Cell activity detection method, indicator and application thereof

Abstract

The invention discloses a method for detecting cell activity, and relates to application of positive expression rate of cytoskeleton protein in the aspect of detecting or evaluating the cell activity, the method comprises the following steps: detecting positive expression of the cytoskeleton protein in a cell, if the expression of the cytoskeleton protein of the cell is positive, indicating that the cell has continuous survival ability, and if the expression of the cytoskeleton protein of the cell is positive, indicating that the cell has continuous survival ability. Through the method, the real activity state of the cells after cryopreservation recovery can be rapidly reflected.

Description

Technical Field

[0001] The present application relates to the biological field, and in particular, to the application of the positive expression rate of cytoskeleton proteins in detecting or evaluating cell viability. Background Art

[0002] Cell therapy products require specialized handling to ensure they are successfully transferred from manufacturing facilities to patients. In order to provide logistics flexibility, many current commercial models use frozen cell products delivered to hospital clinics and stored, and then thawed and injected into patients as needed. However, during the cryopreservation and resuscitation process, the vitality and function of many cells will be affected to varying degrees. The evaluation results of the cryopreservation effect affect the judgment of the effective dose for reinfusion and the subsequent analysis of drug efficacy.

[0003] Due to the delayed cell death caused by freezing, analyzing the cell viability and number immediately after thawing cannot accurately reflect the success of the cryopreservation process. There is a large proportion of false positives in the cell viability measured by immediate counting after thawing, and it cannot accurately indicate the true survival of cells after cryopreservation and recovery. Therefore, to evaluate the viability of frozen cells, it is necessary to measure the cell viability after a certain period of culture. Usually, the cell viability after 24 hours of recovery is examined. The detection methods include trypan blue exclusion staining, AO / PI double staining, MTT colorimetry, etc. However, this method also has some problems, including the cumbersome and time-consuming evaluation process, the strict environmental requirements, and the final measured cell viability may be affected by the culture conditions after recovery, so that it cannot accurately reflect the state of cell recovery at that time. Therefore, how to evaluate cell viability and function more quickly and accurately is an urgent problem to be solved. Summary of the invention

[0004] The purpose of this application is to provide a new method for detecting cell viability, which provides a certain indication for predicting the survival of cells in the later stage by characterizing cytoskeletal protein (F-actin) and determining its positive ratio. Specifically, the technical solution of this application includes:

[0005] 1. A method for detecting cell viability, comprising detecting the positive expression of cytoskeleton proteins in the cells. If the cytoskeleton proteins in the cells are positively expressed, it indicates that the cells have the ability to survive continuously.

[0006] 2. The method according to item 1, wherein the cell is a modified or unmodified immune cell.

[0007] 3. The method according to item 2, wherein the modified or unmodified immune cells are T cells, NK cells, B cells, macrophages, dendritic cells, CAR-T cells, TCR-T cells or CAR-NK cells, preferably T cells, NK cells, CAR-T cells or CAR-NK cells.

[0008] 4. The method according to any one of items 1 to 3, wherein the cells are cells that have been revived after being frozen.

[0009] 5. The method according to any one of items 1 to 4, wherein the cytoskeletal protein is actin.

[0010] 6. The method according to item 5, wherein the actin is in an aggregated or non-aggregated form, preferably an aggregated form of actin.

[0011] 7. The method according to any one of items 1 to 6, wherein the cytoskeletal protein is detected by one or more of the following methods: fluorescent labeling method, chemiluminescent dye labeling method, colorimetric method.

[0012] 8. The method according to item 7, wherein the positive expression of the cytoskeletal protein is detected using a device or instrument that can detect fluorescence, chemiluminescence intensity, or optical density.

[0013] 9. The method according to item 8, wherein the device or instrument comprises a flow cytometer, a laser confocal microscope, a fluorescence microscope, an ELISA reader or a cell analyzer, or a cell counter.

[0014] 10. The method according to any one of items 1 to 9, wherein the method is used for pharmacological and toxicological evaluation of cell products, viability detection of cell products, determination of active cell content in cell products, quality identification of cell products, or formulation of relevant quality standards for cell products.

[0015] 11. The method according to item 10, wherein the cell product is a cell product that has been revived after being frozen.

[0016] 12. The method according to item 11, wherein the detection is performed after the cell product is revived, preferably within 24 hours or within 24 hours after the cell product is revived, and more preferably within 2 hours or within 1 hour.

[0017] 13. A method according to any one of items 10-12, wherein the viability test of the cell product includes performing a viability test before infusing the cell product into a subject.

[0018] 14. A method according to any one of items 1 to 13, wherein the sustained survival means that the cell product is still alive 24 hours after resuscitation, is still alive during pharmacological or toxicological evaluation of the cell product, is still alive during quality evaluation of the cell product, or is still alive before infusion into a subject.

[0019] 15. The method according to item 10, wherein the method, when used for quality identification of cell products, comprises determining the ratio of cells in the cell product that positively express cytoskeletal proteins to the total number of cells in the cell product, and a higher ratio indicates a higher quality of the cell product.

[0020] 16. The method according to item 15, wherein the quality of the cell product is considered to meet the use requirements when the ratio of the positive expression is not less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%.

[0021] 17. Use of actin antibodies, actin labeling molecules or actin labeling dyes for identifying cell viability or identifying the quality of cell products.

[0022] 18. The use according to item 17, wherein the actin antibody or actin marker molecule is labeled.

[0023] 19. The use described in item 18, wherein the label is selected from one or more of the following labels: enzymes, biotin, fluorescent indicators, chemiluminescent indicators, isotopes, colloid indicators, latex microspheres, magnetic bead indicators, preferably fluorescent indicators or chemiluminescent labels.

[0024] 20. The use according to item 19, wherein the enzyme is selected from one or more of the following: horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, β-galactosidase-anti-β-galactosidase bridge.

[0025] 21. The use according to claim 19, wherein the fluorescent indicator is selected from one or more of the following: AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, TexasRed or PerCP dye.

[0026] 22. The use according to claim 19, wherein the chemiluminescent indicator is selected from one or more of the following: acridinium esters, acridinium sulfonamides, luminol (3-aminophthalhydrazide), isoluminol (4-aminophthalhydrazide), isoluminol isothiocyanate (ILITC), N-(4-aminobutyl)-N-ethylisoluminol (ABEI), 4,5-diaminophthalhydrazide (DPH), and aminobutylethylbenzohydrazide (ABENH).

