Cell quality detection method and system in clinical research of cellular immunotherapy

Abstract

The invention discloses a cell quality detection method and system in clinical research of cellular immunotherapy, and relates to the technical field of cellular immunotherapy, and the method comprises the following steps: 1, obtaining cells, observing morphological characteristics of the cells through a microscope, and recording health conditions of the cells; according to the invention, comprehensive cell quality detection is carried out on cell morphology, activity, phenotype, cytokine secretion, cytogenetics, microbial contamination, functionality and the like, so that the cell quality is evaluated more accurately, and missing of potential problems is reduced; a plurality of advanced detection technologies and methods such as trypan blue dye refusal method, flow cytometry, ELISA (enzyme-linked immuno sorbent assay), FISH (fluorescence in situ hybridization) and the like are utilized and are matched with one another to comprehensively detect cells, so that the accuracy and the reliability of a detection result are improved; by optimizing the detection process and method, each detection module is clear in division of labor, a large number of cell samples are rapidly analyzed, the requirement for rapid detection of cell quality in clinical research is met, and the treatment cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of cell immunotherapy, and in particular to a method and system for detecting cell quality in clinical research of cell immunotherapy. Background Art

[0002] As an emerging tumor treatment method, cell immunotherapy has attracted widespread attention in the medical field in recent years. Its basic principle is to activate or enhance the patient's own immune system so that immune cells can recognize and attack tumor cells, thereby achieving the purpose of treating tumors. In clinical research on cell immunotherapy, cell quality detection is a crucial link. The quality of cells is directly related to the safety and effectiveness of treatment. If poor quality cells are used for treatment, it may lead to poor treatment effects and may even cause serious adverse effects on the patient's body. However, existing cell quality detection methods often have the following shortcomings: 1. The testing content is not comprehensive enough. Some traditional testing methods only focus on one aspect of cell characteristics, such as cell morphology and activity, but ignore other important characteristics of cells, such as cell phenotype, cytokine secretion, cell genetics, microbial contamination, and functionality. Such testing methods cannot fully evaluate the quality of cells and may miss some potential problems; Second, the accuracy of detection needs to be improved. Some existing detection technologies may have problems such as large errors and poor repeatability, resulting in inaccurate test results. For example, some cell activity detection methods may be interfered by external factors, thus affecting the reliability of the test results; 3. Low detection efficiency. Some detection methods require a lot of time and manpower, and cannot meet the needs of rapid cell quality detection in clinical research. This will not only affect the timeliness of treatment, but may also increase treatment costs; Therefore, a method and system for detecting cell quality in clinical research of cell immunotherapy was proposed. Summary of the invention

[0003] The purpose of the present invention is to provide a method and system for detecting cell quality in clinical research of cell immunotherapy, so as to solve one of the problems raised in the above background technology.

[0004] In order to solve the above technical problems, a technical solution adopted in this application is: a method for detecting cell quality in clinical research of cell immunotherapy, comprising the following steps: Step 1: Obtain cells, observe the morphological characteristics of the cells under a microscope, and record the health status of the cells; Step 2: Use trypan blue exclusion method to detect cell activity, calculate cell activity rate, and use flow cytometry to detect cell apoptosis rate and proliferation activity; Step 3: Analyze cell surface markers using flow cytometry to determine cell type and differentiation status, and detect activation markers and inhibition markers on the cell surface to evaluate immune function status; Step 4: Based on the cell culture supernatant, the cytokines secreted by the cells were detected by enzyme-linked immunosorbent assay to evaluate the immune activity and function; Step 5: Use chromosome karyotype analysis or fluorescence in situ hybridization to detect chromosomes of cells and evaluate genetic stability; Step 6: Use bacteria and fungi culture methods to detect microbial contamination in the cell culture process, and use polymerase chain reaction technology to detect the nucleic acid of microorganisms in the cells; Step 7: Based on the cytotoxicity experiment, detect the killing activity of cells against tumor cells and evaluate the immunotherapy effect of cells in vivo.

[0005] As a further preferred embodiment of the present technical solution: in step 1, the microscope is an optical microscope or an electron microscope; the morphological characteristics of the cells include cell morphology, cell size and cell structure.

