Assay method and device for the detection of rare events in cells

Abstract

Described are systems for cell culture analysis and methods for the use thereof, wherein the system comprises a plurality of cell culture chambers and a receptacle for receiving the cell culture chambers, v herein the receptacle comprises one or more receptacle chambers each having a. wail and a. floor. Each cell culture chamber comprises a side wall, an opening on the top of the cell culture chamber, and a semi-permeable membrane on the bottom of the cell culture chamber, and i) wherein the membrane is a small distance from the floor of the receptacle chamber and / or ii) wherein the total surface area of the membrane on the plate is 50% or more of the surface area of the receptacle bottom. The systems and methods allow the detection of rare cell types, rare events, and / or higher cell numbers, including the ability to contact the cells with different analytes for extended times and / or for multiple treatment cycles.

Description

[0001]ASSAY METHOD AND DEVICE FOR THE DETECTION OF RARE EVENTS IN CELLS RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No.63 / 659,016 filed June 12, 2024. The entire contents of the above application are incorporated by reference herein. BACKGROUND OF THE INVENTION One limitation in the detection of rare events in live cell culture is that current microscopy tools and plastic / glass consumables are usually optimized for working with the round wells and homogenous cell cultures. Round wells correspond to the round fields for view of the microscopes, and the homogenous cell cultures generally do not require measuring events in more than 5,000-500,000 cells simultaneously because lower cell numbers provide similar responses to high cell numbers. Flow cytometry permits detection of events as rare as 30 per 1 million cells but the cells are usually discarded after use. Very rare cell types and subpopulations play important roles in contemporary immunology. However, studying these rare cells can be problematic, especially when tracking cellular response to a series of treatments, screening the potential responses to multiple treatments (e.g., screen libraries), and / or tracking the response during extended times in live cell culture. Microfluidic chips can partially address the described needs, but there remain multiple limitations associated with their use. For example, processes using such chips are hard to automate for high-throughput screening, the individual costs per microchip can be prohibitive for scaling up the research, and the numbers of cells loaded in the microchips rarely are higher than 200,000. Non-adherent primary immune cell cultures add an additional layer of complexity compared to adherent cells and organoids because medium exchange can be associated with cell loss. These cells are obtained in limited numbers (as compared to the cell lines), especially when collected from the patients with a specific disease or treatment course; and especially when isolated from the tissues with low immune cell infiltrations (for example, so- called, “cold tumors”). The ability to efficiently work with such cell cultures is essential for the development of advanced immune medicines. Therefore, there remains a need in the improved high-throughput methods and devices for identifying rare events in the live primary cell cultures at scale. SUMMARY OF THE INVENTION The present invention encompasses cell culture and analysis devices and methods of use thereof that allow the detection of rare cell types, rare events, and / or higher cell numbers, including the ability to contact the cells with different analytes for extended times and / or for multiple treatment cycles. The described systems and methods enable maintenance of the cell culture with the ability to add and exchange the analyte fluids for extended periods of time, and for multiple treatment cycles. The systems and methods provide compatibility with continuous fluorescence microscopy, label-free or labeled-based readouts, and / or working with high cell numbers and low volumes of analytes as compared to current devices and methods. As discussed herein, the systems and methods allow researchers to (1) easily exchange the treatment in contact with the cell culture without losing the cells and / or without affecting their viability and functionality; and / or 2) easily observe the cell culture using microscopy (e.g., fluorescence microscopy) rapidly obtaining large amounts of visual data (e.g., scanning maximum number of cells per cultural vessel, i.e. dish / plate / chip / cartridge). The lack of ability to obtain large amounts of data (visual data about large numbers of cells) using currently available cell culture tools impedes the ability to detect and study rare events with good statistical power. The invention encompasses systems for cell culture analysis (also referred to herein a plate or a microplate) and methods for the use thereof, wherein the system comprises a plurality of cell culture chambers and a receptacle for receiving the cell culture chambers, wherein the receptacle comprises one or more receptacle chambers each having a wall and a floor, wherein each cell culture chamber comprises a side wall, an opening on the top of the cell culture chamber, and a semi-permeable membrane on the bottom of the cell culture chamber, and i) wherein the membrane is a small distance from the floor of the receptacle chamber (e.g, less than 2 mm, less than 1 mm, or less than 0.75 mm from the floor) and / or ii) wherein the total surface area of the membrane on the plate is increased, e.g, is 50% or more of the surface area of the receptacle bottom, or wherein the diameter or width of the membrane inside the receptacle chamber is more than 75% of the diameter or width of the receptacle chamber at the terminal location. The small distance between the membrane and the floor of the receptacle chamber decreases the volume of fluid (e.g., media or analyte fluid) needed in the receptacle chamber to cover the membrane, and / or decreases the distance between the bottom of the receptacle chamber and cells on the membrane. In certain aspects, the decreased distance between the bottom of the receptacle chamber and the membrane decreases the focal distance for microscopy. In additional aspects, the surface area of the membrane on the device or plate is such that the surface area for cell culture available for the analysis is optimized; for example, in certain aspects, the surface area can be large enough to support at least about 1 million cells, 2 million cells, 5 million cells, 10 million cells, 20 million cells, or 30 million cells. The system can also be used to culture a lower number of cells. The devices / systems described herein as well as their individual components can, in certain embodiments, be disposable and made, for example, from plastic or glass. The invention encompasses a system that comprises a removable cell culture insert and a receptacle for receiving the cell culture insert, wherein: a. the cell culture insert comprises one or more cell culture chambers, each cell culture chamber comprising a side wall that encloses a semi-permeable membrane on the bottom of the cell culture chamber and an opening on the top of the cell culture chamber, wherein the membrane is capable of supporting the growth of cultured cells on the apical surface of the membrane, and wherein the basal side of the membrane faces the bottom of the receptacle; b. the receptacle comprises a plurality of receptacle chambers, wherein each receptacle chamber holds the one or more of the cell culture chambers, wherein each receptacle chamber is arranged to retain a liquid (e.g., cell culture media) that covers the basal and apical surfaces of the semi-permeable membrane, and wherein each receptacle chamber has a wall, an opening, and a floor; and c. wherein the membrane is positioned inside the receptacle chamber such that the apical side of the membrane is at a terminal location above the floor of the receptacle chamber, and wherein the terminal location is less than 1 mm from the floor of the receptacle chamber; and wherein the surface area of the membrane inside the receptacle chamber is more than about 50% of the cross-sectional area of the receptacle chamber at the terminal location, and / or wherein the diameter or width of the membrane inside the receptacle chamber is more than 75% of the diameter or width of the receptacle chamber at the terminal location. In certain aspects, the surface area of the membrane inside the receptacle chamber is more than 60% of the cross-sectional area of the receptacle chamber at the terminal location. The invention also includes systems and methods for cell culture analysis, wherein the system comprises a plurality of cell culture chambers and a receptacle, wherein: a. the receptacle comprises one or more receptacle chambers, each receptacle chamber having a wall, an opening and a floor; b. wherein a semi-permeable membrane covers the entire opening of the receptacle chamber, wherein the membrane is capable of supporting the growth of cultured cells on the apical surface of the membrane, and wherein the basal side of the membrane faces the floor of the receptacle chamber; optionally the semi-permeable membrane covers the entire top of the receptacle; c. each cell culture chamber is arranged on the apical surface of the membrane over a receptacle chamber, and wherein each cell culture chamber comprises a side wall, has an opening on the top of the cell culture chamber, and the semi- permeable membrane on the bottom of the cell culture chamber; d. wherein the receptacle chambers and cell culture chambers are arranged to retain a liquid (e.g., cell culture media) that covers the basal and apical surfaces of the semi-permeable membrane or in a volume sufficient to provide effective diffusion through the membrane; for example, the receptacle chamber is configured to contain a fluid level such that the fluid diffuses through the membrane; e. wherein the receptacle and the side walls of the cell culture chambers are attachable and detachable from the semi-permeable membrane; for example, the receptacle is attachable / detachable from the basal side of the membrane and the side walls are attachable / detachable from the apical side of the membrane; f. wherein the membrane is less than 2 mm from the floor of the receptacle chamber that holds that cell culture chamber; in certain aspects, the membrane within the cell culture chamber is less than 1 mm from the floor of the receptacle chamber. The invention also encompasses a lid that can, for example, be used with the systems / devices described herein comprising an inlet and an outlet (or inlet and outlet ports) for the transfer of liquid (e.g., media or analyte / candidate agent) into one or more receptacle chambers. The lid releasably engages the receptacle to enclose the receptacle chambers and / or the cell culture chambers. The lid can be in fluid communication with receptacle chamber(s), e.g., by tubing and / or can have entry ports for tubing or for pipetting. The lid can be made of a rigid material including, for example, plastic, silicone, or other polymers or a combination thereof, as well as flexible materials comprising plastics, plastic, silicone, or other polymers or a combination thereof. In certain aspects, the lid is optically transparent. In other aspects, the lid is non-reflective. In yet other aspects, the lid is highly reflective, for example, allowing illumination of the cell culture analysis system from above or below (e.g., above the lid or below the receptacle chamber). In some cases, the lids has internal or connected manifold systems for the inlets, outlets, or a combination thereof. In yet further aspects, the lids has one or more structural features that decreases evaporation from the cell culture chambers. In some embodiments, the lids has one or more structural features that fix or hold the cell culture insert in the receptacle chamber, e.g., decreasing movement during longitudinal imaging. The invention additionally includes a pedestal for the cell culture insert or cell culture chamber of the system described herein, wherein the pedestal is placed on the floor of the receptacle chamber or is attached to the wall of the receptacle chamber, and wherein contact between the pedestal and the cell culture chamber supports or holds the cell culture insert in the receptacle chamber, for example, the pedestal can fix the cell culture insert in place and / or decrease movement. In certain embodiments, the pedestal is made of rigid material including, for example, plastic, silicone, or other polymers or a combination thereof, as well as flexible materials comprising plastics, plastic, silicone, or other polymers or a combination thereof. In yet additional aspects, the pedestal adheres to the cell culture insert or cell culture chamber (e.g., the support has a sticky surface that adheres to the cell culture insert or cell culture chamber) or the pedestal affixes the cell culture insert or cell culture chamber (e.g., by a clicking mechanism). In certain aspects, the pedestal is optically transparent. In other aspects, the pedestal is non-reflective. It should be understood that the pedestal can have any shape and the dimensions of the pedestal, including the height, can be adjusted; for example, the pedestal can be adjusted to have different heights so as to allow the cell culture chamber to sit at different positions in the receptacle chamber. In some embodiments, one or more pedestals can be used in a receptacle chamber. In other embodiments, the pedestal can be shaped such that only one pedestal need be used in a receptacle chamber. The invention additionally encompasses a method of detecting a cell response to a candidate agent, the method comprising culturing cells in a system described herein, contacting the cells with a candidate agent and detecting a response to the candidate agent. In certain embodiments, the methods can be used to detect a cell response to a plurality of different candidate agents. Also encompassed are methods of identifying a candidate agent that elicits a response in a cell, the method comprising: a first cycle comprising: a. contacting a sample comprising cells with an analyte fluid, wherein the sample comprising the cells is in a cell culture chamber of a system described herein, and wherein the analyte fluid comprises a candidate agent in a liquid medium; b. detecting the response by detection of an optical signal in the sample in the cell culture chamber; and c. removing the analyte fluid from the cell culture chamber thereby removing the candidate agent while retaining the sample comprising the cells on the membrane of the cell culture chamber; a subsequent cycle comprising: d. contacting the retained sample comprising the cells of the preceding cycle with a new analyte fluid, wherein the new analyte fluid comprises a new candidate agent in a liquid medium, wherein the new candidate agent is different from the candidate agent of the preceding cycle; e. detecting the response by detection of an optical signal in the retained sample in the cell culture chamber; and f. removing the analyte fluid from the cell culture chamber thereby removing the candidate agent while retaining the sample comprising the cells on the membrane of the cell culture chamber; repeating the subsequent cycle at least N times, wherein N is an integer greater than or equal to 0, wherein each new analyte fluid of each subsequent cycle comprises a different candidate agent from that of the preceding cycle. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. FIG.1 shows a well of a prior art plate holding a cell culture insert, for example, a transwell system. The opening of the well is at the top of figure, and the bottom of the well is labeled “B.” As shown in FIG.1, evaporation may occur from the opening of the cell culture insert (cell culture chamber) and is labeled “E”. This evaporation can present a problem for environmental stability of the cells. “S” is the surface area of the membrane in the cell culture chamber and represents the cell growth area. In FIG.1, the surface area of the membrane is small relative to the cross-sectional area of the well (and thus relative to the area of the receptacle) and thus is not an optimal use of space. The volume of media that covers the apical surface of the cell membrane where the cell culture is present is represented by “V.” The volume of media required to cover the membrane is greater when the distance “D” of the membrane from the floor of the well (apical side of the line labeled “B”) is greater. “A” represents distance from the opening of the well to the floor of the well within the space between the wall of the cell culture chamber and the wall of the well (this space provides access from the opening of the well, for example, for media exchange and / or for the addition of an analyte). The larger the distance “A,” the greater the difficulty for transmitted light imaging. In the prior art systems, the bottom of the receptacle is often not glass, and thus suboptimal for imaging. In addition, the membrane is difficult to detach from the cell culture chamber. In addition, in prior art systems, the wall(s) of the receptacle are usually transparent while black would be optimal. FIG.2A is a schematic showing an embodiment of the present invention. The figure shows a receptacle chamber holding a cell culture chamber that addresses many of the limitations discussed with respect to the design shown in FIG.1. The surface area (“S”) of the membrane is increased. The increased surface area of the membrane increases the area for cell culture and utilizes more of the available space in the receptacle chamber for cell culture. The distance “D” of the membrane to the floor of the receptacle chamber is decreased as compared to FIG.1, thus minimizing the volume of media required to cover the apical surface of the cell membrane. Evaporation can optionally be prevented by covering the opening, for example, with a lid or other seal (e.g, a film seal). Optionally, the color of the wall(s) of the receptacle are black and / or anti-reflective and / or the bottom of the receptacle / receptacle chamber can be made from glass or plastic. The receptacle can, for example, be a multi-well plate (wherein the wells of the multi-well plate are the receptacle chambers). When the receptacle is a multi-well plate, access to the receptacle for media exchange and treatment occurs from the top of the chamber at the opening of the receptacle chamber. In other aspects, the receptacle chamber has horizontal chambers as described in more detail herein. FIG.2B shows a design of a receptacle with horizontal chambers. Media exchange and / or analyte treatment can occur in the vicinity of the horizontal chamber on the side of the receptacle, rather than from the opening at the top of the receptacle chamber. This design can simplify experiments with transmitted light. The receptacle plate can optionally include a water jacket for heating. In certain embodiments, the receptacle with horizontal chambers can be used with cell culture inserts, for example, each horizontal chamber can hold one or more cell culture chambers. In certain additional aspects, a membrane can cover one or more of the channels, or the receptacle opening or a part thereof, thus maximizing the surface area of the membrane. The cell culture chamber(s) are arranged over the receptacle chamber on the membrane (e.g., by attaching the walls of the cell culture chamber thereto). In some examples, the membrane can be attachable / detachable from the receptacle chamber and / or from the walls of the cell culture chambers. When using the receptacle with one or more horizontal chambers, an extra wash between treatments (e.g., with different analytes or candidate agents) may be required. FIG.2C is a schematic showing the receptacle plate with and without a water jacket. The circles depict a center of the flow stream across the enclosed channel. FIG.3 shows a well of another prior art plate holding a cell culture insert, for example, a transwell system. The cell culture chamber has a structure (e.g., a ring) that rests on a support at the top of the plate. The opening of the well is at the top of figure, and the bottom of the well is labeled “B.” As shown in FIG.3, evaporation may occur at the opening of the cell culture insert (cell culture chamber) and is labeled “E”. This evaporation can present a problem for environmental stability of the cells. “S” is the surface area of the membrane in the cell culture chamber and represents the cell growth area. In FIG.3, the surface area of the membrane is small relative to the cross-sectional area the well (and thus relative to the area of the receptacle) and thus is not an optimal use of space. The volume of media that covers the apical surface of the cell membrane where the cell culture is present is represented by “V.” The volume of media required to cover the membrane is greater when the distance “D” of the membrane from the floor of the well (apical side of the line labeled “B”) is greater. When “D” is too large, there is a higher objective requirement and is not possible to use a transparent heated glass incubation or a heated water jacket (because the focal distance is a limiting factor). “A” represents distance from the opening of the well to the floor of the well within the space between the wall of the cell culture chamber and the wall of the well (this space provides access from the opening of the well, for example, for media exchange and / or for the addition of an analyte). The larger the distance “A,” the greater the difficulty for transmitted light imaging. In the prior art systems, the bottom of the receptacle is often not glass, and thus suboptimal for imaging. In addition, the membrane is difficult to detach from the cell culture chamber. In addition, in prior art systems, the wall(s) of the receptacle are usually transparent while black would be optimal. FIG.4A is a schematic showing a receptacle chamber holding a cell culture chamber that addresses many of the limitations discussed with respect to the design shown in FIG.3. One or more pillars (“P”) is placed inside the receptacle chamber and the cell culture chamber rests on the pillar(s) thus reducing the distance “D” and reducing the diameter / dimensions of the receptacle chamber. Because the pillars are placed in the receptacle chamber, the surface area (“S”) of the membrane relative to the dimensions of the receptacle chamber is increased (because some of the space is occupied by the pillar(s)) and this decreases the volume of media required to cover the apical surface of the cell membrane. Evaporation can optionally be prevented by a lid or film seal, for example. Optionally, the color of the wall(s) of the receptacle are black or anti-reflective, the bottom of the receptacle / receptacle chamber can be made from glass or plastic, and the membrane is difficult to detach. Media exchange and / or analyte treatment can occur on the sides or in narrow channel between the cell culture chamber and the pillar, and between the cell culture chamber and the bottom of the plate. FIG.4B shows how the design of FIG.4A can be used with a receptacle having horizontal chambers. Media exchange and / or analyte treatment can occur at the horizontal chamber rather than from the opening of the receptacle chamber. This design can simplify experiments with transmitted light. The receptacle plate can optionally include a water jacket for heating. In certain aspects, a membrane can cover the surface of one or more of the channels thus maximizing the surface area of the membrane. In some examples, the membrane can be attachable / detachable from the receptacle chamber. An extra wash between treatments (e.g., with different analytes or test agents) may be required. FIG.4C is a schematic showing the receptacle plate with and without a water jacket. Circles depict a center of flow stream across the enclosed channel. In certain aspects, the height of the water jacket is between 2-3 mm. In additional examples, the height of the water jacket is 2 mm or less. FIG.5 is a drawing showing an alternative design of the receptacle plate. The left side of the FIG.5 shows an alternative design of the receptacle plate that allows maintenance of the cell culture on the top of membrane and ensures unidirectional flow of the exchanging liquid. The liquid is exchanged below a single transwell insert of a squared shape, located on the top of rectangular well, that allows multiple cycles of addition of the new treatment (liquid) on one side of the insert (left side on the figure), and removal on another side. The bottom left part of the figure shows the membrane, and arrows show the flow of liquid. The right side of FIG.5 shows an alternative design of the receptacle plate that allows maintenance of the cell culture in its bottom containing multiple microwells with diameters and heights from 20 to 200 micrometers divided by narrow shared walls. This plate is divided into chambers or channels for the cell culture, covered by the top inserts that are filled or hollow structures that direct the laminar flow above the grid of the microwells containing the cells, and may or may not be inserted and removed. This design can allow to couple the flow plate directly on the top of the optical chip sensors. On the bottom right part of the FIG.5 the grid is showing the microwells, and arrows show the flow of liquid; small black rectangles are the pillars that hold the top inserts that prevent evaporation, level and direct the flow of liquid. The invention encompasses a receptacle as shown in FIG.5 and described herein. DETAILED DESCRIPTION OF THE INVENTION As used herein, the words “a” and “an” are meant to include one or more unless otherwise specified. The term “about” as used herein, in reference to a numerical value or range, allows for a degree of variability in the value or range, for example, within 20%, within 10%, within 5%, or within 4%, or within 2% of the value or range, depending on the context. A “cell culture” is the growth of live cells under controlled conditions. The term “primary cell culture” implies that the cell culture is derived from a live organism in contrast to immortal cell lines also studied in vitro. In further aspects, the biological sample is dissociated tissue culture, primary cell culture, cell line, or mixtures of thereof. In certain specific aspects, the biological sample is a mixed culture of T-cells and antigen-presenting cells. The cell culture can, for example, be adherent, partially adherent, or suspension culture. The biological sample or cell culture can be pre-conditioned by a variety of methods including, but not limited to, mechanical or enzymatic dissociation, centrifugation, magnetic or other tools for specific cell types, isolation, separation, enrichment or depletion, freezing / thawing, pre-treatment or incubation at different culture conditions, transfection and other genetic modifications. Any positioning terminology, such as “left”, “right”, “top”, “bottom”, “above”, “below”, “upper”, “lower”, etc., may be used herein for convenience to describe one element's or feature's relationship to one or more other elements or features, or methods of use, or in reference to the figures. The terms “top”, “bottom”, “apical”, “basal”, “side”, or “sides” and the like with respect the cell culture analysis system / plate / microplate / device described herein or any component thereof is intended to mean the orientation when the cell culture is located on top of the membrane and / or wherein the apical side of the membrane faces up, e.g., when the system is resting on a surface. It should be apparent that the positioning terminology is intended to encompass different orientations of the structure and device disclosed herein, in addition to the orientation(s) depicted in the figures. As an example, if one imaginatively rotates the structure or device in the figures 90 degrees clockwise, elements or features described as “top” and “bottom” relative to other elements or features would then be oriented, respectively, “right” and “left” relative to the other elements or features. Therefore, the positioning terminology used herein should not be construed as any limitation of the present disclosure. The term “sample” as used herein can be used to refer to a specific portion of the specimen, biological sample, or cell culture on the solid support. For example, when the support is a 6-well plate, the plate can be described as having a sample in each well. The sample in each well can be derived from the same biological sample or from different biological samples. In certain specific aspects, the sample is a cell culture. The terms “analyte fluid,” “analyte composition,” “analyte liquid” and “analyte solution,” are used interchangeably herein to mean a liquid composition comprising a candidate agent. The candidate agent can, for example, be a small molecule or a biologic. A non-limiting example of the analyte fluid is the peptide diluted in DMSO and further diluted in the optimized T-cell culture medium. The “candidate agent” is the agent being tested or screened for biological response. Non-limiting examples of candidate agents are small molecule drug candidates, peptides, antibodies, and libraries of any of thereof. A “candidate library” or “candidate agent library” is a mixture of 2 or more (usually up to several hundred) individual candidate agents. A “specimen,” or “biological sample” as used herein, is a biospecimen or biological sample obtained from experimental animals, humans, in vitro cell lines, or other sources. An essential feature of the specimens described here is that they comprise live and functional cells capable of responding to diverse treatments. Non-limiting examples of biological samples include tissue, fluid, and other sample taken directly from a subject, as well as sample resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector encoding a recombinant chimeric receptor), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, saliva sweat, and tissue and organ sample (e.g., sample from a tissue or organ containing a tumor), including processed sample derived therefrom. In some embodiments, the biological sample is a biological fluid sample or a biological tissue sample. In some embodiments, the biological sample is a biological tissue. In some aspects, the biological sample from which the immune cells are derived or isolated is blood or a blood-derived sample, or is derived from an apheresis or leukapheresis product. When blood is used as the biological sample, an anticoagulant such as heparin, citrate or ethylenediaminetetraacetate may be added to the blood, as necessary. A fraction containing a T-cell and an antigen- presenting cell prepared from blood by a conventional method may be used as the biological sample. The cells cultured and / or analyzed herein can be derived from the sample. “Single-cell sensitivity,” as used herein, means that even if one single cell responds to treatment, the method or