[0027] 23. The use according to item 19, wherein the isotope is selected from one or more of the following: 125 I / 131 I / 124 I. 3 H. 14 C. 111 In, 89 Zr, 32 P.

[0028] 24. The use according to item 19, wherein the colloidal indicator is selected from one or more of the following: colloidal gold, colloidal carbon, colloidal selenium.

[0029] 25. The use according to item 17, wherein the actin marker molecule is phalloidin-FITC.

[0030] 26. The use according to item 17, wherein the actin labeling dye is CELL MASK.

[0031] The new method for reflecting cell viability provided by the present application, especially for frozen cells, can quickly reflect the true viability state of cells after cryopreservation and resuscitation within 2 hours;

[0032] The nodes and methods for quality control of cell therapy products provided in this application provide a basis for the actual reinfusion dose of cell therapy products and ensure the effective efficacy of cell therapy;

[0033] The cell viability indicator provided in the present application can be used as a method for evaluating the viability of CAR-T cells after cryopreservation and resuscitation, and can quickly confirm the true proportion of viable CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application.

[0035] Figure 1 Schematic diagram of the laser confocal staining results of cells

[0036] Figure 2 Schematic diagram of the analysis of flow cytometry results for 0h cytoskeleton positive rate

[0037] Figure 3 Schematic diagram of the correlation between the cytoskeleton positive rate at 0 hours of recovery and the cell viability counted by AOPI at 24 hours of recovery

[0038] Figure 4 Schematic diagram of the correlation between CAR-T cell skeleton and cell viability

[0039] Figure 5 Schematic diagram of the correlation between NK cell skeleton and cell viability

[0040] Figure 6 Killing capacity of viable cells calculated for different viability assays

[0041] Figure 7 Schematic diagram of the correlation between the positive rate of skeleton proteins detected by different detection instruments and cell activity DETAILED DESCRIPTION

[0042] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0043] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0044] Generally speaking, the terms used in this specification have the following meanings.

[0045] Cell viability described in this application, also referred to as cell activity, refers to the proportion of healthy cells in a sample population. Cell viability is an important indicator for determining whether in vitro cultured cells can grow normally under certain conditions, such as drug treatment, radioactivity or ultraviolet irradiation, changes in culture conditions, etc. Cell viability detection indicators generally include cell membrane permeability to nucleic acid dyes, metabolic activity, membrane potential, etc. Live / dead cell counting detection is a common method for assessing cell viability. This type of detection uses cell permeability vital dyes, cell impermeable dyes, or a combination of the two to simultaneously mark live and dead cells in a cell population. For example, trypan blue is an azo dye that cannot penetrate the cell membrane and therefore does not enter healthy cells. However, dead cells with membrane damage absorb trypan blue, so the number of dead (blue) and surviving (colorless) cells can be counted using a hemocytometer.

[0046] The cytoskeleton described in this application refers to the network structure of fibers composed of proteins in the cytoplasm of cells. The cytoskeleton is mainly composed of three types of protein fibers, namely microtubules, microfilaments and intermediate filaments (IF).

[0047] The cytoskeleton proteins described in the present application are various proteins constituting the cytoskeleton, including tubulin (α-tubulin and β-tubulin), the basic components of microtubules, actin (Actin), the basic components of microfilaments, vimentin (vimentin), keratin (keratin), desmin, neurofibrillary protein, and glial fibrillary protein, which constitute intermediate filaments. In addition, it also includes many accessory proteins of structural units.

[0048] The non-aggregated form of actin described in this application is the spherical molecular monomer of actin, which is called globular actin (G-actin); the aggregated form of actin described in this application is the polymer form of actin, which is also called fibrous actin (F-actin). Both are essential for important cell functions such as cell movement and contraction during cell division.

[0049] The positive expression of cytoskeleton proteins described in this application refers to the ability to detect the presence of cytoskeleton proteins in cells in conventional detection. That is, it is considered that cells in which cytoskeleton proteins can be detected have the vitality to achieve cell functions. Further, it is considered that cells with positive expression of cytoskeleton proteins have complete cytoskeleton proteins.

[0050] The above-mentioned detection does not limit the detection method or detection instrument, and can be detected using a device / instrument that can detect fluorescence or chemiluminescence intensity, including but not limited to a flow cytometer, a laser confocal microscope, a fluorescence microscope, an ELISA reader or a cell analyzer.

[0051] The positive expression rate of the cytoskeleton protein, or the positive expression ratio, mentioned in the present application refers to the ratio of cells expressing the cytoskeleton protein positive to the total number of cells to be tested.

[0052] The cell cryopreservation described in this application is to place the cells in a protective liquid and store them at low temperature for a long time. The cell cryopreservation technology can temporarily remove the cells from the growth state and preserve their cell characteristics, and then revive the cells for experiments when needed. Some cell therapy preparations are processed by cell cryopreservation technology as frozen cell products and delivered to hospitals and clinics for storage, and then thawed and revived according to needs and injected into patients.

[0053] The cell recovery described in this application is the process of thawing frozen cells and resuming their growth.

[0054] The flow detection method described in this application, i.e., flow cytometry, is a technical method for rapid, one-by-one quantitative analysis and sorting of cells by detecting the fluorescent signal of the marker for single cells or other biological particles in suspension. One or more fluorescently labeled antibodies are added to the single cell suspension, and the antibodies bind to the antigens on the cells through antigen-antibody reaction, while the instrument emits one or more laser beams to excite the fluorescent substances marked on the cells. The light emitted from the fluorescent substances is collected, separated, and detected, and the data is transmitted to the computer controlling the flow cytometer for analysis.

[0055] AOPI described in this application, i.e., AOPI double staining method, uses a DNA probe to double-stain the nucleus to detect the state of the cell. Among them, Acridine Orange (AO for short) is a DNA-binding dye that can emit green fluorescence, and Propidium Iodide (PI for short) is a DNA-binding dye that can emit red fluorescence. AO can be embedded in the nuclei of all cells (living cells and dead cells) through the complete cell membrane, showing green fluorescence; PI can only be embedded in the nuclei of all dead cells through the incomplete cell membrane, i.e., the cell membrane of dead cells, showing red fluorescence. When both dyes are present in the nucleus, under the appropriate AO and PI ratios, the two dyes undergo energy resonance transfer, and dead cells excite red fluorescence under the green channel (EX: 525 / 30, EM: 600LP).

[0056] Based on the findings of the present application, the cytoskeleton protein can be detected by fluorescence labeling, chemiluminescent dye labeling, or colorimetry.

