Central memory T cell body and in-vitro culture method

An in vitro culture and cell culture technology, which is applied to cell culture active agents, tissue culture, animal cells, etc., can solve the problems of low in vitro expansion purity of central memory T cells, easy conversion into effector cells, etc., and achieves low cost and high efficiency. The effect of increasing the killing effect and facilitating the promotion

Active Publication Date: 2018-06-15
BEIHAO STEM CELL & REGENERATIVE MEDICINE RES INST CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of low purity of central memory T cells in vitro expansion and easy transformation into effector cells, one of the objects of the present invention is to provide a method for culturing central memory T cells in vitro, which improves central memory T cells. Cell Scale and Functionality

Method used

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  • Central memory T cell body and in-vitro culture method
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  • Central memory T cell body and in-vitro culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Method for Proliferating Central Memory T Cells

[0035] 1. Isolation of PBMC and acquisition of autologous supernatant

[0036] Volunteers were recruited, and 50ml of fresh blood was drawn into a collection tube by a collection nurse using a standard blood drawing method. Use a 10ml pipette to draw the whole blood into two 50ml centrifuge tubes that have been previously added with 14ml of lymphocyte separation medium, and add 25ml of whole blood to each tube. Set the centrifuge temperature at 20°C, centrifugal force at 400g, acceleration 1, deceleration 5, and centrifugation for 30 minutes. After centrifugation, the upper layer of autologous plasma was aspirated and frozen. Take the buffy coat cells in the middle into a new centrifuge tube filled with 40ml DPBS, blow evenly, set the temperature of the centrifuge at 20°C, 300g, accelerate 5 decelerate 5, and centrifuge for 8 minutes. After the centrifugation is completed, remove the supernatant, scrape up ...

Embodiment 2

[0043] Example 2: Flow cytometry detection of central memory T cells

[0044] CD4+T cells sorted in Example 1, TCM cells on day 7 of culture and TCM cells on day 14 were subjected to flow cytometric detection using CD4 antibody, CD95 antibody, CD45RO antibody and CD62L antibody. All antibodies were purchased from BD Company. The amount of cells required for detection is 300,000-500,000 cells per tube. Add 5 microliters of each antibody, stain at room temperature in the dark for 15 minutes, wash and centrifuge twice with DPBS solution, and finally use 400 microliters of DPBS Resuspend the machine for detection. Central memory T cells are CD45RO + CD62L + CD95 + cell. Test results such as figure 1 , figure 2 with image 3 As shown, it can be seen that adding IL-21 in the early stage of culture can increase the proportion of cultured TCM cells, and adding rapamycin in the later stage of culture can inhibit the transformation of central memory T cells into effector cells a...

Embodiment 3

[0048] Example 3: Comparison of the killing effect of TCM cells on tumor cells under different culture conditions

[0049] After the central memory T cells were cultured for 14 days, the cultured HCC827 cell line was taken and digested with 0.05% trypsin at 37° C. for 5 minutes. After the digestion was terminated with serum, it was centrifuged at 500 g for 5 minutes. Remove the supernatant, resuspend in DPBS and count, take 5*10 6 cells, centrifuged at 500 g for 5 min. Remove the supernatant and resuspend in 500 μl DPBS solution. Take another 1.5ml, add 500 microliters of DPBS solution and 1 microliter of CFSE (fluorescent dye) solution with a concentration of 5mM to it, mix well and gently add the 500 microliters of CFSE solution to another tube of 500 microliters Incubate in the cell suspension at 37°C in the dark for 10-20min. Then 2ml of FBS serum was added to stop the staining, and centrifuged at 500g for 5min. Remove the supernatant, add 10ml DPBS solution to wash tw...

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Abstract

The invention discloses an in-vitro culture method of a central memory T lymphocyte. The method comprises the following steps: adding IL-21 into an initial medium, and allowing the oxygen concentration in a culture environment to be 1 + / - 0.1%; and adding the IL-21 and rapamycin into an amplification medium when the culturing is performed for 7 days. The in-vitro culture method using the combination of the rapamycin, interleukin 21 and low-oxygen environment in a proper culture process to improve the proportion of the central memory T cell, inhibit the differentiation of the central memory T cell into effector cells and enhance the killing effect on tumor cells is disclosed for the first time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a central memory T lymphocyte and an in vitro culture method thereof. Background technique [0002] Adoptive immune cell therapy refers to the infusion of autologous or allogeneic immune effector cells activated in vitro into patients to kill tumor cells in patients. Adoptive immune cell therapy is a new and effective treatment method in current tumor immunotherapy. Among them, T cell adoptive immunotherapy is the most widely used and most effective immunotherapy method in the world. Its principle of action is to use the specific T cells activated in vitro to carry out the killing function of antigens. [0003] There are two main types of specific T cells, CD4 + T cells and CD8 + T cells. In adoptive immunotherapy, CD4 + In addition to its own killing ability, T cells can also assist the body's own immune cells and co-adopted immune cells. They have strong versatilit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/2321C12N2501/999
Inventor 吴海涛聂慧蓉施念沈政吴灵
Owner BEIHAO STEM CELL & REGENERATIVE MEDICINE RES INST CO LTD
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