CROSS-REFERENCE TO RELATED APPLICATION

This International patent application claims priority to United States Provisional patent application No. 63/350,830, filed June 9, 2022, which is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure provides for methods and systems for single-cell analysis. More particularly, the disclosure provides for methods and systems for analyzing cocultures of a single cancer-targeting cell (such as a T-cell) and one or more cancer cells. A single cancer-targeting cell may generally be cocultured with one or more cancer cells to evaluate the behavior of the cancer-targeting cell. Generally, the single cancer-targeting cell (i.e. T- cell) and one or more cancer cells are cocultured in microwells of a microwell array. Useful cell behavior data may be obtained by time-averaging or group-averaging techniques to overcome noise issues typically associated with single-cell techniques.

BACKGROUND

Robust determination of single-cell kinetics including killing rate, proliferation rate, engaging time, among others, are useful to compare and predict in-vivo performances among different cell clones. This is especially relevant in chimeric antigen receptor T-cell (CAR-T) development or T-cell receptor T (TCR-T) identification. At least conceptually, single-cell analysis might seem to be a viable option for comparing different cell clones. For example, in theory, performance of clones as a single cell could possibly correlate to their behavior in bulk. However, single-cell analysis is complex and poses several challenges.

In existing single cell monitoring platforms, analysis shows a greater spread among single cells carrying the same DNA materials than inter-genotype differences. This makes comparisons between cells with different genotypes, and correlation to bulk properties, unfeasible. Such spread is attributable to noise such as, individual cell health, environmental turbulence, and even the starting distances between T cells and tumor cells. Simply put, a modest clone i might easily outperform a relatively better clone due to random or systematic factors. Such measurement would cause ambiguity in identifying phenotypes and comparing different genotypes (DNA construct). In comparison, measurements in bulk are rather robust and are considered the standard and preferred methodology due to the unreliability of current single-cell technologies.

Such discrepancies or noise may follow typical gaussian distribution(s). For any observables with gaussian randomness, the bigger the sampling size, the less spread (sharper peaks) will be observed in the measurement. For example, gaussian parameters such as the full width at half maximum (FWHM) will be smaller with larger sample sizes. To make meaningful comparison between two distinct groups, adequate sample size is typically required to achieve a minimal peak spread. Single cell measurements (e.g. killing time, doubling time) obtained from a single event by a short period of observation are usually too noisy to advise isolation of single clones from thousands of clones, instead, such platforms are better at assessing one construct (such as one genotype) with lots of wells.

To suppress single cell noise, different strategies to increase sample size may be effective as provided by the present disclosure, including time average and micro-group average. The former (i.e. time average) includes a long-term observation over a single clone, and calculating the average kinetical properties of these events. The latter (i.e. micro-group averaging) requires a cell proliferation step, so that multiple cells (>3) originated from one single clone can be obtained, which can be followed by monitoring and calculation the cumulative kinetical behavior by this micro-group. In various embodiments, cells can be primary cells, genetically modified cells or cell lines. The present disclosure provides systems and methods for single-cell analysis which suppress noise so that effective analysis can be performed on single cells.

SUMMARY

The present disclosure provides for methods and systems for single-cell analysis. More particularly, the disclosure provides for methods and systems for analyzing cocultures of a single cancer-targeting cell (such as a T-cell) and one or more cancer cells.

In an embodiment, the present disclosure provides for methods for analyzing longterm behavior of single cancer-targeting cells, comprising the following steps in any useful order: plating cancer-targeting cells, optionally T-cells, on a microwell array having a plurality of microwells to provide about one cancer-targeting cell per microwell; plating cancer cells on the microwell array to provide a mixture of the cancertargeting cell with one or more cancer cells in each microwell; optionally, staining the cancer-targeting cells and/or cancer cells with a fluorescent marker; coculturing the mixtures of plated cancer-targeting and cancer cells for a period of time of at least about 96 hours; and imaging each of the plurality of microwells during coculturing to observe one or more long-term cell behaviors, wherein the long-term cell behaviors occur during a period of time of at least about 96 hours.