[0006] As a further preferred embodiment of the present technical solution: in step 2, the method for detecting cell activity using trypan blue exclusion method comprises the following steps: Step 101, taking a cell sample to be tested and mixing it with a trypan blue solution, wherein the concentration of the trypan blue solution is 0.4%; Step 102, observing the dead cells stained blue and the live cells not stained blue under a microscope, and counting the number of live cells and dead cells; Step 103, calculating the cell activity rate using an activity rate calculation formula, wherein the cell activity rate calculation formula is: cell activity rate = number of live cells / (number of live cells + number of dead cells) × 100%; The method for detecting cell apoptosis rate and proliferation activity by flow cytometry comprises the following steps: Step 201, obtaining a cell sample to be tested, and labeling apoptosis and proliferation-related markers on the cell surface with fluorescently labeled antibodies; Step 202, analyzing the fluorescence intensity of the cell sample based on flow cytometry to determine the number of apoptotic cells and the number of cells in different cell cycle stages; Step 203: Calculate the cell apoptosis rate and proliferation activity using a formula.

[0007] As a further preferred embodiment of the present technical solution: the calculation formula of the cell apoptosis rate is: Cell apoptosis rate = number of apoptotic cells / total number of cells × 100%; The cell proliferation activity is represented by the proliferation index PI, and the calculation formula is: PI=(S+G2 / M) phase cell number / (G0 / G1+S+G2 / M) phase cell number; Among them, the S phase and G2 / M phase represent the cell cycle stages in a proliferative state, and the G0 / G1 phase represents the quiescent or non-proliferative state cells.

[0008] As a further preferred embodiment of the present technical solution: in step 4, the method of detecting the concentration of cytokines secreted by cells by using enzyme-linked immunosorbent assay (ELISA) comprises the following steps: Step 301, adding the cell culture supernatant into the ELISA plate wells to bind with the antibodies on the plate wells; Step 302, adding enzyme-labeled antibodies to react with cytokines on the plate wells; Step 303, adding a substrate solution to produce a color reaction; Step 304: Calculate the concentration of cytokines based on the intensity of the color development reaction measured by the microplate reader.

[0009] As a further preferred embodiment of the present technical solution: in step five, the chromosome karyotype analysis utilizes conventional chromosome banding technology or high-resolution chromosome banding technology.

[0010] As a further preferred embodiment of the present technical solution: in step six, the bacteria and fungi culture method utilizes a culture medium for detection, and the culture medium includes a blood plate culture medium, a chocolate plate culture medium and a Sabouraud dextrose agar culture medium.

[0011] As a further preferred embodiment of the present technical solution: in step seven, the method for detecting the killing activity of immune cells against tumor cells comprises the following steps: Step 401, obtaining a cell sample to be tested and a tumor cell sample, wherein the tumor cell sample includes lung cancer cells, breast cancer cells and liver cancer cells; Step 402, mixing and culturing the cell sample and the tumor cell sample according to a certain proportion; Step 403: After a predetermined period of time, the survival rate of the tumor cells is detected, and the killing activity of the cell sample is calculated.

[0012] As a further preferred embodiment of the present technical solution: in the process of analyzing the fluorescence intensity of cells based on flow cytometry, multiple control samples are set, including cell samples not labeled with fluorescent antibodies and cell samples labeled with only a single fluorescent antibody.

[0013] To solve the above technical problems, another technical solution adopted in this application is: a cell quality detection system in clinical research of cell immunotherapy, including: a morphological detection module, an activity detection module, a phenotypic analysis module, a cytokine detection module, a genetic detection module, a microbial detection module and a functional detection module; The morphological detection module is used to observe the morphological characteristics of cells through a microscope to determine whether the cells are intact and have abnormal morphology, and to record the morphological characteristics of the cells and compare them with normal cells to assess health status; The activity detection module is used to detect cell activity using the trypan blue exclusion method, calculate the cell activity rate, and detect the cell apoptosis rate and proliferation activity by flow cytometry; The phenotypic analysis module is used to analyze the expression of cell surface markers using flow cytometry, determine cell types and differentiation states, detect activation markers and inhibition markers on the cell surface, and evaluate immune function status; The cytokine detection module is used to collect cell culture supernatant, detect the concentration of cytokines secreted by cells using ELISA, analyze cytokine secretion, and evaluate immune activity and function; The genetic detection module is used to detect chromosomal abnormalities of cells using chromosome karyotype analysis or FISH technology, detect gene mutations and epigenetic changes, and evaluate genetic stability; The microbial detection module is used to detect microbial contamination during cell culture using bacterial and fungal culture methods, and to detect whether there is microbial nucleic acid in the cells using PCR technology; The functional detection module is used to conduct cytotoxicity experiments to detect the killing activity of immune cells on tumor cells, and to conduct in vivo experiments using animal models to evaluate the immunotherapy effect of cells in vivo.

