Method for evaluating in-vitro lymphocyte proliferation inhibition of mesenchymal stem cells
By using commercially established mixed-source PBMC and CFSE staining techniques, the inhibitory ability of mesenchymal stem cells on lymphocyte proliferation was evaluated, and the problem of instability of existing detection methods was solved, and more reliable evaluation results were achieved, providing a basis for the clinical application of mesenchymal stem cells.
Patent Information
- Application Number
- CN202510060194.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-04-15
AI Technical Summary
There are problems that existing detection methods for mesenchymal stem cells to inhibit lymphocyte proliferation in vitro have large data differences and are not stable enough.
The commercially-built mixed-source PBMCs were used to expand the sample size of PBMCs in the same batch to reduce batch-to-batch differences, and the proliferation inhibitory ability of MSCs to the PBMCs was evaluated through CFSE staining and flow cytometry detection.
A more comprehensive and reliable assessment of the ability to inhibit lymphocyte proliferation in vitro is achieved, providing the basis for the clinical application of mesenchymal stem cells.
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Figure CN119824067A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and in particular to a method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro. Background Art
[0002] MSC can have a wide range of effects on immune cells through contact inhibition paracrine effects, inhibiting the over-activation of the immune system and maintaining the stability of the immune system. MSC cells can actively migrate to the inflammatory environment in the body, and under the action of inflammatory factors, they can inhibit contact with immune cells and produce TGF-β, PGE2, IDO, HGF, NO, etc. to inhibit the activity of immune cells through paracrine effects, thereby inhibiting the over-activation of the body's immune system. An important application of MSC is in the field of immune regulation, and inhibiting the proliferation of peripheral blood mononuclear cells (PBMC) is an important manifestation of MSC's immune regulation ability.
[0003] CFSE is a cell staining reagent that can fluorescently label living cells. It can permeate the cell membrane. After entering the cell, it can occasionally and irreversibly bind to the Lysine residues or other amino groups of intracellular proteins and label these proteins. CFSE can be excited by a 490nm laser to emit 520nm light and can be observed using a flow cytometer. The fluorescence of CFSE-labeled cells is very uniform and stable. During cell division and proliferation, the CFSE fluorescent label can be evenly distributed to two daughter cells. The fluorescence of the daughter cells will be weakened by half each time they divide. In this way, cells that have not divided, cells that have divided once, cells that have separated twice, cells that have divided three times, and cells with similar other division times can be detected by flow cytometry. By detecting the different division generations of cells and their ratios, the cell division rate can be obtained.
[0004] The lymphocytes are labeled with CFSE, and the labeled lymphocytes are stimulated to proliferate, and a group of MSCs are co-cultured with lymphocytes. By setting up a control group and a co-culture group, after a certain period of culture, the cells are collected and detected by flow cytometry, and the difference in the proliferation rate of the two groups of lymphocytes is analyzed to determine whether MSCs have a promoting or inhibiting effect on lymphocyte proliferation.
[0005] Due to the different donors, each batch of allogeneic PBMC is unique. At present, the detection methods of MSC in vitro inhibition of PBMC proliferation in various laboratories generally have the characteristics of large data differences and instability.
[0006] In view of this, it is necessary to further improve the problems in the existing technology of mesenchymal stem cells inhibiting lymphocyte proliferation in vitro. Summary of the invention
[0007] In view of this, the present invention aims to address the deficiencies in the prior art, and its main purpose is to provide a method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro. The method adopts commercially established mixed-source PBMCs to expand the sample size of the same batch of PBMCs so as to reduce the differences between batches. Compared with traditional evaluation methods, the method provided by the present invention can more comprehensively and reliably evaluate the ability to inhibit lymphocyte proliferation in vitro, thereby providing a basis for the clinical application of mesenchymal stem cells.