In an embodiment, the methods further comprise, prior to plating, combining the cancer-targeting cells, optionally T-cells, with the one or more cancer cells in a predetermined ratio for microwell plating and coculturing.

In an embodiment, the methods further comprise analyzing or determining one or more long-term cell behavior parameters based upon imaging collected at periods of time of at least 96 hours

In an embodiment, the methods further comprise selecting, based upon the analyzed or determiend cell behavior parameters at periods of at least 96 hours, cancer-targeting cells which effectively neutralize cancer cells.

In an embodiment, the cancer cell expresses a fluorescent marker (GFP)

In an embodiment, the cell behaviors include cell growth of cancer-targeting cells, killing of cancer cells, cell-cell interaction, trogocytosis, phagocytosis, endocytosis, apoptosis, necrosis, cell migration, cell fusion, secretion of cellular materials, division of cell chromosome, and surface expression of proteins/reporter genes.

In an embodiment, the imaging is microscopic imaging.

In an embodiment, the microscopic imaging is fluorescent microscopy.

In an embodiment, the cancer-targeting cells are T-cells.

In an embodiment, each of the plurality of microwells in the microwell array are generally rectangular or square.

In an embodiment, each of the plurality of microwells have a square or rectangular dimension in a range from about 50 pm to about 2 mm.

In an embodiment, each of the plurality of microwells have a height of about 100 - 500 pm.

In an embodiment, the methods further comprise one or more additional occurrences or steps of plating cancer cells on the microwell array during coculturing.

In an embodiment, the present disclosure provides for methods for analyzing single cancer-targeting cell behavior, comprising: plating cancer-targeting cells, optionally T-cells, on a microwell array having a plurality of microwells to provide about one cancer-targeting cell per microwell; cloning each cancer-targeting cell until at least about four identical clones are obtained in each microwell; plating cancer cells on the microwell array to provide a mixture of the cloned cancertargeting cells with one or more cancer cells in each microwell; optionally, staining the cancer-targeting cells and/or cancer cells with a fluorescent marker; coculturing the mixtures of the plated cancer-targeting and cancer cells; and imaging each of the plurality of microwells during coculturing for a period of time sufficient to observe at least one cell behavior.

In an embodiment, the methods further comprise selecting, based upon an analyzed cell behavior, cancer-targeting cells which effectively neutralize cancer cells.

In an embodiment, the cancer cell expresses a fluorescent marker (GFP).

In an embodiment, the cell behaviors include cell growth of cancer-targeting cells, killing of cancer cells, cell-cell interaction, trogocytosis, phagocytosis, endocytosis, apoptosis, necrosis, cell migration, cell fusion, secretion of cellular materials, division of cell chromosome, and surface expression of proteins/reporter genes.

In an embodiment, the imaging is microscopic imaging.

In an embodiment, the microscopic imaging is fluorescent microscopy.

In an embodiment, the methods further comprise one or more additional occurrences of plating cancer cells on the microwell array during coculturing.

In an embodiment, the present disclosure provides for systems for single-cell analysis with reduced noise, comprising: a microwell array containing a plurality of microwells, wherein the plurality of microwells are each generally rectangular or square with a dimension of about 50 pm - 2 mm, and with a height of about 100 - 500 pm; and an imaging apparatus for obtaining single-cell images of each of the plurality of microwells; wherein the single-cell analysis with reduced noise comprises: plating cancer-targeting cells, optionally T-cells, on a microwell array having a plurality of microwells to provide about one cancer-targeting cell per microwell; plating cancer cells on the microwell array to provide a mixture of the cancertargeting cell with one or more cancer cells in each microwell; optionally, staining the cancer-targeting cells and/or cancer cells with a fluorescent marker; coculturing the mixtures of plated cancer-targeting and cancer cells for a period of time of at least about 96 hours; and imaging each of the plurality of microwells to observe one or more long-term cell behaviors, wherein the long-term cell behaviors occur during a period of time of at least about 96 hours.

