Multiplex cell assays for screening and quality assessment of engineered cells

Abstract

The present invention relates to methods and systems for performing one or more highly multiplex cellular assays. In some embodiments, one or more channels of a fluidic device are provided with a photopolymerizable polymer precursor and cells randomly arranged on a surface, followed by measuring the location of the cells by a detector, synthesizing hydrogel chambers by photopolymerization to encapsulate individual cells, and loading assay reagents to the channels. An assay signal indicative of a desired cell characteristic may be generated for each of the encapsulated cells.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 322,601, filed on March 22, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by reference

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. Background Art

[0005] Therapies based on engineered cells offer promising new approaches for treating complex diseases, as cells have the ability to sense and integrate a wide range of signals, actively move to specific tissue compartments, and initiate proximity-dependent responses, e.g., Fischbach et al., Science Transl. Med., 5:1797 (2013). Such cell-based approaches offer novel therapeutic devices that address the obstacles currently faced by small molecules and biologics, such as poor target specificity, suboptimal tissue compartment localization, lack of personalization, and limited potential for the effects of drugs to be modified spatially and temporally or in response to changing clinical manifestations once administered to patients. These issues can reduce the pharmacological utility of such compounds. Cytotoxic lymphocytes (CLs), such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, are excellent platforms for building cell-based therapeutic systems for several reasons: (i) CLs have a unique cell-to-target cell molecular transfer system in the granzyme-perforin pathway; (ii) the T cell receptor (TCR) or related chimeric antigen receptor (CAR) endows CLs with a high degree of specificity in targeting cell populations presenting major histocompatibility complex (MHC)-bound cognate antigens, or in the case of CARs, arbitrary surface antigens; (iii) activated CLs differentially express cytokines and tissue-specific receptors, enabling lymphocytes to selectively home to target tissues throughout the body; and (iv) laboratory and clinical protocols for lymphocyte modification and therapeutic administration have been developed in the field of adoptive cell therapy, e.g., Restifo et al., Nature Reviews Immunology, 12:269-281 (2012). Given these advantages and positive clinical results, a number of cell-based therapies have been approved for the treatment of a range of cancers and other conditions.

[0006] However, with the success of cell-based therapies, there are also major challenges in development, manufacturing, and quality assurance due to the complexity of using living organisms as drugs. Tests must be performed to select appropriate cell subpopulations for engineering and to ensure that there are no harmful changes in the identity, purity, or operability of the engineered subpopulations at each step of the manufacturing process, for example, Tanna et al., Cytotherapy, 21: 278-288 (2019); Wang et al., Molecular Therapy, 3: 16015 (2016); Levine et al., Molecular Therapy: Methods & Clinical Development, 4: 92-101 (2017). Therefore, the availability of cell analysis platforms (including methods and systems) will promote the field of cell-based therapies, which are used to perform a wide range of highly multiplexed cell assays related to the development and manufacture of cell-based therapies. Summary of the invention

[0007] The present invention relates to methods and systems for performing large-scale multiplexed single-cell assays, particularly for characterizing population engineered cells developed for medical or industrial applications such as cell-based therapies. Exemplary single-cell assays include, but are not limited to, cytotoxicity, proliferation capacity, activation state, vector copy number of transformed cells, and insertion site analysis, among others.

[0008] In one aspect, the present invention provides a method for determining one or more cell characteristics, the method comprising: (a) synthesizing one or more hydrogel chambers, wherein the hydrogel chambers in the one or more hydrogel chambers encapsulate cells arranged on the surface of a channel; (b) lysing the cells so that the genomic DNA of the cells is released into the hydrogel chambers of the cells; (c) amplifying the genomic DNA of the cells to obtain amplified genomic DNA; and (d) using the amplified genomic DNA, measuring the viral copy number, viral integration site or genomic copy number variation of the cells.

[0009] In some cases, the measurement further comprises: (i) annealing a vector-specific primer to the amplified genomic DNA; (ii) extending the vector-specific primer to obtain an extension product comprising a copy of a segment of the genomic DNA; and (iii) identifying one or more integration sites of the vector-specific primer and the genomic DNA from the segment. In some cases, the cell is a mammalian cell, and the measurement further comprises: (i) sequencing one or more fragments of the amplified genomic DNA to obtain the sequence of the one or more fragments; and (ii) determining the genomic copy number variation of the cell from the sequence of the one or more fragments. In some cases, sequencing includes obtaining a sequence coverage of the amplified genomic DNA of 0.25X or greater. In some cases, the determination of the genomic copy number variation has a resolution of 3 megabases or higher.

[0010] On the other hand, the present invention provides a method for determining one or more cellular characteristics of one or more cells, the method comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel, the channel comprising a first surface, one or more cells and one or more polymer precursors, wherein the one or more cells are arranged on or near the first surface; (ii) a spatial energy modulation element, the spatial energy modulation element being optically connected to the first surface; and (iii) a detector; (b) identifying the position of one or more cells in the channel with the detector; (c) using the spatial energy modulation element, projecting energy into the channel so that the projected energy causes the one or more polymer precursors to form a polymer matrix wall of one or more chambers, wherein the one or more chambers at least partially encapsulate the one or more cells at the position identified by the detector; (d) loading one or more assay reagents into the channel; and (e) incubating the one or more cells under assay conditions to generate signals from the one or more chambers indicating one or more cellular characteristics of the cells encapsulated by the one or more chambers.

[0011] In some cases, (i) one or more cell characteristics include proliferation rate, (ii) incubation under assay conditions includes incubation under growth conditions, and (iii) the method further includes: after incubation, determining the proliferation rate of one or more cells at least partially encapsulated by one or more chambers. In some cases, the determination of the proliferation rate includes counting one or more cells at least partially encapsulated by one or more chambers. In some cases, one or more cell characteristics further include a spectrum of cell membrane proteins, and the method further includes incubating one or more cells with antibodies, each of which is specific to different cell surface proteins, the relative expression of the different cell surface proteins allows identification of one or more cells, and each of such antibodies has a different label. In some cases, the first surface includes one or more capture elements, which are used to capture one or more assay components or one or more components of one or more cells. In some cases, each of the antibodies has an oligonucleotide tag, which includes an antibody-specific barcode that can be captured by one or more capture elements.

[0012] In some cases, the method further includes: (i) providing an oligonucleotide tag, wherein the oligonucleotide tag is attached to the antibody via a cleavable linkage; (ii) loading a release reagent into the channel to cleave the cleavable linkage so that the oligonucleotide tag is released and captured by the capture element; (iii) replicating the captured oligonucleotide tag to produce complementary DNA of the captured oligonucleotide tag; and (iv) sequencing the complementary DNA to identify the captured oligonucleotide tag.

[0013] In some cases, the first surface includes one or more capture elements for capturing one or more assay components or components of one or more cells, wherein the one or more cell characteristics further include a cellular transcriptome, and the method further includes: (i) loading a lysis reagent into the channel so that messenger RNA of the one or more cells is released and captured by the one or more capture elements; (ii) loading a reverse transcription reagent into the channel to replicate the captured messenger RNA to produce complementary DNA; and (iii) sequencing the complementary DNA.

[0014] In some cases, (i) the one or more cellular characteristics further include a profile of proteins secreted by the one or more cells, (ii) the channel further includes a protein capture surface comprising a protein affinity reagent that binds to proteins secreted by the one or more cells, and (iii) the method further includes using an amount of a labeled protein detection antibody to detect proteins secreted by the one or more cells that are bound to the protein capture surface adjacent to the one or more cells.

[0015] In some cases, (i) one or more cell characteristics include cytotoxicity, (ii) the first surface includes target cells arranged on the first surface, (iii) the method further includes loading the channel with effector cells so that the effector cells are arranged on or near the first surface, (iv) the step of incubating includes incubating the effector cells and the target cells with a vital stain that generates an optical signal in dead cells but not in live cells; and (v) the dead cells in each of the one or more chambers are counted to determine the cytotoxicity value of the effector cells encapsulated by each of the one or more chambers. In some cases, the first surface includes one or more capture elements, which are used to capture one or more assay components or one or more components of one or more cells. In some cases, one or more cell characteristics further include a spectrum of cell membrane proteins, and the method further includes incubating one or more cells with antibodies, each of which is specific to a different cell surface protein, the relative expression of the different cell surface proteins allows identification of cells, and each of such antibodies has a different label. In some cases, each of the antibodies has an oligonucleotide label, which includes an antibody-specific barcode that can be captured by one or more capture elements. In some cases, the method further includes: (i) providing an oligonucleotide tag, wherein the oligonucleotide tag is attached to the antibody via a cleavable linkage; (ii) loading a release reagent into the channel to cleave the cleavable linkage so that the oligonucleotide tag is released and captured by the capture element; (iii) replicating the captured oligonucleotide tag to produce complementary DNA of the captured oligonucleotide tag; and (iv) sequencing the complementary DNA to identify the captured oligonucleotide tag.

[0016] In some cases, the first surface includes one or more capture elements for capturing one or more assay components or components of one or more cells, wherein the one or more cell characteristics further include a cell transcriptome, and the method further includes: (i) loading a lysis reagent into the channel so that messenger RNA of the one or more cells is released and captured by the one or more capture elements; (ii) loading a reverse transcription reagent into the channel to replicate the captured messenger RNA to produce complementary DNA; and (iii) sequencing the complementary DNA. In some cases, (i) the one or more cell characteristics further include a spectrum of proteins secreted by the one or more cells, (ii) the channel further includes a protein capture surface, the protein capture surface includes a protein affinity reagent that binds to proteins secreted by the one or more cells, and (iii) the method further includes using a certain amount of labeled protein detection antibodies to detect proteins secreted by the one or more cells that bind to the protein capture surface adjacent to the one or more cells.

[0017] In some cases, one or more cell characteristics include the number of copies of one or more nucleotide sequences in the genomic DNA of one or more cells, and the method further includes: (a) lysing one or more cells to release genomic DNA; (b) amplifying one or more nucleotide sequences to obtain one or more amplified nucleotide sequences; (c) capturing the amplified one or more nucleotide sequences with one or more capture elements arranged on the channel; (d) replicating the captured one or more amplified nucleotide sequences to produce complementary DNA of the captured one or more amplified nucleotide sequences; and (e) sequencing the complementary DNA to identify the number of copies of the one or more nucleotide sequences. In some cases, each of the one or more nucleotide sequences is a barcode. In some cases, each barcode includes a unique molecular identifier. In some cases, the unique molecular identifier identifies viral DNA integrated into the genomic DNA, and many different unique molecular identifiers identified in a single cell indicate the number of viral copies of such a single cell.

[0018] In some cases, one or more cell characteristics include the copy number of one or more nucleotide sequences in the genomic DNA of one or more cells, and the method further includes: (a) lysing one or more cells to release genomic DNA; (b) loading an amplification reagent to the channel, the amplification reagent generates a signal proportional to the copy number of one or more nucleotide sequences; and (c) amplifying one or more nucleotide sequences to generate a light signal proportional to the copy number of one or more nucleic acid sequences. In some cases, the amplification reagent is a quantitative PCR reagent, and the signal is a light signal. In some cases, (i) the first surface includes a bridge PCR primer, (ii) the amplification reagent includes a bridge PCR reagent, and (iii) the signal is a number of clusters formed by bridge PCR. In some cases, the amplification reagent includes a rolling circle amplification reagent, and the signal is a number of DNA nanoballs.

[0019] In some cases, one or more cells are randomly arranged on the first surface. In some cases, each of the one or more chambers encapsulates a single cell in the one or more cells. In some cases, the one or more cells include cells transduced by a vector, and the one or more cell characteristics include vector integration sites of cells transduced by the vector, and the method further includes: (a) lysing the cells transduced by the vector to release the genomic DNA of each cell into the corresponding chamber of each cell; (b) amplifying the released genomic DNA; (c) annealing the vector-specific primer to the amplified genomic DNA; (d) extending the vector-specific primer to obtain an extension product including a copy of a segment of the genomic DNA; and (e) identifying one or more integration sites of the vector-specific primer and the genomic DNA from the segment.

[0020] In some cases, one or more cells are mammalian cells, and one or more cell characteristics include genomic copy number variation, and the method further includes: (a) lysing one or more cells to release genomic DNA of each cell into a corresponding chamber of each cell; (b) amplifying the released genomic DNA; (c) sequencing the amplified genomic DNA fragments to obtain the sequence of the genomic DNA fragments; and (d) determining the genomic copy number variation of each cell from the sequence of the genomic DNA fragments. In some cases, sequencing includes obtaining a sequence coverage of 0.25X or greater of the genomic DNA fragments, and the determination of the genomic copy number variation has a resolution of 3 megabases or higher.

[0021] On the other hand, the present invention provides a system for measuring single-cell properties of a cell population, the system comprising: (a) one or more channels, each of the one or more channels comprising a surface, a plurality of cells arranged on the surface, and one or more polymer precursors; (b) at least one spatial energy modulation element, the at least one spatial energy modulation element being optically connected to the surface of each channel; (c) at least one detector, the at least one detector being optically connected to the surface of each channel and being operably associated with the at least one spatial energy modulation element, wherein the detector is configured to detect each of the plurality of cells and determine the position of each of the plurality of cells on the surface of at least one channel; and (d) a plurality of gel chambers in each channel, wherein each gel chamber encapsulates one or more cells of the plurality of cells, wherein the gel chamber is synthesized by projecting light into the channel using at least one spatial energy modulation element so that the projected light causes one or more polymer precursors to form a polymer matrix wall of the gel chamber, and wherein the position of the synthesized chamber is determined in part by the position of the cell encapsulated by the synthesized chamber as identified by the detector.

[0022] In some cases, the gel chambers are randomly arranged on at least one of the surfaces. In some cases, each of the gel chambers encapsulates a single cell. In some cases, the polymer matrix wall has a molecular weight of less than 3×10 6 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 6 Dalton molecules are impermeable. In some cases, the polymer matrix wall is impermeable to molecules with a molecular weight of less than 3×10 5 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 5 Dalton molecules are impermeable. In some cases, the polymer matrix wall is impermeable to molecules with a molecular weight of less than 3×10 4 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 4Dalton molecules are impermeable. In some cases, the polymer matrix wall is impermeable to molecules with a molecular weight of less than 3×10 3 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 3 Dalton's molecules are impermeable.

[0023] In some cases, the gel chamber is a degradable hydrogel chamber. In some cases, the gel chamber is hollow and encapsulates an area of ​​the surface. In some cases, the surface includes a capture element configured to capture nucleic acids.

[0024] On the other hand, a method for determining one or more cell characteristics of one or more cells is provided herein, the method comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel, the channel comprising a first surface, one or more cells and one or more polymer precursors, wherein the one or more cells are arranged on or near the first surface; (ii) a spatial energy modulation element, the spatial energy modulation element being optically connected to the first surface; and (iii) a detector; (b) identifying the position of one or more cells in the channel with the detector; (c) using the spatial energy modulation element, projecting energy into the channel so that the projected energy causes the one or more polymer precursors to form a polymer matrix wall of one or more chambers, wherein the one or more chambers at least partially encapsulate the one or more cells at a position identified by the detector; and (d) performing one or more assays on one or more cells in the channel to determine one or more cell characteristics, wherein the one or more cell characteristics are selected from cytotoxicity, viability, proliferation rate, phenotype, vector copy number, vector integration site, transcriptome, and genomic copy number variation. In some cases, the fluid device further comprises a plurality of channels, and performing one or more assays comprises performing a plurality of assays, wherein each different assay in the one or more assays is performed in a different channel in the plurality of channels. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A Diagrammatically illustrates the processing steps in an exemplary cell-based therapy, such as autologous CAR-T cell therapy.

[0026] Figure 1B Contains a table listing combinations of assays that can be performed according to the present invention.

[0027] Figure 2A-2F Shown are steps for an exemplary assay for determining the proliferative capacity of cells in a population after identification of the cells by cell surface protein expression and protein secretion.

[0028] Figure 3A-3B The steps of an assay for determining the cytotoxicity of a population of cells are shown. Figure 2B- A subsequent assay to identify cells with desired cytotoxicity values ​​is performed as shown in Figure 2G.

[0029] Figure 4A-4B An assay for determining the number of integrated vector copies in transduced cells is shown.

[0030] Figure 4C-4F An assay for determining the copy number and insertion site of integrated vectors in transduced cells is shown.

[0031] Figure 4G-4H An assay for determining single cell genome copy number variation is shown.

[0032] Figure 5A-Figure 5B An embodiment of a system for carrying out the method of the present invention is shown diagrammatically.

[0033] Figure 6A-6B One embodiment of a flow cell comprising a plurality of channels is shown.

[0034] Figure 7A-7B Exemplary steps are shown for preparing cDNA from a single-cell target template (such as mRNA), and its sequencing either at the site of the hydrogel chamber ("in situ" or "internal" sequencing) or after elution from the channel in an external sequencing instrument. DETAILED DESCRIPTION

[0035] Unless otherwise stated, the practice of the present invention may adopt conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant technology), cell biology and biochemistry within the technical scope of the art. Such conventional techniques include, but are not limited to, preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, etc. Suitable techniques may be specifically described by reference to the examples below this article. However, other equivalent routine procedures may also be used, of course. Such conventional techniques and descriptions may be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Renault and Duchateau, ed., Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer, ed., Protein Engineering Handbook (Wiley-VCH, 2009); and the like. Guidance for selecting materials and components to perform particular functions can be found in available treatises and references on scientific instrumentation, including but not limited to: Moore et al., Building Scientific Apparatus, 3rd ed. (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd ed. (Academic Press, 2013); and similar references.

