CN-121384765-B - Method for quantifying and distinguishing MSC immunocompetence by using CD82 and application

Abstract

The invention discloses a method for quantifying and distinguishing MSC immunocompetence by using CD82 and application thereof, wherein the method comprises the steps of collecting cells, using fluorescent labeled antibody to label membrane protein CD82 on the surface of the MSC, detecting the positive rate and MFI of the CD82 of the cells by using a flow cytometry, quantifying the MSC immunocompetence by using the expression level of the CD82, wherein the immunocompetence refers to the inhibition capability of the MSC on activated PBMC to secrete inflammatory cytokines TNF-alpha and IFN-gamma, and meanwhile, the MSC cell population with high immunocompetence can be distinguished by detecting the level of the CD82 expressed by the MSC.

Inventors

• DU WENJING
• MAO AIBIN
• LIU XUEYAN
• WEI ZHE
• HAN ZHIBO
• JIA HONGHONG
• LI WENXUE
• NIE FANYU
• ZHAO MENG
• YANG ZICONG
• YAN SHULIN
• LIU XINXIN

Assignees

• 天津昂赛细胞基因工程有限公司

Dates

Publication Date

20260505

Application Date

20251224

Claims (6)

1. 1. A method for quantifying and differentiating MSC immunocompetence by using CD82, characterized in that, by collecting cells, labeling membrane protein CD82 on the surface of MSC with fluorescent labeled antibody, detecting the positive rate and MFI of CD82 on the surface of MSC by using flow cytometry, quantifying the immunocompetence of MSC by detecting the expression level of CD82 on the surface of MSC, when the positive rate and MFI of CD82 on the surface of MSC are positively correlated, the inhibition ability of activated PBMCs to secrete TNF- α and IFN- γ is greater than 50% on the premise that the positive rate and MFI of CD82 on the surface of MSC are positive >54% and MFI >70, the immunocompetence of different MSC cell populations can be quantified by detecting the level of CD82 expression by different donor sources by flow cytometry, and differentiating MSC cell populations with high immunocompetence, wherein the immunocompetence refers to the inhibition ability of activated PBMCs to secrete inflammatory cytokines TNF- α and IFN- γ.
2. 2. The method of claim 1, wherein the MSCs are derived from bone marrow, fat, perinatal tissue including umbilical cord, placenta, amniotic membrane or amniotic fluid or induced differentiation from iPSC/ESC.
3. 3. A method for quantifying and differentiating MSC immunocompetence according to claim 2, comprising in particular the steps of: s1, collecting cells to be detected, namely digesting and collecting cultured MSC, counting the cells and transferring the cells into a flow tube; S2, labeling a flow type antibody, namely centrifuging a cell suspension, removing supernatant, respectively adding fluorescent labeled antibodies into cell precipitates, uniformly mixing by vortex, placing in a room temperature light-shielding refrigerator for incubation or a 4 ℃ refrigerator for incubation, adding flow type cell washing liquid for washing out unbound antibodies, centrifuging, adding flow type cell washing liquid for resuspension of cells, filtering cell aggregates by a filter membrane, and waiting for detection; S3, detecting by using a flow cytometer, namely detecting the positive rate and MFI of CD82 on the surface of the MSC by using the flow cytometer; s4, quantitatively analyzing the immunocompetence of the MSC cell population; And S5, differentiating the high-activity MSC cell population, wherein the MSC surface CD82 positive rate is higher than 70% and is regarded as the high-activity MSC cell population, and the CD82 positive rate is lower than 40% and is regarded as the low-activity MSC cell population.
4. 4. Use of a method according to any one of claims 1-3 for quantifying and differentiating MSC immunocompetence using CD82 in the formulation of cell banks and quality detection criteria for cell injection.
5. 5. Use of a method according to any one of claims 1-3 for quantifying and differentiating MSC immunocompetence using CD82 in screening a pool of high immunocompetent MSC seeds.
6. 6. Use of a method of quantifying and differentiating MSC immunocompetence with CD82 according to any of claims 1-3 for the preparation of a high immunocompetence cell injection for the treatment of inflammation, immune diseases.