[0008] To achieve the above purpose, the present invention adopts the following technical solutions:
[0009] A method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro comprises the following steps:
[0010] S1. Prepare MSC cell suspension;
[0011] S2. Prepare PBMC cell suspension: prepare commercially established mixed-source PBMCs;
[0012] S3, MSC inoculation and adherence culture: Take MSC cell suspension, take an appropriate amount of MSC cell suspension in the culture medium according to the inoculation ratio, and inoculate it into the well plate; after MSC inoculation, culture until the cells adhere to the wall;
[0013] S4, CFSE staining and inoculation: CFSE was added to the PBMC cell suspension, incubated in the dark, and free CFSE was neutralized; after the MSC cells adhered to the wall, the original MSC culture medium was discarded, and the CFSE-stained PBMCs were inoculated into the negative control group, the positive control group, and the MSC experimental group, respectively; an appropriate amount of RPMI 1640 complete culture medium was added with a corresponding proportion of PHA-P, and the RPMI 1640 complete culture medium containing PHA-P was supplemented into the positive control group and the MSC experimental group, respectively, for cell stimulation and activation; the above-mentioned inoculated cells were cultured;
[0014] S5. MSC labeling of PBMC proliferating cells: After the culture is completed, add cell activity detection reagent, incubate in the dark, detect and perform statistical analysis.
[0015] As a preferred solution: in step S5, the sum of the proportions of cells of each proliferation generation in the positive control group and the MSC experimental group is statistically analyzed, and the average value of the duplicate well results is calculated; the negative control results do not need to be statistically analyzed; MSC inhibition rate of PBMC proliferation = (P proliferation amount - T proliferation amount) / P proliferation amount × 100%, wherein:
[0016] P proliferation amount: the sum of the proportions of PBMC cells at each proliferation generation in the positive control, = P total;
[0017] T proliferation amount: the sum of the proportions of cells in each proliferation generation of PBMC in the MSC experimental group, = P total.
[0018] As a preferred solution: the MSC-PBMC proliferating cell labeling in step S5 is specifically as follows: after the culture is completed, the well plate is taken out, and the corresponding liquid is transferred to a flow tube, centrifuged and the supernatant is discarded, the cells are resuspended with PBS, a cell activity detection reagent is added, and the cells are incubated at 2-8°C in the dark for 10 minutes; after the incubation is completed, PBS is added, the supernatant is discarded by centrifugation, and the cells are washed twice; the supernatant is discarded, PBS is added, and the cells are tested and statistically analyzed by the machine.
[0019] As a preferred solution, the preparation of MSC cell suspension in step S1 specifically comprises: obtaining umbilical cord, preparing cell suspension, and co-culturing lymphocyte proliferation inhibition using fourth-generation MSC.
[0020] As a preferred solution: the culture medium in step S3 is MSC complete culture medium; after MSC inoculation, the culture medium is cultured in a 37° C., 5% CO 2 incubator for 6 hours until the cells adhere to the wall.
[0021] As a preferred solution: the final concentration of CFSE used in the CFSE staining in step S4 is 5 μM.
[0022] As a preferred solution, the dark-protected incubation in step S4 is specifically as follows: incubating the labeled cells at 37°C in the dark for 10 min; after the incubation, the RPMI 1640 complete medium is placed in a 2-8°C drug storage box in the dark for 5 min; the inoculation density of PBMC is: 3×10 5 cells / well; the final concentration of PHA-P was 6-10 μg / mL.
[0023] As a preferred solution: in step S4, the inoculated cells are cultured in a 37° C. 5% CO 2 incubator in the dark for 3-5 days.
[0024] As a preferred solution: the cell activity detection reagent in step S5 includes 7-AAD.
[0025] As a preferred embodiment, the MSC cells are at least one selected from umbilical cord-derived mesenchymal stem cells, amnion-derived mesenchymal stem cells and placenta-derived mesenchymal stem cells.
[0026] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that by adopting commercialized established mixed-source PBMCs, the sample size of the same batch of PBMCs is expanded to reduce the difference between batches; the PBMC cells of mixed sources are subjected to a proliferation inhibition co-culture experiment with mesenchymal stem cells, and a relatively stable proliferation inhibition rate is obtained. Compared with traditional evaluation methods, the method provided by the present invention can more comprehensively and reliably evaluate the ability to inhibit lymphocyte proliferation in vitro, thereby providing a basis for the clinical application of mesenchymal stem cells.