[0014] Advantages of the present invention: 1. The present invention conducts comprehensive cell quality testing on multiple aspects such as cell morphology, activity, phenotype, cytokine secretion, cytogenetics, microbial contamination and functionality, so as to more accurately assess cell quality and reduce the omission of potential problems; 2. The present invention improves the accuracy and reliability of the test results by utilizing a variety of advanced detection technologies and methods such as trypan blue exclusion, flow cytometry, ELISA, FISH, etc., and cooperating with each other to conduct comprehensive detection of cells; 3. The present invention optimizes the detection process and method, and each detection module has a clear division of labor. At the same time, it adopts efficient technical means, such as flow cytometry, ELISA, etc., which can quickly analyze a large number of cell samples, thereby improving the detection efficiency. It can meet the needs of rapid detection of cell quality in clinical research, does not affect the timeliness of treatment, and reduces the cost of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 This is a schematic diagram of the process of the cell quality detection method in the clinical study of cell immunotherapy of the present invention; Figure 2 The figure is a schematic diagram of the process of detecting cell activity by trypan blue exclusion method of the present invention; Figure 3 The figure is a schematic diagram of the process of the method for detecting cell apoptosis rate and proliferation activity by flow cytometry of the present invention; Figure 4 The figure is a schematic diagram of the process of the method for detecting the concentration of cytokines secreted by cells by using ELISA of the present invention; Figure 5 This is a schematic diagram of the process of the method for detecting the killing activity of immune cells against tumor cells of the present invention; Figure 6 This is a schematic diagram of the functional modules of the cell quality detection system in the clinical research of cell immunotherapy of the present invention. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Embodiment 1 Figure 1 is a flow chart of a method for detecting cell quality in a clinical study of cell immunotherapy according to an embodiment of the present invention. It should be noted that if there are substantially the same results, the method of the present application is not regarded as Figure 1 The process sequence shown is limited. Figure 1-Figure 5 As shown: The cell quality detection method in the clinical study of cell immunotherapy includes the following steps: Step 1: Obtain cells, observe the morphological characteristics of the cells under a microscope, and record the health status of the cells; specifically: First, take the cell sample to be tested and prepare the cell smear; Then, observe the cell smear under a microscope and record the cell's morphology, size, structure and other characteristics; Finally, the health of the cells was assessed by comparing them with normal cells.

[0019] Step 2: Use the trypan blue exclusion method to detect cell activity, calculate the cell activity rate, and use flow cytometry to detect the cell apoptosis rate and proliferation activity; specifically: Specific steps for detecting cell activity using the trypan blue exclusion method: First, take an appropriate amount of the cell sample to be tested and mix it with 0.4% trypan blue solution; Then, observe under a microscope and count the number of live and dead cells; Finally, the cell activity rate was calculated according to the formula.

[0020] Specific steps for detecting cell apoptosis rate and proliferation activity by flow cytometry: First, the cell samples to be tested are collected and labeled with apoptosis- and proliferation-related markers using fluorescently labeled antibodies; Then, the cell fluorescence intensity was analyzed by flow cytometry to determine the number of apoptotic cells and cells in different cell cycle stages; Finally, the cell apoptosis rate and proliferation index PI were calculated according to the formula.

[0021] Step 3: Analyze the expression of cell surface markers using flow cytometry to determine cell type and differentiation status, detect activation markers and inhibition markers on the cell surface, and evaluate the immune function status; specifically: Specific steps to determine cell type and differentiation status: First, collect the cell samples to be tested and label the markers on the cell surface with fluorescently labeled antibodies; Then, the fluorescence intensity of the cells was analyzed by flow cytometry to determine the cell type and differentiation status.

[0022] Specific steps to assess immune status: First, fluorescently labeled antibodies are used to label activation markers and inhibitory markers on the surface of immune cells; Then, the fluorescence intensity of the cells was analyzed by flow cytometry to evaluate the immune function status of the cells.

[0023] Step 4: Based on the cell culture supernatant, use enzyme-linked immunosorbent assay (ELISA) to detect the cytokines secreted by the cells to evaluate the immune activity and function; specifically: First, cell culture supernatant was collected; Then, the supernatant was added to the wells of the ELISA plate to bind to the antibody; Next, enzyme-labeled antibodies and substrate solution are added to produce a color reaction; Finally, the color reaction intensity was measured by an enzyme reader and the cytokine concentration was calculated.

