CN-121999862-A - Method for allelic pairing of donor mesenchymal stem cells and receptor

Abstract

The invention provides a method for matching alleles of donor mesenchymal stem cells and recipients, which comprises the following steps of S10, S20, extracting genomic DNA of the mesenchymal stem cells and conducting HLA genotyping, S30, conducting HLA genotyping on the DNA of the recipients, S40, comparing the HLA genes of the recipients with the HLA genes of the mesenchymal stem cells, calculating the matching degree, S50, judging the matching degree of the recipients and the mesenchymal stem cells according to the matching degree, S60, storing the HLA genotyping result and the matching degree of the mesenchymal stem cells in a database, and establishing a matching library for matching the donor mesenchymal stem cells and the recipients. The method establishes a standardized system, aims at realizing compatibility evaluation of the donor and the receptor at the allele level, effectively predicts and avoids immune rejection risks caused by HLA mismatch before treatment, and improves the in vivo survival and implantation efficiency of MSCs.

Inventors

• HUANG BISHENG
• ZHU YABIN
• XU MENGYAO
• HU ZEMING
• LIU GUANGCHENG
• JI JIALI

Assignees

• 浙江天远生物科技有限公司

Dates

Publication Date

20260508

Application Date

20251203

Claims (10)

1. 1. A method for allelic profiling of donor mesenchymal stem cells with a recipient, the method comprising the steps of: S10, separating mesenchymal stem cells from perinatal tissues of a donor; s20, extracting genome DNA of the mesenchymal stem cells and carrying out HLA genotyping; s30, carrying out HLA genotyping on the receptor DNA; s40, comparing the HLA genes of the receptor with the HLA genes of the mesenchymal stem cells in an allele spectrum, and calculating the matching degree; And S50, judging the matching degree of the receptor and the mesenchymal stem cells according to the matching degree. And S60, storing HLA genotyping results of the mesenchymal stem cells and the matching degree in a database, and establishing a matching library for matching the donor mesenchymal stem cells with the receptor.
2. 2. The method according to claim 1, wherein the step S10 comprises the steps of: S110, isolating the mesenchymal stem cells from the perinatal tissue; S120, performing quality detection on the separated mesenchymal stem cells to obtain qualified mesenchymal stem cells; and S130, performing amplification culture on the mesenchymal stem cells with qualified quality to obtain amplified mesenchymal stem cells.
3. 3. The method of claim 2, wherein the expanded mesenchymal stem cells are expanded mesenchymal stem cells of passage 2 to passage 6.
4. 4. The method of claim 1, wherein the HLA genotyping determines loci of at least HLA-a, HLA-B and HLA-C.
5. 5. The method of claim 4, wherein the HLA genotyping further confirms loci of HLA-DR, HLA-DQ.
6. 6. The method of claim 1, wherein the classification criteria for the degree of matching are: Perfect match 12 alleles are all identical; Partial matches, 6 to 11 alleles are identical; Low match, 0 to 5 alleles identical.
7. 7. The method as recited in claim 1, further comprising: S70, performing stimulation culture on the mesenchymal stem cells by using an immune stimulation factor to induce HLA antigen expression; And S80, detecting the HLA antigen expression level of the mesenchymal stem cells before and after the stimulated culture.
8. 8. The method of claim 7, wherein the immunostimulatory factor is selected from one or more of gamma interferon, immune cell conditioned culture supernatant.
9. 9. The method of claim 8, wherein the conditions for stimulating the culture using the gamma interferon are: And adding the gamma-interferon with the concentration of 10-50ng/mL into the culture medium, and continuously culturing for 48-72 h.
10. 10. The method of claim 8, wherein the immune cells in the immune cell conditioned supernatant comprise one or more of T cells, natural killer cells, monocytes, macrophages and dendritic cells.