[0027] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a schematic diagram of the effect of MSC on PBMC proliferation detected by flow cytometry in the negative control group of the present invention;
[0029] Figure 2 This is a schematic diagram of the effect of MSC on PBMC proliferation detected by flow cytometry in the positive control group of the present invention;
[0030] Figure 3 This is a schematic diagram of the effect of MSC on PBMC proliferation detected by flow cytometry in the MSC experimental group of the present invention;
[0031] Figure 4 This is a schematic diagram of the effect of MSC on PBMC proliferation observed under a biological microscope in the negative control group of the present invention;
[0032] Figure 5 This is a schematic diagram of the effect of MSC on PBMC proliferation observed under a biological microscope in the positive control group of the present invention;
[0033] Figure 6 This is a schematic diagram of the effect of MSC on PBMC proliferation observed under a biological microscope in the MSC experimental group of the present invention. DETAILED DESCRIPTION
[0034] The present invention Figures 1 to 6 As shown, a method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro comprises the following steps:
[0035] S1. Prepare MSC cell suspension;
[0036] S2. Prepare PBMC cell suspension: prepare commercially established mixed-source PBMCs;
[0037] S3, MSC inoculation and adherence culture: Take MSC cell suspension, take an appropriate amount of MSC cell suspension in the culture medium according to the inoculation ratio, and inoculate it into the well plate; after MSC inoculation, culture until the cells adhere to the wall;
[0038] S4, CFSE staining and inoculation: CFSE was added to the PBMC cell suspension, incubated in the dark, and free CFSE was neutralized; after the MSC cells adhered to the wall, the original MSC culture medium was discarded, and the CFSE-stained PBMCs were inoculated into the negative control group, the positive control group, and the MSC experimental group, respectively; an appropriate amount of RPMI 1640 complete culture medium was added with a corresponding proportion of PHA-P, and the RPMI 1640 complete culture medium containing PHA-P was supplemented into the positive control group and the MSC experimental group, respectively, for cell stimulation and activation; the above-mentioned inoculated cells were cultured;
[0039] S5. MSC labeling of PBMC proliferating cells: After the culture is completed, add cell activity detection reagent, incubate in the dark, detect and perform statistical analysis.
[0040] In step S5, the sum of the proportions of cells of each proliferation generation in the positive control group and the MSC experimental group is statistically analyzed, and the average value of the duplicate well results is calculated; the negative control results do not need to be statistically analyzed; MSC inhibition rate of PBMC proliferation = (P proliferation amount - T proliferation amount) / P proliferation amount × 100%, where:
[0041] P proliferation amount: the sum of the proportions of PBMC cells at each proliferation generation in the positive control, = P total;
[0042] T proliferation amount: the sum of the proportions of cells in each proliferation generation of PBMC in the MSC experimental group, = P total.
[0043] In step S5, the MSC labels the proliferating PBMC cells as follows: after the culture is completed, the well plate is taken out, and the corresponding liquid is transferred to a flow tube, the supernatant is discarded after centrifugation, the cells are resuspended with PBS, a cell activity detection reagent is added, and the cells are incubated at 2-8° C. in the dark for 10 minutes; after the incubation is completed, PBS is added, the supernatant is discarded after centrifugation, and the cells are washed twice; the supernatant is discarded, PBS is added, and the cells are tested and statistically analyzed by the machine.
[0044] The preparation of the MSC cell suspension in step S1 specifically includes: obtaining the umbilical cord, preparing the cell suspension, and co-culturing the lymphocyte proliferation inhibitor using the fourth-generation MSC.
[0045] The culture medium in step S3 is MSC complete culture medium; after MSC inoculation, the cells are cultured in a 37° C., 5% CO 2 incubator for 6 hours until the cells adhere to the wall.
[0046] The final concentration of CFSE used in the CFSE staining in step S4 is 5 μM.
[0047] The dark-protected incubation in step S4 is specifically as follows: incubating the labeled cells at 37°C in the dark for 10 minutes; after the incubation, the RPMI1640 complete medium is placed in a 2-8°C drug storage box in the dark for 5 minutes; the inoculation density of PBMC is: 3×10 5 cells / well; the final concentration of PHA-P was 6-10 μg / mL.
[0048] In step S4, the inoculated cells are cultured in a 37° C. 5% CO 2 incubator in the dark for 3-5 days.
[0049] In step S5, the cell activity detection reagent includes 7-AAD.
[0050] The MSC cells are selected from at least one of umbilical cord-derived mesenchymal stem cells, amnion-derived mesenchymal stem cells and placenta-derived mesenchymal stem cells.