[0057] Fluorescence labeling is an analytical method that uses fluorescent substances such as fluorescent proteins as specific markers to label the research object. Fluorescent substances are compounds with a conjugated double bond system chemical structure. When exposed to ultraviolet light or blue-violet light, they can be excited to an excited state, and when they return to the ground state from the excited state, they emit fluorescence. Fluorescent substances are covalently bound or physically adsorbed to a certain group of the molecule to be studied, and their fluorescence properties are used to provide information about the object of study.

[0058] In a specific embodiment of the present application, a substance that can bind to a skeleton protein is coupled with a fluorescent substance to obtain a fluorescent dye that can mark the skeleton protein; the cells are then marked with the skeleton protein fluorescent dye; the fluorescent substance is excited by a laser to emit fluorescence, and the corresponding fluorescence intensity of a single cell is detected to determine the proportion of skeleton protein-positive cells; based on the proportion of skeleton protein-positive cells, the actual survival status of the cells is predicted.

[0059] Chemiluminescent dye labeling method, also known as chemiluminescent immunoassay, combines the chemiluminescent system with the immune reaction, labels the antibody or antigen with a chemiluminescent-related substance, reacts with the antigen or antibody to be tested, separates the free chemiluminescent marker, and adds other related substances of the chemiluminescent system to produce chemiluminescence, and conducts qualitative or quantitative detection of the antigen or antibody. Commonly used labeling substances such as isoluminol and acridinium ester can produce special luminescent groups and directly participate in the reaction during the analysis process.

[0060] In a specific embodiment of the present application, a substance that can bind to a skeleton protein is coupled with a chemiluminescent substance to obtain a chemiluminescent dye that can mark the skeleton protein; the cells are then marked with the skeleton protein chemiluminescent dye; the luminescence principle of the chemiluminescent substance is utilized to make the cells expressing the skeleton protein glow; and the proportion of luminescent cells is detected to determine the proportion of skeleton protein-positive cells, thereby predicting the actual survival of the cells.

[0061] The colorimetric method is based on the color reaction that produces colored compounds. It is a method of determining the content of the component to be tested by comparing or measuring the color depth of the colored substance solution.

[0062] In a specific embodiment of the present application, a substance that can bind to a skeleton protein is coupled with a color-developing substance to obtain a color-developing dye that can mark the skeleton protein; the cells are then marked with the skeleton protein color-developing dye; the color-developing principle of the color-developing substance is utilized to allow cells expressing the skeleton protein to develop color; and the proportion of colored cells is detected to determine the proportion of skeleton protein-positive cells, thereby predicting the actual survival status of the cells.

[0063] Based on the findings of the present application, a device / instrument capable of detecting fluorescence or chemiluminescence intensity can be used to detect actin as described in the present application. In some embodiments, the device / instrument is a flow cytometer, laser confocal microscopy, or fluorescence microscopy, microplate reader, cell analyzer, etc.

[0064] Flow cytometer detection principle: Flow cytometer is a device for automatic analysis and sorting of cells. It can quickly measure, store and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and can sort out specified cell subpopulations according to the preselected parameter range. Flow cytometer can measure multiple parameters at the same time, and the information mainly comes from specific fluorescence signals and non-fluorescence scattering signals. In flow cytometer measurement, two scattering directions of scattered light measurement are commonly used, namely forward scattering (FSC) and side scattering (SSC). Generally speaking, the intensity of FSC is related to the size of the cell. Although SSC is also related to the shape and size of the cell, it is more sensitive to the refractive index of the cell membrane, cytoplasm, and nuclear membrane, and can also give a sensitive response to larger particles in the cytoplasm. Therefore, the measurement of side scattered light is mainly used to obtain relevant information about the particle properties of the fine structure inside the cell. In actual use, the instrument must first measure the light scattering signal. When light scattering analysis is used in conjunction with fluorescent probes, stained and unstained cells in the sample can be identified. In some embodiments, the specific application scheme is: after the frozen cells are thawed, the cells expressing the skeleton protein positively are marked with a cytoskeleton dye with a fluorescent indicator (if the cytoskeleton dye is a membrane-impermeable dye, the cells are marked after fixation and membrane permeation; if the cytoskeleton dye is a membrane-permeable dye, the cells can be directly marked without fixation and membrane permeation), after the marking is completed, the excess skeleton dye is washed away, and then the marked cells are tested on a flow cytometer. The excitation wavelength and detection channel of the flow cytometer are determined according to the fluorescent label of the cytoskeleton dye, the cell population is delineated by FSC and SSC, and the skeleton protein positivity rate of the cell population is determined according to the fluorescence expression.

[0065] Fluorescence microscope uses ultraviolet light as a light source to illuminate the object to be inspected, causing it to emit fluorescence, and then observes the shape and location of the object under a microscope, captures the fluorescence signal and performs quantification. In some embodiments, the specific application scheme is: after the frozen cells are revived, the cytoskeleton-positive expression cells are marked with a cytoskeleton dye with a fluorescent indicator (if the cytoskeleton dye is a membrane-impermeable dye, it is marked after the cells are fixed and broken, and if the cytoskeleton dye is a membrane-permeable dye, it can be directly marked without fixing the cells and breaking the membrane). After the marking is completed, the excess skeleton dye is washed away, and then the marked cells are examined and photographed under a fluorescence microscope. The fluorescence exciter and filter are selected according to the fluorescent label carried by the cytoskeleton dye, and the total number of cells is obtained by white field counting. The same field of view is converted to a fluorescence exciter, and the cytoskeleton-positive expression cell population can be counted. By counting and averaging multiple fields of view, the cytoskeleton expression positive rate is obtained.