In an embodiment, the microwell array supports long-term cell growth for a period of time of at least about 96 hours.

In an embodiment, the present disclosure provides for systems for single-cell analysis with reduced noise, comprising: a microwell array containing a plurality of microwells, wherein the plurality of microwells are each generally rectangular or square with a dimension of about 50 pm - 2 mm, and with a height of about 100 - 500 pm; and an imaging apparatus for obtaining single-cell images of each of the plurality of microwells; wherein the single-cell analysis with reduced noise comprises: plating cancer-targeting cells, optionally T-cells, on a microwell array having a plurality of microwells to provide about one cancer-targeting cell per microwell; cloning each cancer-targeting cell until at least about four identical clones are obtained in each microwell; plating cancer cells on the microwell array to provide a mixture of the cloned cancertargeting cells with one or more cancer cells in each microwell; optionally, staining the cancer-targeting cells and/or cancer cells with a fluorescent marker; coculturing the mixtures of the plated cancer-targeting and cancer cells; and imaging each of the plurality of microwells for a period of time sufficient to observe at least one cell behavior.

In an embodiment, the present disclosure provides for kits for single-cell analysis with reduced noise, comprising: a microwell array containing a plurality of microwells, wherein the plurality of microwells are each generally rectangular or square with a dimension of about 50 pm - 2 mm, and with a height of about 100 - 500 pm; and wherein the single-cell analysis with reduced noise comprises: plating cancer-targeting cells, optionally T-cells, on a microwell array having a plurality of microwells to provide about one cancer-targeting cell per microwell; plating cancer cells on the microwell array to provide a mixture of the cancertargeting cell with one or more cancer cells in each microwell; optionally, staining the cancer-targeting cells and/or cancer cells with a fluorescent marker; coculturing the mixtures of plated cancer-targeting and cancer cells for a period of time of at least about 96 hours; and imaging each of the plurality of microwells to observe one or more long-term cell behaviors, wherein the long-term cell behaviors occur during a period of time of at least about 96 hours.

In an embodiment, the present disclosure provides for kits for single-cell analysis with reduced noise, comprising: a microwell array containing a plurality of microwells, wherein the plurality of microwells are each generally rectangular or square with a dimension of about 50 pm - 2 mm, and with a height of about 100 - 500 pm; and wherein the single-cell analysis with reduced noise comprises: plating cancer-targeting cells, optionally T-cells, on a microwell array having a plurality of microwells to provide about one cancer-targeting cell per microwell; cloning each cancer-targeting cell until at least about four identical clones are obtained in each microwell; plating cancer cells on the microwell array to provide a mixture of the cloned cancertargeting cells with one or more cancer cells in each microwell; optionally, staining the cancer-targeting cells and/or cancer cells with a fluorescent marker; coculturing the mixtures of the plated cancer-targeting and cancer cells; and imaging each of the plurality of microwells for a period of time sufficient to observe at least one cell behavior. BRIEF DESCRIPTION OF THE DRAWINGS

Aspects and advantages of the present disclosure will become apparent from the following exemplary embodiments taken in conjunction with the accompanying drawings, of which:

FIG. 1 depicts a progression six independent coculture of T-cells with tumor cells in six respective microwells, labeled wells 1 - 6, over time. The fluorescent microscopy images were obtained at representative time points of 0, 1, 4, 7, and 8 days.

FIG. 2 depicts generation of monoclonal T cell groups (mono-T) in a grouping or representative microwells. The two images at the left, where the bottom image is a magnified view of the top image, were collected at day 0. The two images at the right, where the bottom image is a magnified view of the top image, were collected at day 3.

