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Culture medium and its application in the culture of central memory T-lymphocytes

A culture medium and basal medium technology, applied in the direction of cell culture active agent, animal cells, tissue culture, etc., can solve the problem of dependence on anti-tumor effect

Active Publication Date: 2020-10-30
赛德特(北京)生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effective anti-tumor effect in vivo depends on the joint action of CD4+ and CD8+ central memory cells. Therefore, the development of simple and effective isolation and in vitro expansion technology of CD4+ and CD8+ central memory T cells still needs to be further optimized.

Method used

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  • Culture medium and its application in the culture of central memory T-lymphocytes
  • Culture medium and its application in the culture of central memory T-lymphocytes
  • Culture medium and its application in the culture of central memory T-lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 The acquisition of blood mononuclear cells

[0054] ⑴ Use a heparin anticoagulant tube to collect about 30-100ml of peripheral blood, transfer the peripheral blood to a 50ml centrifuge tube, add an equal amount of normal saline to dilute, mix well, add dropwise to an equal amount of lymphocyte separation medium, and be careful not to damage the interface. The ratio of peripheral blood, normal saline, and lymphocyte separation fluid is 1:1:1.

[0055] (2) Centrifuge at 1500-2000rpm / min for 20-30min. Due to different densities, the centrifuge tube is divided into four layers from top to bottom: the first layer is the plasma layer. The second layer is a ring-shaped milky mononuclear cell layer (PBMC). The third layer is a transparent separation liquid layer. The fourth layer is the red blood cell layer.

[0056] (3) Use a straw to carefully draw the second layer of milky white mononuclear cell layer (PBMC) into another 50ml centrifuge tube, add physiological s...

Embodiment 2

[0059] Example 2 Isolation and cultivation of human central memory T lymphocytes

[0060] First prepare the culture medium, the formula is shown in Table 2:

[0061] Table 2 medium formula

[0062] Reagent name Medium 1 Medium 2 Medium 3 GT-T551 100ml 100ml 100ml interleukin-2 5×10 4 U / L

5×10 5 U / L

1×10 6 U / L

interleukin-7 1ng / ml 30ng / ml 60ng / ml Interleukin-12 1ng / ml 50ng / mL 100ng / ml Interleukin-15 1ng / ml 30ng / ml 60ng / ml interleukin-1a 1ng / ml 30ng / ml 60ng / ml Anti-CD3 antibody 1ng / ml 50ng / mL 100ng / ml Anti-CD28 antibody 1ng / ml 50ng / mL 100ng / ml Everolimus 0.1μM 5μM 10μM

[0063] The preparation method of the culture medium is as follows: mix the components evenly, and prepare immediately after use.

[0064] 1. the mononuclear cell suspension that embodiment 1 is made is respectively inoculated in above-mentioned medium culture, concrete steps

[0065] ...

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Abstract

The invention relates to the technical field of stem cell culture, in particular to a medium and application thereof in central memory T lymphocyte culture. The medium provided by the invention is composed of a basic medium, an mTOR inhibitor, interleukin-2, interleukin-7, interleukin-12, interleukin-15, interleukin-1a, an Anti-CD3 antibody and an Anti-CD28 antibody. By means of the medium and a culture method provided by the invention, great numbers of high-proportional central memory T lymphocytes can be provided, experiments show that by using the medium provided by the invention, TCMs (central memory T cell) can amplify around 100 times, and in 20 days of culture, the number of the cells is the highest on the twentieth day, while the purity of the TCMs (sample B) is the highest on thetwelfth day.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a culture medium and its application in the culture of central memory T lymphocytes. Background technique [0002] The immune system can help the body resist bacterial infection and eliminate tumors, so as to maintain the homeostasis of the body's internal environment, and the basis for its function lies in immune cells. Immune cells include antigen-presenting cells, natural killer cells, B lymphocytes, and T lymphocytes, which are distributed in both central immune organs and peripheral immune organs. The central immune organs generally refer to the bone marrow and thymus, which are the places where B cells and T cells develop respectively; the peripheral immune organs generally refer to the spleen, tonsil, lymph nodes, etc., which are the places where B cells and T cells station and patrol. Disturbances in the homeostasis of the immune system can lead to a variety of dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N2501/2301C12N2501/2302C12N2501/2307C12N2501/2312C12N2501/2315C12N2501/51C12N2501/515C12N2501/727
Inventor 孙忠杰陈立功郭潇薛庆磊左慧晶刘庆军马骏凡
Owner 赛德特(北京)生物工程有限公司
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