[0036] The present invention relates to methods and systems for performing one or more large-scale multiple determinations (particularly for the purpose of developing cell-based therapies and ensuring their quality) for simultaneously evaluating multiple characteristics of cells. In some embodiments, the present invention includes methods and systems for simultaneously performing multiple determinations to determine multiple cell characteristics important to cell-based therapies, including but not limited to cell-based therapies for, for example, using CAR-T cells to treat cancer or for, for example, using induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, etc. to treat other conditions (such as spinal cord injury, stroke, diabetes) based on cell therapy. In some embodiments, the present invention relates to a system for simultaneously performing multiple single-cell determinations, which is designed to detect or measure selected or predetermined cell characteristics of a cell population. In some embodiments, one or more such cell determinations rely on or utilize a hydrogel chamber (sometimes referred to herein as an "analysis chamber", or simply referred to as a "chamber" or "hydrogel cage"). In some embodiments, such cell determinations are single-cell determinations, so that one or more hydrogel chambers used in such determinations each encapsulate only single cells. In some embodiments, such single-cell determinations may include a single engineered cell and one or more target cells, i.e., one or more cells that can be acted on (e.g., killed) by engineered cells.

[0037] "Cells" that can be determined by the methods and systems of the present invention include any biological cells, including but not limited to vertebrate cells, invertebrate cells, eukaryotic cells, mammalian cells, microbial cells, protozoan cells, prokaryotic cells, bacterial cells, insect cells or fungal cells. In some embodiments, mammalian cells are determined by the methods and systems of the present invention. In particular, any mammalian cell population that can or has been induced, processed, modified or genetically altered (i.e., genetically engineered) to be used in medicine, industry, environment or treatment processes can be analyzed by the methods and systems of the present invention. In some embodiments, "cells" as used herein include genetically modified mammalian cells. In some embodiments, "cells" include stem cells that have been induced to differentiate. In some embodiments, "cells" refer to cells modified by CRISPR Cas9 technology. In some embodiments, "cells" refer to cells of the immune system, including but not limited to cytotoxic T lymphocytes, regulatory T cells, CD4+T cells, CD8+T cells, natural killer cells, antigen presenting cells or dendritic cells. Of particular interest are cytotoxic T lymphocytes engineered for therapeutic applications (such as cancer therapy).

[0038] As used herein, the term "determination" refers to a process for detecting or measuring a cell property or property of a single cell or a cell group. Typically, the process steps of the determination include chemical, biochemical or molecular reactions (such as the cutting of a bond, specific binding of complementary components, enzyme reactions, dissolution of complementary components, etc.) or changes in physical state (such as the increase or decrease of temperature, changes in energy levels, etc.), and result in the generation of one (or more) signals, from which the presence, absence or size of a cell-related amount can be inferred. The properties of the signal generated by the determination can vary greatly, and may include, but are not limited to, electrical signals, optical signals, chemical signals or material signals. Material signals include the generation of a substance, which includes information that can be extracted. For example, a material signal can be an amplification of a polynucleotide, the length, quantity, composition or nucleotide sequence of which indicates a cell property. For example, a barcode oligonucleotide can be a material signal. The characteristics or properties of cells detected or measured can vary widely and include, but are not limited to, cytotoxicity, viability, ability to proliferate under selected conditions, size, shape, motility, type and profile of cell surface or membrane proteins, type and profile of secreted proteins, metabolite production, transcriptome, gene copy number, gene or allele identity, chromatin accessibility profile, vector copy number of engineered or infected cells, etc. Assays of particular interest for cell-based therapies include, but are not limited to, measurement of the characteristics listed in Table 1.

[0039] Table 1

[0040]

[0041] As mentioned above, CAR-T cell therapy (e.g., for the treatment of cancer) is an exemplary application of the present invention. The main steps in the production of autologous CAR-T cells for therapy are Figure 1A, and described in many publications, such as Wang et al., Molecular Therapeutics—Oncolytics, 3: 16015 (2016); Barrett et al., Annu. Rev. Med., 65: 333-347 (2014). The figure shows where quality assurance testing may occur during production. Leukocytes containing lymphocytes are separated from other blood components of the patient (100) by apheresis (102), for example, Allen et al., Transfusion, 57 (5): 1133-1141 (2017). Further enrichment (104) of specific lymphocyte subsets can be performed by using magnetic beads or other techniques to deplete unwanted subsets or enrich the required subsets, for example, to remove T regulatory cells that can inhibit the activity required for CAR-T cells. After selecting the appropriate subset, the T cells are activated and transduced (106); that is, they have the ability to perform immune functions and express CAR. Activation can be achieved by a variety of treatments (e.g., exposure to anti-CD3 / anti-CD28 beads). Transduction can also be achieved by a variety of techniques (e.g., lentiviral transduction and integration into the T cell genome), for example, Wang et al. (cited above); Barrett et al. (cited above). After such transformation, the T cells (108) are tested, for example, using the present invention, to determine whether there are desired properties for CAR-T cell therapy, such as sufficient degree of cytotoxicity to the desired target cells, viability, proliferation capacity, identity, absence of contaminants, viral copy number values ​​that are not too large, etc. If the activated and transduced cells do not have acceptable therapeutic properties (109), new cells must be prepared. If they have acceptable therapeutic properties, the cells are expanded (110); that is, they are cultured to increase their number. Since the conditions of expansion can change the cells, their desired properties are tested again after expansion (112). Successfully expanded cells can be formulated (114) for infusion into the patient (100) and / or stored (116) for subsequent infusion. If subsequent infusions are required, cells removed from storage are tested again to ensure that they possess the desired therapeutic properties. Figure 1B Exemplary single cell assays that can be performed using the methods and systems of the present invention are listed. In some embodiments, consecutive assays can be performed on the same cell or cells in a chamber of a channel, with each assay acquiring additional information.

[0042] The development and manufacture of CAR-T cells or therapeutic stem cells are exemplary applications of the present invention. In some embodiments, the method of the present invention for determining one or more cell characteristics (such as cell characteristics related to the development or production of CAR-T cells or therapeutic stem cells) can be performed by the following steps: (a) providing a fluid device, the fluid device comprising: (i) one or more channels, each of the one or more channels comprising a first surface and an optional assay component for detecting or measuring cell characteristics; (ii) a spatial energy modulation element, the spatial energy modulation element being optically connected to each first surface; and (iii) a detector, the detector identifying the position of cells in each channel based on one or more light signals from the cells; (b) arranging cells on the first surface of the channel; (c) loading one or more polymer precursors into each channel; (d) synthesizing one or more chambers in the channel by projecting light into each channel with a spatial energy modulation element so that the projected light causes one or more polymer precursors to cross-link to form a polymer matrix wall of the chamber, each chamber encapsulating a cell, wherein the position of the synthesized chamber in each channel is determined by the position of the cell encapsulated or wrapped by the synthesized chamber identified by the detector; and (e) incubating cells in each channel under assay conditions to generate a signal indicative of cell characteristics from the chamber of each channel. In some embodiments, incubating cells under assay conditions includes performing one or more assays on cells in the channel to determine one or more of the following cell characteristics: cytotoxicity, viability, proliferation rate, phenotype, vector copy number, vector integration site, transcriptome, and genomic copy number variation. In some embodiments, the polymer matrix wall of the chamber includes a degradable hydrogel. In some embodiments, incubation includes lysing cells and capturing one or more cell components on the first surface by a capture agent. In some embodiments, incubation further includes degrading the degradable polymer matrix wall after capturing such one or more cell components. As used herein, the terms "capture element" and "capture agent" are used interchangeably. In some embodiments, a capture agent (or capture element) is covalently attached to the surface of the channel. In some embodiments, a capture agent (or element) includes an oligonucleotide having a sequence that can directly hybridize with a cell component (e.g., a polyA tail of an mRNA) or having a sequence that can indirectly hybridize with a cell component (e.g., an adapter chain of a genomic DNA fragment produced by labeling). In some embodiments, a capture agent (or element) includes one or more antibodies. In some embodiments, a capture agent (or element) includes a spatial barcode. In some embodiments, the capture agent (or element) comprising an oligonucleotide further comprises a primer for solid phase amplification, such as bridge PCR. In some embodiments, the fluidic device comprises a plurality of channels. In some embodiments, a plurality of cells are arranged on the surface of each channel.In some embodiments, such plurality of cells comprises 100 or more cells, or 1000 or more cells, or 10,000 or more cells, or between 100 and 10. 6 Many cells within the range of 1 cell.

[0043] In some embodiments, the step of arranging cells and loading polymer precursors is performed by mixing cells and polymer precursors outside the fluid device, and then loading the mixture of cells and polymer precursors to the channel. In some embodiments, cells are delivered to the first surface in the fluid, and they are randomly dispersed on the first surface in the fluid. In some embodiments, after the cells are delivered to the surface of the channel, they are randomly arranged on the surface. In some embodiments, the step of incubation under the determination conditions can only include a single step of the determination comprising more than one step, for example, such as the step of detecting a signal (or in the case of a material signal, generating sequencing-ready nucleic acid), or such steps of incubation under the determination conditions can include multiple steps of multi-step determination. In some embodiments, before being loaded into the channel of the fluid device, cells can be processed or cells can be subjected to a determination step, so that the step of incubation can only include a single step of multi-step determination, such as signal generation and / or signal collection. In other embodiments, the step of incubation can include a part of implementing a determination step or a determination, such as cDNA synthesis, second chain synthesis, the capture of a determination component or a cell component, etc. In some embodiments, the assay conditions may include a series of steps, each with different conditions (e.g., temperature, pH, presence or absence of a particular reagent (such as a primer or an enzyme, e.g., a ligase, a polymerase, a transposase, etc.)). For example, such steps may include loading primers so that they diffuse across the chamber wall and anneal to target sites on a template strand in a sample in the chamber, extending the annealed primers, heating the channel so that the extended primers melt from their template strands, capturing the melted extended primers by a capture agent attached to the first surface, and the like.

[0044] In some embodiments, cells are adherent relative to the first surface, so that even in the presence of fluid movement, cells will remain on the surface after sedimentation and contact. In other embodiments, cells are non-adhesive relative to the first surface, so that although they can be deposited on the first surface, they can move or return to a suspended state in the presence of fluid movement. It will be appreciated by those of ordinary skill in the art that the first surface can be processed (for example, by attaching appropriate capture elements) to retain non-adherent cells on the entire first surface (or polymer matrix wall or second surface) or a selected portion thereof. In some embodiments, the assay components that can be provided together with the channel or as a part of the channel include but are not limited to capture elements, such as capture oligonucleotides, primers for surface amplification, antibodies, functional groups that can react with hydrogel components, beads, etc. In some embodiments, such assay components can be attached separately to any one of the first surface, the second surface, or the polymer matrix wall, or attached separately or in combination with other reagents to a combination of such surfaces. In some embodiments, the assay components that can be provided after the synthesis of the gel chamber include but are not limited to cleavage reagents, transcription reagents, reverse transcription reagents, antibodies, polymerases, primers, beads, etc. As used herein, "channel" means a container capable of containing a fluid (which may be static or flowing) and having at least one surface on which a cell assay can be performed. In some embodiments, a channel may have a first surface and / or a second surface on which a chamber may be synthesized and / or on which cells or assay components may be attached. In addition, in some embodiments, cells or assay components may be attached or captured by capture elements on a polymer matrix wall. As used herein, reference to a "first surface" (e.g., as a surface comprising a capture element) may include a second surface, or, where appropriate, a polymer matrix wall. As used herein, reference to a "surface" without reference to a "first" or "second" is intended to include a first surface or a second surface. In some embodiments, a channel may limit the flow of a fluid through a channel from an inlet to an outlet. In other embodiments, a channel may include a non-flowing volume of fluid that may be removed, replaced, or added through an opening or an inlet; that is, in some embodiments, the channel of the present invention may be a groove or a groove-like structure, such as a groove. In some embodiments, a hydrogel chamber is synthesized in the method of the present invention; and in other embodiments, a degradable hydrogel chamber is synthesized in the method of the present invention. In some embodiments, particularly for CAR-T cells, the multiple cell characteristics can be selected from the following characteristics: cytotoxicity, proliferation capacity or proliferation rate, activation state, cell identity, purity, gene expression profile or transcriptome, epigenetic profile, sequence copy number (e.g., the number of integrated viral copies of transduced cells, the number of plasmid copies of transiently transfected cells, the number of gene copies, etc.).Additional assays may include culture contamination assays, including but not limited to virus, bacteria, yeast, mold or mycoplasma assays, endotoxin assays, and cell morphology assays.

[0045] Another exemplary application of the present invention includes screening for modified cell populations based on CRISPR / Cas9. For example, Shifrut et al. (Cell, 175 (7): 1958-1971 (2018)) screened T cell populations transduced by lentivirus, which produced a single guide RNA targeting a library of known T cell genes. The purpose is to identify genes that enhance T cell responses when knocked out, which is measured by growth rate. The present invention is very suitable for providing large-scale single cell proliferation rates required for such studies.

[0046] Figure 5A An exemplary system for performing the above method is shown in FIG. A flow cell (500) is a component of a fluidic device that provides channels for performing various assays and liquid handling components for delivering samples and reagents to the channels under programmable control. In this illustration, four channels (502, 504, 506, and 508) are shown, with an enlarged view (512) of a segment (510) of channel 2 (504) shown below. Figure 5A In the abstract view of the flow cell (500) of FIG. 5 , the inlet, outlet, and other features of the channel are not shown. On the first surface (514) of channel 2 (504), a plurality of cells (e.g., (518)) are each encapsulated by a hydrogel chamber (e.g., (516)). In some embodiments, the porosity of the polymer matrix wall of the hydrogel chamber is selected to be impermeable to the cells but permeable to the assay reagents. Thus, reagents can be introduced into and removed from the interior of the hydrogel chamber by flowing the reagents through the channel (520), but the cells remain therein. In some embodiments, the enlarged view (512) below of the channel segment (510) shows an exemplary optical system (521) for photosynthesizing the hydrogel chamber at the location of the cells in the channel and for collecting optical signals including images. One of ordinary skill in the art will recognize that a device having the same optical properties as those of the embodiment of the present invention can be used. Figure 5A and Figure 5B Optical systems of different configurations can perform these functions. In some embodiments, multiple DMD-objective subsystems for synthesizing hydrogel structures can be used to increase the synthesis speed by synthesizing multiple structures simultaneously.

[0047] return Figure 5AFor making the hydrogel chamber perform photosynthesis, the light source (522) generates a beam (523) of light of appropriate wavelength (e.g., UV light), which passes through an appropriate photomask or beam shaping or beam pointing (Galvo) system to shape the beam so as to synthesize the desired one or more structures in the channel. In some embodiments, a digital micromirror device (DMD) (524) is used, and in other embodiments, a physical photomask can be used. The chamber position, shape, and polymer matrix wall thickness are determined at least in part based on the cell position information determined by the image collected by the detector (532). The reflected light from the DMD (524) is shaped using conventional optical devices (e.g., collimating optical devices (528)) and is directed through an objective lens system (534) into the channel 2 section (510). The objective lens (534) and the flow cell (500) move relative to each other in the xy direction (536) to make the chamber perform photosynthesis at any position in any one of the channels. In some embodiments, the flow cell (500) moves while the optical system (521) is stationary. In some embodiments, the objective lens (534) can also direct the light beam (527) from the light source (529) to the target (such as cells) on the first surface (514) and collect the light signal (such as fluorescent signal) from the measurement performed on the first surface (514). Alternatively, the optical system (521) can be used as shown in FIG. Figure 5B The light signal is collected by the independent objective lens shown. Figure 5B The information collected by the counterparts in the embodiments, in particular the cell positions in their corresponding channels, is used by a computer (538) and / or an auxiliary controller to guide the DMD (524) and a translation device that controls the relative positions of the objective lens (534) and the flow cell (500) to synthesize hydrogel chambers of appropriate shape and size at appropriate locations.

[0048] Figure 5B An alternative optical system is shown in which the detection portion (550) of the optical system moves (572) independently of the movement (568) of the combining portion (552) of the optical system. The detection portion (550) of the optical system includes a detector (556), an objective lens (558), a light source (560), and interconnected optical elements, such as a dichroic mirror (562). Figure 5A As in the embodiment of the present invention, the detector (556) is operatively associated with the computer (564) and the synthesis portion (552) of the optical system to provide cell position information to the synthesis portion (522). The computers (564) and (538) are also operatively associated with a stage and / or motor that controls the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the synthesis portion (552) of the optical system is located on the opposite side of the first surface (564) from the detection portion (550). Figure 5ALike the embodiment of the present invention, it includes conventional components: objective lens (574), reflector (576), collimating optics (580), DMD (582) and light source (578).

