against a sieve valve 402 (a valve that blocks only a portion of the channel cross-section). A layer of non-functional beads 403 is stacked against the readout particles 401. Thereby, a barrier is provided. A layer of effector cells 404 is provided and stacked against the layer of non-functional beads 403. As understood from FIG. 40, the effector cells 404 are provided through a supply channel) 405 in one embodiment. An effector cell assay that measures the effect of one or more effector cells 404 on one or more readout particles 401 is performed using a lower bath channel 406 in one embodiment. One advantage of the temporary structure is that the contents of the chamber are recoverable (selectively recoverable in some embodiments) through the lower bath channel by opening one or more sieve valves.

[0441] In addition to microfabricated retention methods and fields, hydrogels such as agarose are suitable for use in localizing effector cells and / or readout particles. For example, in one embodiment, the readout particles and / or effector cells are loaded into a liquid agarose (e.g., photosensitive agarose) that solidifies on-chip. In this way, the movement of effector cells or readout particles is restricted. This simplifies the imaging process while still allowing for diffusive transport. Upon remelting of the agarose (selective remelting in one embodiment), effector cells are recovered using the microfluidic methods described herein. In another embodiment, immobilized cells are selectively recovered using a micromanipulator or robotic method.

[0442] In one embodiment, the separation of effector cells and readout particles within a chamber or a specific zone within a particular chamber is achieved by a specific flow profile, stochastic loading, magnetic, electric or dielectric fields, gravitational fields, deformation of the surface of the microfluidic chamber, and by selecting a specific relative buoyancy of the effector cells, or a combination thereof. For example, in one embodiment, either population of cells is labeled with magnetic particles or the readout particles are magnetic. As a result, providing a magnetic field to the upper surface of a particular chamber pulls the cell population or readout particles upward. Also, the magnetic field repels and attracts either the readout particles when the cells are magnetically labeled or the cell population when the readout particles are magnetically labeled. In another embodiment, the specific gravity of the fluid in which the cell population and readout particles are incubated is selected to facilitate the separation of effector cells and readout particles based on their relative buoyancies. Specific methods for separating particles and cells are described in more detail below.

[0443] In one embodiment, effector cells and readout particles are dispensed into the chamber by functionalizing one or more walls of the chamber. In one embodiment, surface functionalization is performed by grafting, covalent bonding, adsorption, or otherwise attaching one or more molecules to the surface of the chamber, or by modifying the surface of the chamber. As a result, the attachment of cells or particles to the chamber surface is modified. Exemplary, non-limiting examples of functionalization as used herein include non-specific adsorption of proteins, chemical coupling of proteins, non-specific adsorption of polymers, electrostatic adsorption of polymers, chemical coupling of small molecules, chemical coupling of nucleic acids, oxidation of surfaces, etc. PDMS surface functionalization has already been described and these methods are used herein to functionalize the surfaces of the devices provided herein (e.g., Zhou et al. (2010). Electrophoresis 31, pp. 2-16, which is incorporated herein by reference for all purposes). The surface functionalization described herein selectively binds, in one embodiment, one type of effector cell (e.g., effector cells present in a cell population). Or selectively binds one type of readout particle. In another embodiment, surface functionalization is used to sequester all readout particles present within the chamber.

[0444] In yet another embodiment, the surface functionalization or multiple different surface functionalizations are spatially defined within the chamber of the device. Also, the surface functionalization or multiple different surface functionalizations are made across the chamber. Both of these embodiments are useful for the distribution of effector cells, which are readout particles, at distinct locations within the microfluidic chamber. For example, in an embodiment where the entire chamber is functionalized with molecules that bind all types of introduced readout particles, the particles are directed to different regions of the device using the methods described above. They are immobilized on the surface. In another embodiment, the entire chamber may be functionalized to bind only one particular type of readout particle. In this case, in one embodiment, all the particles are first directed to one region using any of the methods described herein. Thereby, a subset of the particles causes them to adhere to the chamber surface in the functionalized region. Subsequently, a force is exerted towards a different region that replaces only the functionalized surface or the particles that do not bind to the surface. In yet another embodiment, regions of the device (e.g., different chambers or regions within a single chamber) are functionalized with different molecules that selectively bind different subsets of effector cells and / or readout particles. As a result, inducing interactions of effector cells and / or readout particles across substantially the entire chamber surface leads to the partitioning of different particles or cell types in different regions. As described herein, surface functionalization may be used alone or in combination ...

Claims

1. A method for identifying antibody-secreting cells that produce antibodies that neutralize a virus, the method comprising: holding a plurality of cell populations in a plurality of microreactors, each microreactor containing 10 to 500 cells, each individual cell population of the plurality of cell populations containing one or more antibody-secreting cells and being held in an individual microreactor of the plurality of microreactors, each individual microreactor of the plurality of microreactors containing one or more readout cells; introducing a plurality of accessory particle populations into the plurality of microreactors, the plurality of accessory particle populations containing a plurality of virus particles operable to infect the one or more readout cells, each individual accessory particle population of the plurality of accessory particle populations being held in the individual microreactor; incubating the individual cell populations, the readout cells, and the accessory particle populations in the individual microreactors for a time sufficient to produce a plurality of antibodies; assaying the individual microreactors to determine whether the plurality of antibodies includes antibodies that neutralize the virus; and identifying the microreactors containing antibody-secreting cells that produce antibodies that neutralize the virus. A method comprising the above steps.

2. The method according to claim 1, wherein the plurality of virus particles are engineered to include a fluorescent protein expressed by the one or more readout cells after viral infection of the one or more readout cells.

3. The method according to claim 2, wherein assaying the individual microreactors includes assaying the fluorescent protein expressed by the one or more readout cells after viral infection of the one or more readout cells.

4. The method according to claim 1, wherein assaying the individual microreactors includes assaying the death of the one or more readout cells after viral infection of the one or more readout cells.

5. The method according to claim 1, further comprising substantially isolating the individual microreactors from the surrounding environment.

6. The method according to claim 2, further comprising substantially isolating the individual microreactors from the surrounding environment.

7. The method according to claim 3, further comprising substantially isolating the individual microreactors from the surrounding environment. **Claim 8** When the individual cell populations comprise antibody-secreting cells that produce antibodies that neutralize the virus, the method further comprises a recovery step of recovering the individual cell populations or a part thereof that comprise antibody-secreting cells that produce antibodies that neutralize the virus to obtain a recovered cell population, the method according to claim 1. **Claim 9** The method according to claim 8, wherein the recovering step comprises positioning an open end of a microcapillary within a microreactor containing a cell population comprising antibody-secreting cells that produce antibodies that neutralize the virus, and aspirating the contents of the microreactor or a part thereof to obtain a recovered aspirated cell population. **Claim 10** The method according to claim 9, wherein the microcapillary is mounted on a robotic micromanipulation system on a microscope or the microcapillary is robotically controlled. **Claim 11** holding a plurality of cell subpopulations derived from the recovered cell population in a plurality of containers, each cell subpopulation being present in an individual container; lysing the individual cell subpopulations of the plurality of cell subpopulations to provide lysed cell subpopulations; and The method of claim 8, further comprising amplifying one or more nucleic acids in each of the lysed cell subpopulations.

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