DETAILED DESCRIPTION

The present disclosure provides for methods and systems for single-cell analysis. More particularly, the disclosure provides for methods and systems for analyzing cocultures of a single cancer-targeting cell (such as a T-cell) and one or more cancer cells. A single cancer-targeting cell may generally be cocultured with one or more cancer cells to evaluate the behavior of the cancer-targeting cell. Generally, the single cancer-targeting cell (i.e. T- cell) and one or more cancer cells are cocultured in microwells of a microwell array. Certain useful microwell arrays and related imaging technologies are described in, for example, PCT/US2019/046752, filed August 16, 2019 and published as WO 2020/037176 Al on February 20, 2020, and PCT/US2022/026955, filed April 29, 2022 and published as WO 2022/235507 Al on November 10, 2022, each of which are incorporated by reference herein in their entireties.

As explained in the foregoing, bulk techniques are typically preferred compared to single cell techniques due to unsolved noise issues with single cell techniques. Therefore, single-cell techniques present significant untapped potential for analyzing single cell behavior, as is disclosed in the present application and embodiments described herein.

Imaging to observe or analyze one or more cell behaviors is generally performed by microscopy which is capable of observing the single cells in microwells. In an embodiment, the microscopy includes fluorescent microscopy. In an embodiment, one or more of the cancer-targeting cells or cancer cells are labeled with a fluorophore (i.e. a fluorescent tag, molecule, or construct including but not limited to green fluorescent protein (GFP)). In an embodiment, the cancer cells express GFP. In an embodiment, antibodies binding the cancer- targeting cells may be utilized as fluorescent labels. In an embodiment, antibodies binding the cancer-targeting cells may be fluorescent-labeled CD4 and/or CD8 antibodies. Antibodies may be labeled with fluorescent tags including but not limited to phycoerythrin cyanine 5.5 (PE-Cyanine5.5) or any other fluorescent tag. Generally, the cancer-targeting cell may be labeled with a fluorophore emitting light in a different wavelength from a fluorophore associated with a cancer cell so that the two cells may be observed and/or quantified separately. Clone kinetics/statistics parameters can be calculated by microscopic image analysis or other non-imaging sensors. That is, in an embodiment, alternative non-imaging sensors may be utilized to observe and/or analyze one or more cell behaviors.

In various embodiments, cell behaviors may be any observable cell behaviors including, but not limited to, cell growth of T cells, killing of tumor cells, cell-cell interaction, trogocytosis, phagocytosis, endocytosis, apoptosis, necrosis, cell migration, cell fusion, secretion of cellular materials, division of cell chromosome, and surface expression of certain proteins/reporter genes. In an embodiment, cell behaviors include cell growth of cancer targeting cells (i.e. T cells) and killing of cancer cells (i.e. tumor cells). One or more cell behaviors may be observed simultaneously. Image data from a well may be analyzed to determine one or more cell behaviors. In an embodiment, long-term image data may be compared to an initial state or short-term image data. In an embodiment, analysis may include quantitative and/or qualitative factors.

In an embodiment, cancer-targeting cells, including monoclonal cells (i.e. T cells), may require initial seeding of single cells in an isolated compartments and cultured for an extended period, optionally with media change. The isolated compartments can be microwells, microgrids, droplets or microgels as long as the cells can be physically isolated and grow. In an embodiment, initial seeding may be utilized prior to coculturing the cancertargeting cells with one or more cancer cells. Generally, the dimension of the microcompartments should be able to accomodate at least 10 cells to allow long term cell growth and culture(>4days).