[0049] In some embodiments, a cell (e.g. Figure 5A In some embodiments, the first surface (514) may include regularly spaced locations or features for capturing cells, such that the cells are substantially arranged only on such locations or features on the first surface. For example, in some embodiments, such locations or features may be a linear or hexagonal array of points. In some embodiments, such locations or features may include capture elements (such as antibodies specific for cell surface proteins) for capturing or fixing cells at the locations or features. In some embodiments, the first surface (514) may include capture elements designed to fix specific cell types. For example, lymphocytes are non-adherent on most surfaces; however, such cells can be settled by gravity on a first surface, or can be fixed on a surface using a capture element comprising antibodies specific for lymphocyte surface proteins (e.g., CD3, CD4, CD8, etc.), such as Sekine et al., J. Immunol. Meth., 313 (1-2): 96-109 (2006); Zhu et al., Anal. Chim. Acta, 608: 186-196 (2008). In some embodiments, for adherent cells, patterned fibronectin or polylysine can be used on the surface to capture such cells at defined locations. In some embodiments, such methods can be used to separate different subsets of cells in different zones within the same channel.

[0050] Cell proliferation assay

[0051] Figure 2A-2F Shown is an exemplary assay for determining the proliferation rate of a cell after the cell is identified based on cell surface proteins, protein secretion profiles, etc. Cells can also be identified or characterized by their transcriptomes determined as described below or by similar techniques. It should be understood that the term "proliferation rate" can include a measure of insufficient proliferation. For example, a chamber encapsulating one or more cells can be exposed to an agent, such as a drug candidate, after which such cells can return to a normal growth state. In some cases, the agent may kill or slow the growth of cells, such as compared to a control group not exposed to the agent. Therefore, in the case of treated cells, a negative "proliferation rate" may occur because the final number of cells counted in the chamber may be less than the original number; or the value of a signal monotonically related to the number of cells may decrease.

[0052] Figure 2AThe steps of a proliferation assay performed by the system of the present invention are shown. Cells and polymer precursors are loaded into a channel (200) and arranged on a first surface (202) (e.g., cells (204)). The position of the cells is determined by a detector (206), which is used by a control system to generate instructions for causing a spatial energy modulation element (208) to generate a light beam to synthesize a hydrogel chamber (210) in the channel (200) around a single cell, as shown by the hydrogel chambers (212, 213, and 214). An enlarged view (215) shows that the solid appearance structure (212, 213, and 214) has an interior (211) and a wall (221) having a predetermined thickness (216). Similarly, the hydrogel chamber has a predetermined shape (e.g., a circle with a diameter (217)) and encapsulates a predetermined area. In the drawings, for convenience, the chambers are shown as being isolated and not connected to adjacent chambers, and are shown as having a cylindrical or ring-like shape; however, the spatial energy modulation element can synthesize chambers of different shapes and sizes, as may be useful for a particular application. In some embodiments of the proliferation assay, each hydrogel chamber synthesized has the same shape and area, for example, the internal area is selected from the range of 0.001 to 0.01 mm 2 or range from 0.001 to 1.0 mm 2quasi-ring. In some embodiments, for each different type of cell measured, each synthesized hydrogel chamber has the same shape and area, for example, cytotoxic T lymphocytes can be confined to a chamber with one area, while helper T lymphocytes can be confined to a chamber with another area. After synthesizing the required number of hydrogel chambers, the cells are incubated under growth conditions for a period of time, and then (for example) the cells in each chamber are counted (220) to give a measure of the proliferation capacity of each cell. In some embodiments, after the synthesis chamber is used to encapsulate selected cells, non-selected cells can be removed by washing steps or by other changes in reagents in the channel. In some embodiments, cells can be stained with membrane or intracellular dyes to determine proliferation by dye dilution, so that an independent measure of cell proliferation can be obtained. Exemplary intracellular dyes for dye dilution include but are not limited to Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), etc. The required number of single cells encapsulated by the chamber depends on the statistical confidence required in the measured value. If the subpopulation of interest accounts for only a small part of the total population, more chambers are required. In some embodiments of assaying mammalian cells, the number of hydrogel chambers synthesized around a single cell can be greater than 100; or greater than 1000; or greater than 10,000; or the number can be in the range of 100 to 100,000; or in the range of 1000 to 100,000. After the counts for each chamber are recorded, the clonal population within the chamber can be further assayed to identify the cell type, for example by assessing cell surface proteins, cellular protein secretion, transcriptome, etc. This approach is particularly useful for assessing immune cell populations, particularly engineered immune cells. In some embodiments, a cell proliferation assay can be performed by the following steps: (a) providing a fluidic device comprising: (i) a channel comprising a first surface, cells arranged on or near the first surface, and one or more polymer precursors; (ii) a spatial energy modulation element optically connected to the first surface; and (iii) a detector that identifies the position of cells in the channel based on one or more light signals from the cells; (b) synthesizing one or more chambers in the channel that encapsulate each of the one or more cells by projecting light into the channel with the spatial energy modulation element so that the projected light causes one or more polymer precursors to crosslink to form a polymer matrix wall of the chamber, wherein the position of the synthesized chamber is determined by the position of the cell encapsulated by the synthesized chamber identified by the detector; (c) incubating the cells under proliferation conditions; and (d) determining the proliferation rate of the cells. In some embodiments, determining the proliferation rate includes counting the cells in each chamber.

[0053] Figure 2BSubsequent determination of cells identified by cell surface markers is shown. It should be noted that if cell surface markers are to be measured directly (without first measuring cell proliferation), cells can be incubated with labeled antibodies before the introduction of the channel, wherein the labeling can vary greatly, including but not limited to fluorescent labels, oligonucleotide labels, colorimetric labels, enzyme labels, etc. In addition, it should be understood that binding compounds other than antibodies or fragments thereof can be used in the present invention, for example, such as aptamers. After determining the proliferation rate of each chamber, a mixture of different labeled antibodies is loaded into the channel (200), and each of the labeled antibodies is specific to different cell surface proteins. Such antibodies can be prepared for any cell surface protein, but the surface proteins of particular interest are those surface proteins (such as CAR) that characterize immune cells and their binding specificity. In some embodiments, the antibody is specific to surface proteins, and the surface proteins include but are not limited to differentiation cluster (CD) markers, such as CD3, CD4, CD5, CD6, CD7, CD8, CD11, CD19, CD20, CD21, CD22, CD23, CD24, CD25, etc. In some embodiments, the labeling of the antibody is an oligonucleotide. In some embodiments, such oligonucleotides can be attached to the antibody by a scissile bond, for example, the scissile bond can be cut by conventional methods, such as chemical cutting by reducing conditions, acid conditions or alkaline conditions, or photocutting by being exposed to the light of appropriate wavelength and intensity. In some embodiments, the cell of the antibody labeling is exposed to a cleavage reagent sufficient to release the antibody-oligonucleotide conjugate to be captured by the capture element, whether or not there is a scissile bond. The oligonucleotide labeling can include a barcode sequence that uniquely identifies the protein to which the antibody has specificity. The oligonucleotide labeling can also include a sequence complementary to the oligonucleotide attached to the first surface as a capture element. In some embodiments, such complementary sequences or the complementary sequences of the capture oligonucleotide can be blocked by mismatching or hybridization of shorter oligonucleotides (for example, more unstable than oligonucleotide labeling-capture oligonucleotide duplexes), to prevent false capture of antibody-oligonucleotide conjugates by the capture oligonucleotides on the first surface when loaded into the channel. Then before releasing the oligonucleotide labeling of the cell surface binding antibody, such blocking oligonucleotides are removed in a washing step. Alternatively, the oligonucleotide tag can be configured to be connected to capture oligonucleotides by splicing oligonucleotides. The splicing oligonucleotide will block the false hybridization of the oligonucleotide tag before releasing from the antibody-surface protein complex. After this release and the introduction of ligase, the oligonucleotide tag will be captured by the free end of the splicing oligonucleotide, and the oligonucleotide tag will be connected to the end of the capture oligonucleotide.

[0054] exist Figure 2A-2BIn some embodiments, after a period of incubation (226) to allow the antibody to bind to its target protein, the oligonucleotide tag is released and captured by the complementary sequence of the capture element. For example, the oligonucleotide tag can be attached via a disulfide linker that can be released by a reducing agent (e.g., Hermanson (cited above)). In some embodiments, after incubation, unbound antibody can be removed, for example, by a washing step. In some embodiments, a reaction mixture including a polymerase, dNTPs, and other components can be loaded (228) into the channel (200), which reaction mixture is necessary for extending the complementary oligonucleotide of the capture element using the captured antibody oligonucleotide as a template. In some embodiments, the polymer matrix wall of the chamber can be degraded or depolymerized (226), after which such extension reagents can be loaded (228) into the channel (200). As explained more fully below, the DNA copies can be sequenced in situ (230), or they can be released from the first surface (possibly after amplification), eluted, and sequenced externally. In the latter process, the complementary oligonucleotides of the capture element include a spatial barcode for identifying the chamber from which the antibody barcode sequence originates. In either case, the number of each antibody oligonucleotide calculated from the sequence data provides a profile of cell surface proteins of each encapsulated cell in the hydrogel chamber (e.g., the relative frequency of each cell surface protein). In addition, this data can be correlated with the proliferation capacity of such cells.

[0055] Figure 2C-Figure 2D One embodiment of a protein secretion assay using commercially available protein capture beads (e.g., Biolegend (San Diego, CA)) is shown. One of ordinary skill in the art will recognize that other surfaces beside (or in addition to) the bead surface can be used to locally capture secreted proteins, such as cytokines. Figure 2C The upper and lower figures are top views of a cross section of channel (200) including chambers (212, 213, and 214). (Thus, Figure 2C-Figure 2D An alternative assay for counting cells is shown ( Figure 2B or, in some embodiments, Figure 2B The determination can be Figure 2C-Figure 2DAfter counting the cells, the channel (200) is loaded (232) with protein capture beads (e.g., 234), which are beads (typically impermeable to the polymer matrix walls of the chamber) with covalently attached antibodies that are specific for predetermined proteins secreted by the cells encapsulated by the chamber. Such beads can be prepared for any secreted protein, but proteins of particular interest are cytokines and immunologically active proteins, including but not limited to interferons (such as interferon-γ (IFN-γ) and interferon-α (IFN-α)), interleukins (such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-15 (IL-15) and interleukin-21 (IL-21), interleukin-23 (IL-23)), colony stimulating factors (CSF) (such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF)), tumor necrosis factor (TNF) (such as TNF-α and TNF-β), and effector molecules (such as granzyme B). After loading such beads, concentric chambers are synthesized around each of the chambers containing cells (236), after which beads outside the concentric chambers are removed (238). In some embodiments, the size of the larger concentric chamber is sufficient to encapsulate many protein capture beads so that there is a high probability (e.g., greater than 90%, or greater than 95%, or greater than 99%) that there is at least one bead for each different cytokine to be measured. One of ordinary skill will recognize that the determination of such size depends on the concentration of beads loaded into the channel (200). After the concentric chambers are synthesized, the polymer matrix wall of the inner chamber is depolymerized (240) so that the cells and beads can mix freely so that the secreted protein can be captured by the beads, after which a labeled antibody ("detection antibody") is loaded into the channel to bind to another epitope of the captured protein (as in a sandwich assay). In some embodiments, the porosity of the inner chamber is selected to prevent or inhibit the secreted protein from passing through its polymer matrix wall. In some embodiments, the porosity of the inner and outer concentric chambers is selected to prevent or inhibit the passage of secreted proteins, and prior to loading the detection antibody, the polymer matrix wall of the outer chamber is depolymerized to remove barriers to binding of the detection antibody to the captured protein. Alternatively, in some embodiments, the porosity of the polymer matrix wall of the inner chamber can be selected to allow secreted proteins to enter the annular region containing the beads. The relative amount of secreted protein can be estimated by counting each type of bead that generates a characteristic fluorescent signal or by integrating each distinct fluorescent signal on all beads in the vicinity of the cell of the chamber.This embodiment can be implemented by the following steps after the cells are initially arranged on the first surface or after another assay that does not destroy the cells: (a) loading the channel with a second reaction mixture including a second polymer precursor and protein capture beads, the protein capture beads including protein capture antibodies that bind to proteins secreted by the cells; (b) synthesizing a second chamber encapsulating each of the one or more chambers by projecting light into the channel with a spatial energy modulation element so that the projected light causes one or more polymer precursors to crosslink to form a polymer matrix wall of the second chamber, wherein the position of the synthesized second chamber is determined by the position of the chamber encapsulated by the synthesized second chamber identified by a detector; and (c) depolymerizing the chamber so that the protein capture beads in each second chamber are mixed with the cells encapsulated therein by the depolymerized chamber. In some embodiments, the (first) polymer precursor may include a precursor that forms a gel linkage that can be degraded by reducing conditions, such as Structure 2 of Table 2B, while the second polymer precursor does not include such a precursor. In some embodiments, the above method further comprises: incubating the cells with protein capture beads; and detecting the proteins secreted by the cells by a certain amount of labeled protein detection antibodies for each protein bound to the protein capture beads adjacent to the cells.

[0056] Those of ordinary skill recognize that the function of a protein capture antibody can be performed by other affinity reagents (such as aptamers) with specific binding capabilities. As used herein, the term "affinity reagent" means a compound that is capable of specifically binding to a target molecule (such as a protein). Affinity reagents include, but are not limited to, antibodies, antibody fragments, aptamers, and similar compounds.

[0057] An alternative embodiment for measuring protein secretion profiles is Figure 2E-2F. In this embodiment, before loading the cells, evenly distributed protein capture beads are attached to the first surface (250). As shown in the enlarged view (244), the even distribution may include beads with antibodies specific to different proteins. After the cells are deposited on the surface (250), the cells are located by a detector (245), and the hydrogel chamber (246) is synthesized by a spatial energy modulation element (247). The cells are incubated under growth conditions, and then the cells in each chamber are counted to determine the proliferation rate (253). As described above, the secreted protein is detected by being captured by the protein capture beads and then labeled with a detection antibody (255). In some embodiments, the porosity of the polymer matrix wall of the chamber is selected to prevent cells from passing through, but allows proteins to pass freely. In other embodiments, such porosity is selected to prevent both cells and proteins from passing through. In the latter embodiment, an additional step of depolymerizing the polymer matrix wall is included. The relative amount of secreted protein is determined by counting different types of labeled beads adjacent to or near the cells in each chamber (257), or by integrating different fluorescent signals from beads adjacent to or near the cells in each chamber (256). As mentioned above, in addition to beads carrying protein capture antibodies, other surfaces capable of carrying secreted protein capture antibodies include but are not limited to the first surface, the polymer matrix wall of the chamber, or the second surface. These surfaces capable of attaching secreted protein capture antibodies are referred to herein as "protein capture surfaces."

[0058] In some embodiments, the above method for associating cell characteristics with proliferation rate may include the following steps: (a) providing a fluidic device, the fluidic device comprising: (i) a channel, the channel comprising a first surface, cells arranged on or near the first surface, and one or more polymer precursors; (ii) a spatial energy modulation element, the spatial energy modulation element being optically connected to the first surface; and (iii) a detector, the detector identifying the position of the cell in the channel based on one or more light signals from the cell; (b) synthesizing one or more chambers in the channel encapsulating each of the one or more cells by projecting light into the channel with the spatial energy modulation element so that the projected light causes the one or more polymer precursors to crosslink to form a polymer matrix wall of the chamber, wherein the position of the synthesized chamber is determined by the position of the cell encapsulated by the synthesized chamber identified by the detector; (c) incubating the cells under proliferation conditions; and (d) counting the cells in each chamber to determine its proliferation rate. In some embodiments, the first surface may include one or more capture elements, the one or more capture elements being used to capture one or more biological components of the cell, such as secreted proteins, messenger RNA, genomic DNA, etc. In some embodiments, the step of synthesizing may be followed by a washing step to remove unreacted polymer precursors. In some embodiments, after such a washing step (or as part of such a washing step), a cell culture medium can be loaded into the channel. In some embodiments, the above method further comprises: (i) incubating cells with antibodies specific for surface proteins whose relative expression allows identification of cells, wherein each of such antibodies has an oligonucleotide tag comprising an antibody-specific barcode capable of being captured by a capture element; (ii) optionally loading a release reagent into the channel so that the oligonucleotide tag of the antibody attached to one or more cells is released and captured by the capture element (if the oligonucleotide tag is attached by a cleavable bond); (iii) optionally depolymerizing the polymer matrix wall of the chamber; (iv) loading reagents into the channel to replicate the captured oligonucleotide tag, thereby generating complementary DNA of the captured oligonucleotide tag; and (v) sequencing the complementary DNA to identify the captured oligonucleotide tag. In some embodiments, the captured labeled cDNA can be sequenced at or near its capture location by the following steps: (a) amplifying the complementary DNA, (b) sequencing the amplified complementary DNA, and (c) determining the relative expression of the surface protein of the cells in each of the chambers. Oligonucleotide labels can be attached to antibodies (or other binding moieties) using cleavable linkages well known in the art.Such oligonucleotide labels attached to antibodies by cleavable linkages can be cut or released from antibodies by cutting agents or cutting agents or (equivalently) releasing agents or releasing agents, which can be chemical, physical or electrical properties. Reagents for copying captured oligonucleotide labels include DNA polymerase, dNTPs and associated buffers and saline solutions to extend capture oligonucleotides using oligonucleotide labels as templates. In some embodiments, the cDNA of the captured label can be sequenced on an external sequencing instrument after (optionally, amplification and) elution from a channel. For the latter sequencing, the capture element on the first surface includes a spatial barcode, and the cDNA of the captured oligonucleotide label each includes a spatial barcode. After the cDNA spatial barcode conjugate is sequenced, cDNA can be assigned to the chamber based on the spatial barcode.