[0066] Laser scanning confocal fluorescence microscopy (LSCM) is an analytical instrument for molecular cell biology that uses computers, lasers and image processing technology to obtain three-dimensional data of biological samples. It is mainly used to observe the biological changes of living cell structures and specific molecules and ions, quantitative analysis, and real-time quantitative determination. Laser confocal microscope uses laser as the light source. On the basis of traditional fluorescence microscope imaging, it adds a laser scanning device and a conjugate focusing device. It is a system for digital image acquisition and processing through computer control. The excitation light of a certain wavelength emitted by the laser is amplified and forms a point light source through the illumination pinhole light bar in the scanner. It is focused on the focal plane of the sample by the objective lens. The corresponding irradiated point on the sample is excited and emits fluorescence. After passing through the detection hole light bar, it reaches the detector and is imaged on the computer monitor screen. In this way, the fluorescence image of each point of the sample on the focal plane forms a complete confocal image, which is called a light slice. In some embodiments, the specific application scheme is: after the frozen cells are revived, all cell groups are marked with a nucleic acid dye with a fluorescent indicator, and the skeleton-positive expression cells are marked with a cytoskeleton dye with a fluorescent indicator (if the cytoskeleton dye is a membrane-impermeable dye, it is marked after the cells are fixed and broken, and if the cytoskeleton dye is a membrane-permeable dye, it can be directly marked without fixing and breaking the cells). After the marking is completed, the excess dye is washed away, and then the marked cells are examined and photographed under a laser scanning confocal fluorescence microscope. The fluorescence exciter and filter are selected according to the fluorescent label of the cytoskeleton dye. After obtaining the confocal photo, the fluorescent dye-positive cells and the fluorescent dye and skeleton dye double-positive cells in the picture are counted according to the expression of the fluorescence situation. The counting result of the fluorescent dye-positive cells is used as the denominator, and the counting result of the fluorescent dye and skeleton dye double-positive cells is used as the numerator, and the cytoskeleton positive expression rate can be calculated.

[0067] The microplate reader is an enzyme-linked immunosorbent assay (ELISA) and its basic working principle and main structure are basically the same as those of a photoelectric colorimeter. The light waves emitted by the light source lamp are converted into a monochromatic light beam through a filter or a monochromator and enter the sample to be tested in the plastic microplate. Part of the monochromatic light is absorbed by the sample, and the other part is irradiated onto the photodetector through the sample. The photodetector converts the light signal of different strengths of the sample to be tested into a corresponding electrical signal. After pre-amplification, logarithmic amplification, analog-to-digital conversion and other signal processing, the electrical signal is sent to the microprocessor for data processing and calculation, and finally the display and printer display the results. It can be widely used in DNA, RNA quantification and purity analysis (A260 / A280) and protein quantification (A280 / BCA / Braford / Lowry) in the low ultraviolet region, enzyme activity and enzyme kinetics detection, enzyme-linked immunosorbent assays (ELISAs), cell proliferation and toxicity analysis, cell apoptosis detection (MTT), reporter gene detection and G protein-coupled receptor analysis (GPCR), etc. In some embodiments, a specific implementation scheme is as follows: an antibody or compound for detecting cytoskeletal proteins with an enzyme-linked label is selected, and then a color developer is combined with the label molecule to develop color, and the data is read on an ELISA instrument, and the expression of the cells is calculated based on a standard curve.

[0068] The present application provides a method for detecting cell viability. Specifically, the method comprises detecting the positive expression of cytoskeleton proteins in the cells.

[0069] In a specific embodiment, the method provided in the present application can calculate the proportion of live cells in a cell sample or a cell product by detecting the number of cells that are positive for cytoskeletal protein expression in the cell sample or the cell product.

[0070] In a specific embodiment, the method provided in the present application uses the positive expression rate value of the cytoskeleton protein in the cell sample or cell product as the cell viability value.

[0071] In a specific embodiment, the method provided in the present application can count or determine the number of live cells in a cell sample.

[0072] In some specific embodiments, the cells detected in the method of the present application are immune cells. Further, the cells are T cells, NK cells, B cells, macrophages, etc.

[0073] In other specific embodiments, the cells detected in the method of the present application are modified immune cells. Further, the cells are CAR-T cells, TCR-T cells or CAR-NK cells.

[0074] In some preferred embodiments, the cell is a T cell, a NK cell, a CAR-T cell or a CAR-NK cell.

[0075] In a specific embodiment of the present application, the cytoskeletal protein is a protein that constitutes the microfilaments in the cytoskeleton. Further, the cytoskeletal protein is an aggregated form of actin, i.e., globular actin (G-actin); or a non-aggregated form of actin, i.e., fibrous actin (F-actin). In a preferred embodiment, the cytoskeletal protein is fibrous actin (F-actin).

[0076] In a specific embodiment, the cells detected in the method of the present application may be cells that have been cryopreserved. Further, the cells are cells that have been revived after being cryopreserved.

[0077] In a specific embodiment, the cells detected in the method of the present application may be a frozen cell product. Further, the cells detected are a partial cell sample in the cell product.

[0078] The cell products described in this application, or "cell drugs" or "cell preparations", include cell therapy products used for cell or gene therapy, such as genetically modified or non-genetically modified cells, such as autologous or allogeneic immune cells, stem cells, tissue cells or cell lines.

[0079] In a specific embodiment, the method for detecting cell viability is to detect the frozen cells / cell products / cell preparations / cell drugs after resuscitation. In some embodiments, the detection is performed 24 hours after resuscitation. In some preferred embodiments, the detection is performed within 24 hours, 12 hours, 6 hours, 3 hours, 2 hours, or 1 hour after resuscitation. In another preferred embodiment, the measurement is performed immediately at the beginning of resuscitation.

[0080] In a specific embodiment, the method provided in the present application can detect the viability of the cell product before infusing it into a subject.

[0081] In an embodiment of the present application, a protein or compound capable of binding to a cytoskeleton protein may be used to label the cytoskeleton protein, so as to detect the positive expression of the cytoskeleton protein in the cell.

[0082] In some specific embodiments, the positive expression rate of the cytoskeleton protein can be marked by fluorescent labeling or chemiluminescent dye labeling or color reaction or antibody binding to detect the positive expression of the cytoskeleton protein in the cell.

[0083] In an embodiment of the present application, a device / instrument capable of detecting fluorescence or chemiluminescence intensity may be used to detect the positive expression of the cytoskeletal protein.

[0084] In some specific embodiments, the positive expression of the cytoskeleton protein can be detected using devices / instruments including but not limited to flow cytometer, laser confocal microscope, fluorescence microscope, microplate reader or cell analyzer.

[0085] In a specific embodiment, the method provided in the present application can characterize the cytoskeleton proteins at the time of cell recovery or within a period of time and determine the positive ratio of the cytoskeleton proteins through the instrument to predict the survival status and cell quality of the cells in actual use after cell recovery.

[0086] The present application provides a specific method for detecting cell viability, comprising using an automatic cell counter to detect the positive rate of cytoskeleton proteins in a cell sample to characterize the proportion of live cells and obtain the cell viability.