system can discriminate this response from the background. In certain aspects, the methods described herein have single-cell sensitivity. In certain aspects, the treatment cycling systems and methods described herein have single-cell sensitivity. “Laminar cell wash,” as used herein, is a method to exchange the liquid component of the cell suspension or cultured monolayer by creating a liquid shear flow that does not impact the attachment of the cells to the bottom of the cell culture vessel. A “downstream workflow,” “downstream assay,” “downstream method,” or the like as used herein, is a method applied after the treatment cycling. “Direct readout” is the direct detection of a cell response, e.g., by a change in their optical property. “Indirect readout,” is the indirect detection of a cell response, e.g, their interactions with neighboring cells or the responses of a neighboring cell. A positive readout indicates that the cell response (e.g., T-cell activation) was detected. A negative readout (e.g., lack of T-cell activation) indicates that cell response was not detected. The invention also encompasses methods of cell culture analysis using the systems described herein. In certain aspects, the devices described herein allow researchers to easily exchange the treatment (e.g., media or analyte fluid) in contact with the cell culture without losing the cells and / or affecting their viability and functionality and / or easily observe the cell culture using (fluorescent) microscopy rapidly obtaining large amounts of visual data (scanning maximum number of cells per cultural vessel, i.e., dish / plate / chip / cartridge). Currently, the lack of ability to obtain large amounts of data (visual data about large numbers of cells) makes it difficult to study rare events with good statistical power. Many prior art cell culture systems use transwell inserts placed in conventional cell culture plates, e.g., 6-well, 24-well or 96-well plates. The inserts maintain the cell layer on the top of the membrane, and allow exchange of the treatments below the membrane. The cell layer can be observed with microscopy after any number of treatment exchanges. However, such current systems are associated with numerous disadvantages. One issue is that the surface of the membranes (that corresponds to the scannable area of the cell culture layer) is small relative to the total area of the plate. Our current estimate is one can continuously observe 1-2 million cells with the current technology, whereas it is desirable to be able to observe 10-20 million cells to reliably capture rare events. The systems described herein improve at least one or several of the following parameters: area of cell culture available for the analysis, ease of access to exchange the treatments for the cells, decreasing or preventing evaporation of the cell culture medium, reduction in the volume of the treatment required to achieve contact between the cells and the treatment, decrease in the distance between the floor of the receptacle chamber and cell culture layer preventing increase of the possible focal distance for microscopy, and / or improving imaging and / or preventing reflection by making the receptacle a color that prevents reflection and / or optimizing the material of the receptacle bottom. Optionally, the receptacle, for example, the bottom of the receptacle is heated and optionally, this heating is provided by a thin water jacket or heated glass. In certain aspects, the system includes a cell culture chamber that sits deeper in the well and has a larger membrane surface than current transwell systems. In some examples, the cell culture chamber has a lid or seal that prevents evaporation. In certain aspects, the bottom of the receptacle is made of glass and / or the receptacle walls are black. In some examples, the cell culture chamber is placed in a receptacle that has channels (instead of wells, for example). In such an example, several cell culture chambers (or inserts) can have a shared receptacle chamber (channel). In certain aspects, the channels can be narrow and / or have dimensions that can fix the inserts firmly to decrease / prevent movement. The horizontal channels allow medium submission and removal at the ends of channels, on the sides of the plate. Channels can be produced separately and inserted inside an existing plate that has no wells (e.g., a plate tray). In a further example, a receptacle with horizontal channels as described herein has continuous membrane support over the channels, e.g., at the top of the receptacle, dividing the top part that contains cells from the lower part that allows the medium exchange. The top and bottom parts of the plate are attached to the continuous membrane that covers all plate surfaces, e.g., with the detachable glue. Therefore, after the cells are bound to the membrane (by hydrogel layer or by other means), both the top and bottom plate parts can be removed to allow working with the cells affixed to the membrane. The systems described herein can be used to seed live cell culture on the top of membranes, and the cultures can be continuously observed by microscopy. Using the systems described herein, it is possible to scan large numbers of cells per plate (10-20 million) and run multiple exchanges of treatment (up to 100 and more) for these cells below the membranes, without losing the cells or affecting their viability or functionality. One or more advantages of the systems described herein include larger area of cell culture available for the analysis, ease of access to exchange the treatments for the cells, prevention of evaporation of the cell culture medium, reduced volume of the treatment required to achieve cell contact with the treatment, decreased distances between the overall vessel bottom and cell culture layer preventing increase of the possible focal distance for microscopy, plate color that prevents reflection, plate bottom material improving the imaging. As discussed above, the invention encompasses systems for cell culture analysis and methods for the use thereof, wherein the systems (e.g., cell culture plates) provide greater surface area for the cell culture than conventional plates that are currently utilized and / or that decrease the volume of media or analyte needed for culture and analysis. In certain aspects, the system comprises a plurality of cell culture chambers and a receptacle, wherein the receptacle comprises one or more receptacle chambers and wherein a porous membrane covers one or more of the receptacle chambers; in such a system, the one or more cell culture chambers are arranged on the membrane over the receptacle chamber, and the membrane is less than 2 mm from the floor of the receptacle chamber. In additional embodiments, the system comprises a receptacle with one more receptacle chambers and a cell culture insert comprising a cell culture chamber that includes a porous membrane, wherein each receptacle chamber holds at least one cell culture chamber, wherein the membrane is inside the receptacle chamber and is less than 1 mm from the floor of the receptacle chamber and wherein the surface area of the membrane inside the receptacle chamber is more than about 50% or 60% of the cross-sectional area of the receptacle chamber at the terminal location, or wherein the diameter or width of the membrane inside the receptacle chamber is more than 75% of the diameter or width of the receptacle chamber at the terminal location. A “cell culture insert” is a support that includes a surface (e.g., a porous, semi- permeable or permeable membrane) that supports cell culture, wherein the insert is semi- permeable to air and liquid and wherein the insert can be placed inside a receptacle / receptacle chamber (e.g., a well or a channel). The insert may optionally also be removed from the receptacle / receptacle chamber. The cell culture insert includes one or more chambers for cell culture. For example, the insert can have only a single cell culture chamber (and optionally, multiple inserts can be placed into a receptacle). In another example, a single insert can have a plurality of cell culture chambers (and optionally the single insert is placed into a receptacle). An example of a cell culture insert is the Transwell® permeable supports sold by Corning. Such commercially available inserts are available in a range of sizes. A receptacle is configured to retain fluid, such as cell culture media, and can comprise one or more receptacle chambers. Each receptacle chamber is configured to retain the fluid and / or to hold one or more cell culture chambers. The receptacle chamber can comprise a floor at the bottom of the chamber, an opening at the top of the chamber, and a wall that surrounds or encloses the interior of the receptacle chamber. When the system comprises a cell culture insert, the insert or cell culture chamber of the insert (or a portion thereof) is placed inside the receptacle chamber and the receptacle chamber “holds” the cell culture chamber. The receptacle has a design that allows for ease of medium / liquid addition, removal, or exchange. The media added to the receptacle chamber (or the receptacle) can be added in a volume sufficient to cover the basal and apical surfaces of the membrane of the insert placed therein. A non-limiting example of a receptacle that can be used with the cell culture insert is a multi-well plate, e.g., a 6-well plate, a 12-well plate, a 24-well plate, a 96-well plate, or a 384-well plate, wherein each well holds one cell culture chamber. In another example, the receptacle has one or more horizontal channels and each channel holds one or more cell culture chambers. When the system comprises a porous membrane that covers one or more of the receptacle chambers, the cell culture chambers are arranged on the membrane above the channels. The receptacle has a design that allows for ease of medium / liquid addition, removal, or exchange. The cell culture media added to the receptacle chamber (or the receptacle) can be added in a volume sufficient to provide effective diffusion through the membrane. A non-limiting example of a receptacle with a membrane that covers one or more of the receptacle chambers is a multi-well plate, e.g., a 6-well plate, a 12-well plate, a 24-well plate, a 96-well plate, or a 384-well plate, wherein the membrane covers all of the wells, and wherein each well has one cell culture chamber above it. In another example, the receptacle has one or more horizontal channels, wherein the membrane covers the one or more channels, and each channel has one or more cell culture chambers arranged on the membrane above the receptacle chamber. Each cell culture chamber comprises an opening at the top, a semi-permeable or microporous membrane on the bottom of the cell culture chamber, and a side wall that encloses the interior of the cell culture chamber including the membrane at the bottom. In this context, a side wall “encloses” the membrane when the wall is capable of enclosing or separating the cells cultured on the apical surface of the membrane from the cells of another cell culture chamber or from another part of the device. As described herein, the porous membrane is configured to include / harbor / accommodate / hold cells cultured thereon. In some embodiments, the porous membrane is positioned a small distance from the floor of the receptacle chamber (either as part of a cell culture insert or covering a receptacle chamber). Each cell culture chamber can have its own wall(s) or can share a wall or a portion thereof with an adjacent cell culture chamber. The number of and shape of the side wall depends on the shape of cell culture chamber. For a round cell culture chamber (e.g, a round well), the membrane is enclosed by a round side wall. For a rectangular cell culture chamber, the membrane can be enclosed by four side walls. As will be understood, the pore size of membrane can be chosen based on the specific cell analysis of interest, e.g., the specific cells and the specific treatments and / or analytes. For example, smaller pore sizes (e.g., 0.1 to 3 μm) can be used for drug transport studies. In certain examples, the openings or pores of the membrane can be about 0.1 μm to 1 mm in size; about 0.1 μm to 30 μm in size; about 0.1 μm to about 15 μm in size; about 0.1 μm to about 10 μm in size; about 0.3 μm to about 1 mm in size; about 0.3 μm to about 30 μm in size; about 0.3 μm to about 15 μm in size; or about 0.3 μm to about 10 μm in size. In certain aspects, the openings or pores of the membrane can be about 0.1 μm to 12 μm in size. The semi-permeable or permeable membrane can, for example, be a mesh with an open weave with openings through which liquids can pass, formed as a woven material, a perforated material or the like. In certain aspects, the mesh is a nylon mesh. In some embodiments, these inserts allow cells to migrate via the semi- permeable membrane. In other embodiments, the membrane or mesh is not permeable to cells. The membrane can optionally be treated for optimal cell attachment. Non-limiting examples of material that can be used to make the membrane material are polypropylene, polycarbonate, polyester, polyethersulfone, polyethylene terephthalate, polyimide, polytetrafluoroethylene, silicone, silicon nitride, or a combination thereof. The receptacle chamber can be any shape including a cylinder (e.g, a round well; wherein the opening of the well is a circle) or rectangular prism (e.g., shaped as channel; wherein the opening of the chamber is a rectangle). As discussed above, the receptacle has a bottom, a side wall and a top. The receptacle chambers are positioned within the side wall, and the opening of the receptacle chamber(s) can be located on the top of the receptacle. In certain aspects, at least the bottom of the receptacle is optically transparent. In some embodiments, the entire receptacle is optically transparent. In additional aspects, the walls of the receptacle (the walls that enclose the receptacle bottom) are non-reflective. In certain aspects, the receptacle is made from plastic or glass, or a combination thereof. As discussed above, media can be added or removed from the opening of the receptacle chamber. For example, when the receptacle is a multi-well plate that holds the cell culture inserts, media can be added or removed from the opening of the wells with a pipette tip. Specifically, the pipette can be inserted into the small space between the wall of the cell culture chamber and the wall of the receptacle chamber. One thus needs at least a small space between the cell culture insert and the side wall of the well that is at least large enough for a pipette tip. In another example, wherein the receptacle has horizontal channels, a liquid can be added or removed from a the side of the receptacle, e.g., from an inlet or outlet, or from a port. In additional aspects, the receptacle has one or more ports for the addition and / or removal of media, wherein the ports are in fluid communication with the receptacle chambers. In certain cases, each receptacle chamber has its own port. In other examples, more than one receptacle chamber shares a common port. In some examples, the port(s) is located at the top of the receptacle or the side(s) of the receptacle, or a combination thereof. In certain specific aspects, the receptacle chamber has more than one horizontal chambers that holds one or more cell culture chambers and each channel has its own port, for example, on the side of the receptacle. As discussed above, the receptacle holds one or more cell culture chambers or supports one or more