DC-CIK cell culture medium, DC-CIK cell culture method and application

A technology of DC-CIK and culture method, applied in the direction of cell culture active agent, culture process, tissue culture, etc., can solve the problems of small number of cell culture, slow expansion speed, long culture time, etc., to achieve enhanced killing ability, quantity Increase and increase the effect of multiplying multiples

Inactive Publication Date: 2020-05-08
山东龙辰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the defects in the prior art, a DC-CIK cell culture medium, culture method and application are provided, aiming at solving the problems of slow expansion speed, small number of cells cultured and long culture time in the culture process of DC-CIK cells

Method used

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  • DC-CIK cell culture medium, DC-CIK cell culture method and application
  • DC-CIK cell culture medium, DC-CIK cell culture method and application
  • DC-CIK cell culture medium, DC-CIK cell culture method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0141] The preparation of embodiment 1DC-CIK cell

[0142] Specific steps are as follows:

[0143] Preparation of 1DC-CIK cells by conventional methods (control group)

[0144] 1.1 Separation of PBMC from peripheral blood mononuclear cells: Peripheral blood mononuclear cells were collected from 100 mL peripheral blood of the patient's immediate blood relatives, and obtained by density gradient centrifugation.

[0145] 1.2 Culture peripheral blood mononuclear cells using serum-free medium, add IFN-γ (1000IU / mL), IL-12 (500IU / mL), GM-CSF (50IU / mL) at 37°C, 5% CO2 After culturing in the incubator for 24 hours, add IL-2 (100IU / mL), IL-15 (50IU / mL), Anti-CD3 (20IU / mL), continue culturing for 72 hours, add TNF-a (500IU / mL) for To promote the maturation of DC cells. After 96 hours, IL-2 (100IU / mL) was added and IL-15 (50IU / mL) was cultured for another 8 days to obtain DC-CIK cells.

[0146] 2 Preparation of DC-CIK cells optimized by the present invention (experimental group)

[...

experiment example 1CI

[0174] Experimental Example 1 Detection of CIK cells and DC-CIK cells

[0175] 1. Determination of cell proliferation multiples: the above-mentioned experimental group and control group were subjected to cell counting for the two different culture media and culture methods, and the multiplication multiples were measured. The results were as follows: figure 1 Shown (the abscissa indicates the culture time, and the ordinate indicates the amplification factor).

[0176] Determination result analysis of cell proliferation multiple:

[0177] CIK began to proliferate on the 3rd to 4th day of culture, and the number increased significantly on the 7th day. Co-cultivation with DC can significantly increase the proliferation rate of CIK cells. On the 14th day of culture, live cells were counted by trypan blue staining, and the total number of CIK cells expanded to (37.96±4.15) times of the original, while that of DC-CIK cells was (61.49±5.01) times of the original, and the difference ...

experiment example 2

[0180] Experimental example 2 cell phenotype analysis

[0181]CD3+, CD8+, CD56+, CD3-CD56+, CD4 / CD8 amplification number and CIK cell and DC-CIK cell immunophenotype detection, cell immunophenotype CD3+ was 79.2%, CD3+CD56+ was 38.2%, CD3-CD56+ was 43.8%, CD4+CD8+ was 34.8%. With the extension of culture time, the proportion of CD3+CD8+ and CD3+CD56+ cells in CIK cells gradually increased, and the proportion of CD3+CD8+ and CD3+CD56+ double-positive cells in CIK cells co-cultured with DC was significantly higher than that of CIK cells under the same conditions (P<0.05).

[0182] Table 1 Comparison of immunophenotypes of CIK and DC-CIK cells (x±s, %)

[0183] group CD3+ cells CD3+ / CD4+ CD3+ / CD8+ CD3+ / CD56+ CIK cells 65.23±3.31 23.15±2.53 38.41±3.46 27.67±3.67 DC-CIK cells 82.42±4.25 24.27±3.36 52.46±4.05 41.53±4.29

[0184] Note: Compared with the control group, *P<0.05.

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Abstract

The invention discloses a DC-CIK cell culture medium, a DC-CIK cell culture method and application, and relates to the technical field of cell culture. The DC-CIK cell culture medium comprises a DC culture medium containing specific components, a DC maturation factor, a CIK promoter, a CIK bottle enlargement culture medium, a CIK separate culture medium, a coating liquid and a basic culture medium, and all the components have different effects through different compositions. When the DC-CIK cell culture medium is used for culturing the DC-CIK cells, the number of the obtained DC-CIK cells is substantially increased and can reach (1.05-1.56) * 1011/mL, the survival rate is kept at 96.6%-99.7%, meanwhile, the CD3+, CD4+ and CD5+ of the DC-CIK cells can reach 68%.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a DC-CIK cell culture medium, a culture method and application of DC-CIK cells. Background technique [0002] Immune cell therapy refers to the infusion of immune cells with anti-tumor activity to tumor patients, directly killing tumor cells or stimulating the body's anti-tumor immune response, so as to achieve the purpose of treating tumors. Commonly used somatic cell immunotherapy methods include: dendritic cell vaccine (DC), cytokine-induced killer cells (CIK), and DC-stimulated CIK cells (DC-CIK). DCs are known to have the strongest antigen-presenting ability in the body. Through the uptake and presentation of tumor antigens, DCs can effectively induce and activate immune effector cells, thereby mediating powerful and specific tumor immunity. CIK cells are a group of heterogeneous cells obtained by co-culturing human peripheral blood mononuclear cells with various cytok...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0784
CPCC12N5/0638C12N5/0639C12N2501/2304C12N2501/22C12N2501/25C12N2501/515C12N2501/2301C12N2501/2302C12N2501/24C12N2501/2312C12N2500/90C12N2502/1121
Inventor 张守村李树迎孙如东
Owner 山东龙辰生物技术有限公司
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