CD3+TCRValpha7.2 + T cell culture medium and multiplication culture method

Abstract

The invention provides a CD3 + TCRV alpha7.2 + T cell culture medium and a cell multiplication culture method. According to the culture medium, a basic culture medium contains 3%-10% of FBS, 10 U / mL to 50 U / mL of IL-2 and 10 ng / mL to 50 ng / mL of IL-15; the basic culture medium is selected from one or more of an RPMI 1640 culture medium, a T009 culture medium, an AIMV culture medium, a DMEM culture medium and an X-VIVO culture medium. A new technical route for obtaining and purifying the T cell subtype CD3 + TCRV alpha7.2 + T cell is developed, the culture medium is low in economic cost and simple to operate, the cell amplification multiple can be adjusted to be close to that of conventional T cells within a certain time period, and the common problem that the amplification difficulty of the CD3 + TCRV alpha7.2 + T cell is large is solved.

Description

technical field [0001] The invention belongs to the biological field, and in particular relates to a CD3+TCRVα7.2+T cell culture medium and a method for expanding and culturing. Background technique [0002] At present, there are few studies on CD3+TCRVα7.2+ T cells, but MAIT cells (whose phenotype is CD3+TCRVα7.2+CD161+) are mainly obtained and cultured in the following ways: [0003] i. Take fresh peripheral blood (PBMC) from healthy volunteers, use antibody-Microbeads coupling to sort CD161+ cells, immediately stain the separated cells with antibodies, and use flow sorting to obtain CD3+ TCRVα7.2+CD161+ cells are cultured in complete medium (50% AIMV+50% RPMI1640+10% FBS+glutamine+β-mercaptoethanol), and the cell density ranges from 5E+05 to 2E+06 cells / mL , the incubator environment is 37°C, 5% CO 2 (Lu Yongyong, Research on the number, function and anti-tumor effect of mucosa-associated invariant T cells (MAIT) in patients with bladder cancer, 2017 dissertation, Shand...

AI Technical Summary

Problems solved by technology

Generally, MAIT cells are sorted from PBMC, and most of them are enriched by magnetic bead sorting and then flow sorted. However, this method has obvious disadvantages: the proportion of MAIT cells is low, and the flow sorter sorts cells The required experiment is longer, and the cells are exposed to the external environment during the whole process, which is easy to be polluted; the total amount of cells obtained is small, and it is difficult to expand

This kind of basal medium is very difficult to cultivate MAIT cells. According to the relevant literature [3], at present, most MAIT cells can only be expanded by about 30 times within 21 days.

3) In order to increase the amplification factor of cells, various factors (such as IL-2, IL-7, IL-15, IL-12, IL-18) and chemical reagents (such as specific antigen 5- OP-RU / 5-OE-RU, 1% penicillin-streptomycin, 10mM HEPES buffer, 0.1mM MEM non-essential amino acids, 1mM sodium pyruvate and 5.5mM 2-mercaptoethanol, etc.), but these culture methods are expensive High, and it is easy for cells to enter a mature state, which is not conducive to the continuous expansion of cells and later functional tests and clinical research

[0013] In summary, the purification, activation and expansion methods of MAIT cells have the characteristics of high cost and difficult operation

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