[0051] Example 1: A method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro
[0052] CFSE-labeled PBMCs were stimulated with PHA-P, and the proliferation of PBMCs was detected by flow cytometry. The experimental groups included a negative control group, a positive control group, and positive and negative samples of the MSC experimental group. The difference between the three groups was that the negative control group was only PBMCs, the positive control group was PBMCs stimulated with PHA-P, and the MSC experimental group was PBMCs stimulated with PHA-P and co-cultured with MSCs. The PBMCs were commercialized and established with mixed sources to establish a PBMC cell bank and expand the sample size in order to reduce the differences between batches.
[0053] The concentration of the stimulator PHA-P was 8 μg / mL, the culture time was 4 days, and three different MSC:PBMC experimental groups (1:5, 1:10, 1:20) were set up, with 3 replicate wells in each group.
[0054] The steps include:
[0055] S1. Preparation of MSCs; obtaining umbilical cords, preparing cell suspensions, and co-culturing fourth-generation MSCs in lymphocyte proliferation inhibition experiments.
[0056] S2. Prepare PBMC: PBMC from commercially established mixed sources.
[0057] S3. MSC inoculation and adherence culture: Take MSC for experiment, count the cells and take appropriate amount of cell suspension in culture medium according to the inoculation ratio, mix well and inoculate into the well plate, the culture medium is MSC complete culture medium; after MSC inoculation, culture in a 37°C, 5% CO2 incubator for 6 hours until the cells adhere to the wall.
[0058] S4. CFSE staining and inoculation: CFSE was added to the PBMC cell suspension to make the final CFSE concentration of 5 μM, and the cells were incubated at 37°C in the dark for 10 min to label the cells; after the incubation, RPMI 1640 complete medium was placed in a 2-8°C drug storage box in the dark for 5 min to neutralize the free CFSE; the cells were washed twice with RPMI 1640 complete medium, resuspended and counted; after the MSC cells adhered to the wall, the original MSC complete medium was discarded, and the CFSE-stained PBMCs were inoculated into the negative control group, the positive control group, and the MSC experimental group; the inoculation density of PBMCs was: 3×10 5 cells / well. Take an appropriate amount of RPMI 1640 complete medium and add a corresponding proportion of PHA-P to make the final concentration of PHA-P 6-10μg / mL, and supplement the positive control and MSC experimental groups with RPMI 1640 complete medium containing PHA-P for cell stimulation and activation. The above-mentioned inoculated cells were placed in a 37℃ 5% CO2 incubator in the dark for 3-5 days.
[0059] S5. MSC cell labeling for PBMC proliferation experiment: After the culture is completed, the well plate is removed, and the corresponding liquid is transferred to the flow tube, centrifuged and the supernatant is discarded, the cells are resuspended with PBS, 5 μL 7-AAD is added, and the cells are incubated at 2-8°C in the dark for 10 minutes; after the incubation is completed, PBS is added, the supernatant is discarded by centrifugation, and the cells are washed twice; the supernatant is discarded, 300 μL PBS is added, and the test is performed on the machine. The sum of the proportions of cells in each proliferation generation of the positive control group and the MSC experimental group is statistically analyzed, and the average value of the duplicate well results is calculated; the negative control results do not need to be counted; MSC inhibition rate of PBMC proliferation = (P proliferation amount-T proliferation amount) / P proliferation amount × 100%, where:
[0060] P proliferation amount: the sum of the proportions of cells in each proliferation generation of PBMC in the positive control, = P total.
[0061] T proliferation amount: the sum of the proportions of cells in each proliferation generation of PBMC in the MSC experimental group, = P total.
[0062] Results Analysis
[0063] When the culture days were 4 days, the ratio of MSC to mixed PBMC was 1:5, 1:10 and 1:20, the proliferation inhibition rates were (20.06±2.96)%, (17.96±2.23)% and (10.89±1.04)%, respectively, which was in line with expectations. The inhibition rate decreased in a dose-dependent manner. The CV value between duplicate wells was less than 5%, and the data was relatively stable.
[0064] Table 1: PBMC proliferation inhibition rate at different ratios
[0065]
[0066] Example 2
[0067] The present embodiment provides a method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro, wherein CFSE-labeled PBMCs are stimulated with PHA-P, and the proliferation of PBMCs is detected by flow cytometry. The experimental groups include a negative control group, a positive control group, and positive and negative samples of the MSC experimental group. The difference between the three is that the negative control group is only PBMCs, the positive control group is PBMCs stimulated with PHA-P, and the MSC experimental group is PBMCs stimulated with PHA-P and co-cultured with MSCs. The concentration of the stimulator PHA-P is 6 μg / mL, the culture time is 5 days, the MSC:PBMC experimental group is set at 1:5, and 3 replicates are set for each group. The following steps are included:
[0068] S1. Preparation of MSCs: Obtain umbilical cords, prepare cell suspensions, and co-culture with fourth-generation MSCs for lymphocyte proliferation inhibition experiments. Select two MSC samples and perform in vitro lymphocyte proliferation inhibition tests after 2 months of cryopreservation.