cell culture chambers (e.g., the cell culture chamber(s) is arranged over the receptacle chamber). In some aspects, the receptacle holds or supports more than one cell culture chamber. In some examples, the receptacle holds or supports between 1 and 30 cell culture chambers, between 1 and 100 cell culture chambers, between 1 and 400 cell culture chambers, between 1 and 1000 cell culture chambers, between 1 and 2000 cell culture chambers, between 3 and 30 cell culture chambers, between 3 and 100 cell culture chambers, between 3 and 400 cell culture chambers, between 3 and 1000 cell culture chambers, between 3 and 2000 cell culture chambers. In certain specific examples, the receptacle holds 4, 6, 8, 12, 24, 96, 384, 1536 or more cell culture chambers having individual (separate) or shared walls. In certain specific embodiments, the receptacle has one or more horizontal chambers that each holds or supports one or more cell culture chambers. In some examples, the receptacle holds or supports between 1 and 30 cell culture chambers, between 1 and 100 cell culture chambers, between 1 and 400 cell culture chambers, between 1 and 1000 cell culture chambers, between 1 and 2000 cell culture chambers, between 3 and 30 cell culture chambers, between 3 and 100 cell culture chambers, between 3 and 400 cell culture chambers, between 3 and 1000 cell culture chambers, between 3 and 2000 cell culture chambers. In certain specific examples, the receptacle holds or supports 4, 6, 8, 12, 24, 96, 384, 1536 or more cell culture chambers having individual or shared walls. In some examples wherein a membrane covers one or more of the horizontal channels, the receptacle can have parallel horizontal channels that each has one or more cell culture chamber arranged over it. For example, the receptacle can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more parallel horizontal channels, wherein each channel has or supports 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more cell culture chambers. A number of configurations are possible. For example, in one configuration, the receptacle has six cell culture chambers and has two receptacle chambers, wherein the two receptacle chambers are parallel horizontal channels and wherein three of the cell culture chambers are arranged in a row above each channel. In another exemplary configuration, the receptacle has six cell culture chambers and three receptacle chambers, wherein the three receptacle chambers are parallel horizontal channels and wherein two of the cell culture chambers are arranged in a row above each channel. In yet another example, the receptacle has twelve cell culture chambers and has three receptacle chambers, wherein the three receptacle chambers are parallel horizontal channels wherein four of the cell culture chambers are arranged in a row above channel. In another example, the receptacle has twelve cell culture chambers and has four receptacle chambers, wherein the four receptacle chambers are parallel horizontal channels and wherein three of the cell culture chambers are arranged in a row above each channel. In yet another example, the receptacle has twenty-four cell culture chambers and has six receptacle chambers, wherein the six receptacle chambers are parallel horizontal channels and wherein four of the cell culture chambers are arranged in a row above each channel. In a further example, the receptacle has twenty-four cell culture chambers and has four receptacle chambers, wherein the four receptacle chambers are parallel horizontal channels and wherein six of the cell culture chambers are arranged in a row above each channel. In yet another example, the receptacle has 96 cell culture chambers and has eight receptacle chambers, wherein the eight receptacle chambers are parallel horizontal channels and wherein twelve of the cell culture chambers are arranged in a row above each channel. In a further example, the receptacle has 96 cell culture chambers and has twelve receptacle chambers, wherein the twelve receptacle chambers are parallel horizontal channels and wherein eight of the cell culture chambers are arranged in a row above each channel. For systems that include a cell culture insert, the membrane of the cell culture chamber is positioned inside the receptacle chamber such that it is a small distance from the floor of the receptacle chamber, e.g, less than about 1 mm. The location where the membrane sits inside the receptacle chamber (e.g., during cell culture and / or analysis) is referred to herein as the “terminal location.” As described above, the membrane can be placed inside the receptacle chamber such that the apical side of the membrane is at the terminal location (wherein the terminal location is below the opening of the receptacle chamber and above the floor of the receptacle chamber) is less than about 1 mm, less than about 0.9 mm, less than about 0.8, less than about 0.75 mm, less than about 0.7 mm, less than about 0.6 mm, or less than about 0.5 mm from the floor from the floor of the receptacle chamber. Additionally or alternatively, the surface area of the membrane of the cell chamber inside the receptacle chamber cover is more than about 60% of the cross-sectional area of the receptacle chamber, and / or the diameter or width of the membrane (depending on the shape of the membrane / cell culture chamber) is more than 75% of the diameter or width of the receptacle chamber at the terminal location. The “cross-sectional area of the receptacle chamber” is the cross-sectional area of the cross-section of the receptacle chamber at the terminal location that is parallel to the floor of the receptacle chamber. The “width of a rectangular chamber” is understood to be the shortest side, whereas the length is the longer side. For a square, the width is one side of the square. For example, for a round well and a round cell chamber, if the cross-sectional area at the terminal location is 3.5 cm2, then the surface area of the membrane would be at least about 2.1 cm2in order to cover more than 60% of the cross-sectional area of the receptacle chamber. In another example, for a receptacle chamber (e.g., a well or channel) with a rectangular cross-section having a width of 3 cm and a length of 6 cm, then the width or diameter of the membrane (depending on the shape of the membrane) would be at least 2.25 cm in order to cover more than 75% of the width of the receptacle chamber. In certain aspects, the surface area of the membrane is more than about 70% of the cross-sectional area of the receptacle chamber at the terminal location. In additional aspects, the diameter or width of the membrane inside the receptacle chamber is more than 80% of the diameter of width of the receptacle chamber at the terminal location. In certain specific examples of a system that includes a cell culture insert, the receptacle has a plurality of parallel horizontal channels that holds more than one cell culture chamber. For example, the receptacle can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more parallel horizontal channels, wherein each channel holds 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more cell culture chambers. In certain aspects, the receptacle holds six cell culture chambers and has two receptacle chambers, wherein the two receptacle chambers are parallel horizontal channels that each holds three of the cell culture chambers in a row along the length of the channel. In another example, the receptacle holds six cell culture chambers and has three receptacle chambers, wherein the three receptacle chambers are parallel horizontal channels that each holds two of the cell culture chambers in a row along the length of the channel. In yet another example, the receptacle holds twelve cell culture chambers and has three receptacle chambers, wherein the three receptacle chambers are parallel horizontal channels that each holds four of the cell culture chambers in a row along the length of the channel. In another example, the receptacle holds twelve cell culture chambers and has four receptacle chambers, wherein the four receptacle chambers are parallel horizontal channels that each holds three of the cell culture chambers in a row along the length of the channel. In another example, the receptacle holds twenty-four cell culture chambers and has six receptacle chambers, wherein the six receptacle chambers are parallel horizontal channels that each holds four of the cell culture chambers in a row along the length of the channel. In another example, the receptacle holds twenty-four cell culture chambers and has four receptacle chambers, wherein the four receptacle chambers are parallel horizontal channels that each holds six of the cell culture chambers in a row along the length of the channel. In yet another aspect, the receptacle holds 96 cell culture chambers and has eight receptacle chambers, wherein the eight receptacle chambers are parallel horizontal channels that each holds twelve of the cell culture chambers in a row along the length of the channel. In a further example, the receptacle holds 96 cell culture chambers and has twelve receptacle chambers, wherein the twelve receptacle chambers are parallel horizontal channels that each holds eight of the cell culture chambers in a row along the length of the channel. As discussed above, a cell culture insert can have only a single cell culture chamber (and optionally, multiple inserts can be placed into a receptacle) and / or a single insert can have a plurality of cell culture chambers. In some embodiments, a single insert has a plurality of cell culture chambers, wherein the plurality of cell culture chambers are arranged on a support and the support rests on a structure at the top of the receptacle or on a pedestal or shelf in the receptacle chamber(s). In another aspect, the receptacle holds a plurality of individual cell culture inserts, wherein each individual cell culture insert has one cell culture chamber, and wherein each individual insert has a structure (e.g., proximal to the cell culture chamber opening) that rests at the top of the receptacle. In yet another embodiment, the receptacle holds a plurality of individual cell culture inserts wherein each individual cell culture insert has a structure that rests on a pedestal in the receptacle chamber that holds the cell culture chamber. In certain embodiments, the cell culture chamber of the insert has a structure, e.g., a flange, a ring, or a tab, that rests on a support at the top of the plate or on a pedestal inside the well. The pedestal is a structure that can be placed on the floor of the receptacle chamber or can be attached to the wall of the receptacle chamber such that contact between the pedestal and the cell culture chamber supports or holds the cell culture insert in the receptacle chamber, for example, the pedestal can fix the cell culture insert in place and / or decreases movement. In certain embodiments, the pedestal is made of rigid material including, for example, plastic, silicone, or other polymers or a combination thereof, as well as flexible materials comprising plastics, plastic, silicone, or other polymers or a combination thereof. In yet additional aspects, the pedestal adheres to the cell culture insert or cell culture chamber (e.g., the support has a sticky surface that adheres to the cell culture insert or cell culture chamber) or the pedestal affixes the cell culture insert or cell culture chamber (e.g., by a clicking mechanism). In certain aspects, the pedestal is optically transparent. In other aspects, the pedestal is non-reflective. It should be understood that the pedestal can have any shape and the dimensions including the height of the pedestal can be designed with different heights so as to allow the cell culture chamber to sit at different positions in the receptacle chamber. In some embodiments, one or more pedestals can be used in a receptacle chamber. In other embodiments, the pedestal can be shaped such that only one pedestal need be used in a receptacle chamber. Each of the cell culture chambers can be covered with a removable seal, e.g., to decrease evaporation. In some embodiments, the system or plate further comprises a lid that covers the openings of the cell culture chambers. In some embodiments, the lid comprises an inlet and an outlet (or inlet and outlet ports) for the transfer of liquid (e.g., media or analyte / candidate agent) into one or more receptacle chambers. The inlet can be designed to be compatible with fluidics, pipetting or both. The lid releasably engages the receptacle to enclose the receptacle chambers and / or the cell culture chambers. The lid can be in fluid communication with receptacle chamber(s), e.g., by tubing and / or can have entry ports for tubing or for pipetting. The lid can be made of a rigid material including, for example, plastic, silicone, or other polymers or a combination thereof, as well as flexible materials comprising plastics, plastic, silicone, or other polymers or a combination thereof. In certain aspects, the lid is optically transparent. In other aspects, the lid is non-reflective. In yet other aspects, the lid is highly reflective, for example, allowing illumination of the cell culture analysis system from above or below (e.g., above the lid or below the receptacle chamber). In some cases, the lids has internal or connected manifold systems for the inlets, outlets, or a combination thereof. The inlet(s) can be designed to be compatible with fluidics, pipetting or both. In yet further aspects, the lids has one or more structural features that decreases evaporation from the cell culture chambers. In some embodiments, the lids has one or more structural features that fix or hold the cell culture insert in the receptacle chamber, e.g., decreasing movement during longitudinal imaging. The invention additionally encompasses the lid as described herein. The lid can be used for multiplex exchange with conventional cell culture plates (e.g., used without any transwell inserts). For example, the plates used with the lid in this embodiment can be used to culture adherent cells, cells attached to the bottom of the well, or cells embedded in the gel attached to the bottom of the well. The invention additionally encompasses methods of detecting a cell response to a first candidate agent comprising culturing cells in a system described herein, contacting the cells with a candidate agent and detecting a response to the candidate agent. Non-limiting examples of cells that can be used in the systems and methods described herein are cells of multicellular organisms, e.g., cells of invertebrates and vertebrates, such as myoblasts, neutrophils, erythrocytes, osteoblasts, chondrocytes, basophils, eosinophils, adipocytes, invertebrate neurons (e.g., Helix aspera), vertebrate neurons, mammalian neurons, adrenomedullary cells, melanocytes, epithelial cells, and endothelial cells; tumor cells of all types (e.g., melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes); cardiomyocytes, endothelial cells, lymphocytes (e.g., T-cells and B cells), mast cells, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes; stem cells such as hematopoietic stem cells, neural, skin, lung, kidney, liver and myocyte stem cells; osteoclasts, connective tissue cells, keratinocytes, melanocytes, hepatocytes, and kidney cells. Suitable cells also include known cell lines, including, but not limited to, Jurkat T-cells, NIH3T3 cells, CHO, COS, etc. In certain aspects, the cells or culture thereof comprise immune cells including, but not limited to, T-cells, B-cells and NK cells. In additional