[0024] Step 5: Use chromosome karyotype analysis or fluorescence in situ hybridization (FISH) technology to detect chromosomal abnormalities in cells, detect gene mutations and epigenetic changes, and evaluate genetic stability; specifically: Specific steps for using chromosome karyotype analysis to detect chromosomal abnormalities in cells: First, collect the cell samples to be tested and perform cell culture; Then, during metaphase of cell division, the cells were treated with colchicine to stop cell division; Next, the cells are collected and chromosome specimens are prepared; Finally, the morphology and number of chromosomes were observed under a microscope, and chromosomal abnormalities were analyzed.

[0025] Specific steps for using FISH technology to detect gene mutations and epigenetic changes in cells: First, collect the cell samples to be tested and prepare cell smears; Then, a fluorescently labeled probe is hybridized with the DNA on the cell smear; Finally, hybridization signals were observed under a fluorescence microscope to analyze gene mutations and epigenetic changes.

[0026] Step 6: Use bacterial and fungal culture methods to detect microbial contamination during cell culture, and use polymerase chain reaction (PCR) technology to detect the nucleic acid of microorganisms in cells; specifically: Specific steps for detecting microbial contamination during cell culture using bacterial and fungal culture methods: First, take the cell sample to be tested and inoculate it on bacterial or fungal culture medium; Then, culture it at a suitable temperature for a period of time and observe whether bacteria or fungi grow on the culture medium.

[0027] Specific steps for using PCR technology to detect whether microbial nucleic acids exist in cells: First, the nucleic acid in the cell sample to be tested is extracted; Then, primers targeting specific microorganisms are designed and PCR amplification is performed; Finally, the PCR products are analyzed by gel electrophoresis to determine whether nucleic acids of microorganisms such as viruses are present in the cells.

[0028] Step 7: Based on the cytotoxicity experiment, detect the killing activity of cells against tumor cells and evaluate the immunotherapy effect of cells in vivo; specifically: Specific steps of cytotoxicity experiment: First, collect samples of immune cells and tumor cells to be tested; Then, immune cells and tumor cells are mixed and cultured in a certain ratio; Finally, after a preset period of time, the survival rate of tumor cells was detected and the killing activity of immune cells was calculated.

[0029] Specific steps for in vivo experiments using animal models: First, the cells to be tested are transplanted into an animal model; Then, the tumor growth, survival time and other indicators of the animal model were observed to evaluate the immunotherapy effect of the cells in vivo.

[0030] In this embodiment, specifically: in step one, the microscope is an optical microscope or an electron microscope; wherein the optical microscope is suitable for observing the morphology, size and general structure of cells, such as cell membrane, cell nucleus, cytoplasm, etc.; the electron microscope provides higher resolution images, which is suitable for observing the fine structures inside cells, such as organelles, tiny structures on the cell membrane, etc.

[0031] The morphological characteristics of cells include cell form, cell size and cell structure; among them, cell morphology refers to the shape and outline of cells. Cells can have various shapes, such as spherical, elliptical, fusiform, flat, etc. These morphological differences are closely related to the function of the cells and the environment in which they are located; cell size refers to the volume of cells. The sizes of different types of cells vary significantly, ranging from bacterial cells as small as a few microns to plant cells as large as a few millimeters. The size of cells is also related to their functions. For example, red blood cells are smaller so that they can flow smoothly in the blood, while egg cells are larger to store enough nutrients for embryonic development; cell structure refers to the composition and layout inside cells. Cells are composed of many complex structures and organelles, such as cell membranes, nuclei, mitochondria, endoplasmic reticulum, Golgi bodies, etc. These structures and organelles each have different functions and work together to maintain the normal operation of cells.

[0032] In this embodiment, specifically: in step 2, the method for detecting cell activity using trypan blue exclusion method includes the following steps: Step 101, taking a cell sample to be tested and mixing it with a trypan blue solution, wherein the concentration of the trypan blue solution is 0.4%; wherein, when performing the trypan blue exclusion method, it is necessary to ensure that the concentration of the trypan blue solution is appropriate to avoid poor staining effect due to too high or too low concentration; Step 102, observing the dead cells stained blue and the live cells not stained blue under a microscope, and counting the number of live cells and dead cells; wherein the number of live cells and dead cells is counted using a counting plate or an automatic cell counter; Step 103, using an activity rate calculation formula to calculate the cell activity rate, the cell activity rate calculation formula is: cell activity rate = number of live cells / (number of live cells + number of dead cells) × 100%.