[0059] In other embodiments, cell proliferation can be associated with protein secretion by the following steps: (a) providing a channel comprising a protein capture surface, the protein capture surface comprising protein capture antibodies that bind to proteins secreted by the cells; and (b) detecting proteins secreted by the cells by a labeled protein detection antibody for each protein bound to the protein capture surface adjacent to the cells. In some embodiments, the protein capture surface comprises protein capture beads.

[0060] In some embodiments, the cell proliferation rate can be associated with the cell transcriptome following the steps for converting mRNA from the cell into cDNA and sequencing the resulting cDNA. In short, after the counting step, the following further steps can be implemented: (a) loading a lysis reagent to the channel so that the cell's mRNA is released and captured by the capture element; and (b) loading a reverse transcription reagent to the channel to replicate the captured oligonucleotide tag, thereby generating a complementary DNA of the captured oligonucleotide tag; and (c) sequencing the complementary DNA. It should be understood that in certain embodiments, the sequencing step may include additional steps, including but not limited to labeling, adding adapters, cutting cDNA to form an appropriate length for sequencing, etc. In some embodiments, additional steps for depolymerizing or degrading the polymer matrix wall of the chamber after mRNA capture can be implemented. Reverse transcription reagents include conventional reagents for reverse transcription; i.e., reverse transcriptase (such as Moloney murine leukemia virus (MMLV)), dNTPs, optional RNase inhibitors, buffers. The sequencing step can be performed at the site of the captured mRNA (in situ), or the cDNA can include a spatial barcode and be eluted and sequenced on a separate sequencing instrument ("external" sequencing). For in situ sequencing, further steps may include: (i) amplifying complementary DNA, such as by bridge amplification or similar methods; (ii) sequencing the amplified complementary DNA, such as by sequencing by synthesis; and (iii) determining the relative expression of mRNA in cells of each chamber. For external sequencing, further steps may include: (i) providing a capture element including a spatial barcode; (ii) synthesizing cDNA including a spatial barcode; and (iii) eluting and sequencing the cDNA and associating each cDNA with the location of the chamber by its spatial barcode.

[0061] Cytotoxicity assay

[0062] As with cell proliferation, measures of cytotoxicity against cells of a target cell population can be correlated with other cell properties, including but not limited to cell membrane protein expression, cell protein secretion profiles, transcriptome, etc. The target cells can include a tumor cell sample from a patient, or the target cells can be from a cell line, e.g., a tumor cell line, such as the liver tumor cell line SK-HEP-1, Chava et al., J. Vis. Exp., Feb. 22, 2020:(156):10.3791 / 60714. The steps of an exemplary cytotoxicity assay are described in Figure 3A-3B. Target cells (303) are loaded (304) into the channel (300) so that they are arranged on the first surface (302), after which effector cells are loaded (306) and arranged on the first surface (302) together with the target cells. "Effector cells" means cells whose cytotoxicity is being measured. In some embodiments, effector cells are cells engineered for therapeutic purposes (such as treating cancer). Target cells and effector cells are incubated together (308) with a vital dye that generates a light signal in response to the characteristics of viable cells or dead cells, which allows the counting of dead cells. Exemplary vital dyes for live cells include, but are not limited to, Hoechst 33342. Exemplary vital dyes for dead cells include, but are not limited to, 7-AAD, for example, Schmid et al., Cytometry, 15: 12-20 (1994); Bradford et al., poster entitled "Dead cellstains in flow cytometry: a comprehensive analysis", Molecular Probes (Eugene, OR). After such incubation, a measure of cytotoxicity can be determined by counting the number of dead target cells adjacent to or near each effector cell (310). In some embodiments, individual effector cells can be encapsulated by the chamber immediately after loading to ensure that the only dead target cells counted are cells killed by the encapsulated single effector cells. In other embodiments, if the dead target cells are easily associated with the dead target cells, for example, by proximity, the effector cells can be encapsulated by the chamber at a later time. Effector cells can then be identified by, for example, cell surface markers, protein secretion, transcriptome, etc. In order to identify by cell surface markers, effector cells can be encapsulated by the hydrogel chamber (312) (if not already encapsulated), and antibodies with oligonucleotide tags can be loaded into the channel (314). In some embodiments, unencapsulated cells can be removed by washing steps or other changes in reagents in the channel. After incubation (316) to bind the antibody to the target surface protein, the oligonucleotide tag is released and captured by the capture element. After such capture, the chamber is optionally depolymerized and an extension reagent is loaded for replicating the captured oligonucleotide by extending the capture element oligonucleotide using the captured oligonucleotide as a template (318). The resulting cDNA is amplified and sequenced (320) for use in cell proliferation assays as described above.

[0063] Likewise, encapsulated cells from different channels that have been subjected to cytotoxicity measurements as shown in step (312) can be subjected to different assays in those different channels to determine different cell characteristics from cell surface markers. Such different assays include, but are not limited to, protein secretion, vector copy number, transcriptome, etc.

[0064] In some embodiments, additional steps can be implemented to reduce or eliminate false signals generated by proteins or mRNA of target cells. For example, after incubation to kill and after labeled antibodies bind to cell surface proteins, the effector cells in the chamber can be encapsulated or wrapped in a gel (or a second chamber can be synthesized) to separate them from the target cells in the first chamber. After counting, the target cells in the first chamber can then be lysed and removed by washing. After such removal, the gel encapsulating the effector cells can then be depolymerized to allow antibody labels or mRNA to be released and captured by the capture element of the first surface. In some embodiments, the gel encapsulating the effector cells is degradable (e.g., degradable by a reducing agent) and has a sufficiently small average pore size to prevent most mRNAs of interest (e.g., mRNAs of 300 nucleotides or more) from passing through until depolymerization.

[0065] In some embodiments, a method of correlating a cell property with cytotoxicity may include the following steps: (a) providing a fluidic device comprising: (i) a channel comprising a first surface and a target cell population disposed on the first surface; (ii) a spatial energy modulation element optically connected to the first surface; and (iii) a detector that identifies the position of the cells in the channel based on one or more light signals from the cells; (b) loading each channel with effector cells and one or more polymer precursors such that the effector cells are disposed on or near the first surface; (d) synthesizing one or more chambers in the channel by projecting light into the channel using the spatial energy modulation element such that the projected light causes the one or more polymer precursors to cross-link to form a polymer matrix wall of the chamber, the one or more chambers each encapsulating an effector cell, wherein the position of the synthesized chamber is determined by the position of the effector cell encapsulated by the synthesized chamber as identified by the detector; (e) incubating the target cells and the effector cells with a vital stain that identifies dead cells; and (f) counting the dead cells in each chamber to determine a measure of the cytotoxicity of the effector cells encapsulated by each chamber. In some embodiments, the vital dye generates a light signal in dead cells but not in live cells. In some embodiments, the step of synthesizing can be followed by a washing step to remove unreacted polymer precursors. In some embodiments, after such a washing step (or as part of such a washing step), cell culture medium can be loaded into the channel.

[0066] In some embodiments, after measuring the cytotoxic capacity of effector cells, for example by counting dead cells adjacent to or within the chamber, effector cells can be identified by other assays measuring properties such as surface proteins, protein secretion, etc. Assays for identifying effector cells by their cell surface proteins can include the following steps: (i) incubating cells with antibodies specific for surface proteins whose relative expression allows identification of cells, wherein each of such antibodies has an oligonucleotide tag comprising an antibody-specific barcode that can be captured by a capture element; (ii) loading a release reagent into the channel such that the oligonucleotide tags of the antibodies attached to one or more cells are released and captured by the capture element; (iii) optionally depolymerizing the polymer matrix wall of the chamber; (iv) loading reagents into the channel to replicate the captured oligonucleotide tags, thereby generating complementary DNA of the captured oligonucleotide tags; and (v) sequencing the complementary DNA to identify the captured oligonucleotide tags and thereby determine the cell surface protein profile of each effector cell. As described above, in some embodiments, prior to loading the release reagent, the following steps can be performed: (a) encapsulating the effector cells in each chamber in a degradable encapsulation gel to prevent the bound oligonucleotide-labeled antibodies or mRNA from escaping; (b) lysing the target cells by loading the lysis reagent into the channel; and (c) a washing step to remove the oligonucleotide-labeled antibodies and mRNA bound to the target cells.

[0067] In some embodiments, such cDNA can be sequenced in situ by the following steps: (a) amplifying complementary DNA, (b) sequencing the amplified complementary DNA, and (c) determining the relative expression of surface proteins of cells in each chamber. In alternative embodiments, such cDNA can be sequenced externally as follows: providing a spatial barcode for the capture element so that the cDNA synthesized from the captured oligonucleotide tag includes the spatial barcode; amplifying the cDNA; releasing (or dehybridizing) and eluting the amplified cDNA (or their complementary sequences); sequencing the cDNA, wherein the spatial barcode allows identification of the chamber from which the cDNA originated.

[0068] Using the additional step of cell proliferation, the cytotoxicity of the cells can be associated with the protein secretion profile. In one embodiment, such steps may include: (i) loading a second reaction mixture including a second polymer precursor and protein capture beads into the channel, the protein capture beads including protein capture antibodies that bind to proteins secreted by the cells; (ii) synthesizing a second chamber that encapsulates each of the one or more chambers by projecting light into the channel with the spatial energy modulation element so that the projected light causes one or more polymer precursors to crosslink to form a polymer matrix wall of the second chamber, wherein the position of the synthesized second chamber is determined by the position of the (initial) chamber encapsulated by the synthesized second chamber identified by the detector; and (iii) depolymerizing the chamber so that the protein capture beads in each second chamber are mixed with the cells encapsulated by the depolymerized chamber. The above method may further include: incubating the cells with protein capture beads; and detecting the proteins secreted by the cells by a certain amount of labeled protein detection antibodies for each protein bound to the protein capture beads adjacent to the cells.

[0069] In an alternative embodiment, the channel further includes protein capture beads disposed thereon, the protein capture beads including protein capture antibodies that bind to proteins secreted by the cells; and the method further includes detecting the proteins secreted by the cells by means of a certain amount of labeled protein detection antibodies for each protein bound to the protein capture beads adjacent to the cells.

[0070] Vector copy number determination

[0071] The vector copy number in cells engineered for therapy is important because if the copy number is too high, such as above about five copies per cell, the risk of tumorigenesis increases, e.g., Chen et al., Cytotherapy, 22(5), supplement, S142 (2020); Paugh et al., Scientific Reports, 11:389 (2021). According to the present invention, the vector copy number can be measured by single-cell quantitative PCR in a chamber or by counting unique molecular identifiers (UMIs) amplified and sequenced from single cells in a chamber, or similar measurements. For any assay, it can be as follows Figure 4ACells are prepared as shown. That is, engineered or transduced cells can be loaded into a channel (400) so that cells (e.g., 401) are arranged on a first surface (402), after which a detector (404) records the location of the cells on the first surface (402), and a spatial energy modulation element (406) synthesizes (408) a chamber (411) that individually encapsulates a single cell. Polymer precursors and conditions can be selected so that the porosity of the polymer matrix wall of the chamber allows proteins, lysis reagents, mRNA, etc. to pass through, but retains genomic DNA, for example, using the formulations described in Spencer et al., ISME Journal, 10:427436 (2016); and Tamminen et al., Frontiers Microbiology: Methods, 6:195 (2015). The cells can be lysed as described by Tamminen et al. (proteinase K followed by lysozyme) or as described by Zhang et al., Proc. Natl. Acad. Sci., 89:5847-5851 (1992) (alkaline lysis: 200 mM KOH / 50 mM dithiothreitol followed by neutralization with 900 mM Tris-HCL, pH 8.3 / 300 mM KCl / 200 mM HCL); or by similar methods (413). After such treatment, the genomic DNA remaining in the chamber can be further analyzed by qPCR (420) (such as described by Vaninsberghe et al., Plos One, 13(1):e0191601 (2018)), or if the vector includes UMIs, it can be analyzed by Figure 4B The described sequencing (421) was further analyzed, for example, by Porter et al., Genome Biology, 15:R75 (2014).

[0072] For qPCR (420), in some embodiments, a PCR reaction mixture including polymerase, primers, taqman probes, buffers and salts is loaded into the channel (400). In some embodiments, in order to run PCR, the flow cell is moved to a temperature cycle station. As PCR is performed in each chamber, the detector (404) records the signal accumulation from the taqman probe at each chamber, which can be converted into a copy number. In some embodiments, a blocking agent can be loaded into the channel containing the chamber where the qPCR reaction occurs to prevent the labeled taqman fragment from diffusing from the chamber. Such a blocking agent can include a polymer precursor that can be polymerized in the space around the chamber.

[0073] For sequencing (412), optionally, the retained genomic DNA can be pre-amplified by whole genome amplification techniques, e.g., as described in Zhang et al. (cited above), after which the desired sequences (e.g., those comprising viral UMIs) are specifically amplified (e.g., by PCR or linear amplification techniques) and captured by the capture elements (412). In some embodiments, the UMIs are amplified along with a "handle" segment (an oligonucleotide segment complementary to the capture oligonucleotide) to facilitate capture by the capture elements of the first surface. As described above, the chambers can be depolymerized, loaded with extension reagents, and cDNAs containing copies of the UMIs synthesized (416). After amplification, the cDNAs can be sequenced in situ or externally, and the number of UMIs with different sequences determined to give the viral copy number of the cells per chamber.

[0074] The above method can be implemented by first encapsulating the engineered cells in the hydrogel chamber according to the present invention, that is, by the following steps: (a) providing a fluid device, the fluid device comprising: (i) one or more channels, each of the one or more channels comprising a first surface; (ii) a spatial energy modulation element, the spatial energy modulation element being optically connected to each first surface; and (iii) a detector, the detector identifying the position of the cells in each channel based on one or more light signals from the cells; (b) loading cells and one or more polymer precursors into each channel so that the cells are arranged on or near the first surface; (c) projecting light onto each channel by using the spatial energy modulation element (d) loading assay reagents (e.g., such as lysis reagents, followed by amplification reagents, and then cDNA synthesis reagents, etc.) into one or more channels; and (e) incubating the cells in each channel under assay conditions including PCR conditions, rolling circle amplification conditions, and / or sequencing conditions to generate a signal indicating sequence copy number (such as a vector copy number (VCN)) from the chambers of each channel. In some embodiments, to count the number of copies of a specific (or predetermined) nucleotide sequence (such as a UMI), the following steps may be performed: (i) lysing cells to release genomic DNA; (ii) amplifying one or more nucleotide sequences; (iii) capturing the amplified one or more nucleotide sequences by a capture element; (iv) loading reagents into the channel to replicate the captured one or more nucleotide sequences, thereby generating complementary DNA of the captured one or more nucleotide sequences; and (v) sequencing the complementary DNA to identify the number of copies of the one or more nucleotide sequences. Embodiments of particular interest include a predetermined nucleotide sequence as a barcode (particularly a barcode as or including a UMI). The amplified nucleotide sequence typically includes one or two primer binding sites, a barcode, and a sequence complementary to a capture oligonucleotide of a capture element. The amplified nucleotide may include an additional segment that is used to increase the molecular weight of the amplified product to prevent or inhibit passage through the polymer matrix wall of the hydrogel chamber. The number of different UMIs can be identified from the sequencing data, which gives the number of viral copies of the cell.

[0075] In order to count the copy number by quantitative PCR or other amplification methods, in some embodiments, the following steps can be implemented: (i) lysing cells to release genomic DNA; (ii) loading the channel with an amplification reagent that generates a signal proportional to the copy number of one or more nucleotide sequences of the genomic DNA; (iii) amplifying one or more nucleotide sequences to generate a signal monotonically related to the copy number of the one or more nucleotide sequences. In some embodiments, the amplification reagent can be a quantitative PCR reagent, a bridge PCR reagent, or a rolling circle amplification reagent. In some embodiments, the quantitative PCR reagent includes an amplification buffer, a polymerase with 5'→3' exonuclease activity, a primer, a dNTP, and a taqman probe.