In an embodiment, cancer cells which are cocultured with the cancer-targeting cells can be immune cells, tumor cells and other cells types from animal or plant. In an embodiment, the cancer cells are associated with a particular cancer of interest. In an embodiment, the cancer cells are multiple myeloma cancer cells. Time-averaged Analysis

In an embodiment, the single cancer-targeting cell and cancer targeting cell are cocultured in a microwell array for a period of time. In an embodiment, the period of time includes a short-term period (i.e. time periods less than about 96 hours, or less than four days) and a long-term period (time periods of 96 hours or greater, or four days or greater). It has been discovered that a surprising amount of time (i.e. a long-term period of 96 hours or greater) is necessary to reliably observe, typically by imaging or more particularly by microscope imaging, cell behaviors. For example, if cell behaviors (for example killing time, the amount of time it takes for the cancer-targeting cell to kill the cancer cells) are observed during the short-term period of less than 96 hours, the results may not be reliable due to the aforementioned noise issues that single cell techniques face. However, if the cell behaviors are observed in the long-term period of 96 hours or greater, then the results are more reliable. For example, the cell behaviors may be observed at 4 days, or 5 days, or 6 days, or 7 days, or 8 days, or 9 days, or 10 days.

During the long-term period, reliable results may be obtained at discrete time points (i.e. at any time greater than or equal to 96 hours, or 4 days), or may be determined based upon a range of time points (for example, days 4 - 10, or days 5 - 10, or days 4 - 6, or days 5 - 8, etc.) Additionally, observations may be made more than once per day or at certain or random intervals throughout various days.

In some embodiments, one or more additional occurrences of plating (alternatively “seeding”) of cancer cells may be performed at any stage during the coculture. In an embodiment, one or more additional occurrences of plating are performed for the purposes of repeated antigen stimulation. In an embodiment, additional occurrences of plating are performed in the short-term period (at less than about 96 hours). In an embodiment, additional occurrences of plating are performed in the long-term period (at more than about 96 hours). In an embodiment, one, two, three, four, or five or more additional occurrences of plating or seeding are performed. In an embodiment, the one or more additional occurrences of plating are performed with the same cancer cells in the initial coculture. In an embodiment, the one or more additional occurrences of plating are performed with one or more different cancer cells compared to the initial coculture.

Group Average Analysis

In an embodiment, group averages may be used to overcome the noise issues with single-cell analysis techniques. In an embodiment, group averages are generally obtained by isolating single cells and culturing the single cells to obtain a minimum number of cells prior to coculturing the cells with one or more cancer cells. In an embodiment, a group average analysis requires a minimum of 3 cells from the initially isolated single cell. In an embodiment, a group average analysis requires a minimum of 4 cells from the initially isolated single cell.

Cell proliferation to obtain the minimum number of cells from the initially isolated cells can be achieved by inherent growth or by external simulation such as activation antibodies and cytokines / chemokines / chemicals. Group averaging can typically be performed during short-term periods or long-term periods of coculturing the cancer-targeting cells with the one or more cancer cells. In an embodiment, group averaging is performed during a short-term coculturing period (less than about 96 hours). In an embodiment, group averaging is performed during a long-term coculturing period (about 96 hours or greater). In an embodiment, group averaging is performed over both a short-term and long-term period. In an embodiment, group averaging allows for meaningful observation or determination of cell behaviors in a short-term coculturing period which would not have been possible if only a single cancer-targeting cell was cocultured with one or more cancer cells.

In some embodiments, one or more additional occurrences of plating (alternatively “seeding”) of cancer cells may be performed at any stage during the coculture. In an embodiment, one or more additional occurrences of plating are performed for the purposes of repeated antigen stimulation. In an embodiment, one, two, three, four, or five or more additional occurrences of plating or seeding are performed. In an embodiment, the one or more additional occurrences of plating are performed with the same cancer cells in the initial coculture. In an embodiment, the one or more additional occurrences of plating are performed with one or more different cancer cells compared to the initial coculture.