aspects, the cells or culture thereof comprise peripheral blood mononuclear cells (PBMCs). In additional aspects, the cells or culture thereof comprise T-cells. Such T-cells include, for example, CD4+, CD8+, and regulatory T-cells. A suitable example is a Jurkat cell. Jurkat cells are immortalized T lymphocytes first derived from the peripheral blood of a child with T-cell leukemia (Schneider et al., 1977, Int J Cancer 19 (5):621-6). The sample can comprise T-cells and APCs, such as professional APCs. Such professional APCs can be selected from the group consisting of dendritic cells, macrophages, monocytes and B cells. In certain aspects, the candidate agent is a peptide or a library thereof; a disease- related antigen (e.g., a bacterial antigen, a viral antigen, or a cancer antigen). A candidate agent can be an agent from which the effect on T-cell activation and / or differentiation and / or modulation of other cell response is unknown. Candidate agents (or libraries thereof) include for example oligopeptides, polypeptides, proteins, antibodies, (peptide-)mimetics, and small molecules. In some examples, the candidate agent is screened for its ability to activate an immune cell. The candidate agent can, for example, be an infectious disease-associated candidate epitope, an autoimmune disease-associated candidate epitope, or a tumor- associated candidate epitope. An epitope refers to the portion of antigen that is recognized by B cells or T-cells, and the portion of the antigen to which an antibody binds. An epitope refers more specifically to the portion of antigen that is recognized by B cells or T-cells, and the portion of the antigen to which an antibody binds. An epitope is typically a small peptide of 2 or more amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids). Class I epitopes are typically about 8-15 amino acids in length, and more typically about 8-12 amino acids in length. Class II epitopes are typically about 8-24 amino acids in length, and more typically about 15 amino acids in length. A biological sample or sample comprising T-cells can be stored in a suitable solution such that the T-cell and optionally, the antigen-presenting cell are maintained in a viable state. Examples of such a liquid include a medium generally used for culturing an immune cell in vitro. Such a medium is known, and examples thereof include MEM, DMEM, RPMI- 1640, CTL-Test medium (Cellular Technology Limited), and the like. If necessary, the medium may be mixed with an additive such as fetal bovine serum (FBS) or L-glutamine. In a preferred embodiment, the biological sample or specimen is stored in a medium under conditions under which an immune cell can be cultured. Such conditions are known per se, and examples thereof include conditions at 37° C. in a 5% CO2atmosphere. In certain embodiments, the total number of cells cultured in the plurality of cell culture chambers is at least 1000, 10,000, 100,000, 1 million, 5 million, 10 million or more, and optionally the area of the receptacle is substantially similar to that of a conventional cell culture plate, e.g., wherein the length of the receptacle is between 122 mm and 132 mm and the width of the receptacle is between 80 and 90 mm; and / or wherein the surface area of the receptacle is between about 970 cm2and 1200 cm2. In some embodiments, the cells form a monolayer. In other examples, the cells form multiple layers. The cells can be adherent or in suspension. In some examples, the cells are contacted with the analyte or candidate agent prior to culturing on the system described herein. In additional examples, the cells are contacted with the analyte or candidate agent in the cell culture chamber. In some cases, all of the cells in the cell culture chambers are contacted with the same candidate agent. In other examples, cells in different cell culture chambers are contacted with different candidate agents. In certain aspects, the cells are treated with more than one candidate agents. For example, the method can include an optional first washing step that removes the first candidate agent, contacting the cells with a second candidate agent following the first optional wash step, and detecting the cell response to the second candidate agent. The method can further comprise a second washing step that removes the second candidate agent, contacting the cells with a third candidate agent following the second wash step, and detecting the cell response to the third candidate agent. As will be understood, this process can be repeated for one or more additional candidate agents. In some cases, the response is detected by microscopy or high-resolution optical sensing with or without magnification, with or without lenses and optionally, wherein the microscopy is fluorescence microscopy or method of signal detection relies on fluorescence. The systems described herein can also be utilized for treatment cycling as described, for example, in WO2025 / 015241 (PCT Application PCT / US2024 / 037718 filed July 12, 2024); the contents of which are expressly incorporated by reference herein. “Treatment cycling,” is the method described therein that allows repeating treatments of the same sample to significantly improve the chances of obtaining the cellular response at a single-cell sensitivity level. Alternatively, this process can be seen as recycling or re-use of the same samples in a multiplicity of experimental treatments. This process can also be described as re- running an experiment seeking a positive response until it appears. In some embodiments, a positive response can be obtained in multiple cells and / or samples at different treatment cycles. In certain aspects, when a sample is positive for a response (such as T-cell activation) in a cycle, that sample is not subjected to a subsequent cycle. In yet another aspect, when a sample is positive for a cell response (e.g., T-cell activation) in a cycle, that sample is subjected to one or more subsequent cycles. A positive response can, for example, be a fluorescent, luminescent, or other signals above the background from the cells, interaction of the cell with another cell in the sample, or its metabolic product, and / or change of the phenotypic features by optical imaging of the cell(s) of interest or the cell(s) interacting with the cell of interest or its metabolic products. The optical signal can be detected by microscopy, for example, fluorescence microscopy. Treatment cycling combines the quick readouts with gentle wash / analyte exchange. The gentle wash / analyte exchange methods described herein avoid the use of high-speed centrifugation. In certain embodiments, the gentle wash / analyte exchange methods described herein avoid the use of centrifugation. Centrifugation is almost universally utilized for washing cells. Centrifugation is difficult to incorporate in an automated system and method and can negatively affect cell viability and quantity, especially when repeated multiple times. The present invention therefore utilizes methods and techniques that can be readily automated. When the treatment cycling approach is combined with the multiple signals per vessel / well / microchip for the spatial transcriptomics, i.e., Cycle-Location Barcoding (CLB), the efficacy of the specimen use, for example for determining TCR sequences and activation types, may be additionally increased up to 10-fold or more. Applying treatment cycling and CLB together can increase assay efficacy up to 1000 times or more. As described herein, each treatment cycle includes a response readout step (detection of the cell response or detection of T-cell activation) and a removal step wherein the analyte fluid is removed. The removal of the analyte fluid removes all or a portion of the analyte fluid from the solid support (e.g., the multi-well plate) and thus removes the candidate agent but retains the sample comprising the cells on the solid support (e.g., the cell culture). “Gentle analyte removal” as used herein, is a set of methods that allow removal of the analyte fluid from a sample without extreme stress or damage to the live cells, preferably preserving their capability for a functional response. In certain aspects, the removal of the analyte fluid is gentle enough that the cell viability and / or the cell quantity of the retained sample is decreased by less than about 5% after 10 total cycles. The methods used for removal do not include high-speed centrifugation. In certain aspects, the method used for removal of the analyte does not include centrifugation. Standard centrifugation at high centrifugal forces is not considered a gentle cell wash since it can lead to cell loss, death, or dysfunction. In certain aspects, the gentle analyte removal can include analyte exchange wherein previous potentially T-cell activating analyte solution is removed, and the next potentially T-cell activating analyte solution is added. In additional aspects, the gentle wash can comprise a wash step wherein a wash buffer with no components to activate the cells is introduced to the sample. An example of a wash buffer for T-cell activation cycling is a T-cell medium. The gentle wash can be performed at the optimal physiological temperatures, ideally at +37C; however, temperature variation should be possible without affecting the assay. In certain aspects, the temperature is no lower than +20C and no higher than +40C. Ideally, the gentle wash / analyte exchange will retain the cells in the same 2D orientation in the well. For some assay implementations these stable cellular coordinates will be useful. As described above, the invention encompasses a method of identifying a candidate agent that elicits a response in a cell, the method comprising: a first cycle comprising: contacting a sample comprising cells with an analyte fluid, wherein the sample comprising the cells is in a cell culture chamber described herein, and wherein the analyte fluid comprises a candidate agent in a liquid medium; detecting the response by detection of an optical signal in the sample in the cell culture chamber; and removing the analyte fluid from the cell culture chamber thereby removing the candidate agent while retaining the sample comprising the cells on the membrane of the cell culture chamber; a subsequent cycle comprising: contacting the retained sample comprising the cells of the preceding cycle with a new analyte fluid, wherein the new analyte fluid comprises a new candidate agent in a liquid medium, wherein the new candidate agent is different from the candidate agent of the preceding cycle; detecting the response by detection of an optical signal in the retained sample in the cell culture chamber; and removing the analyte fluid from the cell culture chamber thereby removing the candidate agent while retaining the sample comprising the cells on the membrane of the cell culture chamber; repeating the subsequent cycle at least N times, wherein N is an integer greater than or equal to 0, wherein each new analyte fluid of each subsequent cycle comprises a different candidate agent from that of the preceding cycle. The analyte fluid is removed by a gentle analyte removal method described herein. In certain aspects, the removal of the analyte fluid is a method whereby the cell viability and / or the cell quantity of the retained sample is decreased by less than about 5% after 10 total cycles and that does not comprise centrifugation. Treatment cycling includes the first cycle and at least one subsequent cycle. For example, when the total number of cycles is 10 this means that there is a first cycle and 9 subsequent cycles (or in other words, N is 9). In another example, when the total number of cycles is 100, this means that there is a first cycle and 99 subsequent cycles. Each subsequent cycle contacts the sample of the preceding cycle from which the analyte fluid was removed. The preceding cycle is the cycle directly before the cycle being referenced, for example, when the total number of cycles is 3, the first cycle is “the preceding cycle” for the second cycle and the second cycle is “the preceding cycle” for the third cycle. In another example, when the solid support is a 96-well plate wherein each well includes a sample and the total number of cycles is 3, analyte fluid can be removed from each well at the end of first cycle (while retaining the sample comprising the cells in the well), and that retained sample is treated with the new candidate agent in the second cycle. Next, analyte fluid can be removed from each well at the end of second cycle (while retaining the sample comprising the cells in the well), and that retained sample is treated with the new candidate agent (different from the candidate agent of the second cycle) in the third cycle. The analyte fluid can be removed from the system described herein by any method that displaces all or a part of the analyte fluid. For example, the analyte fluid can be aspirated or otherwise removed such that cells are retained in the well with minimal loss in viability and / or function (such methods can include tilting of the plate and / or use of a mesh to cover the cells). In a further method, fluid (e.g., a buffer, other wash fluid or the new analyte fluid) can be added to the solid support thereby displacing the analyte fluid as liquid waste. In certain examples, the analyte fluid is removed by laminar wash. Laminar wash devices are sold by Curiox (curiox.com) and have been described, for example, in WO2020028406A1 and U.S. Pat. No.9557318B2; the contents of which are expressly incorporated by reference herein. In a laminar wash method, buffer or other wash fluid is dispensed and aspirated at opposing locations at a precise rate resulting in laminar flow with high flow rate at the top of the well and a low or static flow rate where the cells are. The systems and methods described herein detect a cell response (e.g., T-cell activation) by detecting an optical signal in the sample on the support, for example, by fluorescence microscopy. For T-cell activation, the optical signal can, for example, indicate calcium flux, cytokine release, or the formation of a complex comprising a T-cell and an antigen-presenting cell (APC). In certain aspects, the optical signal indicates calcium flux. Increased calcium concentration in the T-cell is a rapid and sensitive measure of cell activation. Thus, the optical signal can be provided by a calcium binding moiety (or a calcium probe) that includes a detectable label (e.g., a fluorescent label). A calcium binding moiety with a fluorescent label can be referred to as a fluorescent calcium indicator and can comprise a fluorescent dye coupled to a calcium chelator, and various products are commercially available. Examples of such fluorescent calcium indicators include Fura2, Fluo3, Fluo4, Indo1, Rhod2, and the like. The fluorescent probe may be protected with an acetoxymethyl (AM) group. Protection with an AM group imparts cell permeability to the fluorescent probe. Non-limiting examples of labeled calcium probes / indicators that can be used according to the methods described herein are include x-Rhod-1 (a red fluorescent calcium indicator), Calbryte™ 630 AM (AAT Bioquest), Calbryte™ 520 AM (AAT Bioquest), as well as Fluo-4, Fluo-Gold, Fluo-3, Fluo-2 available from Ion Biosciences. The labeled calcium probe can be added before, after or concurrently with the analyte fluid. For example, the calcium probe can be added to the cells before the analyte, incubated for about 1 hour and then it is washed off. In additional aspects, the optical signal indicates cytokine release. Examples ofcytokines