[0076] In a further embodiment, the copy number can be determined by duplicating the selected genomic sequence, capturing them with a capture element, synthesizing cDNA from the selected genomic sequence, and then forming cDNA clusters by bridge PCR or forming DNA nanoballs by rolling circle amplification, after which the clusters or DNA nanoballs can be counted as a measure of the copy number. When quantitative PCR is used, the signal associated with the copy number can be a light signal or a cycle number. When bridge PCR is used, the first surface can include bridge PCR primers, and the signal associated with the copy number can be the number of clusters formed. When rolling circle amplification is used, the signal associated with the copy number can be many DNA nanoballs formed. Clusters and DNA nanoballs can be detected by a variety of techniques (including but not limited to using fluorescently labeled dNTPs, double-stranded DNA dyes, etc.).

[0077] Integration site analysis

[0078] One or more integration sites of the viral vector in the genome of the therapeutic cell are crucial because the expression of one or more important genes may be disabled or altered, which is sometimes called genotoxicity or insertional mutagenesis, for example Biasco et al., Molecular Therapy: Methods & Clinical Development, 8: 21-30 (2018); Cornetta et al., Molecular Therapy: Methods & Clinical Development, 28: 28-39 (2023); Desfarges et al., Viruses, 2: 111-130 (2010); etc. According to the present invention, the insertion site can be detected in the cell population, such as Figure 4C-4F The general concept of the measurement method is to anneal primers to known vector sequences integrated into the genome, extend the primers into the host's genomic DNA, and then identify the host gDNA and integration site. Figure 4AAs described, individual cells are encapsulated in a hydrogel chamber, wherein the hydrogel porosity (420) is selected so that the walls of the chamber prevent the passage of genomic DNA, but allow the passage of reagents (such as deoxynucleoside triphosphates (dNTPs), DNA polymerases, primers, etc.), for example, as disclosed by Spencer et al., ISME J., 10:427-436 (2010). The cells are lysed (422) to release genomic DNA (gDNA) (424), after which the released genomic DNA is amplified, denatured, and bound to vector-specific primers (e.g., 425). Such lysis can be performed by loading a lysis reagent into the channel using various methods known in the art, for example, Spencer (cited above); Cui et al., Proc. Natl. Acad. Sci., 86:9389-9393 (1989); Deleye et al., Scientific Reports, 5:11711 (2015); etc. After lysis, whole genome amplification (WGA) can be performed using techniques known in the art, such as primer extension pre-amplification PCR (PEP-PCR), degenerate oligonucleotide primer PCR (DOP-PCR), multiple displacement amplification (MDA), multiple annealing and loop-based amplification cycles (MALBAC), etc., which are disclosed in the following references: Volozonoka et al., Int. J. Mol. Sci., 23:4819 (2022); Hou et al., Giga Science, 4:37 (2015); Yu et al., Anal. Chem., 86:9386-9390 (2014); etc. Figure 4CThe integrated vector (426) is shown as a linear segment of DNA flanked by long terminal repeat (LTR) sequences. In some embodiments, other vector regions can be used as primer binding sites. The LTR primer (425) has a 5' oligonucleotide tail (427) having a sequence complementary to the capture oligonucleotide (430) on the first surface (428) in the chamber. The LTR primer is extended by a DNA polymerase in the presence of dNTPs (e.g., 431), and the chamber is then washed with sufficiently high stringency (e.g., salt, heat, etc.) to destabilize and remove the non-extended LTR primer, but not so high as to remove the extended LTR primer (429). The extended LTR primer is then melted from the gDNA and captured (436) by the capture oligonucleotide (430) by hybridizing with the 5' tail (427) of the extended LTR primer. The capture oligonucleotides (430) are attached to the surface (428) via their 3' ends and have a 5'-phosphate group attached to their 5' ends. The capture oligonucleotides may have additional features depending on the embodiment. For example, if the sequence of the extension region of the extended LTR primer is to be determined in situ, the capture oligonucleotide may include a primer for bridge amplification. On the other hand, if the sequence of the extension region is to be determined outside the instrument, the capture oligonucleotide may include a spatial barcode for identifying sequences associated with the same cell. Figure 4D In the embodiment, the surface (428) is shown with the so-called P7' (432) and P5 (434) sequences of the bridge PCR; therefore, the capture oligonucleotide (430) will include the P7 sequence, which is used to initiate the bridge PCR after the LTR primer extension is copied and removed. The actual sequence of the P5 and P7' regions is a matter of design choice for those of ordinary skill in the art.

[0079] After capturing the extended LTR primer (436), the random sequence hexanucleotide primer (441) anneals to the extension region (438), and the random sequence hexanucleotide primer (441) itself is extended (440), after which the synthesized chain is ligated to the 5' end of the capture oligonucleotide, for example using the protocol described by Schmidt et al., Nature Methods, 4(12): 1051-1057 (2007); Kalle et al., U.S. Patent 6514706; which is incorporated herein by reference. Alternatively, the 3' end of the extension region (438) can be extended by terminal deoxynucleotidyl transferase (TdT) to generate a homopolymer tail, and a complementary primer can anneal to the homopolymer tail and extend to the 5' end of the capture oligonucleotide (430). The replicated sequence is then ligated to the capture oligonucleotide (430), after which the resulting construct is treated with a restriction endonuclease to generate a known sequence end distal to the surface (428). The restriction endonuclease is selected to maximize the likelihood of retaining a sufficient extension region for uniquely identifying its location in the engineered cell genome (e.g., about 18 nucleotides in humans). In some embodiments, a restriction endonuclease is selected that has a 4-nucleotide recognition site and leaves a 4-nucleotide overhang after cleavage. After such cleavage, a double-stranded adapter is ligated to the end of the double-stranded fragment attached to the surface (428). Figure 4F As shown in (446), the sequences of the adapter strands can be determined in situ by performing surface amplification followed by a sequencing reaction, or they can be determined off-surface (428) by PCR amplification and eluting the amplicons. In the former case, the capture oligonucleotide (430) includes a P7 sequence at its end near the surface (428). In the latter case, the capture oligonucleotide (430) includes a spatial barcode at its end near the surface (428).

[0080] In some embodiments, the above-mentioned method for determining the vector integration site of a cell population can be implemented in the following manner: (a) synthesizing one or more hydrogel chambers, each of which encapsulates one or more cells arranged on the surface of the channel; (b) lysing cells so that the genomic DNA of each cell is released into its hydrogel chamber; (c) amplifying the genomic DNA of each cell; (d) annealing a vector-specific primer to the amplified genomic DNA; (e) extending the vector-specific primer so that an extension product including a copy of a segment of the genomic DNA is formed; and (f) identifying the site of each vector integrated into the genomic DNA of each of the cells from the segment. In some embodiments, the vector-specific primer includes a primer that is complementary to the vector sequence but not complementary to the genomic sequence of the cell. In some embodiments, whenever the vector is a retrovirus, the vector-specific primer can be complementary to the sequence of a long terminal repeat (LTR) element. Such vector-specific primers are sometimes referred to as "LTR primers" herein. Ordinary technicians will recognize that the extent or length of the vector-specific primer extending into the adjacent region of the genomic DNA must be sufficient to uniquely identify the size of the position of the integration vector in the cell genome. For the human genome, in some embodiments, the length of such an extension includes at least 18 nucleotides of the cell's genomic DNA.

[0081] Genome copy number variation

[0082] An important issue in stem cell therapy is the genetic stability of stem cells, as they can display aneuploidy and genomic copy number variability, which are common features of tumorigenic cells, e.g., Goldring et al., Cell Stem Cell, 8:618-628 (2011). Figure 4G-4H As illustrated, according to the present invention, single cell genome copy number variation can be detected in a cell population. In some embodiments, the methods described herein include the steps described in Example 3 of U.S. Patent Publication US2022 / 0219170 by Khurana et al., which is incorporated herein by reference. As described above, individual cells can be encapsulated in Figure 4A (450) The hydrogel chamber described in claim 450, wherein the porosity of the hydrogel is selected such that the walls of the chamber prevent the passage of genomic DNA but allow the passage of reagents (such as deoxynucleoside triphosphates (dNTPs), DNA polymerase, primers, WGA reagents, labeling reagents, etc.), for example, the porosity of the gel disclosed in Spencer et al., ISME J., 10:427-436 (2010). Figure 4GAs shown, after the cells are encapsulated in the hydrogel chamber, they are lysed to release genomic DNA, and then the WGA reagent is loaded to increase the amount of gDNA in the chamber so that after fragmentation, the sequenced fragments will provide sufficient coverage for copy number variation (CNV) determined at an acceptable resolution. In some embodiments, a sequencing depth of 0.3X produces a 3 megabase (Mb) resolution for CNV determination, for example, Deleye et al., Scientific Reports, 7:3422 (2017). For 0.3X coverage in human cells, sequence reads of approximately 1 billion nucleotides are required. Ordinary technicians will recognize that the degree of amplification from WGA, the surface area encapsulated by the chamber, and the capture oligonucleotide density can be adjusted to achieve the desired CNV resolution. In some embodiments, for CNV measurements, the surface area encapsulated by the chamber is at least 10 3 μm 2 , or at least 10 4 μm 2 , or at least 10 5 μm 2 , or at least 10 6 μm 2 , and the density of capture oligonucleotides is at least 1×10 12 Capture oligonucleotide / cm 2 . In some embodiments, WGA increases the amount of genomic DNA in each chamber by at least 100 times. Based on such genomic sequence data, one can estimate genomic copy number variation by known methods, such as Mallory et al., Genome Biology, 21: 208 (2020); Wang et al. Briefings in Bioinformatics, 19 (5); 731-736 (2018); etc. In some embodiments, the resolution of CNV measurement is 3 megabases (Mb) or higher, or 2 Mb or higher, or 1 Mb or higher (wherein higher resolution corresponds to smaller genome length units, such as smaller Mb values).

[0083] return Figure 4G The amplified gDNA (454) is treated with a labeling agent (e.g., Tn5 transposase (456)) to generate gDNA fragments (466) having upstream (460) and downstream (464) linkers of predetermined sequences, which may include amplification primer binding sites, sequencing primer binding sites, etc.

[0084] The linked tagged fragments are then denatured so that the chains can be captured and extended (467) by a capture oligonucleotide (469). The capture oligonucleotide (469) includes a capture portion ("CM"), a spatial barcode ("sBC"), and an amplification primer ("primer 1"). The depicted capture oligonucleotide (469) is located on a sub-region of the surface (428) encapsulated by the chamber. Of course, the entire surface (428) can have such capture oligonucleotides attached, whether or not encapsulated by the chamber. The captured tagged fragments provide a second amplification primer ("primer 2"). These sequences can be amplified by conventional PCR, and the amplicons are eluted from the channel (470) for sequencing. One of ordinary skill will recognize that such elution and / or amplification can be performed prior to degradation of the hydrogel of the chamber.

[0085] In some embodiments, the above-mentioned method for determining the genomic copy number of a cell population can be implemented in the following manner: (a) synthesizing one or more hydrogel chambers that encapsulate each of one or more cells arranged on the surface of the channel; (b) lysing the cells so that the genomic DNA of each cell is released into the hydrogel chamber of each cell; (c) amplifying the genomic DNA of each cell; (d) sequencing the fragments of the amplified genomic DNA; and (e) determining the genomic copy number variation of each of the cells from the sequence of the genomic DNA fragments. In some embodiments, the step of sequencing includes fragmenting the genomic DNA and attaching the adapter to the genomic DNA fragments by labeling. In some embodiments, such adapters include chains complementary to the oligonucleotides of the capture element. In some embodiments, the step of sequencing includes obtaining a sequence coverage of the cell genomic DNA of 0.25X or greater. In some embodiments, the step of sequencing includes obtaining a sequence coverage of the cell genomic DNA of about 0.25X to about 100X. In some embodiments, the step of sequencing comprises obtaining a sequence coverage of the genomic DNA of the cell of about 0.25X to about 0.5X, about 0.25X to about 1X, about 0.25X to about 10X, about 0.25X to about 30X, about 0.25X to about 100X, about 0.5X to about 1X, about 0.5X to about 1X, about 0.5X to about 30X, about 0.5X to about 100X, about 1X to about 10X, about 1X to about 30X, about 1X to about 100X, about 10X to about 30X, about 10X to about 100X, or about 30X to about 100X. In some embodiments, the step of sequencing comprises obtaining a sequence coverage of the genomic DNA of the cell of about 0.25X, about 0.5X, about 1X, about 10X, about 30X, or about 100X. In some embodiments, the step of sequencing comprises obtaining a sequence coverage of at least about 0.25X, about 0.5X, about 1X, about 10X, or about 30X of the genomic DNA of the cell. In some embodiments, the step of sequencing comprises obtaining a sequence coverage of at most about 0.5X, about 1X, about 10X, about 30X, or about 100X of the genomic DNA of the cell.

[0086] In some embodiments, genome copy number variation is determined as 3 megabases or higher resolution.In some embodiments, genome copy number variation is determined as the resolution of about 1 megabase to about 6 megabases.In some embodiments, genome copy number variation is determined as about 1 megabase to about 2 megabases, about 1 megabase to about 3 megabases, about 1 megabase to about 4 megabases, about 1 megabase to about 5 megabases, about 1 megabase to about 6 megabases, about 2 megabases to about 3 megabases, about 2 megabases to about 4 megabases, about 2 megabases to about 5 megabases, about 2 megabases to about 6 megabases, about 3 megabases to about 4 megabases, about 3 megabases to about 5 megabases, about 3 megabases to about 6 megabases, about 4 megabases to about 5 megabases, about 4 megabases to about 6 megabases or about 5 megabases to about 6 megabases. In some embodiments, the genomic copy number variation is determined to be a resolution of about 1 megabase, about 2 megabases, about 3 megabases, about 4 megabases, about 5 megabases, or about 6 megabases. In some embodiments, the genomic copy number variation is determined to be a resolution of at least about 1 megabase, about 2 megabases, about 3 megabases, about 4 megabases, or about 5 megabases. In some embodiments, the genomic copy number variation is determined to be a resolution of at most about 2 megabases, about 3 megabases, about 4 megabases, about 5 megabases, or about 6 megabases.

[0087] Sequencing, barcoding, genomic fragments and transcriptomes

[0088] Oligonucleotide markers, barcodes, genomic fragments, messenger RNA and similar polynucleotide targets can be sequenced by the method and system of the present invention. In some embodiments, the capture element for this purpose includes an oligonucleotide attached to a surface in a channel, wherein such oligonucleotides include a sequence segment complementary to the sequence segment of the nucleic acid to be captured, which can be a polyA segment of mRNA or an arbitrary sequence "handle" sequence region adjacent to a barcode or oligonucleotide marker. When a sequencing operation is to be performed, the channel is provided with such a capture element. Such capture oligonucleotides can be attached to the first surface by many chemical actions known in the art, such as the Integrated DNA Technologies brochure (2014) entitled "Strategies for attaching oligonucleotides to solid supports". The sequencing step can be performed on the surface of the channel ("in situ" sequencing), or the template can be optionally amplified, released and eluted from the channel and sequenced on an external sequencing instrument ("external" sequencing). In the latter method, the capture element can include a spatial barcode providing channel position information, and allows the sequence determined externally to be associated with each chamber. In some embodiments, the spatial barcode exists at a sufficiently high density so that each chamber covers a first surface area uniquely associated with one or more spatial barcodes (and typically a single spatial barcode). In some embodiments, the preparation of polynucleotides for sequencing operations occurs after the target template (e.g., oligonucleotide markers, mRNA, genomic fragments) is released and captured by the complementary sequence in the capture element. The release step depends on the properties of the target template. For example, the oligonucleotide markers attached to the antibody by disulfide bonding can be released by a reducing agent (which can be the same as a cleavage agent). mRNA can be released by treating cells with conventional cleavage agents. Releasing genomic fragments may require cleavage and pre-amplification steps. Cleavage conditions may vary greatly and may be based on the effects of a combination of heat, detergents, proteases, alkalis, or such factors. The following references provide guidance for selecting lysis reagents or lysis buffers for single cell lysis conditions of mRNA and / or genomic DNA: Thronhill et al., Prenatal Diagnosis, 21:490-497 (2001); Kim et al., Fertility and Sterility, 92:814-818 (2009); Spencer et al., ISME Journal, 10:427-436 (2016); Tamminen et al., Frontiers Microbiol. Methods, 6: article 195 (2015); etc.Exemplary lysis conditions include the following: 1) cells in H2O at 96°C for 15 min, then at 10°C for 15 min; 2) 200 mM KOH, 50 mM dithiothreitol, heated to 65°C for 10 min; 3) for 4 μL of protease-based lysis buffer: 1 μL of 17 μM SDS combined with 3 μL of 125 μg / mL proteinase K, then incubated at 37°C for 60 min, then incubated at 95°C for 15 min (to inactivate proteinase K); 4) for 10 μL of detergent-based lysis buffer: 2 μL of H2O, 2 μL of 10 mM EDTA, 2 μL of 250 mM dithiothreitol, 2 μL of 0.5% N-lauryl sarcosinate solution; 5) 200 mM Tris pH 7.5, 20 mM EDTA, 2% sarcoyl, 6% Ficoll.