Examples:

Use of hydrogel based microwell system and CAR-T/Tumor cell coculture as an example. Microcompartments generation

Microwells were printed followed the current optimal microwell print for coculturing cells. The gel mixture was prepared in 500uL, and contained 20% PEG-DA, 1%LAP, ImM Tartrazine, and PBS to fill. The mixture was then added to the 20mm glass bottom portion of the 35mm dish, and placed in the LASUM (LCD aided selection under microscope) for printing. The parameters for this microwell are 1.5 seconds exposure, 2.0 step, 20mm dish diameter, 250mm wall length, and 100mm wall width. See, e.g., PCT/US2019/046752, filed August 16, 2019 and published as WO 2020/037176 Al on February 20, 2020 and PCT/US2022/026955, filed April 29, 2022 and published as WO 2022/235507 Al on November 10, 2022. After printing, 2mL of pre-warmed 37°C PBS was added to the dish and incubated at 37°C for 5 minutes to wash. This washing step was repeated three times, and then complete medium (RPMI1640, 20%HI-FBS, and 1% Penicillin/Streptomycin) was added for an overnight incubation at 37°C. In general, the microwell is an array of micowells (i.e. microwell array) having a plurality of microwells which are generally rectangular or square having a dimension of 50 pm to 2 mm. In general, the microwells may have a height from about 100 - 500 pm.

Cell Culture:

All work was done in a biosafety cabinet and sterile techniques are followed to maintain sterility throughout the experiment.

Car-T library cells (POP) were thawed the day before setting up the microwell coculture. POP containing 90% complete medium and 10% DMSO in ImL was thawed from liquid nitrogen in a 37°C water bath until ice was almost fully melted. POP was then added dropwise to 9mL of pre-warmed 37°C complete medium. After, the tube containing POP was centrifuged at 400xg for five minutes to pellet the cells. Supernatant was removed and pelleted cells were resuspended in 2mL of complete medium to count. POP is maintained at IxlO ⁶ cells/mL in complete medium with lOOU/mL of IL-2.

Multiple myeloma cell line (OPM2-GFP) was thawed at least a week in advance to increase viability and cell number before setting up the microwell coculture. The thawing process for POP above is followed for OPM2. These cells were maintained at 3xl0 ⁵ - 8xl0 ⁵ cells/mL in complete medium.

Time averaged Tumor cell killing and T cell proliferation.

Microwell Coculture Setup:

The viability of both POP and OPM2-GFP should be at least >70% before setting up the microwell coculture. Having a higher cell viability will yield more accurate results.

POP and OPM2 cells were combined at a 1 : 10 ratio in a microcentrifuge tube at a total volume of 200uL. The cell number plated follows the current most optimal for microwell coculturing, which is POP and OPM2 at 10,000 and 100,000 cells respectively. Once the cell mixture was prepared, the 35mm dish containing the 20mm microwell was removed of all medium. The cell mixture was then added dropwise throughout the 20mm microwell and incubated for 15 minutes at 37°C to allow the cells time to settle into the microwells. After this incubation, 1.5mL of complete medium was added to the dish and it is incubated at 37°C. Cell Staining in Microwell:

Any stain used for a microwell was prepared in a total of 200uL. The stain used for this coculture was luL of both CD4 and CD8 monoclonal antibody (PE-Cyanine5.5) mixed with 198uL of complete medium. The CD4 and CD8 antibodies bind to the POP cells so they fluoresce red when imaging. The 200uL prepared stain was added dropwise to the microwell and incubated at 37°C for 60 minutes. After this incubation, 2mL of complete medium was added and removed from the 35mm dish to wash the microwell.

Microwell Montage (Imaging):

The microwell was montaged daily to catalog the cocultures over an eight-day period. To prepare the microwell for a montage, the microwell was first stained as described above. Then medium was removed from the sides of the 35mm dish without removing the media covering the microwell. The LASUM was then setup for a montage and the dish was placed into the machine. The montage parameters used were 1000ms of red and green fluorescent light exposure, 20mm scan diameter, 0.02 scan offset, and red was used for autofocusing. When the montage was finished, the dish was removed from the LASUM and 1.5mL of complete medium was added. The dish was incubated until the following day for another montage. Long term time averaged coculture results were obtained and quantified to produce distinctive microwell phenotypes, where robust increase of T cells and reduction of tumors can be observed and quantified. Typical microwell images were followed daily to remove ambiguity (Fig. 1). It is evident that prior to day 4, the proliferation of T cells (red and yellow) and the growth of tumor cells ( GFP green) was still ambiguous. Starting from day 4, and further at day 7, the fate of each coculture became clear: In wells 1, 2, and 3, T cells kills almost all tumor cells and proliferated such that the visible dots at days 4 - 8 are predominantly T-cells with little to no remaining tumor (cancer) cells. In wells 4, 5, and 6, tumor cells took over and dominated, indicating a poor killing capacity of the enclosed T cells, such that the visible dots at days 4 - 8 for these wells are predominantly tumor (cancer) cells with little to no remaining T-cells.

