Method for detecting cell stability

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a method for detecting cell stability, a cell sample is detected in three aspects of cell performance, cell mutation and cell stability, and when the cell proliferation effect is good, the cell does not migrate, and the cell activity is gt; the cell of which the cell concentration is 80%, the cell early-stage and late-stage apoptosis ratio is less than or equal to 10%, the cell autophagy ratio is less than 5%, no proto-oncogene is remarkably expressed, the cell aging ratio is less than or equal to 20% and the cell multiplication time is less than 48 hours is a stable cell; if one of the indexes does not reach the standard, it is determined that cell passage is unstable, and the cell culture process is stopped. The method provided by the invention can comprehensively judge the life cycle of the cell, so that the cell is applied to the optimal economic / efficient service life, the cell state can be effectively and accurately judged, and the method has good reliability.

Description

Technical Field

[0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for detecting cell stability. Background Art

[0002] With the advancement of biotechnology, basic scientific research has received extensive attention, especially in the rapid development of organoid culture, cutting-edge medical treatment and cell agriculture. Generally speaking, the application technology of cells in vitro has been widely and rapidly improved. In addition, research related to cell science, cell medicine and cell agriculture has further developed in the direction of cell transformation, which has brought about the discussion and evaluation of cell safety issues. However, there are still major challenges in the development of various directions of cell-based technology, especially in the judgment of cell status, cell mutation and cell stability during in vitro cell culture and use. As a result, the current in vitro cell use process is still in a high-cost and low-efficiency stage. At the same time, it is still a major challenge to complete the evaluation of cells through technical means during commercial use.

[0003] In addition to checking the overall status of the cell population at a certain time point through activity detection, a more comprehensive, real-time, comprehensive and in-depth evaluation method is also needed. Therefore, the present invention has developed a cell stability evaluation scheme for the stability of the cells used in cell-cultured meat, which can be evaluated in real time, continuously and comprehensively, including multiple evaluation factors such as cell proliferation, cell migration, cell adhesion, cell viability, cell apoptosis, cell autophagy, cell carcinogenesis, cell senescence, etc., and can be used for comprehensive cell evaluation technology used in processes such as continuous cell passage, cell experiments, cell organoid culture, cell-cultured meat and cell medical treatment.

[0004] Therefore, cell stability evaluation has not received much attention so far, but it is urgently needed in the field of life science research and the industrial application of cells. A technical solution for cell life activities, functional testing and stability evaluation during the continuous passage of cultivated meat cells is currently needed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting cell stability, which is suitable for detecting stability during cell cultivation. It mainly performs stability detection on continuously passaged cells during cell use, comprehensively judges the life cycle of cells, and enables cells to be used in the best economic / efficiency service life. It can effectively and accurately judge the cell status and has good reliability.

[0006] A method for detecting cell stability in the present invention for solving the above technical problems includes a cell sample, characterized in that: the cell sample includes three aspects of detection of cell function, cell mutation, and cell stability, wherein when the cell proliferation effect is good, the cell does not migrate, the cell viability is >80%, the early and late cell apoptosis ratio is ≤10%, the cell autophagy ratio is <5%, the cell has no significant expression of oncogenes, and the cell karyotype analysis shows that the chromosome number and morphology are not mutated; cell adhesion index: obvious adhesion within 2 hours; cells with cytotoxicity index ≤10%, cell senescence ratio ≤20% and cell doubling time <48 hours are stable cells; if one of the indicators does not meet the standard, the cell passage is unstable and the cell cultivation process is terminated.

[0007] The key indicator for evaluating cell stability is to measure the use cycle of cells related to cell-cultured meat and determine the optimal economic / efficiency service life of cells. It can lay a technical foundation in the direction of cell-cultured meat and has broad application prospects in the fields of cell agriculture, biomedicine and basic research.

[0008] The detection method of the present invention can identify the stability of cells of 0-35 generations or more, screen stable cell lines for the research and development and optimization of cell-cultured meat, lay a technical foundation for the efficient production of cell-cultured meat, evaluate the economic / efficiency value of the cell use process, and screen cells with the optimal service life cycle to realize cultured meat production.