[0089] Figure 7A-7B An exemplary capture and cDNA synthesis method for performing a sequencing operation is shown. Fig. 7AA process starting from capturing the target template and preparing cDNA for external sequencing is shown. Those skilled in the art will recognize that the details of the following examples of target template capture and cDNA synthesis can vary greatly depending on the sequencing system used. In some embodiments, the preparation of cDNA includes a tagging step. Guidance for specific embodiments can be found in the following references: Picelli et al., Genome Research, 24: 2033-2040 (2014); Bose et al., Genome Biology, 16: 120 (2015); Hashimshony et al., Genome Biology, 17: 77 (2016); Yuan et al., Scientific Reports, 6: 33883 (2016); and similar references. Attached to the surface (701) by its 5' end is an oligonucleotide having the following components: a primer binding site P7 (for Illumina sequencers) (702), an optional primer binding site R1 (for Illumina paired end sequencing), a barcode oligonucleotide (706) (which can be or include a spatial barcode), an optional unique molecular identifier (708), and a capture oligonucleotide (710), which can be a polyT segment whenever mRNA is to be captured. The target template (712) is captured by hybridization of a polyA segment or sequence handle (714) to the capture oligonucleotide (710). After capture, the capture oligonucleotide (710) and the polyA segment (714) are extended by a polymerase (e.g., Moloney murine leukemia virus (MMLV) reverse transcriptase), leaving a single-stranded polyC tail (716). In some embodiments, the template switching oligonucleotide (718) is hybridized thereto, and the polyC tail is further extended, as shown in (730), e.g., Zhu et al., Biotechniques, 30:892-897 (2001). The unattached strand is melted, and the attached strand is amplified, e.g., by PCR, and eluted for external sequencing (732). For in situ sequencing, the surface (753) includes an attached P5 primer (Illumina) and a capture oligonucleotide (750), which can have the same structure as that used for external sequencing: primer binding site P7 (for Illumina sequencers) (752), optional primer binding site R1 (for Illumina paired end sequencing), barcode oligonucleotide (756) (which can be or include a spatial barcode), optional unique molecular identifier (758) and capture oligonucleotide (760), which can be a polyT segment whenever mRNA is to be captured. The target template (762) is captured by hybridization of the polyA segment or sequence handle (764) to the capture oligonucleotide (710).After capture, the capture oligonucleotide (710) and polyA segment (764) are extended by a polymerase, leaving a single-stranded polyC tail (766). In some embodiments, a template switching oligonucleotide (768) is hybridized thereto, and the polyC tail is further extended, as shown in (780), after which a double-stranded segment (781) is connected thereto. Segment (781) includes the complementary sequence R2' (765) of the primer binding site R2 and the complementary sequence P5' of the primer binding site P5. The unattached chain of the resulting polynucleotide (785) is melted, and the attached chain of (785) is bridge-amplified. In some embodiments, the DNA of the resulting cluster can be sequenced using sequencing-by-synthesis technology to generate a sequence of fluorescent signals for nucleotide recognition. It should be understood that there are many alternatives to the above-described process steps, which can be replaced by other process steps or added to other process steps while still within the scope of the present invention.

[0090] In embodiments employing spatial barcodes on surfaces, a variety of methods can be used to generate spatial barcodes, including but not limited to the methods described in the following references, which are incorporated by reference: Horgan et al., International Patent Publication WO2022 / 013094; Fan et al., U.S. Patent Publication US2019 / 0360121; Chen et al., bioRxiv (https: / / doi.org / 10.1101 / 2021.01.17.427004); Cho et al., bioRxiv (https: / / doi.org / 10.1101 / 2021.01.25.427807); Quan et al., Nature Biotechnology, 29(5):449-453 (2011); Singh-Gasson et al., Nature Biotechnology, 17:974-(1999); etc.

[0091] In some embodiments, a hydrogel chamber can be synthesized for a single cell on a first surface by the following steps: (a) providing a fluidic device having: (i) a channel comprising a first surface, a biological sample comprising biological cells arranged on or near the first surface; (ii) a spatial energy modulation element optically connected to the first surface; and (iii) a detector that identifies the position of one or more biological components in the channel based on one or more optical signals from the one or more biological components; and (b) synthesizing one or more chambers in the channel that encapsulate each of the biological cells by projecting light into the channel using the spatial energy modulation element so that the projected light causes one or more polymer precursors to cross-link to form a polymer matrix wall of the chamber, wherein the position of each of the synthesized chambers is determined by the position of each encapsulated biological cell in the synthesized chamber identified by the detector. In some embodiments, the first surface includes a capture element, such as a capture oligonucleotide covalently attached to the first surface. Exemplary capture oligonucleotides such as Figure 7A-7B Said. Transcriptome sequencing can be performed by the following additional steps: (i) loading a cleavage reagent into the channel, which ruptures the cell membrane so that messenger RNA (mRNA) is released and captured by the capture element; (ii) loading a transcription reagent into the channel to replicate the captured mRNA, thereby producing a complementary DNA of the captured mRNA; and (iii) sequencing the complementary DNA to identify the captured mRNA. In some embodiments, a washing step can be included to remove the cleavage reagent, for example, before loading the transcription reagent, or before loading the amplification reagent, or before loading the sequencing reagent, etc. In some embodiments, the reverse transcription reagent includes a reverse transcriptase. In some embodiments, such a reverse transcriptase is an MMLV reverse transcriptase. As mentioned above, cDNA sequencing can be performed "in situ" or outside the system. In situ sequencing can be performed by the following additional steps: (a) amplifying complementary DNA; (b) sequencing the amplified complementary DNA, for example, using a sequencing-by-synthesis technique. After sequencing, the relative expression level of the mRNA can be determined, thereby providing a transcriptome. In some embodiments, external or off-channel sequencing requires that the capture element include a spatial barcode as described in the examples above. The cDNAs can optionally be amplified before they are eluted and sequenced by an external sequencing instrument.

[0092] Hydrogel chamber

[0093] FunctionA variety of photosynthesizable gels can be used in conjunction with the present invention. In some embodiments, hydrogels are used with the present invention, particularly because they are compatible with living cells and are widely used in formulating gels with desired properties, including but not limited to porosity (which largely determines the contents and passage of materials through the gel (or polymer matrix) wall), degradability, mechanical strength, ease and speed of synthesis, etc.

[0094] Porosity . In some embodiments, the hydrogel porosity is selected to allow the selected reagent to pass through while preventing other reagents or objects (such as cells) from passing through. In some embodiments, the hydrogel porosity is selected to prevent biological cells from passing through, but allows reagents (including proteins, such as polymerases) to pass through. In some embodiments, such reagents that can penetrate the polymer matrix wall include lysozyme, proteinase K, random hexamer, polymerase, transposase, ligase, deoxynucleotide triphosphate, buffer, cell culture medium or divalent cations. In some embodiments, at least one polymer matrix includes a pore, which is sized to allow the reagent to diffuse through at least one polymer matrix, but is too small to allow the DNA or RNA for analysis to cross the pore (size greater than 100 nucleotides or base pairs, or greater than 300 nucleotides or base pairs). In some embodiments, the polymer chains of the cross-linked hydrogel structure form a hydrogel matrix (i.e., a porous hydrogel matrix) with pores. In some versions, the size of the pores in the hydrogel structure can be regulated or tuned, and can be formulated to encapsulate sufficiently large genetic material (such as cells or nucleic acids) (e.g., greater than about 300 base pairs), but allow smaller substances (such as reagents) or nucleic acids of smaller size (e.g., less than about 50 base pairs) (such as primers) to pass through the pores, thereby entering and exiting the hydrogel structure. In some embodiments, the hydrogel can have any diameter sufficient to allow the reagents listed above to diffuse through the structure while retaining a pore size of nucleic acid molecules greater than 500 nucleotides or base pairs in length. In some embodiments, when the hydrogel is hydrated, the hydrogel structure can expand. Then, the size of the pores can be changed depending on the water content in the hydrogel of the hydrogel structure. In some embodiments, the diameter of the pores is from about 10nm to about 100nm. In some embodiments, the pore size of the hydrogel structure can be tuned by routine experiments by changing the ratio of the concentration of the polymer precursor to the concentration of the cross-linking agent, changing pH, salt concentration, temperature, light intensity, etc. In some embodiments, the average diameter of the pores of the polymer matrix wall prevents the passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater, a molecular weight of 50 kDa or greater, a molecular weight of 75 kDa or greater, a molecular weight of 100 kDa or greater, or a molecular weight of 150 kDa or greater.

[0095] In some embodiments, the length of the retained DNA or RNA can be sequenced using conventional sequencing by synthesis techniques. For example, such DNA or RNA comprises at least 50 nucleotides, or in some embodiments, at least 100 nucleotides. In some embodiments, the average diameter of the pore can be 5nm to 100nm. In some embodiments, the average diameter of the pore can be 5nm to 10nm, 10nm to 20nm, 20nm to 30nm, 30nm to 40nm, 50nm to 60nm, 60nm to 70nm, 70nm to 80nm, 80nm to 90nm, 90nm to 100nm. In some embodiments, the average diameter of the pore can be greater than 100nm. In some embodiments, the average diameter of the pore can be less than 5nm. Reagents can include enzymes or primers with a size less than 50 base pairs (bp). Primers can include single-stranded DNA (ssDNA). In some embodiments, the size of primers can be 5bp to 50bp. In some embodiments, the size of the primer can be 5bp to 10bp, 10bp to 20bp, 20bp to 30bp, 30bp to 40bp or 40bp to 50bp. In some embodiments, the size of the primer can exceed 50bp. In some cases, the size of the primer can be less than 5bp. In some embodiments, the diameter of the pore can be 5nm to 100nm. In some embodiments, the diameter of the pore can be 5nm to 10nm, 10nm to 20nm, 20nm to 30nm, 30nm to 40nm, 50nm to 60nm, 60nm to 70nm, 70nm to 80nm, 80nm to 90nm, 90nm to 100nm. In some embodiments, the diameter of the pore can be greater than 100nm. In some embodiments, the average diameter of the pore can be less than 5nm. The pore size of the polymer matrix can be from about 5 nanometers (nm) to about 100nm.The pore size of the polymer matrix can be from about 5 nm to about 10 nm, from about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, from about 5 nm to about 100 nm, from about 5 nm to about 110 nm, from about 10 nm to about 20 nm, from about 10 nm to about 30 nm, from about 10 nm to about 40 nm, from about 10 nm to about 50 nm, from about 10 nm to about 60 nm, from about 10 nm to about 70 nm, nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 10 nm to about 100 nm, about 10 nm to about 110 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 20 nm to about 110 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 20 nm to about 110 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm about 70nm, about 30nm to about 80nm, about 30nm to about 90nm, about 30nm to about 100nm, about 30nm to about 110nm, about 40nm to about 50nm, about 40nm to about 60nm, about 40nm to about 70nm, about 40nm to about 80nm, about 40nm to about 90nm, about 40nm to about 100nm, about 40nm to about 110nm, about 50nm to about 60nm, about 50nm to about 70nm, about 50nm to about 80nm, about 50nm to about 90nm, about 50nm to about 100nm The pore size of the polymer matrix can be about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 60nm to about 110nm, about 70nm to about 80nm, about 70nm to about 90nm, about 70nm to about 100nm, about 70nm to about 110nm, about 80nm to about 90nm, about 80nm to about 100nm, about 80nm to about 110nm, about 90nm to about 100nm, about 90nm to about 100nm, or about 100nm to about 110nm. The pore size of the polymer matrix can be about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm or about 110nm. The pore size of the polymer matrix may be at least about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, or less.The pore size of the polymer matrix may be up to about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or more.

[0096] Modulation of porosity . The pore size in the polymer matrix can be adjusted using chemical reagents or by applying heat, electric field, light or another suitable stimulus. In other words, the polymer matrix can include tunable properties (e.g., pore size). In some cases, the polymer matrix can include thermoresponsive or temperature responsive polymers. Thermoresponsive polymers (e.g., poly (N-isopropylacrylamide) (NIPAAM)) may phase separate with the solution after heating or after cooling (e.g., polymers showing lower critical solution temperature (LCST) or upper critical solution temperature (UCST)). The polymer matrix can include polymers that can collapse at high temperatures, such as to control the pore size of the hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that can be used to form a hydrogel / polymer matrix with tunable properties can include poly (N-vinyl caprolactam), poly (N-ethyl oxazoline), poly (methyl vinyl ether), poly (acrylic acid-acrylamide) copolymers or combinations thereof. Changes in temperature can enlarge or shrink the average pore size in the polymer matrix to allow selected molecules such as nucleic acid molecules, proteins or any biomolecules or molecules smaller than the adjusted pore size to be released from the hydrogel chamber.

[0097] Size and shape of hydrogel chambers. In some embodiments, the polymer matrix wall of the chamber inhibits the passage of predetermined components, such as mammalian cells, genomic DNA, larger polynucleotides (e.g., mRNA greater than 200 ribonucleotides, greater than 300 ribonucleotides or 500 ribonucleotides), etc. In some embodiments, the polymer matrix wall extends from the first surface to the second surface (parallel to the first surface) to form a chamber in the channel. In some embodiments, the chamber has a polymer matrix wall and an interior. In some embodiments, the size of the interior of the chamber is designed to encapsulate cells. For example, such chambers may include a cylindrical shell or a polygonal shell, which includes an interior space or an interior and a polymer matrix wall. In some embodiments, such chambers have a ring-like cross section. As used herein, the term "ring-like cross section" means a cross section that is topologically equivalent to a ring. In some embodiments, the inner diameter of the inner space or interior of the chamber is 1 μm to 500 μm, and the volume of the inner space or interior of the chamber ranges from 1 picoliter to 200 nanoliters, 100 picoliters to 100 nanoliters, or 100 picoliters to 10 nanoliters. In some embodiments, the thickness of the polymer matrix wall is at least 1 μm (micrometer). In some embodiments, the value of the height of the chamber with a ring-like cross-section ranges from 10 μm to 500 μm or ranges from 50 μm to 250 μm. In some embodiments, the aspect ratio (i.e., height / width) of the polymer matrix wall with a ring-like cross-section is 1 or less. In some embodiments, the aspect ratio and polymer matrix wall thickness are selected to maximize the chamber stability against forces such as reagent flow through the channel, washing, etc. In some embodiments, at least one polymer matrix wall is a hydrogel wall. In some embodiments, at least one polymer matrix is ​​degradable. In some embodiments, the degradation of at least one polymer matrix is ​​"as needed". In some embodiments, the chamber in the channel is discontinuous. In some embodiments, a chamber in a channel can abut an adjacent chamber. In some embodiments, the chambers can share polymer matrix walls with each other. In some embodiments, the chambers can be synthetic, with slits or other orifices large enough to allow certain components (e.g., beads) to pass through, but small enough to prevent other components (e.g., cells) from passing through.

[0098] Hydrogel composition. In some embodiments, the channel of the fluid device of the system of the present invention comprises one or more polymer precursors for forming a chamber. In some embodiments, the one or more polymer precursors comprise a hydrogel precursor. Such precursors can be selected from a variety of compounds, including but not limited to polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), Poly (L-aspartic acid), poly (L-glutamic acid), polylysine, agar, agarose, alginate, heparin, sulfated alginate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate or ethoxylated tetraacrylate pentaerythritol ester or a combination or mixture thereof. In some embodiments, the hydrogel comprises an enzymatically degradable hydrogel, PEG thiol / PEG-acrylate, acrylamide / N, N '-bis (acryloyl) cystamine (BACy) or PEG / PPO. In some embodiments, the following precursors and cross-linking agents can be used to form a chamber with a degradable polymer matrix (hydrogel) wall. The polymer precursor can be formed by using any of the hydrogel precursors and crosslinking agents of Table 2A (columns 1 and 3, respectively). The resulting polymer matrix can be degraded using a degradation agent (column 4) as indicated in Table 2A.

[0099] Table 2A

[0100]

[0101] Table 2B

[0102]

[0103] Hydrogel degradation. In some embodiments, the hydrogel chambers of the present invention are generally degradable or depolymerizable within the channel or "as needed" within the channel. Generally, the degradable hydrogel chambers are degraded by treatment with a degradation agent or equivalently exposed to a depolymerizing agent in all chambers within the channel. Exemplary depolymerizing agents include, but are not limited to, heat, light, and / or chemical depolymerizing agents (sometimes also referred to as cutting agents or degradation agents). In some embodiments, polymer precursors that allow photocrosslinking and photodegradation can be used according to the degradation required, for example, different wavelengths are used for crosslinking and degradation to achieve. For example, Eosin Y can be used for free radical polymerization at a defined area using a 500nm wavelength, after which 380nm irradiation can be used to cut the crosslinker. In other embodiments, a light cage hydrogel cutting agent can be included in the formation of the polymer matrix wall. For example, an acid-labile crosslinker (such as an ester, etc.) can be used to produce a hydrogel, and then UV light can be used to generate local acidic conditions, which in turn degrade the hydrogel. In some embodiments, at least one polymer matrix can be degraded by at least one of the following: (i) contacting at least one polymer matrix with a cleavage agent; (ii) heating at least one polymer matrix to at least 90°C; or (iii) exposing at least one polymer matrix to a certain wavelength of light that cuts a photo-cleavable cross-linking agent that cross-links the polymers of at least one polymer matrix. In some embodiments, at least one polymer matrix comprises a hydrogel. In some embodiments, the cleavage agent degrades the hydrogel. In some embodiments, the cleavage agent includes a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage agent, or a combination thereof. In some embodiments, the cleavage agent includes dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THP), or a combination thereof. In some embodiments, the surface of the polymer matrix or hydrogel can be functionalized by coupling functional groups to the polymer matrix or hydrogel. Some non-limiting examples of functional groups can include capture reagents (e.g., picolinaldehyde (PCA)), acrylamide, agarose, biotin, streptavidin, strep-tag II, linkers, functional groups comprising aldehydes, phosphates, silicates, esters, acids, amides, aldehyde dithiolanes, PEGs, thiols, olefins, alkynes, azides, or combinations thereof. In some cases, the functionalized polymer matrix can be used to capture biomolecules within the polymer matrix compartments formed adjacent to (e.g., around or on) the biological component. The biomolecules can be produced by the biological component (e.g., from the secretome of the cell). The functionalized surface of the polymer matrix within the compartment can be used to capture reagents or molecules from outside the compartment. The functionalized surface can increase the surface area covered by reagents, molecular sensors, or any molecules of interest (e.g., antibodies).