This surprising result indicates that relatively long coculture time is required to suppress noise in single-cell analysis. Unlike existing methods, meaningful single-cell analysis results can be obtained which overcome noise limitations. For example, where existing methods may rely upon coculture times of less than 96 hours (often significantly less than 96 hours such as only 24 - 48 hours) reliable results can be obtained by monitoring cocultures over a period of at least about 96 hours or greater, such as 4, 5, 6, 7, 8, 9, or 10 days or more. In some cases, the period of time to observe long-term cell behavior may be slightly shorter than 96 hours and it should be appreciated that a period of “about 96 hours” is intended to have some flexibility due to the daily sampling frequency. For example “about 96 hours” may be inclusive of periods close to 24 hours shorter than 96 hours, eg. about 75 hours, about 80 hours, about 85 hours, about 90 hours, etc. The practitioner running the experiment may, in relying upon their judgement, determine a reasonable sampling frequency to maintain long enough coculturing periods to observe the desired long-term cell behavior.

Monoclonal T cell (monoT) group generation in microwell.

POP and 0PM2 cells were combined at a 1 : 10 ratio in a microcentrifuge tube at a total volume of 200uL. The cell number plated followed the current most optimal for microwell coculturing, which is POP and 0PM2 at 10,000 and 100,000 cells respectively. Once the cell mixture was prepared, the 35mm dish containing the 20mm microwell was removed of all medium. The cell mixture was then added dropwise throughout the 20mm microwell and incubated for 15 minutes at 37°C to allow the cells time to settle into the microwells. After this incubation, 1.5mL of complete medium was added to the dish and it is incubated at 37°C. 37.5uL human CD3/CD28 T-cell activator (recommended 25uL/mL from protocol) and lOOU/mL IL-2 are added to the culture, the coculture will be Incubated at 37°C for 3 days (recommended maximum incubation time for CD3/CD28 from protocol). Microwell was montaged on Day 0, Day 2, and Day 3. On Day 3, activation medium was removed and 100K OPM2 was added. Dish was then montaged daily, and incubated in complete medium (RPMI1640, 20% FBS, 1% Pen/Strep) at 37°C. monoclonal clusters of T cells were established from single T cell clones. (Fig.2), the killing and proliferation of the monoclonal T cell groups are more robust by averaging out single cell noises.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents, including certificates of correction, patent application documents, scientific articles, governmental reports, websites, and other references referred to herein is incorporated by reference herein in its entirety for all purposes. In case of a conflict in terminology, the present specification controls. EQUIVALENTS

The invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are to be considered in all respects illustrative rather than limiting on the invention described herein. In the various embodiments of the compositions and methods of the present invention, where the term comprises is used with respect to the compositions or recited steps of the methods, it is also contemplated that the compositions and methods consist essentially of, or consist of, the recited compositions or steps or components. Furthermore, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions can be conducted simultaneously. In the specification, the singular forms also include the plural forms, unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control. Furthermore, it should be recognized that in certain instances a composition can be described as being composed of the components prior to mixing, or prior to a further processing step such as drying, binder removal, heating, sintering, etc. It is recognized that certain components can further react or be transformed into new materials.

All percentages and ratios used herein are on a volume (volume/volume) or weight (weight/weight) basis as shown, or otherwise indicated.