[0009] A method for detecting cell stability in the present invention comprises the following specific steps: (1) Obtaining cell samples: the cells are seed cells used for cell-cultured meat; (2) Cultivating cells in vitro; (3) Cell performance testing: testing of cell proliferation, cell migration ability or cell viability, among which good cell proliferation, no cell migration and cell viability>80%; overall cell proliferation, migration and viability are part of the cell performance test; Or detection of cell mutation: detection of cell apoptosis ratio, cell autophagy ratio or cell canceration, among which the early and late apoptosis ratio of cells is ≤10%, the cell autophagy ratio is <5% and the cells have no significant expression of oncogenes; Or detection of cell stability: karyotype analysis, cell adhesion, cell senescence and cytotoxicity, etc.: cell karyotype analysis means no abnormalities in chromosome number and morphology; cell adhesion index: obvious adhesion within 2 hours; cytotoxicity index ≤10%, cell senescence ratio ≤20% and cell doubling time <48 hours; Cytomic analysis can also be performed in the detection of cell stability performance; (4) There is no particular order for the index detection in step (3). If any index fails to meet the standard, it can be judged that the cell stability does not meet the standard; In the optimization scheme, the cell samples include primary cells, bioengineered cells, immortalized cells, etc.; The cell sources include humans, mice, pigs, chickens, cows, sheep and the like.

[0010] In the further optimized scheme, in the detection of cell stability performance in step (3), the cell karyotype analysis detection includes the following steps: performing chromosome karyotype analysis by colchicine to determine the ectopic mutation of cell chromosomes, then mixing the cells with X-Gal substrate for reaction, and then judging whether the cells have β-galactosidase specific for observing aging by observing whether a dark blue product is generated.

[0011] The β-galactosidase catalyzes X-Gal to generate a dark blue product.

[0012] In the detection of cell performance in step (3), the cell migration ability is determined by a cell scratch test, and the cell migration ability is determined by the cell crawling state.

[0013] In the detection of cell mutation in step (3), the cell apoptosis ratio is determined by cell flow cytometry, and the economic / efficiency cell life cycle evaluation is performed by observing the apoptosis ratio of cells of different generations to determine whether the cells are suitable for the large-scale proliferation process.

[0014] In the detection of cell mutation in step (3), the cell autophagy ratio is determined by combining LC3B rabbit polyclonal antibody with phagocytes to produce a marker, which is used as a general marker of autophagic membrane and then the cell autophagy status is determined by fluorescence microscopy and high-content imaging and analysis.

[0015] In the detection of cell stability performance in step (3), the cell adhesion performance is measured by the fluorescent dye calcein acetoxymethyl ester.

[0016] In the detection of cell stability performance in step (3), the cytotoxicity level (≤10%) is determined by transferring the cell aliquots to a new plate and adding the CyQUANT™ LDH Cytotoxicity Assay Kit reaction mixture. Lactate dehydrogenase (LDH) is a cytoplasmic enzyme present in many different cell types. Damage to the plasma membrane will release LDH into the surrounding cell culture medium. The extracellular LDH in the culture medium can then be quantified using a microplate reader through a coupled enzyme reaction, which can effectively reflect the cytotoxicity level.

[0017] The present invention provides the application of the above-mentioned cell stability evaluation method in any of the following: (1) Application in the process of continuous cell passage; (2) Application in cell organoid culture; (3) Application in cell-cultured meat; (4) Preoperative safety evaluation of advanced cellular medicine.

[0018] The detection method of the present invention solves the problem that there is no comprehensive evaluation technology and scheme at present. At the same time, it can be used as a commercial cell evaluation scheme and cell detection and evaluation method to achieve cell status evaluation, with good efficiency and safety, and can better support cells for related applications. It can be used for continuous detection and comprehensive evaluation of amplified cells in various cell culture devices such as culture dishes and bioreactors, and can be used for continuous cell passage and cell culture meat applications. The specific advantages include at least the following aspects: (1) The cell stability evaluation method provided by the present invention can directly evaluate the cell status in all aspects, and can also increase or decrease the evaluation scheme according to specific needs, thus developing a new commercial cell stability evaluation method; (2) The cell stability evaluation method provided by the present invention can maximize the understanding of the cell state and the three-dimensional growth environment of the cell in vitro, which is beneficial to the culture and application of cells in vitro; (3) The cell stability evaluation method provided by the present invention has good biosafety, supports multiple evaluations of cell proliferation, migration, adhesion, vitality, apoptosis, autophagy, carcinogenesis, aging, etc., and presents an intuitive evaluation of cell status; (4) The cell stability evaluation method of the present invention can effectively determine whether the cell state is suitable for large-scale efficient cell expansion and culture, comprehensively evaluate cell cost, time cost, efficiency and titer, etc., ensure efficient and low-cost commercial operation of cells, and support the cell state requirements for continuous passage or other applications; (5) The cell stability evaluation method of the present invention has low cost and can be widely promoted and applied in the fields of life sciences, basic medicine and cell agriculture, which helps to improve the efficiency of cell research and commercial applications, reduce waste and lower costs. Among them, the experimental reagents or kits in the evaluation scheme are easy to obtain, and the idea of ​​the invention has not been proposed yet; (6) The cell stability evaluation method / cell state evaluation scheme of the present invention can be used commercially. The experimental process is simple and convenient, the results are accurate and effective, and the advantages are obvious, which is easy to promote and apply.