Description

Method for quantifying and distinguishing MSC immunocompetence by using CD82 and application Technical Field The invention relates to the technical field of biological medicine, in particular to a method for quantifying and distinguishing MSC (mesenchymal stem cell) immunocompetence by using CD82 and application thereof. Background Mesenchymal Stem Cells (MSC) are a group of cells derived from mesoderm, can be largely isolated and expanded in vitro, are a group of sub-totipotent stem cells with self-renewal and multidirectional differentiation, and have multiple biological functions such as self-renewal, multidirectional differentiation, angiogenesis promotion, hematopoietic support, tissue injury repair promotion, immune regulation and the like. It is worth mentioning that MSC is a heterogeneous population of cells, the biological properties of the MSC cell populations isolated from different donor sources and different tissues are not uniform （Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev Rep. 2013 Feb;9(1):32-43）,, and even the MSC cell populations isolated from the same tissue also contain several subpopulations （VCAM-1+ placenta chorionic villi-derived mesenchymal stem cells display potent pro-angiogenic activity. Stem Cell Res Ther. 2016 Apr 4:7:49.;Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, and placenta. Stem Cell Res Ther. 2016 Nov 10;7(1):163）,, so it is important to find a suitable quantification method to evaluate the biological activity of MSC and to find a population of MSC cells with high immune activity. At present, as a new medicine of stem cell type I, the main mechanism of MSC medicines for treating diseases comprises two aspects, namely immune regulation and tissue injury repair, wherein the immune regulation is a core mechanism for treating various inflammation and immune related diseases. Although research on the immune regulation mechanism of MSC has been continued for more than half a century, researchers in various countries have published many results on the immune regulation of MSC from the standpoint of contact inhibition and paracrine inhibition, for example, known reported soluble factors including TGF-. Beta.1, HGF, IDO, PGE 2, TSG-6, TNFR1, etc., and surface antigen molecules such as VCAM-1, PD-L1, ICAM-1, etc. （Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases.Nat Rev Nephrol.2018 Aug;14(8):493-507）., but since MSC is a group of "living" cells having heterogeneous characteristics, the therapeutic mechanism thereof remains a difficult problem for scientists in various countries. From the microscopic view, MSC cell strains are internally provided with a plurality of different cell subsets, biological characteristics and functions among the cell subsets are different, from the macroscopic view, the cell strains are analyzed to cause heterogeneity difference due to different sources of donors, tissue sources, separation and preparation methods and the like, so that biological attribute variability of different batches of preparations is large, standard substances are extremely difficult to determine, and the action mechanism is complex, and currently, no uniformly determined core efficacy molecule exists, so that quality control, especially biological activity (also called biological efficacy) of MSC drugs becomes a difficult problem for restricting clinical application and curative effect of MSC drugs （MSC-based product characterization for clinical trials: an FDA perspective.Cell Stem Cell. 2014 Feb 6;14(2):141-5）. The biological activity evaluation method for MSC quality detection mainly detects proliferation inhibition of Peripheral Blood Mononuclear Cells (PBMC) and inhibition level of secretion of inflammatory factors, and simultaneously has the effect of an evaluation system for MSC on specific lymphocyte subsets, such as Th1/Treg/Th17 and the like, but the operation content of the evaluation method is complex and the cost is high. To reduce the cost of quality testing, researchers have attempted to evaluate the biological activity of MSCs using alternative functional indicators, which often employ key cytokines or proteins that are expressed by themselves. Currently known methods for evaluating MSC biological activity include ELISA to detect PGE 2 (application number CN 201811394547.9), TNFRI (application number CN 201911149147.6) and expression of cell membrane protein CD276 (patent number ZL 202211276848.8) in MSC supernatant, and also flow to detect MSC surface antigens CD155 (patent number ZL 202211113993.4) and CD273 (application number CN 202410284307.2). Considering that ELISA requires culturing cells for detecting cytokines (such as PGE 2 and TNFRI) in the supernatant, collecting the supernatant takes a long time and the cy