secreted by activated T-cells include interferon gamma (IFN- ) and interleukin-2(IL-2). Accordingly, in some embodiments, the optical signal can be provided by a labeled moiety that binds to a cytokine. An example of such a labeled moiety is a fluorescentlylabeled anti-cytokine antibody, such as an anti-IFN- antibody or an anti-IL-2 antibody. Thelabeled moiety that binds to a cytokine can be added before, after or concurrently with the analyte fluid. In yet further aspects, the optical signal is the formation of an immune synapse, in other words, a complex of a T-cell and an APC. Non-limiting examples for detecting the immune synapse are described, for example, in US20210356454A1 and Calvo et al. (2018), Front Immunol 9: 684; the contents of which are expressly incorporated by reference herein. Representative labels that may be suitable for optical detection (e.g., associated or attached to an anti-cytokine antibody or calcium probe) include radioactive isotopes, fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions and metal sols. In some embodiments, the label is fluorescent. Representative examples of non-proteinaceous fluorescent labels include allophycocyanins (tradename XL665); luminescent organic molecules, such as rhodamines, cyanines (e.g., Cy5), squaraines, coumarins, proflavins, acridines, fluoresceins, boron- dipyrromethene derivatives (commercially available under the tradename “BODIPY”), fluorophores known under the name “Atto”, fluorophores known under the name “DY”, compounds known under the name “Alexa”, and nitrobenzoxadiazole. The “Alexa” compounds are commercially available, e.g., from Invitrogen; the “Atto” compounds are commercially available from Atto-tec; the “DY” compounds are commercially available from Dyomics; and the “Cy” compounds are commercially available from Amersham Biosciences. Fluorescently labeled anti-cytokine antibodies are commercially available. Proteinaceous fluorescent labels may also be useful. Representative examples of fluorescent polypeptides include yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), GFP, mRFP, RFP (tdimer2), and HCRED. Biotin-based labels may also be useful. Biotinylation of target molecules, including antibodies, is well known in the art. Biotinylated anti-cytokine antibodies may be detected by binding of a detectably labeled biotin binding partner, such as avidin or streptavidin. In yet further aspects, the cells express a heterologous reporter gene and the expression of the gene provides the optical signal. Genetic reporters are used widely as indicators to study gene expression and cellular events coupled to gene expression for pharmaceutical and biomedical research. Typically, a reporter gene encoding the reporter is cloned into an expression vector that is then transferred into cells. Expression vectors can include viral vectors, plasmids, mRNA, and others. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. Preferred reporters are those that can be identified easily and measured quantitatively when expressed in the effector T-cells. Many suitable examples are known to those skilled in the art, including fluorescent and luminescent reporters. Optionally the reporter is a bioluminescent reporter, such as a luciferase. A sample that exhibits a positive readout in one or more cycles can be further analyzed or characterized using a downstream assay. Such downstream workflow can include, for example, incubating the cells for several days (e.g, 5 or more days) and evaluating cell response (e.g., cell death, proliferation, cytokine production); collecting cell culture medium for analysis at different time points; sorting of the resulting cells of the specimen by cell type, activation state or other features using magnetic separation or other cell sorting methods; staining for flow cytometry or barcoded antibody staining for sequencing; determining the coordinates of the responded cells and applying an array of methods to perform the spatial transcriptomics for the 2-D specimen, optionally kept attached to the vessel bottom or transwell mesh or between two layers of the mesh; colony-picking by the third-party automated instruments; and / or bulk or single-cell sequencing of DNA or RNA, as the example sequencing of the T-cell receptor (TCR) mRNAs to find their sequence in parallel with the peptide specificity if the treatment included the peptides of peptide pools. The downstream assay can, for example, determine the T-cell subtype and function of the positive sample. The T-cell type and function can be determined from expression of surface proteins (e.g., CD3, CD4, CD8, CD45RA, etc.) and cytokine production (e.g.interferon-gamma (IFN- ), transforming growth factor beta (TGF- ), interleukin (IL)-2, IL-4,IL-17, etc.) by antibody-based methods such as flow cytometry, immunohistochemistry, or immunofluorescence, or by transgenic fluorophore expression. Downstream assays can, for example, include repeated cell activation or use in subsequent experiments alone or in the presence of other cells or tissue culture specimens. As described above, one of the areas of research where the methods described herein may be highly advantageous is T-cell research, specifically the study of peptide specificity of the T-cell receptors, TCRs. Genetic variability of the TCR is enormous; a single person may have up to 108different individual different variants of the TCRs corresponding to individual T-cells or their clones. TCRs specifically (however, with some levels of cross-specificity) interact with the complexes of HLA molecules with peptides, usually short amino-acid sequences generated during the normal protein metabolism in the live cells. In the inflammatory context, TCR may recognize the corresponding non-self peptide presented as an HLA-peptide complex on the surface of the antigen-presenting cell. An outcome of this interaction can comprise T-cell activation, change of phenotype, proliferation, and cytokine secretion; the activated T cells help induce antigen-related antibody responses, participate in antigen-specific signaling, and kill the target cells exposing the non-self peptides on the surface. In many cases, it is enough to determine if any T-cells in the specimen can respond to any random parts of one or more antigens related to some pathogen or gene therapy vector. However, studying the precise peptide specificity of the TCRs and corresponding T-cells may be useful to optimize the response. For example, if simplified, a strong T-cell response would be beneficial in developing vaccines, cancer, and chronic infection immunotherapies. The “low-to-no” T-cell response would be important to demonstrate in developing cell and gene therapies, autoimmunity treatments, and allergy desensitization immunotherapies. Measuring T-cell responses may have high importance in both preclinical and clinical research, as well as in clinical practice as a diagnostic tool. Taking into account the variability of both TCRs (108) and peptides of the length relevant to HLA-peptide-TCR interaction, there may be only a vanishingly slight chance to activate even a single T-cell in a specimen using one single peptide that corresponds to the antigen of interest (protein). In such a case, not only the chance of activation is quite low, but there is also a massive waste of the specimen if the response is negative (and for patients, this specimen is usually blood drained for research). Another area wherein the methods described herein may be advantageous is the research of rare primary cell types activation by the libraries of compounds with low chances of inducing the response of interest. Mixed primary human cell cultures are probably the most relevant ex vivo and in vitro research models able to capture the features of human- specific effects and diversity of human genetic background (as opposed to the use of experimental animals; or animal and human immortal cell lines). Large disadvantages of these models include the lack of standardization and questions regarding the sample's representativeness compared to the general population. Another problem is logistic access to the materials from many donors and the costs of such materials, in parallel with generally limited access to primary cells. The problem becomes even harder when the research has to be done in the rare cell types, which comprise only a small percentage of the total cell population in the tissue. An attempt to screen the extensive library of drug-like molecules (analytes, up to 1000 or more) to see if these molecules activate the rare cell type may be highly wasteful in the case one decides to treat the cell culture once and discard the specimen after the negative response is recorded (which may happen in over 99.99% of times). If 100 cycles are allowable by the model, the treatment cycling approach can help use the rare and primary cells with 100 times efficacy improvement. If these rare cells have highly genetically diverse receptors of interest, a single specimen may comprise a mixture of cells from different patients (while immune responses between them are blocked, for example, immune cells and antibodies are depleted), and later the response features / cycles can be correlated with genetic signatures. Such a mixture of the primary cells from 10 patients may lead to an additional 10-fold increase in the bioassay efficacy, with Cycling leading to a 1000-fold increase in specimen use efficacy. The present invention comprises the devices and methods to cycle the testing of the specimen reactivity against multiple analytes to increase the frequency of detecting low- probability positive responses. A device or a set of devices has the following functionality: (a) allows to add / adds the treatment to the suspension of the cells of interest, (b) measures the cellular response readout as negative or positive if one or more cells activated, (c) removes the excess treatment from the cell suspension, and optionally replenishes it with the washing solution, (d) repeats the cycles of steps (a)-(c), optionally excludes the samples with a positive response from the following cycles. As a result, if the chance of activating any single cell during one treatment cycle is low, the repeated treatment cycles substantially increase the chances of obtaining the response. This device and method advantageously can work with a limited sample supply, especially with the limited proportion of the responsive cells of interest within the sample, wherein their responses should be screened against multiple treatments with a low probability of positive response to any given treatment. The invention encompasses: (1) the treat-read cycling approach allowing the repeated use of the same specimen, in comparison to the industry’s traditional one treatment per one experimental specimen to assess the effects of this treatment, (2) use of the quick readouts to allow cycling in comparison to the industry’s traditional readouts usually recorded in at least hours after the treatment, (3) use of various quick cell washes or treatment exchanges without centrifugation at high centrifugal forces, in comparison to the industry’s traditional approach, (4) reliance on using the single-cell-generated responses and colonies, linked to the specific activating treatment conditions (numbers of cycles or coordinates or responding cell), for the sequencing of their features, in comparison to the industry’s traditional alternatives of single- cell sequencing or bulk sequencing done for the unknown number of proliferated colonies without their individual treatment-response tracking. This invention can utilize a limited specimen supply (limited by cost, logistics, ethical reasons, etc.), especially with the limited proportion of the responsive cells of interest within the sample, where their responses should be screened against multiple treatments with a low probability of positive response to any given treatment. An increase in the bioassay efficacy up to 100-1000 times compared to the traditional approaches can be expected. This increase in efficacy can (a) simplify the research logistics and decrease costs, as a result making previously impossible studies possible, (b) decrease the required volumes of specimens, making the research procedures less harmful or painful for the experimental animals or patients, (c) make the research procedures less wasteful improving the ecological outcomes of research, (d) stimulate new research areas such as the studies of T and B cells, studies of the rare cell types and the compounds (drugs, peptides) interacting with these cells and their receptors in the context of high biological and genetic diversity. In some examples, the method includes one or more cycles of treatment exchange involving staining of RNA or protein using in situ hybridization RNA probes (RNA-ISH) or antibodies, and optionally including cell fixation. The data readouts can characterize cellular phenotypes and expression profiles, be detected by imaging within the same system or a different imaging system to capture fluorescence or luminescence, changes of light transmission properties, or colored spots detection. The treatment can include extended cell resting to upregulate signaling molecules expression, signaling molecules secretion blocking, cell surface staining with antibodies, fixation and permeabilization, and / or staining of signaling molecules within the cells with antibodies. More staining parameters can be captured using antibody systems that allow cleavage or inactivation of fluorescent parts of antibodies, which allows the use of additional antibodies against the new targets with the same set of fluorescent labels as have been used earlier. The methods described herein allow automation of the cycles of antibody treatment, imaging, and antibody removal within the same plates. All references, articles, patent applications, patent publications and patents are incorporated herein by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A system for cell culture analysis, the system comprising a plurality of cell culture chambers, a removable cell culture insert, and a receptacle for receiving the cell culture insert, wherein: a. the cell culture insert comprises one or more of the cell culture chambers, each cell culture chamber comprising a side wall that encloses a semi-permeable membrane on the bottom of the cell culture chamber and an opening on the top of the cell culture chamber, wherein the membrane is capable of supporting the growth of cultured cells on the apical surface of the membrane, and wherein the basal side of the membrane faces the bottom of the receptacle; b. the receptacle comprises a plurality of receptacle chambers, wherein each receptacle chamber holds one or more of the cell culture chambers, wherein each receptacle chamber is arranged to retain a liquid that covers the basal and apical surfaces of the semi-permeable membrane, and wherein each receptacle chamber has a wall, an opening, and a floor; and c. wherein the membrane is positioned inside the receptacle chamber such that the apical side of the membrane is at a terminal location above the floor of the receptacle chamber, and wherein the terminal location is less than 1 mm from the floor of the receptacle chamber; and wherein the surface area of the membrane inside the receptacle chamber is more than about 60% of the cross-sectional area of the receptacle chamber at the terminal location, and / or wherein the diameter or width of the membrane inside the receptacle chamber is more than 75% of the diameter or width of the receptacle chamber at the terminal location.