[0087] Specifically, the present application also provides a method for detecting cytoskeleton integrity using flow cytometry, including water bath recovery, cell fixation and membrane permeation (if the cytoskeleton dye can penetrate the membrane, this step can be omitted), fluorescent dye labeling of the cytoskeleton, flow cytometry detection and result analysis.

[0088] In a specific embodiment, the method comprises:

[0089] 1) Water bath recovery: Recover the frozen cells in a 37°C water bath until the freezing solution is completely dissolved, and wash the cells once with cell washing solution (PBS, saline, cell culture medium or other physiological buffer);

[0090] 2) Cell fixation and membrane permeabilization: Use the fixation and membrane permeabilization agent Cytofix / Cytoperm (BD Bioscience) to fix and permeabilize the cells;

[0091] 3) Fluorescent dye labeling of cytoskeleton: Wash cells with Perm / Wash Buffer (BD Bioscience) to remove the fixative permeabilizing agent. Resuspend cells in resuspension buffer (PBS + 2% FBS), add cytoskeleton fluorescent dye Phalloidin-FITC (ABclonal), label at room temperature for 15 minutes. Wash once with cell washing solution to remove excess fluorescent dye;

[0092] 4) Sample detection and result analysis: Resuspend the labeled cells in resuspension buffer, open the FSC, SSC, and FITC detection channels of the flow cytometer (NovoExpress), adjust the appropriate loading voltage, and load the labeled cell samples for detection. Figure 2 The positive rate of cytoskeleton proteins was determined by the method of drawing. Positive cells indicate that their cytoskeleton proteins are intact.

[0093] In another specific embodiment, the method comprises:

[0094] 1) Water bath recovery: Recover the frozen cells in a 37°C water bath until the freezing solution is completely dissolved, and wash the cells once with cell washing solution;

[0095] 2) Fluorescent dye labeling of cytoskeleton: Wash cells with Perm / Wash Buffer (BD Bioscience) to remove the fixative permeabilizing agent. Resuspend cells in resuspension buffer (PBS + 2% FBS), add cytoskeleton fluorescent dye CELLMASK (manufacturer: Invitrogen, catalog number: A57244), label at room temperature for 15 minutes. Wash once with cell washing solution to remove excess fluorescent dye;

[0096] 3) Sample detection and result analysis: Resuspend the labeled cells in resuspension buffer, open the FSC, SSC, and PE detection channels of the flow cytometer (NovoExpress), adjust the appropriate loading voltage, and load the labeled cell samples for detection. Figure 2 The positive rate of cytoskeleton proteins was determined by the method of drawing, and positive cells indicated that their cytoskeleton proteins were intact.

[0097] The cell viability results obtained by the method provided in the present application at the time of cell recovery or within 24 hours are well correlated with the cell viability count results obtained by the traditional detection method (AOPI double staining method) at 24 hours after cell recovery, and can be used as a detection method to predict the actual survival status of frozen cell products.

[0098] The method for detecting cell viability provided in the present application can be used for the pharmacological and toxicological evaluation of cell products, the viability detection of cell products, the determination of the content of active cells in cell products, the quality identification of cell products or the formulation of relevant quality standards for cell products.

[0099] The method described in the present application can be used for cell sampling before or after cryopreservation of cell products, pharmacological or toxicological evaluation, cell viability detection, active cell content determination, quality identification, quality research, etc.

[0100] In a specific embodiment, the present application provides a method for identifying the quality of a cell product, which comprises determining the ratio of cells in the cell product that positively express cytoskeletal proteins to the total number of cells in the cell product, wherein a higher ratio indicates a higher quality of the cell product.

[0101] In a specific embodiment, the method includes: randomly selecting a cell sample from the cell product, measuring the positive expression ratio of the cytoskeleton protein of the cell sample, and when the positive expression ratio is not less than 5%, 10%, 20%, 30%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99%, it is considered that the quality meets the use requirements. In a specific embodiment, the positive ratio that meets the quality requirements can be determined according to different cell types and different use requirements.

[0102] In some specific embodiments, the cell sample is no less than 1%, 2%, 5% or 10% of the total cell product.

[0103] In a specific embodiment, the cells in the cell product can be T cells, NK cells, CAR-T cells, CAR-NK cells, or other modified or unmodified immune cells.

[0104] In a specific embodiment, the cell product is a cell product that has been revived after being frozen.

[0105] In a specific embodiment, the method for identifying the quality of the cell product is: randomly selecting cell samples from the cell product, measuring the positive expression rate of cytoskeleton protein of the cell sample, and when the positive expression rate is ≥70%, it is considered that the quality of the cell preparation product meets the use requirements,

[0106] Wherein, the cell sample is not less than 5% of the total cell product.

[0107] Preferably, the cell is a CAR-T cell,

[0108] Preferably, the method for measuring the positive expression rate of cytoskeleton proteins in a cell sample is the method for detecting cytoskeleton integrity using flow cytometry provided in the present application.

[0109] Furthermore, the present application also provides a method for evaluating the live cell ratio in a cell product: before the cell product is reinfused, tested for effectiveness or used for other purposes, the live cell ratio of the cell product is evaluated by detecting the positive expression rate of cytoskeletal proteins in the cells in the cell product.

[0110] In a specific embodiment, the method comprises detecting the positive expression rate of cytoskeleton protein in the cell sample within 24 hours after the cryopreserved cell product is thawed.

[0111] Furthermore, the present application also provides a new quality control index for cell products, which can be used to indicate the potential of the prepared CAR-T cells to exert effective functions. Specifically, the index, i.e., the positive expression rate of cytoskeleton proteins of the cell samples to be tested, is associated with multiple batches of data of CAR-T function-related indicators such as killing ability and factor secretion ability, and the corresponding function equation is found, so as to predict the CAR-T function through the positive expression rate of cytoskeleton proteins of the cell samples to be tested, and formulate corresponding quality control standards.

[0112] The present application also provides a marker for identifying cell viability. Specifically, the marker is any protein / antibody or compound that can bind to a cytoskeleton protein.

[0113] In a specific embodiment, the marker is any protein / antibody or compound that can bind to actin or an actin antibody.

[0114] In a specific embodiment, the marker is an actin antibody, an actin labeling compound or an actin labeling dye.

[0115] Furthermore, the present application also provides the use of the marker for identifying cell viability or identifying the quality of cell products.

[0116] In a specific embodiment of the present application, the actin labeling molecule or actin labeling dye is a non-antibody molecule that can specifically recognize and detect actin, or a dye that can visualize it, which is generally composed of a high-affinity actin probe coupled to a fluorescent dye.