[0019] The cell stability evaluation method of the present invention combines the currently widely used cell activity detection methods, and judges the cell potential through multiple aspects, the result is relatively accurate, and has important research significance and application value. To a certain extent, the present invention can become an evaluation scheme for cell stability during the commercial use of cell culture meat, promote cell application efficiency, and improve cell use titer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the present invention are obtained by performing chromosome karyotype analysis on cells; Figure 2 The above are the observation results of the present invention in which cells of different generations were subjected to aging verification (the scale bars are all 100 μm); Figure 3 and Figure 4 The results of cell migration analysis by scratch test in the present invention are shown in Figure 5 (the scale bar is 500 μm); Figure 5 and Figure 6 The results of the present invention are obtained by performing flow cytometry analysis on cells to analyze apoptosis. Figure 7 The results of the observation of cell autophagy in the present invention (the scale bar is 50 μm); Figure 8 The results of the observation of cell adhesion in the present invention (the scale bars are all 100 μm); Fig. 9 These are the results of the stability evaluation of the freshly extracted cells and immortalized cells in the present invention. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific embodiments: Example 1 A method for detecting cell stability, the specific steps are as follows: (1) Obtaining cell samples: Cell samples include but are not limited to primary cells, bioengineered cells, and immortalized cells. Cell sources include humans, mice, pigs, chickens, cattle, sheep, etc.; (2) Cultivating cells in vitro: In vitro culture can be performed using conventional techniques; (3) Testing of cell performance: Testing of cell proliferation, cell migration ability, cell performance or cell viability, among which good cell proliferation effect, no cell migration and cell viability>80%; cell migration ability was determined by cell scratch test, and cell migration ability was judged by the cell crawling state.

[0022] Or detection of cell mutation: detection of cell apoptosis ratio, cell autophagy ratio or cell canceration, among which the early and late apoptosis ratio of cells is ≤10%, the cell autophagy ratio is <5% and the cells have no significant expression of oncogenes; The cell apoptosis ratio is determined by cell flow cytometry. The economic / efficiency cell life cycle evaluation is performed by observing the apoptosis ratio of different generations of cells to determine whether the cells are adapted to the large-scale proliferation process. The cell autophagy ratio is determined by the LC3B rabbit polyclonal antibody binding to phagocytes to produce a marker, which is used as a general marker of the autophagic membrane and then the cell autophagy status is determined by fluorescence microscopy and high-content imaging and analysis; Or detection of cell stability: karyotype analysis, cell adhesion, cell senescence and cytotoxicity, etc.: cell karyotype analysis means no abnormalities in chromosome number and morphology; cell adhesion index: obvious adhesion within 2 hours; cytotoxicity index ≤10%, cell senescence ratio ≤20% and cell doubling time <48 hours.

[0023] The cell karyotype analysis test includes the following steps: using colchicine to perform chromosome karyotype analysis to determine the ectopic mutation of the cell chromosome, then mixing the cell with the X-Gal substrate for reaction, and then observing whether a dark blue product is generated to determine whether the cell has β-galactosidase that is specific for observing aging. β-galactosidase catalyzes X-Gal to display a dark blue product.

[0024] Cell adhesion was measured using the fluorescent dye calcein acetoxymethyl ester.

[0025] The cytotoxicity level (≤10%) was determined by transferring the cell aliquots to a new plate and adding the CyQUANT™ LDH Cytotoxicity Assay Kit reaction mixture. Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types. Damage to the plasma membrane will release LDH into the surrounding cell culture medium. The extracellular LDH in the culture medium can then be quantified using a microplate reader via a coupled enzyme reaction, which effectively reflects the cytotoxicity level.

