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Natural killer T cell culture medium and multiplication culture method for natural killer T cells

A cell culture and killing technology, applied in cell culture active agents, cell culture supports/coatings, animal cells, etc., can solve problems such as hidden safety hazards, insufficient expansion of natural killer T cells, safety problems, etc. , to achieve the effect of promoting activation and proliferation and alleviating potential safety risks

Active Publication Date: 2017-12-19
TIANJIN CHANGHE BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] But above-mentioned these culture methods all have the shortcoming of certain degree, wherein, adopt the method for cultivating with fetal bovine serum because fetal bovine serum is animal source serum, may introduce exogenous virus and mycoplasma, there is certain potential safety hazard; The method of cell culture also has certain safety problems; and the number of natural killer T cells obtained by the above method is insufficient to expand, and the proportion of natural killer T cells is low, which cannot meet the needs of clinical treatment

Method used

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  • Natural killer T cell culture medium and multiplication culture method for natural killer T cells
  • Natural killer T cell culture medium and multiplication culture method for natural killer T cells
  • Natural killer T cell culture medium and multiplication culture method for natural killer T cells

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1: CD16 Antibody Antibody Coating

[0052] The CD16 antibody with a concentration of 50 ng / mL was coated on the cell culture flask, the coating condition was 37° C., and the incubation time was 2 h to obtain the coated cell culture flask.

Embodiment 2

[0053] Example 2: Preparation of autologous plasma and human peripheral blood mononuclear cells

[0054] Collect peripheral blood, transfer the collected peripheral blood to a 50mL centrifuge tube, and centrifuge at 2000rmp for 10 minutes at a temperature of 20°C to collect autologous plasma; then use lymphocyte separation medium to separate and collect human peripheral blood mononuclear cells, and the collected The human-derived peripheral blood mononuclear cells were washed three times with PBS buffer to obtain human-derived peripheral blood mononuclear cells;

[0055] Wherein, the above-mentioned method for separating human-derived peripheral blood mononuclear cells using lymphocyte separation fluid comprises the following steps:

[0056] 1. Add the blood sample after drawing the plasma into PBS at a ratio of 1:1, and mix well;

[0057] 2. Slowly add the diluted blood sample on the surface of the lymphocyte separation liquid, and the ratio of the diluted blood sample to th...

Embodiment 3

[0060] Embodiment 3: induction culture

[0061] Step 1: Use X-VIVO 15 serum-free medium to resuspend the human peripheral blood mononuclear cells obtained in Example 2, and adjust the cell concentration in the medium to 1.5×10 6 / mL, then inoculated into the cell culture flask coated with CD16 antibody obtained in Example 1;

[0062] Step 2: Add the autologous plasma obtained in Example 2 into the cell culture flask of the above step 1, the concentration of the autologous plasma in the natural killer T cell culture medium is 5%, and add the cytokine IL-2 (1000IU / mL) , followed by induction culture for 14 days;

[0063] Step 3: In the first 7 days of the induction culture process, perform rehydration every 3 days, and use the rehydration medium to adjust the cell density to 1.5×10 6 / mL, and supplemented with autologous plasma and cytokine IL-2; after 7 days in the induction culture process, rehydration was performed every 3 days, and the cell density was adjusted to 1.5×10 ...

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Abstract

The invention provides a natural killer T cell culture medium and a multiplication culture method for natural killer T cells and relates to the technical field of cell culture. The natural killer T cell culture medium, disclosed by the invention, is mainly composed of a serum-free medium, autologous plasma and a cell factor IL-2. According to the multiplication culture method for natural killer T cells by using the cell culture medium, human-derived peripheral blood mononuclear cells are induced to release a danger signal by virtue of the antibody binding cell factor IL-2, and further the natural killer T cells are activated. According to the method, high-quality natural killer T cells can be obtained by massive high-efficiency multiplication in a short period on premise of ensuring the security. The potential security risk caused by culture of heterogenous animal serum or tumor cells in the conventional natural killer T cell culture method and the problems that the conventional natural killer T cell culture method is low in multiplication times and low in purity and is difficult to use on a large scale are effectively solved.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a natural killer T cell culture substrate and a natural killer T cell expansion culture method. Background technique [0002] Natural killer T cells (nature killer T cells, NKT cells) are a group of lymphocyte subsets that have both T cell receptor (TCR) and NK cell receptor on the cell surface. The distribution of natural killer T cells is similar to that of natural killer cells, and is also tissue-specific, with different proportions in various tissues, such as liver, bone, spleen, lymph node, peripheral blood, lung and thymus. TCR on the surface of natural killer T cells can recognize glycolipid antigens presented by CD1d molecules on the surface of thymocytes, and can secrete a large number of cytokines to participate in the body's innate and adaptive immunity. Therefore, natural killer T cells play an important role in antiviral immunity, tumor immunity, and autoimmune...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/2312C12N2501/599C12N2533/50
Inventor 倪琳李政楠李艳
Owner TIANJIN CHANGHE BIOLOGICAL TECH
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