2. The system of claim 1, wherein the surface area of the membrane is more than about 70% of the cross-sectional area of the receptacle chamber at the terminal location.

3. The system of claim 1, wherein the diameter or width of the membrane inside the receptacle chamber is more than 80% of the diameter of width of the receptacle chamber at the terminal location.

4. The system of claim 1, wherein the terminal location is less than 0.75 mm from the floor of the receptacle chamber.

5. The system of claim 1, wherein the receptacle or its bottom are optically transparent.

6. The system of claim 1, wherein the receptacle walls are non-reflective.

7. The system of any one of claims 1 and 5 to 6, wherein the receptacle is made from plastic or glass or a combination of thereof.

8. The system of claim 1, wherein the receptacle has one or more ports for the addition and / or removal of liquid, wherein the ports are in fluid communication with the receptacle chambers.

9. The system of claim 8, wherein each receptacle chamber has its own port.

10. The system of any one of claims 8 and 9, wherein each port is on a side or top of the receptacle.

11. The system of claim 1, wherein each receptacle chamber is a well, the receptacle is a multi-well plate and wherein each well holds one cell culture chamber; optionally, wherein the receptacle is a 6-well plate, a 12-well plate, a 24-well plate, or a 96-well plate.

12. The system of claim 1, wherein each receptacle chamber is a horizontal channel and each channel holds one or more cell culture chambers.