[0117] In some specific embodiments, the actin marker molecule is phalloidin.

[0118] In some specific embodiments, the actin labeling dye is CELL MASK.

[0119] In a specific embodiment of the present application, the actin antibody or actin marker molecule is labeled.

[0120] In a specific embodiment, the label is selected from one or more of the following labels: enzymes, biotin (one of the B vitamins, also known as vitamin H, vitamin B7, coenzyme R, etc.), fluorescent indicators, chemiluminescent indicators, isotopes, colloid indicators, latex microspheres, magnetic bead indicators, preferably fluorescent indicators or chemiluminescent markers.

[0121] In a specific embodiment, the enzyme is selected from one or more of the following: horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, β-galactosidase-anti-β-galactosidase bridge.

[0122] In a specific embodiment, the fluorescent indicator is selected from one or more of the following: AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green488, Pacific Blue dye, Pacific Orange dye, TexasRed or PerCP dye.

[0123] In a specific embodiment, the chemiluminescent indicator is selected from one or more of the following: acridinium ester, acridinium sulfonamide, luminol (3-aminophthalhydrazide), isoluminol (4-aminophthalhydrazide), isoluminol isothiocyanate (ILITC), N-(4-aminobutyl)-N-ethylisoluminol (ABEI), 4,5-diaminophthalhydrazide (DPH), aminobutylethylbenzohydrazide (ABENH).

[0124] In a specific embodiment, the isotope is selected from one or more of the following: 125 I / 131 I / 124 I. 3 H. 14 C. 111 In, 89 Zr, 32 P.

[0125] In a specific embodiment, the colloidal indicator is selected from one or more of the following: colloidal gold, colloidal carbon, and colloidal selenium.

[0126] The method and marker provided in the present application can be applied to the viability assessment of cryopreserved cell products to determine the effective reinfusion dose of the cell therapy product when in use.

[0127] Example

[0128] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0129] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0130] Example 1 Indicative properties of cytoskeleton (F-actin) for cryopreserved cell viability

[0131] A certain amount of fresh and frozen CAR-T cells were taken, fixed on a glass slide with 4% paraformaldehyde, and then co-labeled with DNA dye DAPI and cytoskeleton protein dye phalloidin-FITC for laser confocal detection: Figure 1 Red staining represents F-actin staining inside and outside the cell membrane, blue staining represents DAPI staining for the cytoplasm, and cryoresistant cells are cells whose viability is detected by the applicant through traditional methods after resuscitation. Cells with higher viability are cryoresistant cells. On the contrary, cells with significantly reduced viability after resuscitation are non-freeze-resistant cells. Specifically, cells with a decrease in viability of 24h resuscitation compared to 0h resuscitation are cryoresistant cells if the percentage decrease is less than 25%, and cells with a decrease of more than 25% are non-freeze-resistant cells. The staining results are as follows: Figure 1 shown.

[0132] pass Figure 1 It can be seen that the F-actin in the fresh cell group is completely stained, among which the F-actin of the freeze-resistant cells evenly wraps the entire cell, and the F-actin of the non-freeze-resistant cells is more concentratedly expressed in the "lateral section" of the cell; the F-actin structure of the cells after freezing is severely damaged, and the F-actin expression detected in the non-freeze-resistant cells is significantly lower than that in the freeze-resistant cells. F-actin can be used as an indicator of the cryopreservation of freeze-resistant cells and the vitality of frozen cells after recovery.

[0133] Example 2 Cytoskeleton (F-actin) positive rate detection method

[0134] Resuscitate the frozen CAR-T cells in a 37°C water bath until the cryopreservation solution is completely dissolved, wash the cells once with PBS, and then label and detect them. The specific plan is as follows:

[0135] Cell fixation and permeabilization: The cells were fixed and permeabilized using the fixation and permeabilization agent Cytofix / Cytoperm (BD Bioscience), and the operation was performed according to the manufacturer's product instructions.

[0136] Fluorescent dye labeling of cytoskeleton: Wash cells with Perm / Wash Buffer (BD Bioscience) to remove the fixative permeabilizer. Resuspend cells in resuspension buffer (PBS + 2% FBS), add cytoskeleton fluorescent dye Phalloidin-FITC (ABclonal), label at room temperature for 15 minutes. Wash once with PBS to remove excess fluorescent dye.

[0137] Sample testing and result analysis: Resuspend the labeled cells in resuspension buffer, open the FSC, SSC, and FITC detection channels of the flow cytometer (NovoExpress), adjust the appropriate loading voltage, and load the labeled cell samples for testing. Refer to the test results and cytoskeleton positive rate marking screen Figure 2 shown.

[0138] Example 3 Verification of the positive rate of F-actin expression in cells as a cell viability indicator

[0139] In order to verify whether the positive rate of F-actin expression can be used as a detection indicator of cell viability, it was compared with the AOPI detection results used in traditional evaluation methods and correlation analysis was performed.

[0140] (1) AOPI cell viability detection

[0141] CAR-T cells were cryopreserved using different cryopreservatives, and the cells were revived, and the activity of CAR-T cells at different times after resuscitation was detected. Specific experimental methods:

[0142] Before freezing the cells, AOPI was used to detect the cell viability of the unfrozen CAR-T cells (AOPI viability detection principle: AO and PI are both DNA dyes, of which AO is a membrane-permeable dye that can pass through the intact cell membrane and embed into the nuclei of all cells (live and dead cells), showing green fluorescence; PI can only pass through the incomplete cell membrane, that is, the cell membrane of dead cells, and embed into the nuclei of all dead cells, showing red fluorescence. AOPI counting can effectively eliminate the interference of impurities and red blood cells to ensure accurate counting of samples). After freezing the cells with cryoprotectant and FBS+10% DMSO for 1 day, they were quickly revived in a 37°C water bath. A certain amount of cells were immediately taken for cell viability detection using AOPI, and the remaining cells were cultured with pre-warmed 1640+10% FBS+cytokine (IL2, IL7, IL15, IL21 and other cytokines can be used alone or in combination) culture medium, and AOPI was used again for cell viability detection after 24 hours. The test results are shown in Table 1:

[0143] Table 1 AOPI cell viability test results (unit: %)

[0144]

[0145] From Table 1, we can see that although the results of cell viability test showed high cell viability when cryopreserved cells were tested immediately after thawing, the results showed that the cell viability was seriously reduced when tested again after 24 hours of culture. This reflects that the results of cell viability test using AOPI test method immediately after cryopreserved cells were thawed cannot reflect their true viability. When determining the reinfusion dose of cell therapy products, there may be a mismatch between the dose of live cells to be reinfused determined according to the experimental protocol and the actual reinfusion dose, and the actual reinfusion cell dose may be far less than the reinfusion dose required by the protocol, resulting in the treatment effect being not as good as expected.