[0104] Partial degradationIn some embodiments, the existing polymer matrix wall can be partially degraded, for example to change the porosity. In some embodiments, the polymer precursor can include degradable beads that form part of the polymer matrix wall and are embedded therein when synthesized and can then be degraded as needed or routinely, resulting in an increase in porosity.

[0105] Photosynthesis . In some embodiments, generating a polymer matrix in the fluid device includes exposing one or more polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light of 350nm to 800nm. In some embodiments, the light generating device generates light of 350nm to 600nm. In some embodiments, the light generating device generates light of 350nm to 450nm. In some embodiments, the light generating device generates UV light. In some embodiments, generating a polymer matrix in the fluid device is performed using a spatial light modulator (SLM) (i.e., a spatial energy modulation element capable of spatially generating a desired light intensity pattern). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam manipulated using a galvanometer. In some embodiments, the SLM is based on liquid crystal.

[0106] Systems and Instruments

[0107] In some embodiments, the system of the present invention includes: (a) a channel including a first surface, a plurality of cells arranged on the first surface, and one or more polymer precursors; (b) a spatial energy modulation element, the spatial energy modulation element being optically connected to the first surface; (c) a detector, the detector being optically connected to the first surface and operably associated with the spatial energy modulation element, the detector detecting each cell in the plurality of cells and determining its position on the first surface; and (d) a plurality of gel chambers, each gel chamber encapsulating a single cell in the plurality of cells, wherein the gel chamber is synthesized by projecting light into the channel using the spatial energy modulation element so that the projected light causes one or more polymer precursors to crosslink to form a polymer matrix wall of the chamber, wherein the position of the synthesized chamber is determined by the position of the cell encapsulated by the synthesized chamber identified by the detector. It should be understood that the term "detector" as used herein may include, but is not limited to: a microscope element that collects and optionally amplifies an image of a portion of the channel; and an image analysis element that includes software for identifying cells, cell features, chambers, and other objects so as to store such information and associated position information. The computer element uses such information generated by the detectors, as well as user input, to generate commands to other elements (such as the spatial energy modulation element) to perform a variety of functions, including but not limited to synthesis chambers, "on demand" degradation of chambers, photolysis of cells, etc. Exemplary configurations of such embodiments are described above. Figure 5A-Figure 5B In some embodiments, the channel of the fluidic device further includes a second surface, wherein the first surface and the second surface are arranged relative to each other across the channel, and wherein the polymer matrix wall of the chamber extends from the first surface to the second surface to form a chamber each having an interior. In some embodiments, the chambers in the channel each encapsulate a single cell. In some embodiments, both the first wall and the second wall are made of a light-transmitting material (such as glass, plastic, etc.), and are positioned so that the first surface and the second surface are substantially parallel to each other. The vertical distance between the first surface and the second surface can be in the range of 10 μm to 500 μm or in the range of 50 μm to 250 μm. In some embodiments, the vertical distance between the first surface and the second surface can be in the range of twice the average size of the cells to be analyzed to five times the average size of the cells to be analyzed.

[0108] In other embodiments, the first surface may include a capture element for capturing cells at a predetermined location. For example, the capture element may include, but is not limited to, a capture antibody specific for all cells or a subset of cells. The capture element may also include, but is not limited to, a non-specific capture material such as polylysine, fibronectin, treated plastics (e.g., Maxysorb TMPlastics, ThermoFisher), etc. In some embodiments, such cell capture moieties (e.g., antibodies) can be confined to points or reaction sites arranged in a regular pattern on the first surface; therefore, cells captured at such reaction sites can be arranged in a regular pattern on the first surface, which may be more effective than random arrangement for chamber synthesis and / or optical signal detection. Guidance on providing cell capture antibodies for surfaces can be found in the following references: Zhu et al., Analytica Chemica Acta, 608: 186-196 (2008); Sekine et al., J. Immunol. Methods, 313 (1-2): 96-109 (2006); etc. In some embodiments, the diameter of such reaction sites or points is in the range of 5-500 μm or in the range of 10-1000 μm. In some embodiments, such points or reaction sites are arranged in a linear array, or in a hexagonal array. In some embodiments, the density of such arrays of such points or reaction sites ranges from 10 to 2500 sites / mm 2 , 10 to 1000 parts / mm 2 , 10 to 500 parts / mm 2 or 10 to 100 sites / mm 2 .

[0109] In some embodiments, the cells may be randomly arranged on the first surface. In some embodiments, the cells are randomly arranged on the first surface in a Poisson distribution. In some embodiments, in such a Poisson distribution, the nearest neighbor distance of the cells is equal to or greater than 10 μm, equal to or greater than 20 μm, equal to or greater than 30 μm, equal to or greater than 40 μm, equal to or greater than 50 μm, or equal to or greater than 100 μm. In some embodiments, a subset of such Poisson distributed cells is each encapsulated by a quasi-annular chamber with a diameter in the range of 10-500 μm. In some embodiments, the cells are randomly arranged on the first surface in a Poisson distribution with a density of 10 to 2500 cells / mm 2 , or 10 to 1000 cells / mm 2 , or 10 to 500 cells / mm 2 , or 10 to 100 cells / mm 2 within the range.

[0110] In some embodiments, multiple channels can be Figure 6A-6B6. The plurality of channels may be arranged together in a flow channel as shown. In some embodiments, the plurality of channels may be in the range of 2 to 12, or 2 to 8, or 2 to 6, or in the range of 2 to 4. An exemplary flow cell (600) is shown in a cross-sectional view and a top view. The flow cell (600) has: a bottom wall or first wall (606) having a first surface (605); a top wall or second wall (602) having a second surface (601); and a spacer (604) sealingly sandwiched between the bottom wall or first wall (606) and the top wall or second wall (602), the longitudinal holes of the spacer (604) forming channels 1-6, one of which is represented by (608) in the cross-sectional view and by (612) in the top view. In some embodiments, the thickness of the spacer (604) can be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm, which determines the internal height of the channel. The top wall (602) includes an inlet (614) and an outlet (616), which are used to load and remove reagents and cells from channel 1-channel 6 individually or collectively. In some embodiments, at least one of the wall (602) and the wall (606) is made of a light-transmitting material (such as glass, plastic, etc.). The flow cell (600) can be operationally associated with a fluid device that delivers reagents and cells to any one of channel 1-channel 6 under program control. Guidelines for specific designs (including fluid handling and valve adjustment of such fluid systems) can be found in U.S. Patents 8921073, 8173080 and 8900828, etc., which are incorporated herein by reference. Figure 6B A channel of a flow cell (600) is shown having randomly distributed (not to scale) hydrogel chambers having a quasi-ring-shaped cross-section, such as (620), on a first surface thereof.

[0111] As described above, any of the first surface, second surface, or polymer matrix wall of the chamber may include capture elements and other functional groups for performing various operations, including but not limited to capturing cells, capturing components of cells (such as mRNA, secreted proteins, intracellular proteins, or genomic sequences), capturing components of analytical reagents (such as oligonucleotide tags from antibodies), etc. Derivatized surfaces for such purposes are well known to those skilled in the art, as demonstrated by the following exemplary references: Zhu et al. (cited above); Sekine et al. (cited above); Integrated DNA Technologies brochure (cited above); Hermanson (cited above); etc.

[0112] As described above, in some embodiments, the fluid device of the method includes a detector or is operationally associated with a detector, which can share the optical path of the spatial energy modulation element, or can be arranged as follows in an embodiment (such as a groove) having only a first wall and a first surface: near the second wall or the first wall opposite to the spatial energy modulation element. The detector is positioned so that it can detect light signals from cells in the channel or near cells in the channel, for example, the cells are distributed on the first surface in the chamber. In some embodiments, the first wall and the second wall each include a light-transmitting material, for example, so that the spatial energy modulation element can project light energy into the interior of the channel, and so that the detector can detect light signals (such as fluorescent emission or reflected light from biological components). In some embodiments, the energy projected from the spatial energy modulation element is light energy from a light beam. In some embodiments, the light beam projected by the spatial energy modulation element can have a complex cross-section, thereby allowing (in various embodiments) to synthesize multiple chambers simultaneously. Light-transmitting materials include, but are not limited to, materials such as glass, quartz, plastic, etc. In some embodiments, the step of synthesizing the chamber includes positioning the chamber so that they encapsulate one or more biological components based on the light signal detected by the detector. That is, in some embodiments, the detector is operatively associated with the spatial energy modulation element to selectively project one or more light beams to locations where detected light signals indicate the presence of cells of interest. In such embodiments, the detector and the spatial energy modulation element are operatively associated so that the spatial energy modulation element is configured to generate an energy beam having predetermined light beam characteristics. For example, one such characteristic may be a beam cross-section that results in the biological component of interest being enclosed by a toroidal-shaped chamber. In such an operative association, the light signals detected by the detector may include, but are not limited to: the morphology of the biological component, such as cell morphology; cell motility; the interaction of one cell type with another cell type, such as the binding of one cell type to another cell type; the presence, absence or quantity of markers on cells, etc.

[0113] The spatial energy modulation element for achieving polymerization using light energy may include a physical photomask or a virtual photomask, such as a digital micromirror device (DMD). The following references are incorporated by reference to provide guidance in selecting and operating a DMD for achieving photopolymerization of gels: Chung et al., U.S. Patent 10464307; Hribar et al., U.S. Patent 10351819; Das et al., U.S. Patent 9561622; Huang et al., Biomicrofluidics, 5:034109 (2011); etc.

[0114] Although the invention has been described with reference to several specific example embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the invention. The invention is applicable to various sensor embodiments and other subject matter in addition to those discussed above.

[0115] definition

[0116] Unless otherwise specifically defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow standard treatises and texts in the field, e.g., Kornberg and Baker, DNA Replication, 2nd edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd edition (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immuology, 6th edition (Saunders, 2007).

[0117] "Chimeric antigen receptor T cell" (CAR-T cell) means a T cell engineered to express an antigen-specific receptor formed by fusing an antigen-binding component with a transmembrane and signaling component, which is capable of initiating a cytotoxic T cell response whenever the antigen-specific component binds to its target. Exemplary references for teaching the manufacture and use of CAR-T cells include the following references incorporated herein by reference: U.S. Patent 8,822,647; and U.S. Patent Publications 2014 / 0134142, 2014 / 0314795, 2012 / 0148552, 2013 / 0288368; etc.

[0118] "Cleavable linkage" or "cleavable nucleotide" means any of a variety of cleavable linkages, or more specifically, cleavable nucleotides can be used with embodiments of the present invention. As used herein, the term "cleavable site" refers to a nucleotide or backbone linkage of a single-stranded nucleic acid sequence that can be excised or cut under predetermined conditions, thereby dividing the single-stranded nucleic acid sequence into two parts. In some embodiments, the step of cutting the cleavable nucleotide or cleavable linkage leaves a free 3'-hydroxyl on the cut chain, thereby, for example, allowing the cut chain to be extended by a polymerase. The cutting step can be performed chemically, thermally, enzymatically, or by light-based cutting. Sometimes the term "release" can be used to refer to the cutting of an oligonucleotide tag, such as by a releasing agent or a releasing agent, which can be one or more of those listed above. In some embodiments, the cleavable nucleotide can be a nucleotide analog (such as deoxyuridine or 8-oxodeoxyguanosine) that is recognized by a specific glycosylase (e.g., uracil deoxyglycosylase, followed by endonuclease VIII and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, cleavage by glycosylases and / or endonucleases may require a double-stranded DNA substrate. Methods for synthesizing and cleaving nucleic acids containing chemically cleavable, thermally cleavable, and photolabile groups are described, for example, in U.S. Pat. No. 5,700,642, which is incorporated herein by reference. Further cleavable linkages are disclosed in the following references: Pon, R., Methods Mol. Biol. 20: 465-496 (1993); Verma et al., Ann. Rev. Biochem. 67: 99-134 (1998); U.S. Patent Nos. 5,739,386, 5,700,642 and 5,830,655; and U.S. Patent Publication Nos. 2003 / 0186226 and 2004 / 0106728; Urdea et al., U.S. Patent No. 5367066, which are incorporated herein by reference. The synthesis and cleavage conditions of chemically cleavable oligonucleotides are described in U.S. Patent Nos. 5,700,642 and 5,830,655. Phosphorothioate internucleotide linkages can be selectively cleaved under mild oxidative conditions. Selective cleavage of phosphoramide bonds can be performed under mild acidic conditions (such as 80% acetic acid). Selective cleavage of the ribose can be performed by treatment with dilute ammonium hydroxide. In another embodiment, the cleavable linking portion can be an amino linker. The resulting oligonucleotide bound to the linker via a phosphoramidite linkage can be cleaved with 80% acetic acid to produce a 3'-phosphorylated oligonucleotide, which can be removed (if necessary) by a phosphatase. In some embodiments, the cleavable linking portion can be a photocleavable linker, such as an o-nitrobenzyl photocleavable linker.For example, in Venkatesan et al., J.Org.Chem.61:525-529 (1996), Kahl et al., J.Org.Chem.64:507-510 (1999), Kahl et al., J.Org.Chem.63:4870-4871 (1998), Greenberg et al., J.Org.Chem.59:746-753 (1994), Holmes et al., J.Org.Chem.62:2370-2380 (1997) and U.S. Patent No. 5,739,386, the synthesis and cleavage conditions of light-labile oligonucleotides on solid supports are described. Linkers based on o-nitrobenzyl (such as hydroxymethyl, hydroxyethyl and Fmoc-aminoethyl carboxylic acid linkers) can also be obtained commercially. In some embodiments, ribonucleotides can be used as cleavable nucleotides, wherein the cleavage step can be implemented using ribonucleases (such as RNase H). In other embodiments, the cleavage step can be performed by treatment with a nicking enzyme.

[0119] "Hydrogel" means a gel comprising a cross-linked hydrophilic polymer network that can absorb and retain large amounts of water (e.g., 60% to 90% water, or 70% to 80%) without dissolving due to the establishment of physical or chemical bonds (which can be covalent, ionic or hydrogen bonds) between polymer chains. Hydrogels exhibit high permeability to oxygen and nutrients, making them attractive materials for cell encapsulation and culture applications. Hydrogels can comprise natural or synthetic polymers and can be reversible (i.e., degradable or depolymerizable) or irreversible. Exemplary synthetic hydrogel polymers include polyethylene glycol (PEG), poly (2-hydroxyethyl methacrylate) and poly (vinyl alcohol). Exemplary natural hydrogel polymers include alginate, hyaluronic acid and collagen. The following references describe hydrogels and their biomedical uses: Drury et al., Biomaterials, 24:4337-4351 (2003); Garagorri et al., Acta Biomatter, 4(5):1139-1147 (2008); Caliari et al., Nature Methods, 13(5):405-414 (2016); Bowman et al., U.S. Patent 9631092; Koh et al., Langmuir, 18(7):2459-2462 (2002).

[0120] The term "as needed" means that operations can be directed to individual, discrete, selected locations (e.g., spatial location of a polymer precursor solution; or a selected polymer matrix chamber). Such selection can be based on manual observation of optical signals or data collected by a detector, or such selection can be based on a computer algorithm that operates on optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include real-time detection or detection of a certain period of time before modulating unit energy to polymerize a polymer precursor or degrading a chamber. For example, a subset of chambers (all formed with photodegradable polymer matrix walls) can be preselected based on position information and the value of the optical signal from the analytical determination performed in the chamber for release and removal of its contents. The preselected chamber can be photodegraded by selectively projecting a beam of light of appropriate wavelength characteristics (e.g., with a spatial energy modulation element) to degrade the polymer matrix wall of the preselected chamber. In another example, multiple chambers can be observed in real time (e.g., via fluorescence microscopy) to detect an analyte of interest, and one or more chambers in multiple chambers can be selected in real time after detecting the analyte of interest for degradation.

[0121] A "physical photomask" generally refers to a physical structure having a plurality of openings or holes through which light can be projected. A physical photomask can be used to produce a hydrogel matrix as described herein by polymerizing a polymer precursor solution and forming a three-dimensional structure corresponding to the pattern on the photomask. A physical photomask can be patterned with a specific layout or geometric pattern. A physical photomask can be adhered to the upper surface of a flow cell.