[0033] The method for detecting cell apoptosis rate and proliferation activity by flow cytometry includes the following steps: Step 201, obtaining a cell sample to be tested, and labeling apoptosis and proliferation-related markers on the cell surface with fluorescently labeled antibodies; wherein the labeling process must follow the antibody instructions to ensure labeling efficiency and specificity, and the selection of labeled antibodies should be based on the cell type to be tested and the markers to be detected to ensure the accuracy and specificity of the results; Step 202: Analyze the fluorescence intensity of the cells based on flow cytometry to determine the number of apoptotic cells and the number of cells in different cell cycle stages; wherein apoptotic cells are usually manifested by enhanced signals in a specific fluorescence channel; Step 203: Calculate the cell apoptosis rate and proliferation activity using a formula.

[0034] The cell activity, apoptosis rate and proliferation activity were detected by trypan blue exclusion method and flow cytometry, respectively, providing key data for cell quality assessment.

[0035] In this embodiment, the specific calculation formula of cell apoptosis rate is: Cell apoptosis rate = number of apoptotic cells / total number of cells × 100%; The number of apoptotic cells is the number of cells in apoptotic state identified and counted by flow cytometry or other apoptosis detection methods; the total number of cells is the total number of all cells in the sample, including living cells, apoptotic cells and possible necrotic cells; the number of apoptotic cells is divided by the total number of cells and then multiplied by 100% to obtain the percentage of cell apoptosis rate.

[0036] The cell proliferation activity is expressed by the proliferation index PI, and the calculation formula is: PI=(S+G2 / M) phase cell number / (G0 / G1+S+G2 / M) phase cell number; Among them, the S phase and G2 / M phase represent the cell cycle stages in a proliferative state, and the G0 / G1 phase represents the quiescent or non-proliferative state cells; Among them, the S phase is the synthesis phase of the cell cycle, when the cell is replicating its DNA; The G2 / M phase is the pre-mitotic and mitotic phases of the cell cycle, when the cell has completed DNA replication and is preparing for or undergoing cell division; The G0 / G1 phase is the quiescent or non-proliferative state in the cell cycle, when cells do not undergo DNA replication and cell division; The number of cells in the (S+G2 / M) phase is the number of cells in the proliferation state (S phase and G2 / M phase) identified and counted by flow cytometry or other cell cycle analysis methods; The number of cells in the (G0 / G1+S+G2 / M) phase is the total number of cells in the sample at different cell cycle stages.

[0037] In this embodiment, specifically: in step 4, the method of detecting the concentration of cytokines secreted by cells using enzyme-linked immunosorbent assay (ELISA) includes the following steps: Step 301, adding the cell culture supernatant into the ELISA plate wells to bind with the antibodies on the plate wells; Specifically: add the cell culture supernatant to the ELISA plate wells, add an appropriate amount of sample to each well to ensure that the sample is fully combined with the specific antibody coated on the plate wells; incubate for a period of time (usually several hours) to allow the cytokines in the sample to form an antigen-antibody complex with the coated antibody; Step 302, adding enzyme-labeled antibodies to react with cytokines on the plate wells; Specifically: washing the ELISA plate to remove unbound sample components; adding enzyme-labeled specific antibodies that can specifically bind to cytokines bound to the plate wells; incubating again to allow the enzyme-labeled antibodies to bind to the cytokine-coated antibody complex; Step 303, adding a substrate solution to produce a color reaction; Specifically: wash the ELISA plate to remove unbound enzyme-labeled antibodies; add substrate solution, the substrate undergoes a chemical reaction under the action of the enzyme to produce a detectable color change; incubate for a period of time to allow the color development reaction to fully proceed; Step 304, based on the intensity of the color development reaction measured by the microplate reader, the concentration of the cytokine is calculated; Specifically: Use a microplate reader to measure the intensity of the color reaction in each well, usually by determining the optical density (OD) value at a specific wavelength; convert the OD value into the concentration of the cytokine according to the standard curve or the calculation method provided by the kit; the standard curve is established through a series of cytokine samples of known concentration and the intensity of the color reaction, which is used to calibrate and calculate the cytokine concentration of unknown samples.

[0038] In this embodiment, specifically: in step 5, the method used for chromosome karyotype analysis is conventional chromosome banding technology or high-resolution chromosome banding technology; Among them, conventional chromosome banding technology is one of the commonly used methods in chromosome karyotype analysis. It uses specific chemical reagents to treat chromosomes so that they present specific banding patterns, so that each chromosome can be identified and distinguished more precisely. The specific steps are as follows: First, the cell suspension is subjected to a series of treatments, such as cell culture, fixation, and slide preparation, to obtain chromosome specimens that can be used for banding; Then, specific chemical reagents (such as Giemsa dye) are used to stain the chromosomes, making them appear as alternating dark and light horizontal stripes, namely G bands; Finally, the banding characteristics of the chromosomes are observed under a microscope, and the chromosomes are identified and paired based on their length, centromere position, arm ratio, satellites and other characteristics to obtain the chromosome karyotype of the cell.