13. The system of claim 12, wherein each channel holds more than one cell culture chamber.

14. The system of claim 13, wherein each channel has its own port.

15. The system of claim 14, wherein each port is on the side of the receptacle.

16. The system of claim 1, wherein the system has 4, 6, 8, 12, 24, 96, 384, 1536 or another number of cell culture chambers having their individual or shared walls.

17. The system of claim 16, wherein the system has six cell culture chambers and two receptacle chambers, wherein the two receptacle chambers are parallel horizontal channels that each holds three cell culture chambers in a row along the length of the channel.

18. The system of claim 16, wherein the system has six cell culture chambers and three receptacle chambers, wherein the three receptacle chambers are parallel horizontal channels that each holds two cell culture chambers in a row along the length of the channel.

19. The system of claim 16, wherein the system has 96 cell culture chambers and eight receptacle chambers, wherein the eight receptacle chambers are parallel horizontal channels that each holds twelve cell culture chambers in a row along the length of the channel.

20. The system of claim 16, wherein the system has 96 cell culture chambers and twelve receptacle chambers, wherein the twelve receptacle chambers are parallel horizontal channels that each holds eight cell culture chambers in a row along the length of the channel.

21. The system of any one of claims 17 to 20, wherein each channel has its own port.

22. The system of claim 21, wherein each port is on the side of the receptacle.

23. The system of claim 1, wherein one or more of the openings of the cell culture chamber are covered by a removable seal, optionally wherein all of the openings are covered by a removable seal.

24. The system of claim 1, wherein the plurality of cell culture chambers are arranged on a support and the support rests on a structure at the top of the receptacle.

25. The system of claim 1, wherein the support rests on a pedestal in at least one receptacle chamber.

26. The system of claim 1, wherein the system comprises a plurality of individual cell culture inserts, wherein each individual cell culture insert has one cell culture chamber, and wherein each individual insert has a structure proximal to the cell culture chamber opening that rests on a structure at the top of the receptacle.

27. The system of claim 1, wherein the system comprises a plurality of individual cell culture inserts and wherein each individual cell culture insert has a structure proximal to the cell culture chamber opening that rests on a pedestal in the receptacle chamber that holds the cell culture chamber.

28. The system of any one of claims 25 and 27, wherein the pedestal rests on the floor of the receptacle chamber or is attached to the wall of the receptacle chamber and wherein contact between the pedestal and the cell culture chamber supports or holds the cell culture insert in the receptacle chamber.

29. The system of claim 1, further comprising a water jacket.

30. The system of claim 1, further comprising a lid.

31. The system of claim 29, wherein the lid comprises an inlet and an outlet, or inlet and outlet ports, for the transfer of liquid, into one or more receptacle chambers.

32. A system for cell culture analysis, comprising a plurality of cell culture chambers and a receptacle for receiving the cell culture insert, wherein: a. the receptacle comprises one or more receptacle chambers, each receptacle chamber having a wall, an opening and a floor;b. wherein a semi-permeable membrane covers the entire opening of the receptacle chamber, wherein the membrane is capable of supporting the growth of cultured cells on the apical surface of the membrane, and wherein the basal side of the membrane faces the floor of the receptacle chamber; optionally the semi-permeable membrane covers the entire top of the receptacle; c. each cell culture chamber is arranged on the apical surface of the membrane over a receptacle chamber, and wherein each cell culture chamber comprises a side wall that has an opening on the top of the cell culture chamber and the semi-permeable membrane on the bottom of the cell culture chamber; d. wherein the receptacle chambers and the cell culture chambers are arranged to retain a liquid that covers the basal and apical surfaces of the semi-permeable membrane or in a volume sufficient to provide effective diffusion through the membrane; for example, the receptacle chamber is configured to contain a fluid level such that the fluid contacts or diffuses through the membrane; e. wherein the receptacle and the side walls of the cell culture chambers are attachable and detachable from the semi-permeable membrane; for example, the receptacle is attachable / detachable from the basal side of the membrane and the side walls are attachable / detachable from the apical side of the membrane; and f. wherein the membrane is less than 2 mm from the floor of the receptacle chamber that holds that cell culture chamber; in certain aspects, the membrane within the cell culture chamber is less than 1 mm from the floor of the receptacle chamber.

33. The system of claim 32, wherein the membrane is less than 1 mm from the floor of the receptacle chamber.

34. The system of claim 32, wherein the receptacle is optically transparent.

35. The system of claim 32, wherein the receptacle walls are non-reflective.

36. The system of any one of claims 32 to 35, wherein the receptacle is made from plastic or glass or a combination of thereof.

37. The system of claim 32, wherein the receptacle has one or more ports for the addition and / or removal of the liquid, wherein the ports are in fluid communication with the receptacle chambers.

38. The system of claim 37, wherein each receptacle chamber has its own port.

39. The system of any one of claims 35 and 36, wherein each port is on the side of the receptacle.

40. The system of claim 32, wherein each receptacle chamber is a well, and the receptacle is a 6-well plate, a 12-well plate, a 24-well plate, or a 96-well plate.

41. The system of claim 32, wherein the system has 4, 6, 8, 12, 24, 96, 384, 1536 or another number of cell culture chambers having their individual or shared walls.

42. The system of claim 41, wherein the system has six cell culture chambers and two receptacle chambers, wherein the two receptacle chambers are parallel horizontal channels and three cell culture chambers are arranged in a row above the channel.

43. The system of claim 41, wherein the system has 96 cell culture chambers and eight receptacle chambers, wherein the eight receptacle chambers are parallel horizontal channels that each holds twelve cell culture chambers in a row above the channel.

44. The system of any one of claims 42 and 43, wherein each channel has its own port.

45. The system of claim 44, wherein each port is on a side or top of the receptacle.

46. The system of claim 34, wherein one or more of the openings of the cell culture chamber are covered by a removable seal.

47. The system of claim 46, wherein all of the openings are covered by a removable seal.

48. The system of claim 32, wherein the side walls of the plurality of cell culture chambers are arranged on a support to form a grid and wherein the grid is attachable and detachable from the membrane.

49. The system of claim 32, further comprising a water jacket.

50. The system of claim 32, further comprising a lid.

51. A method of detecting a cell response to a first candidate agent, the method comprising culturing cells in a system of any one of claims 1 to 50, contacting the cells with the first candidate agent and detecting a response to the first candidate agent.

52. The method of claim 51, wherein the cells are mammalian cells.

53. The method of claim 52, wherein the cells are immune cells, tumor cells, or stem cells.

54. The method of claim 53, wherein the immune cells are selected from the group consisting of B cells, T cells, innate lymphoid cells, natural killer cells, macrophages, monocytes, dendritic cells, neutrophils, myeloid derived suppressor cells, hematopoietic stem cells, and mesenchymal stem cells.

55. The method of claim 51, wherein the cells form a monolayer.

56. The method of claim 51, wherein the cells form multiple layers.

57. The method of claim 51, wherein the cells are adherent cells.

58. The method of claim 51, wherein the cells are in suspension.

59. The method of claim 51, wherein the cells are contacted with the analyte prior to culturing on the system.

60. The method of claim 51, wherein the cells are contacted with the first candidate agent in the cell culture chamber.

61. The method of claim 60, wherein all of the cells in each of the cell culture chambers are treated with the first candidate agent.

62. The method of claim 61, further comprising an optional first washing step that removes the first candidate agent, contacting the cells with a second candidate agent following the first optional wash step, and detecting the cell response to the second candidate agent.

63. The method of claim 61, further comprising a second washing step that removes the second candidate agent, contacting the cells with a third candidate agent following the second wash step, and detecting the cell response to the third candidate agent.

64. The method of claim 51, wherein the cells of a first cell culture chamber are contacted with the first candidate agent, the method further comprising contacting the cells of a second cell culture chamber with the second candidate agent, and further detecting the cell response to the second candidate agent, optionally at the same time as detecting the response to the first candidate agent.

65. The method of claim 64, further comprising contacting a third cell culture chamber with a third candidate agent, and further detecting the cell response to the third candidate agent, optionally at the same time as detecting the response to the first and second candidate agents.

66. The method of claim 51, wherein the candidate agent is a small molecule or biologic.

67. The method of claim 51, wherein the total number of cells cultured in the plurality of cell culture chambers is at least 5 million, and optionally wherein the length of the receptacle is between 122 mm and 132 mm and the width of the receptacle is between 80 and 90 mm.

68. The method of claim 67, wherein the total number of cells cultured is at least 10 million.

69. The method of claim 51, further comprising detecting the response by microscopy or high-resolution optical sensing with or without magnification, with or without lenses.

70. The method of claim 69, wherein the microscopy is fluorescence microscopy or method of signal detection relies on fluorescence.

71. The method of claim 54, wherein the cells are T cells and the cell response is T-cell activation.

72. The method of claim 51, wherein the first candidate agent is a positive control.

73. The method of claim 51, wherein the first candidate agent is a negative control.

74. The method of claim 51, wherein the first candidate agent is a library of candidate agents.

75. The method of claim 51, wherein the cell response produces an optical signal.

76. The method of claim 75, wherein the optical signal is fluorescence.

77. The method of claim 51, wherein the method is automated.

78. The method of claim 51, wherein the method is high-throughput.

79. A method of identifying a candidate agent that elicits a response in a cell, the method comprising: a first cycle comprising: a. contacting a sample comprising cells with an analyte fluid, wherein the sample comprising the cells is in a cell culture chamber of any one of claims 1 to 48, and wherein the analyte fluid comprises a candidate agent in a liquid medium; b. detecting the response by detection of an optical signal in the sample in the cell culture chamber; and c. removing the analyte fluid from the cell culture chamber thereby removing the candidate agent while retaining the sample comprising the cells on the membrane of the cell culture chamber; a subsequent cycle comprising: d. contacting the retained sample comprising the cells of the preceding cycle with a new analyte fluid, wherein the new analyte fluid comprises a new candidate agent in a liquid medium, wherein the new candidate agent is different from the candidate agent of the preceding cycle; e. detecting the response by detection of an optical signal in the retained sample in the cell culture chamber; and f. removing the analyte fluid from the cell culture chamber thereby removing the candidate agent while retaining the sample comprising the cells on the membrane of the cell culture chamber;repeating the subsequent cycle at least N times, wherein N is an integer greater than or equal to 0, wherein each new analyte fluid of each subsequent cycle comprises a different candidate agent from that of the preceding cycle.

80. The method of claim 79, wherein the removal of the analyte fluid is a method whereby the cell viability and / or the cell quantity of the retained sample is decreased by less than about 5% after 10 total cycles and that does not comprise centrifugation.

Images