[0026] (4) There is no particular order for the index detection in step (3). If any index fails to meet the standard, it can be judged that the cell stability does not meet the standard.

[0027] Further, the stability of the cells was determined by the specific detection methods in the following Examples 2-10: The present invention provides a complete performance evaluation method for seed cells of cell-cultured meat through the following examples.

[0028] The cell samples in the following examples are primary chicken muscle cells, and the primary chicken muscle cell samples used are obtained in the following steps: (1) Disinfect fertilized eggs of about 9 weeks old; (2) Carefully cut open the eggshell, take out the embryo, cut off the head and kill the egg, then take the skeletal muscle from the leg, making sure to remove all the bones; (3) Place the cells in a dissociation tube, cut the tissue into small pieces with scissors, and wash 2-3 times; (4) Digest and dissociate to obtain cells.

[0029] Example 2 This example provides a cell karyotype analysis detection method for determining cell stability, and the required reagents include colchicine, potassium chloride, methanol and glacial acetic acid; in the raw material mixed aqueous solution, the working concentration of colchicine is 0.1μg / ml, and methanol and glacial acetic acid are the raw materials of the fixative, and the ratio is 3:1.

[0030] This embodiment also provides the above-mentioned cell karyotype detection method, comprising the following steps: (1) Cell pretreatment: Add colchicine to the culture medium to inhibit cells in metaphase division, and culture at 37°C, 5% CO2, and 100% humidity for 3.5 to 4 hours until a round, shiny sphere appears in the cell nucleus; (2) Cell digestion: Wash the adherent cells in the culture flask with PBS, then add 0.25% trypsin + 0.02% EDTA for digestion, add cell culture medium to terminate digestion, then gently blow on the cell growth surface, transfer the cell suspension to centrifuge and discard the supernatant; (3) Cell fixation: Add potassium chloride solution and place in a 37°C water bath for hypotonic treatment. After the hypotonic treatment, add the above-mentioned methanol and glacial acetic acid mixed fixative solution, mix well, centrifuge and discard the supernatant, leaving a small amount of liquid; (4) Drop staining: Drop the centrifuged cells onto a glass slide pre-cooled with ice water (0-4°C) and tilted at about 45°. Quickly blow off the cells on the drop, gently pass them over an alcohol lamp 2-3 times with the cells facing up, remove excess liquid and dry them at room temperature, then stain with Giemsa stain for 15 minutes. (5) Observation.

[0031] Example 3 This example provides a method for detecting cell aging, which has different treatment methods for iron wall cells and suspension cells. This method takes the method for detecting iron wall cell aging as an example. The required raw materials include β-galactosidase staining solution and X-Gal solution.

[0032] This embodiment includes the following steps: (1) Cell fixation: Collect and wash the cells, add β-galactosidase staining fixative, and fix at room temperature for 15 minutes; (2) Cell staining: Aspirate the cell fixative, wash the cells three times with PBS, then add the staining working solution and incubate at 37°C overnight; (3) Cell fixation: Add potassium chloride solution and place in a 37°C water bath for hypotonic treatment. After the hypotonic treatment, add the above-mentioned methanol and glacial acetic acid mixed fixative solution, mix well, centrifuge and discard the supernatant, leaving a small amount of liquid; (4) Observation: Take some of the stained cells and drop them onto a glass slide or a 6-well plate and observe under an ordinary optical microscope. Remove the staining solution and add PBS. It can be stored at 4°C for several days.

[0033] Example 4 This example provides a method for detecting cell autophagy. The LC3B protein plays a key role in autophagy. Normally, this protein is present in the cytoplasm, but after cleavage and lipidation with phosphatidylethanolamine, LC3 binds to phagocytic cells. This localization can be used as a general marker of autophagic membranes. The specific steps are as follows: (1) Fixation and permeabilization: Fix and permeabilize the cells with 3.7% formaldehyde, 0.2% Triton® X-100 and PBS; (2) Primary antibody incubation: remove the permeabilization buffer, add diluted primary antibody to the cells, and incubate at room temperature for 1 hour; Remove the cell fixative solution, wash the cells 3 times with PBS, then add the staining solution and incubate at 37°C overnight; (3) Cell staining: remove the washing solution and incubate the cells with secondary antibody overnight; (4) Observation: Rinse the cells with PBS and take some of the stained cells, drop them onto a slide or a 6-well plate, and observe under an ordinary optical microscope. The samples can be stored at 4°C for several days.