[0122] "Polymerase chain reaction" or "PCR" means a reaction in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary DNA strands. In other words, PCR is a reaction for preparing multiple copies or replications of a target nucleic acid flanked by primer binding sites, such a reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing the primer to the primer binding site, and (iii) extending the primer by a nucleic acid polymerase in the presence of nucleoside triphosphates. Typically, the reaction is cycled in a thermal cycler at different temperatures optimized for each step. The specific temperature, duration of each step, and rate of change between steps depend on many factors well known to those of ordinary skill in the art, such as those illustrated in the following references: McPherson et al., eds., PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in conventional PCR using Taq DNA polymerase, double-stranded target nucleic acid can be denatured at a temperature of>90°C, primers are annealed at a temperature in the range of 50-75°C, and primers are extended at a temperature in the range of 72-78°C. The term "PCR" covers derivative forms of reactions, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, etc. The reaction volume ranges from several hundred nanoliters (e.g., 200 nL) to several hundred microliters (e.g., 200 μL). "Reverse transcription PCR" or "RT-PCR" means PCR performed before the reverse transcription reaction, which converts the target RNA into a complementary single-stranded DNA, which is then amplified, such as Tecott et al., U.S. Patent No. 5,168,038, which is incorporated herein by reference. "Real-time PCR" or "quantitative PCR" means PCR that monitors the amount of the reaction product (i.e., amplicon) as the reaction proceeds. There are many forms of real-time PCR, which differ primarily in the detection chemistry used to monitor the reaction products, such as Gelfand et al., U.S. Pat. No. 5,210,015 ("taqman"); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons); these patents are incorporated herein by reference. Detection chemistry for real-time PCR is reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002), which is also incorporated herein by reference.

[0123] "Polymer matrix" generally refers to a phase material (e.g., continuous phase material) comprising at least one polymer. In some embodiments, polymer matrix refers to at least one polymer and interstitial space not occupied by polymer. Polymer matrix can be composed of one or more types of polymers. Polymer matrix can include straight chain, branched and cross-linked polymer units. Polymer matrix can also include non-polymer species inserted into the interstitial space not occupied by polymer chains. The inserted species can be solid, liquid or gas species. For example, the term "polymer matrix" can encompass dry hydrogels, hydrated hydrogels and hydrogels containing glass fibers. Polymer matrix can include polymer precursors, which generally refer to one or more molecules that can trigger or initiate polymerization when activated. Polymer precursors can be activated by electrochemical energy, photochemical energy, photons, magnetic energy or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (which are polymerized to produce polymer matrix) and cross-linked compounds, which can include photoinitiators, other compounds necessary or useful for generating polymer matrix (especially the polymer matrix of hydrogel).

[0124] "Transfection" and / or "conversion" and / or "transduction" are used synonymously herein, meaning that exogenous genetic material is transferred to a target mammalian cell. Such transfer may result in temporary or transient expression of a transgenic or temporary or transient transcription of RNA, for example due to exhaustion of genetic material, loss or degradation of genetic material, lack of replication of genetic material, etc. In some embodiments, "transfection" means "stable transfection", because the latter term is a commonly used term, such as Kim et al., Anal. Bioanal. Chem., 379: 3173-3178 (2010). Exogenous genetic material may include plasmids, viral vectors, transgenics, transposons, etc. "Stable" as used herein means that exogenous genetic material persists through multiple cell divisions or persists throughout the life of a cell host. Exogenous genetic material may be integrated into the genome of a target mammalian cell, or it may include episomal DNA, such as a plasmid.

Claims

1. A method for determining one or more cell characteristics, the method comprising: (a) synthesizing one or more hydrogel chambers, wherein a hydrogel chamber of the one or more hydrogel chambers encapsulates cells disposed on a surface of a channel; (b) lysing the cells so that the genomic DNA of the cells is released into the hydrogel compartment of the cells; (c) amplifying the genomic DNA of the cell to obtain amplified genomic DNA; as well as (d) using the amplified genomic DNA, measuring the viral copy number, viral integration site or genomic copy number variation of the cell.

2. The method according to claim 1, wherein the measuring further comprises: (i) annealing vector-specific primers to the amplified genomic DNA; (ii) extending the vector-specific primer to obtain an extension product comprising a copy of the segment of the genomic DNA; and (iii) identifying one or more integration sites of the vector-specific primers and the genomic DNA from the segments.

3. The method of claim 1, wherein the cell is a mammalian cell, and wherein the measuring further comprises: (i) sequencing one or more fragments of the amplified genomic DNA to obtain sequences of the one or more fragments; and (ii) determining the genomic copy number variation of the cell from the sequence of the one or more fragments.

4. The method of claim 3, wherein the sequencing comprises obtaining a sequence coverage of the amplified genomic DNA of 0.25X or greater.

5. The method of claim 3, wherein the determination of the genomic copy number variation has a resolution of 3 megabases or higher.

6. A method of determining one or more cellular characteristics of one or more cells, the method comprising: (a) providing a fluidic device, the fluidic device comprising: (i) a channel, the channel comprising a first surface, the one or more cells, and one or more polymer precursors, wherein the one or more cells are arranged on or near the first surface; (ii) a spatial energy modulation element, the spatial energy modulation element being in optical communication with the first surface; and (iii) a detector; (b) identifying the location of the one or more cells in the channel with the detector; (c) projecting energy into the channel using the spatial energy modulation element such that the projected energy causes the one or more polymer precursors to form polymer matrix walls of one or more chambers, wherein the one or more chambers at least partially enclose the one or more cells at the locations identified by the detector; (d) loading one or more assay reagents into the channel; and (e) incubating the one or more cells under assay conditions to generate signals from the one or more compartments indicative of one or more cellular characteristics of the cells encapsulated by the one or more compartments.

7. The method of claim 6, wherein (i) the one or more cell characteristics comprise proliferation rate, (ii) the incubation under the assay conditions comprises incubation under growth conditions, and (iii) the method further comprises: After said incubating, a proliferation rate of said one or more cells at least partially enclosed by said one or more chambers is determined.

8. The method of claim 7, wherein the determining of the proliferation rate comprises counting the one or more cells at least partially enclosed by the one or more chambers.

9. The method of claim 7, wherein the one or more cell characteristics further comprise a profile of cell membrane proteins, and wherein the method further comprises incubating the one or more cells with antibodies, each of the antibodies being specific for a different cell surface protein, the relative expression of the different cell surface proteins allowing identification of the one or more cells, and wherein each of such antibodies has a different label.

10. The method of claim 9, wherein the first surface comprises one or more capture elements for capturing one or more assay components or one or more components of the one or more cells.

11. The method of claim 10, wherein each of the antibodies has an oligonucleotide tag comprising an antibody-specific barcode capable of being captured by the one or more capture elements.

12. The method according to claim 11, wherein the method further comprises: (i) providing the oligonucleotide tag, wherein the oligonucleotide tag is attached to the antibody via a cleavable linkage; (ii) loading the channel with a release agent to cleave the scissile linkage such that the oligonucleotide tag is released and captured by the capture element; (iii) replicating the captured oligonucleotide tag to generate complementary DNA of the captured oligonucleotide tag; and (iv) sequencing the complementary DNA to identify the captured oligonucleotide tag.

13. The method of claim 7, wherein the first surface comprises one or more capture elements for capturing one or more assay components or components of the one or more cells, wherein the one or more cell characteristics further comprise a cell transcriptome, and wherein the method further comprises: (i) loading a lysis reagent into the channel so that messenger RNA of the one or more cells is released and captured by the one or more capture elements; (ii) loading a reverse transcription reagent into the channel to replicate the captured messenger RNA to produce complementary DNA; and (iii) sequencing the complementary DNA.

14. A method according to claim 7, wherein (i) the one or more cellular characteristics further include a spectrum of proteins secreted by the one or more cells, (ii) the channel further includes a protein capture surface, the protein capture surface includes a protein affinity reagent that binds to the proteins secreted by the one or more cells, and (iii) the method further includes using a certain amount of labeled protein detection antibodies to detect the proteins secreted by the one or more cells and bound to the protein capture surface adjacent to the one or more cells.

15. The method of claim 6, wherein (i) the one or more cellular properties include cytotoxicity, (ii) the first surface includes target cells disposed on the first surface, (iii) the method further includes loading the channel with effector cells such that the effector cells are disposed on or near the first surface, (iv) the incubating step includes incubating the effector cells and the target cells with a vital dye that generates an optical signal in dead cells but not in live cells; and (v) the dead cells in each of the one or more chambers are counted to determine a cytotoxicity value for the effector cells encapsulated by each of the one or more chambers.

16. The method of claim 15, wherein the first surface comprises one or more capture elements for capturing one or more assay components or one or more components of the one or more cells.

17. The method of claim 16, wherein the one or more cell characteristics further comprise a profile of cell membrane proteins, wherein the method further comprises incubating the one or more cells with antibodies, each of which is specific for a different cell surface protein, the relative expression of which allows identification of the cells, and wherein each of such antibodies has a different label.

18. The method of claim 17, wherein each of the antibodies has an oligonucleotide tag comprising an antibody-specific barcode capable of being captured by the one or more capture elements.

19. The method according to claim 18, wherein the method further comprises: (i) providing the oligonucleotide tag, wherein the oligonucleotide tag is attached to the antibody via a cleavable linkage; (ii) loading the channel with a release agent to cleave the scissile linkage such that the oligonucleotide tag is released and captured by the capture element; (iii) replicating the captured oligonucleotide tag to generate complementary DNA of the captured oligonucleotide tag; and (iv) sequencing the complementary DNA to identify the captured oligonucleotide tag.

20. The method of claim 10, wherein the first surface comprises one or more capture elements for capturing one or more assay components or components of the one or more cells, wherein the one or more cell characteristics further comprise a cell transcriptome, and wherein the method further comprises: (i) loading a lysis reagent into the channel so that messenger RNA of the one or more cells is released and captured by the one or more capture elements; (ii) loading a reverse transcription reagent into the channel to replicate the captured messenger RNA to produce complementary DNA; and (iii) sequencing the complementary DNA.

21. A method according to claim 10, wherein (i) the one or more cellular characteristics further include a spectrum of proteins secreted by the one or more cells, (ii) the channel further includes a protein capture surface, the protein capture surface includes a protein affinity reagent that binds to the proteins secreted by the one or more cells, and (iii) the method further includes using a certain amount of labeled protein detection antibodies to detect the proteins secreted by the one or more cells and bound to the protein capture surface adjacent to the one or more cells.

22. The method of claim 6, wherein the one or more cellular characteristics comprise the copy number of one or more nucleotide sequences in the genomic DNA of the one or more cells, and wherein the method further comprises: (a) lysing the one or more cells to release the genomic DNA; (b) amplifying the one or more nucleotide sequences to obtain one or more amplified nucleotide sequences; (c) capturing the amplified one or more nucleotide sequences with one or more capture elements disposed on the channel; (d) replicating the one or more amplified nucleotide sequences captured to produce complementary DNA of the one or more amplified nucleotide sequences captured; and (e) sequencing the complementary DNA to identify the copy number of the one or more nucleotide sequences.

23. The method of claim 22, wherein the one or more nucleotide sequences are each a barcode.

24. The method of claim 23, wherein the barcodes each comprise a unique molecular identifier.

25. The method of claim 24, wherein the unique molecular identifier identifies viral DNA integrated into the genomic DNA, and wherein a number of different said unique molecular identifiers identified in a single cell are indicative of the viral copy number of such single cell.

26. The method of claim 6, wherein the one or more cellular characteristics comprise the copy number of one or more nucleotide sequences in the genomic DNA of the one or more cells, and wherein the method further comprises: (a) lysing the one or more cells to release genomic DNA; (b) loading an amplification reagent into the channel, the amplification reagent generating a signal proportional to the copy number of the one or more nucleotide sequences; and (c) amplifying the one or more nucleotide sequences to generate an optical signal proportional to the copy number of the one or more nucleic acid sequences.

27. The method of claim 26, wherein the amplification reagents are quantitative PCR reagents, and wherein the signal is an optical signal.

28. The method of claim 26, wherein (i) the first surface comprises bridge PCR primers, (ii) the amplification reagents comprise bridge PCR reagents, and (iii) the signal is a plurality of clusters formed by bridge PCR.

29. The method of claim 26, wherein the amplification reagent comprises a rolling circle amplification reagent, and wherein the signal is a plurality of DNA nanoballs.

30. The method of claim 6, wherein the one or more cells are randomly arranged on the first surface.

31. The method of claim 6, wherein each of the one or more chambers encapsulates a single cell of the one or more cells.

32. The method of claim 6, wherein the one or more cells comprise cells transduced by a vector, and wherein the one or more cellular characteristics comprise a vector integration site of the cells transduced by the vector, and wherein the method further comprises: (a) lysing the cells transduced by the vector to release the genomic DNA of each cell into the corresponding compartment of each cell; (b) amplifying the released genomic DNA; (c) annealing vector-specific primers to the amplified genomic DNA; (d) extending the vector-specific primer to obtain an extension product comprising a copy of the segment of the genomic DNA; and (e) identifying one or more integration sites of the vector-specific primers and the genomic DNA from the segments.

33. The method of claim 6, wherein the one or more cells are mammalian cells and the one or more cell characteristics comprise genomic copy number variation, and wherein the method further comprises: (a) lysing the one or more cells to release the genomic DNA of each cell into a corresponding compartment of each cell; (b) amplifying the released genomic DNA; (c) sequencing the fragments of the amplified genomic DNA to obtain the sequences of the genomic DNA fragments; and (d) determining the genomic copy number variation of each cell from the sequences of the genomic DNA fragments.

34. The method of claim 33, wherein the sequencing comprises obtaining a sequence coverage of the genomic DNA fragments of 0.25X or greater, and wherein the determination of the genomic copy number variation has a resolution of 3 megabases or greater.

35. A system for measuring a single cell property of a cell population, the system comprising: (a) one or more channels, each of the one or more channels comprising a surface, a plurality of cells disposed on the surface, and one or more polymer precursors; (b) at least one spatial energy modulation element in optical communication with the surface of each channel; (c) at least one detector in optical communication with the surface of each channel and operably associated with at least one spatial energy modulation element, wherein the detector is configured to detect each of the plurality of cells and determine a location of each of the plurality of cells on the surface of at least one channel; as well as (d) a plurality of gel chambers in each channel, wherein each gel chamber encapsulates one or more cells of the plurality of cells, wherein the gel chambers are synthesized by projecting light into the channels using the at least one spatial energy modulation element such that the projected light causes the one or more polymer precursors to form a polymer matrix wall of the gel chamber, and wherein the position of the synthesized chamber is determined in part by the position of the cell encapsulated by the synthesized chamber as identified by the detector.

36. The system of claim 35, wherein the gel chambers are randomly arranged on at least one of the surfaces.

37. The system of claim 35, wherein each of the gel chambers encapsulates a single cell.

38. The system of claim 35, wherein the polymer matrix wall has a molecular weight less than 3×10 6 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 6 Dalton's molecules are impermeable.

39. The system of claim 35, wherein the polymer matrix wall has a molecular weight less than 3×10 5 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 5 Dalton's molecules are impermeable.

40. The system of claim 35, wherein the polymer matrix wall has a molecular weight less than 3×10 4 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 4 Dalton's molecules are impermeable.

41. The system of claim 35, wherein the polymer matrix wall has a molecular weight less than 3×10 3 Dalton molecules are permeable and are not accessible to molecules with molecular weights greater than 3 × 10 3 Dalton's molecules are impermeable.

42. The system of claim 35, wherein the gel chamber is a degradable hydrogel chamber.

43. The system of claim 35, wherein the gel chamber is hollow and encapsulates an area of ​​the surface.

44. The system of claim 35, wherein the surface comprises capture elements configured to capture nucleic acids.

45. A method of determining one or more cellular characteristics of one or more cells, the method comprising: (a) providing a fluidic device, the fluidic device comprising: (i) a channel, the channel comprising a first surface, the one or more cells, and one or more polymer precursors, wherein the one or more cells are arranged on or near the first surface; (ii) a spatial energy modulation element, the spatial energy modulation element being in optical communication with the first surface; and (iii) a detector; (b) identifying the location of the one or more cells in the channel with the detector; (c) using the spatial energy modulation element, projecting energy into the channel such that the projected energy causes the one or more polymer precursors to form polymer matrix walls of one or more chambers, wherein the one or more chambers at least partially enclose the one or more cells at the locations identified by the detector; and (d) performing one or more assays on the one or more cells in the channel to determine the one or more cellular characteristics, wherein the one or more cellular characteristics are selected from the group consisting of cytotoxicity, viability, proliferation rate, phenotype, vector copy number, vector integration site, transcriptome, and genomic copy number variation.

46. ​​The method of claim 45, wherein the fluidic device further comprises a plurality of channels, and wherein performing the one or more assays comprises performing a plurality of the assays, wherein each different one of the one or more assays is performed in a different channel among the plurality of channels.

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