CAR-NK cell culture method

A technology of NK cells and culture methods, applied in the direction of cell culture active agent, animal cells, culture process, etc., can solve the problems of complexity, insufficient cell activity, and small number, and achieve a large number, enhance proliferation, and promote rapid proliferation. Effect

Pending Publication Date: 2021-01-26
广东康盾创新产业集团股份公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the preparation of CAR-NK cells in the prior art has the problems of c...

Method used

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  • CAR-NK cell culture method

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Embodiment 1

[0023] A method for culturing CAR-NK cells, comprising the following steps:

[0024] S1. Collect peripheral blood from healthy donors and separate peripheral blood mononuclear cells;

[0025] S2. Dilute the cell concentration of peripheral blood mononuclear cells to 1×10 7 cells / mL, pipette 3 mL of diluted cells into a culture flask coated with 10 μg / mL anti-CD16 monoclonal antibody, and place the culture flask at a temperature of 32°C, CO 2 After culturing in an incubator with a volume concentration of 5% for 3 days, supplemented with 120ng / mL interleukin-12, 55ng / mL interleukin-3, 0.5g / L lecithin, and 0.01g / L glutathione, and continued to culture for 5 days , sort out NK cells, and detect the purity of NK cells;

[0026] The sorted NK cells were cultured with serum-free DMEM medium for 24 hours, the cell density of NK cells was adjusted to 3×10 cells / mL, and then transferred to NK cell expansion medium for expansion culture. placed at a temperature of 37 °C, CO 2 Cultiva...

Embodiment 2

[0031] The difference between embodiment 2 and embodiment 1 is:

[0032] The differentiation culture described in step S2 is to place the culture bottle at a temperature of 37 ° C, CO 2 Cultured in an incubator with a volume concentration of 4%.

Embodiment 3

[0034] The difference between embodiment 3 and embodiment 1 is:

[0035]The expansion culture in step S2 is to culture the sorted NK cells with serum-free DMEM medium for 36 hours, adjust the cell density of NK cells to 1.5×10 cells / mL, and then transfer them to the NK cell expansion medium For expansion culture, the culture medium was placed at a temperature of 37°C, CO 2 Cultivate in an incubator with a volume concentration of 5% for 7 days, and detect the expansion factor of NK cells; the NK cell expansion medium contains 10% volume ratio of autologous inactivated serum, 140ng / mL interleukin-2, 0.4g / L galactose in DMEM medium.

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Abstract

The invention provides a CAR-NK cell culture method. The culture method comprises the following steps of S1, collecting peripheral blood of a healthy donor, and separating to obtain peripheral blood mononuclear cells; S2, sorting out NK cells from peripheral blood mononuclear cells; S3, infecting the NK cells in the step S2 with a lentiviral vector with a chimeric antigen receptor gene; and S4, carrying out multiplication culture on the CAR-NK cells by using a culture medium containing interleukin-2, inositol, quercetin glucoside, formononetin, sheep placenta and galactose to obtain a large number of CAR-NK cells. The CAR-NK cell culture method disclosed by the invention is simple to operate, and the cultured NK cells are high in purity, large in quantity, large in quantity of CAR-NK cellsand high in killing activity.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for culturing CAR-NK cells. Background technique [0002] Chimeric antigen receptor (chimeric antigen receptor, CAR) modified immune cells use genetic engineering to modify immune cells to express exogenous anti-tumor genes. The CAR gene mainly includes an extracellular recognition domain and an intracellular signal transduction domain: the former is used to recognize specific molecules on the tumor surface, and the latter is used to initiate an immune cell response after recognizing tumor surface molecules to exert cytotoxicity. This is a new type of cell therapy that has been around for many years, but has only been improved and used clinically in recent years. It has remarkable efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment methods. [0003] Natural killer (NK) cells are an importa...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867
CPCC12N5/0646C12N15/86C12N2740/15043C12N2510/00C12N2500/35C12N2500/34C12N2501/2302C12N2500/84C12N2500/30C12N2501/2312C12N2501/2303C12N2501/999
Inventor 湛振键齐国光刘世豪
Owner 广东康盾创新产业集团股份公司
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