[0034] Example 5 This example provides a method for detecting cell adhesion. Calcein AM does not emit fluorescence, but once loaded into cells, it is cleaved by endogenous esterases to produce highly fluorescent calcein. Calcein provides a bright fluorescent, pH-independent cytoplasmic cell marker that can minimize interference with the cell adhesion process. The steps for the calcein cell adhesion detection are as follows: (1) Fixation and permeabilization: Add calcein AM stock solution to the cell suspension and incubate at 37°C for 30 min. (2) Sample preparation: Wash cells twice with pre-warmed RPMI, suspend cells in RPMI, and add the calcein-labeled cell suspension to prepared microplate wells containing confluent cells (medium removed) or coated with ECM proteins or antibodies; Remove the cell fixative solution, wash the cells 3 times with PBS, then add the staining solution and incubate at 37°C overnight; (3) Fluorescence measurement: Wash four times and add PBS to each well. Measure fluorescence using a fluorescein filter set (the maximum absorbance of calcein is 494 nm and the maximum emission is 517 nm). (4) Result processing: The percentage of adherence was determined by dividing the corrected (background-subtracted) fluorescence of adherent cells by .

[0035] Example 6 This example provides a method for detecting cytotoxicity. Lactate dehydrogenase (LDH) is a cytoplasmic enzyme present in many different cell types and is a clear and reliable indicator for detecting cytotoxicity. Damage to the plasma membrane releases LDH into the surrounding cell culture medium, where LDH catalyzes the conversion of lactate to pyruvate through the reduction of NAD+ to NADH. NADH causes the reduction of tetrazolium salts (INT) to a red formazan product, which can be measured by spectrophotometry at 490nm. The level of formazan formation is proportional to the amount of LDH released into the culture medium, which can indicate cytotoxicity, as follows: (1) Sample preparation: Prepare cell dilutions (0-10,000 cells / 100 µL medium) in triplicate wells of two groups of 96-well tissue culture plates (the first group is used to measure maximum LDH release, and the second group is used to measure spontaneous LDH release), and incubate the cells overnight in a 37°C incubator with an appropriate level of CO2; (2) Sample lysis: Add 10X lysis buffer to dilute to the maximum LDH release, then gently tap to mix, and then place the plate in the incubator and incubate at 37°C with an appropriate level of CO2 for 45 minutes; (3) Sample grouping: transfer 50 µL of each sample medium into a 96-well plate in triplicate, mix well, and incubate the plate at room temperature in the dark for 30 min; (4) Termination: Add 50 µL of stop solution to each sample well and tap gently to mix. After adding the stop solution, measure the absorbance (at 490 nm and 680 nm) within 1-2 hours. (5) Measurement: Plot the relationship between the maximum LDH release control absorbance value minus the spontaneous LDH release control absorbance value and the cell number to determine the linear range and optimal cell number of the LDH cytotoxicity assay.

[0036] Example 7 This example provides a two-color fluorescence detection method for cell viability. The two-color fluorescence detection method can distinguish metabolically active cells from injured cells and dead cells. In metabolically active cells, C12-resazurin is reduced to red fluorescent C12-resorufin and absorbed by the cell permeabilizer; in cells with damaged plasma membranes (usually late apoptotic and necrotic cells), the green fluorescent nucleic acid stain SYTOX® Green dye is used. The specific operation is as follows: (1) Experimental preparation: Incubate cells with 2 mM hydrogen peroxide at 37°C for 4 h as a negative control; untreated cells should be used as a positive control for C12-resazurin staining; dissolve C12-resazurin in 100 µL of dimethyl sulfoxide to obtain a 1 mM C12-resazurin stock solution; dilute the C12-resazurin stock solution with dimethyl sulfoxide to prepare a 50 µM C12-resazurin new working solution; prepare a 1 µM SYTOX Green stain working solution; (2) Cell staining: First, dilute the cells to 1×106 cells / mL with PBS, add 1µL of 50µM C12-rasapurine working solution and 1µL of 1µM SYTOX Green staining working solution per 100µL of cell suspension, and incubate at 37°C, 5% CO2 for 15 min. (3) Dilution: After incubation, add 400 µL of PBS, mix gently, and place the sample on ice. (4) Observation and analysis: Analyze the stained cells using a flow cytometer and measure fluorescence emission at wavelengths of 530 nm and 575 nm. The cell population should be divided into two groups: live cells with low green fluorescence content and high orange fluorescence content, and necrotic cells with high green fluorescence content and low orange fluorescence content.