Description

Method for allelic pairing of donor mesenchymal stem cells and receptor Technical Field The invention relates to the fields of regenerative medicine and biotechnology, in particular to a method for allelic profiling of donor mesenchymal stem cells and recipients. Background Mesenchymal Stem Cells (MSCs) are a class of pluripotent stem cells with self-renewal capacity and multipotent differentiation potential. MSCs can be isolated from a variety of tissue sources including bone marrow, adipose tissue, and perinatal tissue such as placenta, umbilical cord, amniotic membrane, and the like. Among them, perinatal MSCs have the advantages of high proliferation potential, low immunogenicity, enhanced therapeutic utility, and the like, and are becoming an important resource for cell therapy. Despite extensive research, MSCs-based clinical therapeutic results are not consistent, due in large part to differences in immune compatibility and variability between different donor sources. In the traditional solid organ transplantation or hematopoietic stem cell transplantation field, high-resolution HLA matching is a necessary prerequisite for determining whether a graft can be successfully implanted and survived for a long time, and the technology can reduce the rejection risk of a host immune system on the graft to the greatest extent and is a foundation stone for successful transplantation. However, in the field of MSCs transplantation, in MSCs treatment, a standardized set of systems has not been established for compatibility assessment of donor and recipient at the allele level. Due to the lack of this key link, there is significant blindness in treatment, the risk of immune rejection caused by HLA mismatch cannot be effectively predicted and avoided before treatment, and the in vivo survival and implantation efficiency of MSCs may be severely impaired. Disclosure of Invention The invention aims to provide a method for matching the alleles of donor mesenchymal stem cells and recipients, establish a standardized system, aim to realize compatibility assessment of the donor and the recipients at the allele level, effectively predict and avoid immune rejection risks caused by HLA mismatch before treatment, and improve the in vivo survival and implantation efficiency of MSCs. And by constructing a ligand database and a cell library, basic support is provided for subsequent rapid retrieval and intelligent donor screening. In order to solve the problems, the invention provides a method for matching alleles of a donor mesenchymal stem cell and a recipient, which comprises the following steps of S10, S20, extracting genomic DNA of the mesenchymal stem cell and carrying out HLA genotyping, S30, carrying out HLA genotyping on the DNA of the recipient, S40, comparing the HLA gene of the recipient with the HLA gene of the mesenchymal stem cell, calculating the matching degree, S50, judging the matching degree of the recipient and the mesenchymal stem cell according to the matching degree, and S60, storing the HLA genotyping result and the matching degree of the mesenchymal stem cell in a database, and establishing a matching library for matching the donor mesenchymal stem cell and the recipient. By extracting genome DNA from donor MSCs and carrying out HLA genotyping, comparing HLA genes of the receptor with HLA genes of mesenchymal stem cells, a set of standardized system is established, the aim of realizing compatibility assessment of the donor and the receptor at the level of the allele is achieved, the immune rejection risk caused by HLA mismatch is effectively predicted and avoided before treatment, and the in vivo survival and implantation efficiency of the MSCs are improved. The system improves the safety, the precision and the expandability of MSCs in organ transplantation, autoimmune diseases and regenerative medicine application based on cell therapy, is beneficial to promoting the precise medical development in the field of cell therapy, and supports the establishment of a large-scale stem cell bank for transplantation and precise cell therapy. Further, the step S10 specifically comprises the steps of S110, separating mesenchymal stem cells from perinatal tissues, S120, detecting the quality of the separated mesenchymal stem cells to obtain qualified mesenchymal stem cells, and S130, amplifying and culturing the qualified mesenchymal stem cells to obtain amplified mesenchymal stem cells. The technical effect achieved by adopting the technical scheme is that the mesenchymal stem cells with sufficient quantity, activity and purity meeting clinical standards can be stably obtained through the complete process of tissue separation in perinatal period, strict quality detection and in vitro amplification, so that high-quality and standardized cell sources are provided for the follow-up accurate immune compatibility evaluation and transplantation treatment, and the reliability of the evaluation result and the effectiveness an