[0039] Among them, high-resolution chromosome banding technology is a more sophisticated chromosome banding technology formed in the 1970s. It can display more chromosome bands and improve the accuracy and resolution of chromosome analysis. The specific steps are as follows: First, the cells are synchronized to prometaphase using reagents such as methotrexate and thymidine to obtain more clearly visible chromosomes; Then, the cells are subjected to special chemical or physical treatments, such as hot phosphate treatment, hot alkali treatment, etc., to obtain different types of banding (such as R bands, C bands, etc.); Next, high-resolution staining techniques, such as fluorescence in situ hybridization (FISH) combined with specific DNA probes, are used to more precisely mark and observe chromosomes; Finally, observation and analysis are performed under a microscope or using advanced image processing technology to identify the banding characteristics of each chromosome, and they are numbered and sorted.

[0040] In this embodiment, specifically: in step six, the bacteria and fungi culture method uses a culture medium for detection, and the culture medium includes a blood plate culture medium, a chocolate plate culture medium, and a Sabouraud dextrose agar culture medium; Among them, blood plate culture medium is mainly composed of basic culture medium (such as nutrient agar) and a certain proportion of sterile blood. It is mainly used to separate and culture bacteria, especially those bacteria that need blood components to grow well, such as streptococci and Staphylococcus aureus. At the same time, blood plate culture medium can also be used to identify the hemolytic nature of streptococci by observing the lysis of red blood cells around the colonies. Chocolate plate medium contains peptone, beef powder, sodium chloride, agar, and slightly heated defibrated sheep blood. It may also contain nutrients such as V and X factors. It is mainly used to isolate and culture aerobic bacteria such as Neisseria (such as Neisseria meningitidis and Neisseria gonorrhoeae) and Haemophilus. These bacteria can grow rapidly and form visible colonies on chocolate plate medium. Sabouraud dextrose agar medium (SDA medium) mainly contains glucose, peptone, agar and a certain amount of antibacterial agents (such as chloramphenicol and cycloheximide), which is used to inhibit the growth of bacteria and contaminating fungi. It is a general fungal culture medium, mainly used for culturing and identifying skin fungi and other fungi, such as yeasts and molds; at the same time, Sabouraud dextrose agar medium can also be used to separate pathogenic fungi from bacterial materials and detect microbial contamination in cosmetics.

[0041] In this embodiment, specifically: in step seven, the method for detecting the killing activity of immune cells against tumor cells includes the following steps: Step 401: Obtain immune cell samples and tumor cell samples to be tested, wherein the tumor cell samples include lung cancer cells, breast cancer cells, liver cancer cells, etc.; these tumor cells can be separated from the patient's tumor tissue, or obtained from a cell bank, ensuring that the tumor cell line is representative and in a logarithmic growth phase, so as to better simulate the in vivo environment; Step 402: Mix and culture the cell sample and tumor cells according to a certain ratio; select a suitable mixing ratio of immune cells and tumor cells according to the experimental purpose and cell characteristics. Usually, this ratio will be adjusted according to the previous experimental results and literature reports; ensure that the culture environment (such as temperature, humidity, gas concentration, etc.) is suitable for cell growth and survival; and select a suitable culture medium and additives according to the cell type; Step 403: After a predetermined period of time, the survival rate of the tumor cells is detected and the cell killing activity is calculated; based on the detected tumor cell survival rate, an appropriate formula is used to calculate the killing activity of the immune cells. The commonly used formula is: cell killing rate = (1-cell survival rate of the treatment group / cell survival rate of the control group) × 100%.

[0042] In this embodiment, specifically: in the process of analyzing the fluorescence intensity of cells based on flow cytometry, multiple control samples are set, including cell samples not labeled with fluorescent antibodies and cell samples labeled with only a single fluorescent antibody; Among them, the cell sample without fluorescent antibody labeling is used as a negative control to evaluate the background fluorescence concentration generated by the cells themselves; the cell suspension is mixed with a buffer solution without fluorescent antibody without any fluorescent labeling treatment; it is used to determine the baseline fluorescence intensity, thereby distinguishing the fluorescence generated by the cells themselves from the fluorescence generated by the specific binding of the fluorescent antibody; Among them, cell samples labeled with only a single fluorescent antibody are used to confirm the specific and nonspecific binding of the fluorescent antibody; the cell suspension is mixed with a single fluorescent antibody for labeling. Each fluorescent antibody should be tested separately; observe whether a specific fluorescent antibody binds only to the expected target cell subpopulation; by comparing cell samples labeled with different fluorescent antibodies, it is possible to evaluate whether there is nonspecific fluorescent binding, that is, the antibody binds to nonspecific sites on the cell surface.