[0146] (2) Comparison and verification of F-actin expression positivity as a cell viability indicator with AOPI detection results

[0147] Based on the above problems existing in AOPI detection and the findings of Example 1, the feasibility of replacing the existing cell viability AOPI detection scheme with the positive rate of F-actin expression was verified. The specific experimental method is as follows.

[0148] F-actin detection group: The F-actin detection group was set up according to the method of Example 2, and the positive rate of F-actin expression was detected on the frozen CAR-T cells at 0 h of recovery.

[0149] AOPI detection group: The frozen CAR-T cells were revived in a 37°C water bath until the cryopreservation solution was completely dissolved. The cryopreservation solution was replaced with complete culture medium and then placed in a 37°C, 5% CO2 incubator. After culturing for 24 hours, the cells were mixed and counted. The counting dye was AOPI, and the cell viability obtained by counting was recorded as the cell viability at 24 hours of recovery.

[0150] The experiment was conducted in seven groups. The results are shown in Table 2 below:

[0151] Table 2 Results of cell viability test of F-actin and AOPI after cryopreservation and resuscitation

[0152] Cryopreserved cell groups F-actin positive rate at 0h after resuscitation (%) AOPI detection survival rate after 24h resuscitation (%) 1 50.76 58.17 2 35.51 38.59 3 44.01 46.92 4 60.79 66.55 5 29.83 37.04 6 34.19 40.97 7 57.93 59.54

[0153] The results showed that the F-actin positive rate detected immediately after recovery was comparable to the viability of the frozen cells 24 hours after recovery using the conventional AOPI detection method. It should be noted that the 24h AOPI viability detection preserves the truly healthy cells after cell recovery by extending the observation time. However, this method is affected by cell proliferation and cell death and cannot fully reflect the true viability of cells at the time of recovery. (3) Correlation analysis between the F-actin expression positive rate and AOPI detection results

[0154] Furthermore, the correlation analysis was conducted on the cytoskeleton positive rate (F-actin) of CAR-T cells in different groups at 0h of recovery and the cell viability counted by AOPI at 24h of recovery. Figure 3 As shown. The horizontal axis is the expression rate of F-actin detected immediately after the cryopreserved CAR-T cells were revived, and the vertical axis represents the cell viability detected by traditional AOPI after 24 hours of culture of cryopreserved CAR-T cells. Correlation curve: y = 0.9431x + 7.511, R2 = 0.9621.

[0155] The experimental results showed that the cytoskeleton positivity rate (F-actin) at 0h after CAR-T cell recovery was significantly positively correlated with the cell viability counted by AOPI at 24h after recovery. Therefore, the cytoskeleton (F-actin) positivity rate can be used to indicate the viability of CAR-T cells after cryopreservation and recovery, and the expression rate detected immediately can more accurately reflect the viability of CAR-T cells.

[0156] Example 4 Indicative effect of cytoskeletal protein (F-actin) on cell killing function

[0157] Further verify whether the positive rate of F-actin expression can indicate the cell killing ability. Using the cytoskeletal protein (F-actin) expression rate and the conventional AOPI cell viability detection method, CAR-T cell killing detection was performed according to the AOPI cell viability at 0h of recovery, the cytoskeletal protein (F-actin) expression rate at 0h of recovery, and the AOPI cell viability at 24h of recovery to verify the important influence of the cell viability detection method on the effectiveness of CAR-T cells: the cell viability detected above was used to calculate the number of live cells for plating, and the effector-target ratio was set to 1:1 for cell killing and factor secretion detection. The in vitro killing results are as follows: Figure 4 shown.

[0158] Figure 4 The vertical axis represents the cell killing rate, and the horizontal axis represents the cell plating scheme for calculating the effector-target ratio according to different cell viability detection schemes: 0h_0h AOPI for recovery, which means that AOPI is used to detect the cell viability immediately after recovery, and the viable cells for plating are calculated according to the detected cell viability; 0h_24h AOPI for recovery, which means that AOPI is used to detect the cell viability 24 hours after recovery and culture, and the viable cells for plating are calculated according to the detected cell viability; 0h_0h cytoskeleton for recovery, which means that the positive rate of F-actin is detected immediately after recovery, and the viable cells for plating are calculated according to the positive rate of F-actin as the cell viability. Figure 5It can be seen that the killing ability of CAR-T cells calculated according to the cell viability at 0h-AOPI of resuscitation is significantly lower than that of resuscitation 24h-AOPI and resuscitation 0h-cytoskeleton at the same effect-target ratio, and the killing ability of resuscitation 24h-AOPI and resuscitation 0h-cytoskeleton is equivalent. This shows that the cell viability calculated by the cell viability calculation method of resuscitation 0h-AOPI is higher than the actual cell viability, so the actual number of cells that exert cell viability is lower than the theoretical number of cells, so the killing effectiveness is weak. The F-actin positivity rate of cells 0h after resuscitation can be used as one of the quality control standards for cell therapy products such as CAR-T products and CAR-NK products.

[0159] From the above examples, it can be found that cell viability is a prerequisite for ensuring cell efficacy. It can also be considered that cell viability and function are consistent. Therefore, in the quality control process of therapeutic cells such as immune cells, genetically modified immune cells or engineered immune cells and cell products, cell viability detection is very important. Considering that it is not realistic to calculate the viability of clinically applied products after culturing for 24 hours after recovery, and the traditional scheme for detecting viability immediately after recovery is inaccurate, based on the F-actin positive rate detected immediately after recovery in part (2) of Example 2 and this part, it is equivalent to the viability rate of frozen cells 24 hours after recovery using the conventional AOPI detection method; the conclusion that the cytoskeleton (F-actin) positive rate can be used to indicate the viability of CAR-T cells after cryopreservation and recovery, F-actin can be used to identify the quality of cell preparation products. The specific method includes: before the cell product is reinfused, tested for effectiveness or used for other purposes, the cell sample is tested for the positive expression rate of cytoskeleton protein within 24 hours after recovery to evaluate the viable cell ratio of the cell preparation.