[0069] S2. Prepare PBMC: PBMC should be commercially available mixed-source PBMC that has been established. The sample size of the same batch of PBMC should be increased to minimize the differences between batches.
[0070] S3. MSC inoculation and adherence culture: Take MSC for experiment, count the cells and take appropriate amount of cell suspension in culture medium according to the inoculation ratio, mix well and inoculate into the well plate, the culture medium is MSC complete culture medium; after MSC inoculation, culture in a 37°C, 5% CO2 incubator for 6 hours until the cells adhere to the wall.
[0071] S4. CFSE staining and inoculation: CFSE was added to the PBMC cell suspension to make the final CFSE concentration of 5 μM, and the cells were incubated at 37°C in the dark for 10 min to label the cells; after the incubation, RPMI 1640 complete medium was placed in a 2-8°C drug storage box in the dark for 5 min to neutralize the free CFSE; the cells were washed twice with RPMI 1640 complete medium, resuspended and counted; after the MSC cells adhered to the wall, the original MSC complete medium was discarded, and the CFSE-stained PBMCs were inoculated into the negative control group, the positive control group, and the MSC experimental group; the inoculation density of PBMCs was: 3×10 5 cells / well. Take an appropriate amount of RPMI 1640 complete medium and add a corresponding proportion of PHA-P to make the final concentration of PHA-P 6-10μg / mL, and supplement the positive control and MSC experimental groups with RPMI 1640 complete medium containing PHA-P for cell stimulation and activation. The above-mentioned inoculated cells were placed in a 37℃ 5% CO2 incubator in the dark for 3-5 days.
[0072] S5. MSC cell labeling for PBMC proliferation experiment: After the culture is completed, the well plate is removed, and the corresponding liquid is transferred to the flow tube, centrifuged and the supernatant is discarded, the cells are resuspended with PBS, 5 μL 7-AAD is added, and the cells are incubated at 2-8°C in the dark for 10 minutes; after the incubation is completed, PBS is added, the supernatant is discarded by centrifugation, and the cells are washed twice; the supernatant is discarded, 300 μL PBS is added, and the test is performed on the machine. The sum of the proportions of cells in each proliferation generation of the positive control group and the MSC experimental group is statistically analyzed, and the average value of the duplicate well results is calculated; the negative control results do not need to be counted; MSC inhibition rate of PBMC proliferation = (P proliferation amount-T proliferation amount) / P proliferation amount × 100%, where:
[0073] P proliferation amount: the sum of the proportions of cells in each proliferation generation of PBMC in the positive control, = P total.
[0074] T proliferation amount: the sum of the proportions of cells in each proliferation generation of PBMC in the MSC experimental group, = P total.
[0075] Example 3
[0076] A method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells, the specific implementation of which is the same as that of Example 2, except that two MSC samples are selected in S2 and the in vitro inhibition of lymphocyte proliferation is detected after being cryopreserved for 3 months.
[0077] Example 4
[0078] A method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells, the specific implementation of which is the same as that of Example 2, except that two MSC samples are selected in S2 and the in vitro inhibition of lymphocyte proliferation is detected after being cryopreserved for 6 months.
[0079] Table 2: Stability evaluation test results
[0080]
[0081]
[0082] As can be seen from Table 2, the CV values of the three parallel test results of the positive control total, the proliferation inhibition rate of sample 1 and the proliferation inhibition rate of sample 2 in each embodiment are all less than 10%, and the same sample is evaluated by this method at different times, and the CV values of the three parallel test results are all less than 10%. Therefore, the present invention can be stably applied to the detection of mesenchymal stem cells inhibiting lymphocyte proliferation inhibition in vitro.
