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Multiplication culture method of peripheral blood CIK cells

A technology of expansion culture and cell culture, which is applied in the field of expansion culture of peripheral blood CIK cells, can solve the problems of poor cell activity and low amplification rate, and achieve the effects of simple operation, strong killing activity, and convenient and simple acquisition

Pending Publication Date: 2021-02-26
海南优尼科尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of cytokine-induced culture of CIK cells is currently the most widely used and most convenient culture method, but the number of CIK cells, expansion efficiency and killing rate of CIK cells obtained by using different combinations of factors and the specific content of factors are also very different. There is a big difference. The peripheral blood CIK culture method in the prior art generally has problems such as low amplification rate and poor cell activity.

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  • Multiplication culture method of peripheral blood CIK cells

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Embodiment 1

[0022] A method for expanding and culturing peripheral blood CIK cells, comprising the following steps:

[0023] 1) After the peripheral blood is initially centrifuged to obtain plasma, the blood cell components are mixed with normal saline and added to the Ficoll cell separation medium, after centrifugation, the buffy coat layer is collected, and then washed and centrifuged to obtain mononuclear cells;

[0024] 2) Culture the mononuclear cells obtained in step 1), and culture the mononuclear cells at 1.0×10 6 cells / mL was added to the initial culture medium to obtain a cell suspension; the initial medium was GT-T551H3 medium containing 10% plasma, and the initial culture medium also included IFN-γ, anti-CD3 monoclonal antibody and IL -2, wherein, IFN-γ, anti-CD3 monoclonal antibody and IL-2 are commercially available products, and the working concentrations of IFN-γ, anti-CD3 monoclonal antibody and IL-2 are respectively: 1000U / mL, 50ng / mL, 1000U / mL;

[0025] 3) placing the...

Embodiment 2

[0029] A method for expanding and culturing peripheral blood CIK cells, comprising the following steps:

[0030] 1) After the peripheral blood is initially centrifuged to obtain plasma, the blood cell components are mixed with normal saline and added to the Ficoll cell separation medium, after centrifugation, the buffy coat layer is collected, and then washed and centrifuged to obtain mononuclear cells;

[0031] 2) Culture the mononuclear cells obtained in step 1), and culture the mononuclear cells at 0.5×10 6 The density of each / mL was added to the initial culture medium to cultivate a cell suspension; the initial medium was GT-T551H3 medium containing 5% plasma, and the initial culture medium also included IFN-γ, anti-CD3 monoclonal antibody and IL-2, among them, IFN-γ, anti-CD3 monoclonal antibody and IL-2 are commercially available products, and the working concentrations of IFN-γ, anti-CD3 monoclonal antibody and IL-2 are: 800U / mL, 80ng / mL , 1200U / mL;

[0032] 3) placin...

Embodiment 3

[0036] A method for expanding and culturing peripheral blood CIK cells, comprising the following steps:

[0037]1) After the peripheral blood is initially centrifuged to obtain plasma, the blood cell components are mixed with normal saline and added to the Ficoll cell separation medium, after centrifugation, the buffy coat layer is collected, and then washed and centrifuged to obtain mononuclear cells;

[0038] 2) Culture the mononuclear cells obtained in step 1), and culture the mononuclear cells at 1.2×10 6 cells / mL was added to the initial culture medium to obtain a cell suspension; the initial medium was GT-T551H3 medium containing 12% plasma, and the initial culture medium also included IFN-γ, anti-CD3 monoclonal antibody and IL -2, wherein, IFN-γ, anti-CD3 monoclonal antibody and IL-2 are commercially available products, and the working concentrations of IFN-γ, anti-CD3 monoclonal antibody and IL-2 are respectively: 600U / mL, 50ng / mL, 800U / mL.

[0039] 3) placing the ce...

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Abstract

The invention discloses a multiplication culture method of peripheral blood CIK cells. The multiplication culture method comprises the following steps of: preparing peripheral blood mononuclear cellsfrom peripheral blood through centrifugal extraction, taking the mononuclear cells, and adding the mononuclear cells into a complete initial culture solution containing IFN-gamma, IL-2 and anti-CD3 monoclonal antibodies for induction culture, wherein the complete initial culture medium is a GT-T551H3 cell culture solution; and, after the cells are cultured for 3 days, adding a complete fluid infusion culture medium to continuously perform multiplication culture, wherein the fluid infusion culture medium is added once every 2-3 days; the whole induction culture time is 15-21 days; and the CIK cells are obtained. The invention belongs to the technical field of cell culture, and particularly provides the multiplication culture method of the peripheral blood CIK cells, which has a good multiplication effect and can effectively improve the multiplication activity and killing activity of the cells.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and specifically refers to a method for expanding and culturing peripheral blood CIK cells. Background technique [0002] In recent years, with the in-depth study of immunology and molecular biology on tumor treatment decision-making, immune effector cells have become the fourth major technology in the treatment of tumor diseases, and have attracted extensive attention from the medical community. It has become a research hotspot in the field of tumor treatment and breakthrough. CIK cells are the immune effector cells with the strongest cytotoxic activity and the most promising immune cells, which have been widely developed in clinical applications. [0003] CIK cells are cytokine-induced killer cells, which have both the non-major histocompatibility complex-limited tumor-killing effect of NK cells and the high tumor-killing activity of T cells, and can simultaneously express two membrane pr...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/078
CPCC12N5/0638C12N2501/2302C12N2501/24C12N2501/515
Inventor 王晓宇林冠妃郭康合李崴蔡开婷吴清毅陈多峰
Owner 海南优尼科尔生物科技有限公司