[0160] Example 5 Selection of reagents for labeling cytoskeletal proteins

[0161] In addition to actin F-actin, there is also tubulin Tublin, which represents cytoskeletal proteins. The following reagents are selected to perform labeling detection of skeleton proteins on CAR-T cells frozen with different cryopreservatives: Phalloidin-FITC (trade name: Phalloidin-FITC, manufacturer: ABclonal, article number: RM02836), CELL MASK (trade name: CELL MASKTM Actin Tracking stains, manufacturer: Invitrogen, article number: A57244), Tublin Tracker (trade name: Tublin Tracker TM Deep Red, manufacturer: Invitrogen, article number: T34077). Among the above reagents, Phalloidin-FITC and CELL MASK are different reagents for marking F-actin, and Tublin Tracker is a reagent for marking skeleton protein Tublin. The AOPI detection results at 0h and 24h after cell recovery were used as cell viability controls. The specific staining and labeling methods refer to Examples 2 and 3. The results are shown in Table 3.

[0162] Table 3 Indications of cell viability by different scaffold protein labeling reagents:

[0163]

[0164] From the results in Table 3, it can be seen that Phalloidin-FITC and CELL MASK labeled F-actin show excellent coincidence, that is, any dye and / or reagent labeled F-actin can be used as a cell viability indicator to reflect the true cell viability after the cryopreserved cells are revived. However, the labeling reagent using the skeleton protein Tublin cannot indicate the cell viability (all cells express 100%). Therefore, it is believed that labeling F-actin can quickly detect the viability of frozen cells after resuscitation, and the use of any labeling dye or reagent can achieve the effect of indicating cell viability.

[0165] Example 6 Correlation analysis between F-actin positive rate and cell viability of different cell types

[0166] (1) Correlation between F-actin positive rate and cell viability of CAR-T cells

[0167] Summarize the recovery counts and flow cytometry data of multiple batches of CAR-T cells (the recovery 0h cytoskeleton positive rate detection and recovery 24h cell viability detection are performed simultaneously during each recovery evaluation), and count the recovery 0h cytoskeleton positive rate and recovery 24h cell viability. The experimental method refers to Examples 2 and 3. The statistical results are shown in Table 4. Draw a scatter plot with the data in Table 4, as shown in Figure 5 shown.

[0168] Table 4 F-actin positive rate of CAR-T cells at 0h recovery and cell viability counted by AOPI at 24h recovery

[0169]

[0170]

[0171] According to Table 4 and Figure 5 The results showed that there was a positive correlation between the cytoskeleton positivity rate of CAR-T cells at 0 h of recovery and the cell viability at 24 h of recovery, and there was a highly linear relationship (|r|>0.8).

[0172] (2) Correlation between F-actin positivity and cell viability of CAR-NK cells

[0173] The correlation between the F-actin positive rate of CAR-NK cells after cryopreservation and 24h cell viability was further verified. The experimental method was referred to Examples 2 and 3. The experimental results are shown in Tables 5 and Figure 6 shown.

[0174] Table 5 F-actin positive rate of CAR-NK cells at 0 h of recovery and cell viability counted by AOPI at 24 h of recovery

[0175]

[0176]

[0177] According to Table 5 and Figure 6 The results showed that there was a significant correlation between the cytoskeleton positive rate of CAR-NK cells at 0 h of recovery and the cell viability at 24 h of recovery (|r|>0.7).

[0178] Therefore, immune cells include but are not limited to T cells and NK cells and immune cell products such as CAR-T, CAR-NK, etc. The positivity rate of the cytoskeletal protein F-actin can indicate the cell viability of immune cells and immune cell products.

[0179] Example 7 Correlation analysis between F-actin positive rate and cell viability detected by different instruments

[0180] When evaluating the recovery of each batch of frozen cells, AOPI cell counts were performed at 0h and 24h of recovery, and F-actin was labeled with a cytoskeleton dye at 0h of recovery. After labeling, the F-actin positivity rate was detected by flow cytometry and cell analyzer. The experimental method refers to Examples 2 and 3. The skeleton detection and cell viability counting data after freezing CAR-T cells were summarized, and the statistical results are shown in Table 6. A scatter plot was drawn with the data in Table 6, as shown in Table 6. Figure 7 shown.

[0181] Table 6 Correlation between skeleton detection results and cell viability of different detection instruments

[0182]

[0183] According to Table 6 and Figure 7 The results showed that the AOPI cell viability at 0h of CAR-T cell recovery was not correlated with the AOPI cell viability at 24h of recovery, but the F-actin positivity rate detected by different instruments at 0h of recovery was significantly correlated with the cell viability at 24h of recovery (|r|>0.7).

[0184] Therefore, any method for detecting the F-actin positivity rate, including but not limited to: flow cytometry, laser confocal microscopy, fluorescence microscopy, microplate reader or cell analyzer, cell counter, etc., for detecting the F-actin positivity rate has a certain indicative effect on the true viability of cells.

Claims

1. A method for detecting cell viability, comprising detecting the positive expression of cytoskeleton proteins in the cells. If the cytoskeleton proteins in the cells are positively expressed, it indicates that the cells have the ability to survive continuously.

2. The method according to claim 1, wherein: The cells are modified or unmodified immune cells.

3. The method according to claim 1 or 2, wherein: The cells are cells that have been revived after being frozen.

4. The method according to any one of claims 1 to 3, wherein: The cytoskeletal protein is actin.

5. The method according to any one of claims 1 to 4, wherein: The cytoskeleton protein is detected by one or more of the following methods: fluorescent labeling method, chemiluminescent dye labeling method, and colorimetric method.

6. The method according to any one of claims 1 to 5, wherein: The method is used for pharmacological and toxicological evaluation of cell products, viability detection of cell products, determination of active cell content in cell products, quality identification of cell products or formulation of relevant quality standards for cell products.

7. The method according to claim 6, wherein: When the method is used for quality identification of cell products, it includes determining the ratio of cells with positive expression of cytoskeleton protein in the cell product to the total number of cells in the cell product. The higher the ratio, the higher the quality of the cell product.

8. Use of actin antibodies, actin labeling molecules or actin labeling dyes for identifying cell viability or identifying the quality of cell products.

9. The use according to claim 8, wherein The actin antibody or actin marker molecule is labeled.

10. The use according to claim 9, wherein The label is selected from one or more of the following labels: enzyme, biotin, fluorescent indicator, chemiluminescent indicator, isotope, colloid indicator, latex microsphere, magnetic bead indicator, preferably fluorescent indicator or chemiluminescent agent label.

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