[0083] The design focus of the present invention is to expand the sample size of the same batch of PBMCs by using commercially established mixed-source PBMCs, so as to reduce the differences between batches; the PBMCs of mixed sources were subjected to a proliferation inhibition co-culture experiment with mesenchymal stem cells, and a relatively stable proliferation inhibition rate was obtained. Compared with traditional evaluation methods, the method provided by the present invention can more comprehensively and reliably evaluate the ability to inhibit lymphocyte proliferation in vitro, thereby providing a basis for the clinical application of mesenchymal stem cells.
[0084] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro, characterized in that: The steps include: S1. Prepare MSC cell suspension; S2. Prepare PBMC cell suspension: prepare commercially established mixed-source PBMCs; S3, MSC inoculation and adherence culture: Take MSC cell suspension, take an appropriate amount of MSC cell suspension in the culture medium according to the inoculation ratio, and inoculate it into the well plate; after MSC inoculation, culture until the cells adhere to the wall; S4, CFSE staining and inoculation: CFSE was added to the PBMC cell suspension, incubated in the dark, and free CFSE was neutralized; after the MSC cells adhered to the wall, the original MSC culture medium was discarded, and the CFSE-stained PBMCs were inoculated into the negative control group, the positive control group, and the MSC experimental group, respectively; an appropriate amount of RPMI 1640 complete culture medium was added with a corresponding proportion of PHA-P, and the RPMI 1640 complete culture medium containing PHA-P was supplemented into the positive control group and the MSC experimental group, respectively, for cell stimulation and activation; the above-mentioned inoculated cells were cultured; S5. MSC labeling of PBMC proliferating cells: After the culture is completed, add cell activity detection reagent, incubate in the dark, detect and perform statistical analysis.
2. A method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells according to claim 1, characterized in that: In step S5, the sum of the proportions of cells of each proliferation generation in the positive control group and the MSC experimental group is statistically analyzed, and the average value of the duplicate well results is calculated; the negative control results do not need to be statistically analyzed; MSC inhibition rate of PBMC proliferation = (P proliferation amount - T proliferation amount) / P proliferation amount × 100%, where: P proliferation amount: the sum of the proportions of PBMC cells at each proliferation generation in the positive control, = P total; T proliferation amount: the sum of the proportions of cells in each proliferation generation of PBMC in the MSC experimental group, = P total.
3. The method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells according to claim 1, characterized in that: The specific method of MSC labeling PBMC proliferating cells in step S5 is as follows: after the culture is completed, the well plate is taken out, and the corresponding liquid is transferred to a flow tube, centrifuged and the supernatant is discarded, the cells are resuspended with PBS, a cell activity detection reagent is added, and the cells are incubated at 2-8° C. in the dark for 10 minutes; after the incubation is completed, PBS is added, the supernatant is discarded by centrifugation, and the cells are washed twice; the supernatant is discarded, PBS is added, and the cells are tested and statistically analyzed by the machine.
4. The method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells according to claim 1, characterized in that: The step S1 of preparing the MSC cell suspension specifically comprises: obtaining the umbilical cord, preparing the cell suspension, and co-culturing the lymphocyte proliferation inhibitor using the fourth generation MSC.
5. The method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells according to claim 1, characterized in that: The culture medium in step S3 is MSC complete culture medium; after MSC inoculation, the cells are cultured in a 37° C., 5% CO 2 incubator for 6 hours until the cells adhere to the wall.
6. A method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells according to claim 1, characterized in that: The final concentration of CFSE used in the CFSE staining in step S4 is 5 μM.
7. A method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro according to claim 1, characterized in that: The dark-protected incubation in step S4 is specifically as follows: incubating the labeled cells at 37°C in the dark for 10 min; after the incubation, the RPMI1640 complete medium is placed in a 2-8°C drug storage box in the dark for 5 min; the inoculation density of PBMC is: 3×10 5 cells / well; the final concentration of PHA-P was 6-10 μg / mL.
8. The method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells according to claim 1, characterized in that: In step S4, the inoculated cells are cultured in a 37° C. 5% CO 2 incubator in the dark for 3-5 days.
9. A method for evaluating the ability of mesenchymal stem cells to inhibit lymphocyte proliferation in vitro according to claim 1, characterized in that: The cell activity detection reagent in step S5 includes 7-AAD.
10. The method for evaluating the in vitro inhibition of lymphocyte proliferation by mesenchymal stem cells according to claim 1, characterized in that: The MSC cells are selected from at least one of umbilical cord-derived mesenchymal stem cells, amnion-derived mesenchymal stem cells and placenta-derived mesenchymal stem cells.