[0037] Example 8 This example provides a method for detecting cell canceration. Taking pig cells as an example, p21 is a pig tumor suppressor gene. The degree of cell canceration of pig cells can be determined by measuring the content of p21. The specific operation is as follows: (1) Preparation: Determine the number of microplates required to test the desired number of samples and set up each sample, standard, and blank control. Remove particulates from the cell suspension by centrifugation and assay immediately, or aliquot and store the samples at -20°C or -80°C to avoid loss of biological activity and contamination; (2) Add samples: Add 50 μL of standard or sample to the corresponding wells of the antibody pre-coated microplate and mix gently. Incubate at 37°C for 45 minutes; (3) Washing: remove the liquid, shake and dry, add washing buffer diluted 30 times with distilled water to each well, let it stand for 30 seconds and then remove it. Repeat 4 times; (4) Reaction: Add 50 μL of diluted biotinylated anti-IgG to all wells and incubate at 37°C for 30 min. Then add 50 μL of streptavidin-HRP to all wells, mix gently, and incubate at 37°C for 15 min. (5) Color development: Add color development solution and incubate at 37°C for 15 min. (6) Stop reaction: Add 50 μL of stop solution to each well to stop the reaction (the blue color immediately turns to yellow); (7) Detection: Taking the blank well as zero, measure the optical density at a wavelength of 450 nm within 15 minutes after adding the stop solution.

[0038] Example 9 In this example, the cell migration ability was determined by a scratch test.

[0039] An artificial blank area is created on the fused monolayer of cells, called a "scratch / wound". The cells at the edge will gradually enter the blank area to heal the "scratch / wound". Because it is similar to the in vitro wound healing process, it is also called the wound healing experiment, and is widely used to observe the effects of exogenous factors such as drugs and genes on cell migration and repair. The specific operation is as follows: (1) Cell inoculation: Use a marker to draw horizontal lines on the back of a 6-well plate. Draw at least 5 lines across each well, and each line should be even and parallel. Then inoculate about 5-10*105 cells in the well. The principle is that the cells can grow completely overnight. (2) Cell streaking: The next day, use a 20uL pipette tip (sterilized) or toothpick to scratch the black line on the back of the well plate vertically, so that the scratch is the same as the marked line. After streaking, wash the cells 2-3 times with sterile PBS to remove the streaked cells so that the gap left is clearly visible to the naked eye, and then replace with fresh serum-free or low-serum (<2%) culture medium; (3) Observation: Place the cells in a 37°C, 5% CO2 incubator. Then, take out the cells at appropriate time points, such as 0, 6, 12, and 24 hours, and observe and photograph them under a microscope; (4) Analysis: After opening the image using ImageJ software, 6 to 8 horizontal lines were randomly drawn and the average distance between cells was calculated.

[0040] Example 10 This example provides a method for detecting cell apoptosis, which has the same principles and steps as the method for detecting cell autophagy in Example 3, except that the primary antibody and the secondary antibody need only be replaced according to specific circumstances and observation can be performed.

[0041] There is no strict order requirement for the above cell detection embodiments.

[0042] The key indicators for stability evaluation are: good cell proliferation effect; no cell migration; cell viability above 80%; early and late cell apoptosis ratio not exceeding 10%; cell autophagy ratio less than 5%; no significant expression of oncogenes in cells; karyotype analysis shows no abnormalities in chromosome number and morphology; cell adhesion index: obvious adhesion within 2 hours; cytotoxicity index ≤10%; cell senescence ratio not exceeding 20%; cell doubling time less than 48 hours; there is no order of priority for the above evaluation indicators, and if any indicator does not meet the standard, it can be judged that the cell stability does not meet the standard.

[0043] Through the above-mentioned Examples 2-10, the present invention summarizes the stability evaluation of newly extracted cells and immortalized cells. The results show that when the newly extracted immortalized cells are continuously passaged to the 25th generation, all cell parameters are normal, but at the 35th generation, the proliferation ability, autophagy and other functions and stability of the cells begin to be affected, while the immortalized cells can still maintain normal parameters at the 60th generation. The above examples show that the present invention provides an evaluation scheme and cell state measurement standard for seed cell proliferation efficiency, safety and stability in terms of cell stability detection, provides a cell monitoring and evaluation system for the manufacture of cell-cultured meat, and provides technical support for the industrial production of cell-cultured meat.