[0043] In summary, the cell quality detection method in the clinical research of cell immunotherapy provided in this embodiment can provide a scientific basis for the development, production and clinical application of cell therapy products by comprehensively evaluating the quality of cells, and help improve the effectiveness and safety of cell therapy; at the same time, this method also provides strong technical support for research in the field of cell immunotherapy, and helps promote the rapid development of this field.

[0044] Embodiment 2 Figure 6 Schematic diagram of the functional modules of the cell quality detection system in the clinical study of cell immunotherapy in the embodiment of the present application. Figure 6 Shown: Cell quality detection system in clinical research of cell immunotherapy, including: morphology detection module, activity detection module, phenotypic analysis module, cytokine detection module, genetic detection module, microbial detection module and functional detection module; The morphological detection module is used to observe the morphological characteristics of cells through a microscope to determine whether the cells are intact and have abnormal morphology, and to record the morphological characteristics of cells and compare them with normal cells to assess health status; Activity detection module, used to detect cell activity using trypan blue exclusion method, calculate cell activity rate, and detect cell apoptosis rate and proliferation activity by flow cytometry; Phenotypic analysis module, used to analyze the expression of cell surface markers using flow cytometry, determine cell type and differentiation status, detect activation markers and inhibition markers on the cell surface, and evaluate immune function status; Cytokine detection module, used to collect cell culture supernatant, detect the concentration of cytokines secreted by cells using ELISA, analyze cytokine secretion, and evaluate immune activity and function; Genetic testing module, used to detect chromosomal abnormalities of cells using chromosome karyotype analysis or FISH technology, detect gene mutations and epigenetic changes, and evaluate genetic stability; Microbial detection module, used to detect microbial contamination during cell culture using bacterial and fungal culture methods, and to detect the presence of microbial nucleic acid in cells using PCR technology; The functional detection module is used to conduct cytotoxicity experiments, detect the killing activity of immune cells against tumor cells, and conduct in vivo experiments using animal models to evaluate the immunotherapy effect of cells in vivo.

[0045] In summary, the cell quality detection system for cell immunotherapy clinical research provided by the embodiment of the present invention realizes a comprehensive and accurate evaluation of cell quality by integrating a variety of advanced detection technologies, which helps to ensure the safety and effectiveness of cell immunotherapy products and improve the success rate of clinical treatment; at the same time, the system also provides strong technical support and guarantee for the research and development and production of cell immunotherapy products.

[0046] For other details about the technical solutions for implementing each module in the cell quality detection system in the cellular immunotherapy clinical research in the above embodiment, please refer to the description of the cell quality detection method in the cellular immunotherapy clinical research in the above embodiment, which will not be repeated here.

[0047] It should be noted that each embodiment in this specification is described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same or similar parts between the embodiments can be referred to each other. For system embodiments, since they are basically similar to method embodiments, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting cell quality in clinical research on cell immunotherapy, characterized in that: The following steps are involved: Step 1: Obtain cells, observe the morphological characteristics of the cells under a microscope, and record the health status of the cells; Step 2: Use trypan blue exclusion method to detect cell activity, calculate cell activity rate, and use flow cytometry to detect cell apoptosis rate and proliferation activity; Step 3: Analyze cell surface markers using flow cytometry to determine cell type and differentiation status, and detect activation markers and inhibition markers on the cell surface to evaluate immune function status; Step 4: Based on the cell culture supernatant, the concentration of cytokines secreted by the cells was detected by enzyme-linked immunosorbent assay to evaluate the immune activity and function; Step 5: Use chromosome karyotype analysis or fluorescence in situ hybridization to detect chromosomes of cells and evaluate genetic stability; Step 6: Use bacteria and fungi culture methods to detect microbial contamination in the cell culture process, and use polymerase chain reaction technology to detect the nucleic acid of microorganisms in the cells; Step 7: Based on the cytotoxicity experiment, detect the killing activity of cells against tumor cells and evaluate the immunotherapy effect of cells in vivo.

2. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 1, characterized in that: In step one, the microscope is an optical microscope or an electron microscope; the morphological characteristics of the cells include cell morphology, cell size and cell structure.

3. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 1, characterized in that: In step 2, the method for detecting cell activity using trypan blue exclusion method comprises the following steps: Step 101, obtaining a cell sample to be tested and mixing it with a trypan blue solution, wherein the concentration of the trypan blue solution is 0.4%; Step 102, observing the dead cells stained blue and the live cells not stained blue under a microscope, and counting the number of live cells and dead cells; Step 103, calculating the cell activity rate using an activity rate calculation formula, wherein the cell activity rate calculation formula is: cell activity rate = number of live cells / (number of live cells + number of dead cells) × 100%; The method for detecting cell apoptosis rate and proliferation activity by flow cytometry comprises the following steps: Step 201, obtaining a cell sample to be tested, and labeling apoptosis and proliferation-related markers on the cell surface with fluorescently labeled antibodies; Step 202, analyzing the fluorescence intensity of the cell sample based on flow cytometry to determine the number of apoptotic cells and the number of cells in different cell cycle stages; Step 203: Calculate the cell apoptosis rate and proliferation activity using a formula.

4. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 3, characterized in that: The calculation formula of the cell apoptosis rate is: Cell apoptosis rate = number of apoptotic cells / total number of cells × 100%; The cell proliferation activity is represented by the proliferation index PI, and the calculation formula is: PI=(S+G2 / M) phase cell number / (G0 / G1+S+G2 / M) phase cell number; Among them, the S phase and G2 / M phase represent the cell cycle stages in a proliferative state, and the G0 / G1 phase represents the quiescent or non-proliferative state cells.

5. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 1, characterized in that: In step 4, the method of detecting the concentration of cytokines secreted by cells using an enzyme-linked immunosorbent assay comprises the following steps: Step 301, adding the cell culture supernatant to the wells of an ELISA plate to bind to the antibodies on the wells; Step 302, adding enzyme-labeled antibodies to react with cytokines on the plate wells; Step 303, adding a substrate solution to produce a color reaction; Step 304: Calculate the concentration of cytokines based on the intensity of the color development reaction measured by the microplate reader.

6. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 1, characterized in that: In step five, the chromosome karyotype analysis is performed using conventional chromosome banding technology or high-resolution chromosome banding technology.

7. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 1, characterized in that: In step six, the bacteria and fungi culture method utilizes culture medium for detection, and the culture medium includes blood plate culture medium, chocolate plate culture medium and Sabouraud dextrose agar culture medium.

8. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 1, characterized in that: In step seven, the method for detecting the killing activity of cells against tumor cells comprises the following steps: Step 401, obtaining a cell sample to be tested and a tumor cell sample; Step 402, mixing and culturing the cell sample and the tumor cell sample in proportion; Step 403: After a predetermined period of time, the survival rate of the tumor cell sample is detected, and the killing activity of the cell sample is calculated.

9. The method for detecting cell quality in clinical research of cell immunotherapy according to claim 3, characterized in that: In the process of analyzing the fluorescence intensity of cells based on flow cytometry, multiple control samples are set, including cell samples not labeled with fluorescent antibodies and cell samples labeled with only a single fluorescent antibody.

10. A cell quality detection system in a cell immunotherapy clinical study, applied to a cell quality detection method in a cell immunotherapy clinical study according to any one of claims 1 to 9, characterized in that: include: Morphological detection module, activity detection module, phenotypic analysis module, cytokine detection module, genetic detection module, microbial detection module and functional detection module; The morphological detection module is used to observe the morphological characteristics of cells through a microscope and record the health status of cells; The activity detection module is used to detect cell activity using the trypan blue exclusion method, calculate the cell activity rate, and detect the cell apoptosis rate and proliferation activity by flow cytometry; The phenotypic analysis module is used to analyze the expression of cell surface markers using flow cytometry, determine cell types and differentiation states, detect activation markers and inhibition markers on the cell surface, and evaluate immune function status; The cytokine detection module is used to collect cell culture supernatant, detect the concentration of cytokines secreted by cells using enzyme-linked immunosorbent assay, analyze cytokine secretion, and evaluate immune activity and function; The genetic detection module is used to detect chromosomal abnormalities of cells, detect gene mutations and epigenetic changes, and evaluate genetic stability using chromosome karyotype analysis or fluorescence in situ hybridization technology; The microbial detection module is used to detect microbial contamination during cell culture using bacterial and fungal culture methods, and to detect whether there is microbial nucleic acid in the cells using polymerase chain reaction technology; The functional detection module is used to conduct cytotoxicity experiments, detect the killing activity of immune cells on tumor cells, and evaluate the immunotherapy effect of cells in vivo.

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