Culture method for umbilical cord blood CIK cells

A culture method and cord blood technology, which is applied in the field of cord blood CIK cell culture, can solve the problems of low proliferation rate and killing activity of CIK lymphocytes, complicated operation, etc., and achieve the promotion of adhesion and proliferation, high expansion, and avoid rejection The effect of the reaction

Active Publication Date: 2020-05-19
中科细胞科技(广州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The Chinese patent application publication number CN 105695404 A discloses a method for the expansion and activation of CIK lymphocytes, which requires transfection, complicated operation, and the obtained CIK lymphocytes have a low proliferation rate and killing activity

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  • Culture method for umbilical cord blood CIK cells
  • Culture method for umbilical cord blood CIK cells
  • Culture method for umbilical cord blood CIK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The method of this embodiment is as follows: extract umbilical cord blood mononuclear cells (CBMC) with lymphocyte separation medium, count the cells and divide them into six groups on average, including groups A, B, C, D, E, and F. Group A was cultured by the traditional method without RN, group B was cultured with RN (final concentration: 6.25 μg / mL), group C was cultured with RN (final concentration: 9.25 μg / mL), group D was cultured with RN ( The final concentration was 12.5 μg / mL) for coating culture, group E was used for coating culture (final concentration was 15.5 μg / mL), group F was cultured for coating with RN (final concentration was 18.5 μg / mL), trypan blue was used every 3 days The cell viability of each group was detected by staining, and the absolute number of cells was recorded. On the 7th, 14th, and 21st days of culture, the immunophenotypes of cells in each group were measured by flow cytometry. MTT assay was used to measure the killing rate of cells ...

Embodiment 2

[0111] This example is carried out with reference to step 6 of Example 1, the difference is that the final concentration of IFN-γ in the culture system is 500 IU / mL, and the final concentrations of IL-2 and IL-1a in the culture system are both 500 IU / mL, The final concentration of anti-CD3 monoclonal antibody in the culture system was 30ng / mL.

[0112] Table 5 Two groups of cell phenotypes under different culture time

[0113] CD3+CD56+ double positive D0 D3 D6 D9 D12 D15 H1 group 1％ 5.8％ 9.1％ 27.3％ 49.8％ 66.4％ H2 group 1％ 6％ 11.5％ 30.5％ 57.3％ 75.3％

[0114] It can be seen from Table 5 that the final concentration of IFN-γ, IL-2 and IL-1α in the culture system is 500IU / mL, and when the final concentration of anti-CD3 monoclonal antibody in the culture system is 30ng / mL, the The final concentration of γ, IL-2, and IL-1α in the culture system was 1000IU / mL, and the final concentration of anti-CD3 monoclonal antibody in the culture syste...

Embodiment 3

[0119] This example is carried out with reference to Example 1, the difference is that the final concentration of IFN-γ in the culture system is 1500IU / mL, the final concentrations of IL-2 and IL-1a in the culture system are both 1500IU / mL, anti-CD3 monoclonal The final concentration of the cloned antibody in the culture system was 70ng / mL.

[0120] Table 7 Two groups of cell phenotypes under different culture time

[0121] CD3+CD56+ double positive D0 D3 D6 D9 D12 D15 H1 group 1％ 6.2％ 9.4％ 26.9％ 50.9％ 68.8％ H2 group 1％ 6.3％ 11.5％ 32％ 59.1％ 76.9％

[0122] It can be seen from Table 7 that the final concentration of IFN-γ, IL-2 and IL-1α in the culture system is 1500IU / mL, and when the final concentration of anti-CD3 monoclonal antibody in the culture system is 70ng / mL, it can react with IFN-γ The final concentration of IL-2 and IL-1α in the culture system was 1000IU / mL, and the final concentration of anti-CD3 monoclonal antibody in the...

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Abstract

The invention provides a culture method for umbilical cord blood cytokine induced killer (CIK) cells. The method comprises the following steps: adding an umbilical cord blood karyocyte suspension intoa fibronectin-coated culture container for culture, and adding IFN-gamma, IL-2, IL-1a and an anti-CD3 monoclonal antibody, and continuing culture to obtain the CIK cells. The method successfully obtains the umbilical cord blood CIK cells with higher proliferation, a higher percentage of double positive cells and stronger killing activity, and the method is a culture method worthy of clinical recommendation and capable of amplifying the umbilical cord blood CIK cells in large quantities.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing umbilical cord blood CIK cells. Background technique [0002] Cytokine induced killer cells (CIK) are immune effector cells with the strongest cytotoxic activity, and have both the high tumoricidal activity of T lymphocytes and the non-toxicity of NK cells (natural killer cells, natural killer cells). Restricted tumoricidal effects of the major histocompatibility complex (MHC). CIK cells express CD3 and CD56 two membrane protein molecules at the same time, have the advantages of fast proliferation, high tumor killing activity, and broad tumor killing spectrum, and are considered to be a new hope for adoptive immunotherapy of tumors. Most of the CIK cell preparation methods in the prior art use the patient's peripheral blood mononuclear cells, first add IFN-γ, and then add other cytokines such as IL-2, IL-Iα, CD3 monoclonal antibody after 24 hours, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2533/56C12N2501/24C12N2501/2301C12N2501/2302C12N2501/2315C12N2501/515
Inventor 孙丹唐忆琳陈巧林
Owner 中科细胞科技(广州)有限公司
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