[0044] The detection method of the present invention can be used to comprehensively evaluate the life cycle of primary seed cells related to cell-cultured meat based on safety considerations, can also be used in the continuous cell passaging process in scientific research, can be used in organoid culture in life science and medical use; and can provide the optimal use cycle and economic value for seed cell raw materials in the process of cell-cultured meat manufacturing.

[0045] The above implementation / test examples are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for detecting cell stability, comprising a cell sample, characterized in that: The cell samples are tested for three aspects: cell function, cell mutation, and cell stability. When the cell proliferation effect is good, the cells do not migrate, the cell viability is >80%, the early and late apoptosis ratios of the cells are ≤10%, the cell autophagy ratio is <5%, and the cells have no significant expression of oncogenes; the cell karyotype analysis shows that there is no mutation in the number and morphology of chromosomes; the cell adhesion index: obvious adhesion within 2 hours; cells with a cytotoxicity index of ≤10%, a cell senescence ratio of ≤20%, and a cell doubling time of <48 hours are stable cells; if one of the indicators does not meet the standard, the cell passage is unstable and the cell cultivation process is terminated.

2. A method for detecting cell stability according to claim 1, characterized in that: The specific steps are as follows: (1) Obtain cell samples; (2) Cultivating cells in vitro; (3) Testing of cell performance: testing of cell proliferation, cell migration ability or cell viability, where good cell proliferation, no cell migration and cell viability>80%; Or detection of cell mutation: detection of cell apoptosis ratio, cell autophagy ratio or cell canceration, among which, the early and late apoptosis ratio of cells is ≤10%, the cell autophagy ratio is <5% and the cells have no significant expression of oncogenes; Or detection of cell stability: detection of karyotype analysis, cell adhesion, cell senescence or cytotoxicity, where karyotype analysis means no abnormalities in chromosome number and morphology; cell adhesion index: obvious adhesion within 2 hours; cytotoxicity index ≤10%, cell senescence ratio ≤20% and cell doubling time <48 hours; (4) There is no particular order in which the indicators are tested in step (3). If any indicator does not meet the standard, it is determined that the cell stability does not meet the standard.

3. A method for detecting cell stability according to claim 1 or 2, characterized in that: The cell samples include primary cells, bioengineered cells and immortalized cells.

4. A method for detecting cell stability according to claim 1 or 2, characterized in that: The cell sources include humans, mice, pigs, chickens, cows and sheep.

5. A method for detecting cell stability according to claim 2, characterized in that: In the detection of cell stability performance in step (3), the cell karyotype analysis includes the following steps: performing chromosome karyotype analysis by colchicine to determine the ectopic mutation of cell chromosomes, then mixing the cells with X-Gal substrate for reaction, and then judging whether the cells have β-galactosidase specific for observing aging by observing whether a dark blue product is generated.

6. A method for detecting cell stability according to claim 5, characterized in that: The β-galactosidase catalyzes X-Gal to generate a dark blue product.

7. A method for detecting cell stability according to claim 2, characterized in that: In the detection of cell performance in step (3), the cell migration ability is determined by a cell scratch test, and the cell migration ability is determined by the cell crawling state.

8. A method for detecting cell stability according to claim 2, characterized in that: In the detection of cell mutation in step (3), the cell apoptosis ratio is determined by cell flow cytometry, and the economic / efficiency cell life cycle evaluation is performed by observing the apoptosis ratio of cells of different generations to determine whether the cells are suitable for the large-scale proliferation process.

9. A method for detecting cell stability according to claim 2, characterized in that: In the detection of cell mutation in step (3), the cell autophagy ratio is determined by combining LC3B rabbit polyclonal antibody with phagocytes to produce a marker, which is used as a general marker of autophagic membrane and then the cell autophagy status is determined by fluorescence microscopy and high-content imaging and analysis; in the detection of cell stability performance, the cell adhesion performance is measured by the fluorescent dye calcein acetoxymethyl ester.

10. Use of the method for detecting cell stability according to claim 1 or 2, characterized in that: The method is applicable to any one of the processes of continuous cell passage, cell organoid culture, preparation of cell cultured meat and preoperative safety evaluation of advanced cell medicine.

Images