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Method for preparing CIK cells

A cell and nuclear cell technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of limited tumor treatment effect, low tumor killing ability of CIK cell viability, slow cell proliferation rate, etc. The effect of high level, fast proliferation and high lethality

Active Publication Date: 2018-05-04
暨赛国际再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the induced CIK cells have low cell viability and tumor killing ability, and the therapeutic effect on tumors is very limited.
In addition, the mononuclear cells of a small number of blood donors are not sensitive to the induction of CD3 monoclonal antibody and some cytokines, and the cell proliferation rate is slow. Active CIK cell preparation technology

Method used

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  • Method for preparing CIK cells
  • Method for preparing CIK cells
  • Method for preparing CIK cells

Examples

Experimental program
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Effect test

Embodiment 1

[0047] A method for preparing CIK cells, the method comprising the steps in the following order:

[0048] (1) Preparation stage:

[0049] ① Preparation of coating solution: Add 20 mL of D-PBS buffer into a 50 mL centrifuge tube, add 400 μL of recombinant human fibronectin (purchased from TAKARA) (final concentration 20 μg / mL), mouse CD3 monoclonal antibody (trade name: Aioshan ) 200 μL (final concentration 10 μg / mL);

[0050] ② Coating: add the coating solution prepared above into a T175 cell culture flask, lay it flat, mix well, and coat overnight at 4°C in the dark;

[0051] (2) Induction culture of peripheral blood mononuclear cells (PBMC):

[0052] ①Blood collection: collect 50mL of peripheral blood;

[0053]②Preparation of autologous plasma: Centrifuge the peripheral blood collected above at 700 g at room temperature for 20 minutes, the upper layer is the plasma layer, and the lower layer is the white blood cell and red blood cell layer;

[0054] Gently aspirate the u...

Embodiment 2

[0066] A method for preparing CIK cells, the method comprising the steps in the following order:

[0067] (1) Preparation stage:

[0068] ① Preparation of coating solution: Add 20 mL of D-PBS buffer into a 50 mL centrifuge tube, add 100 μL of recombinant human fibronectin (purchased from TAKARA) (final concentration 5 μg / mL), mouse CD3 monoclonal antibody (trade name: Aioshan ) 400 μL (final concentration 20 μg / mL);

[0069] ② Coating: add the coating solution prepared above into a T175 cell culture flask, lay it flat, mix well, and coat overnight at 4°C in the dark;

[0070] (2) Induction culture of peripheral blood mononuclear cells (PBMC):

[0071] ①Blood collection: collect 50mL of peripheral blood;

[0072] ②Preparation of autologous plasma: Centrifuge the peripheral blood collected above at 700 g at room temperature for 20 minutes, the upper layer is the plasma layer, and the lower layer is the white blood cell and red blood cell layer;

[0073] Gently aspirate the u...

Embodiment 3

[0085] A method for preparing CIK cells, the method comprising the steps in the following order:

[0086] (1) Preparation stage:

[0087] ① Preparation of coating solution: Add 20 mL of D-PBS buffer to a 50 mL centrifuge tube, add 250 μL of recombinant human fibronectin (purchased from TAKARA) (final concentration 12.5 μg / mL), mouse CD3 monoclonal antibody (trade name: Aiou Shan) 100 μL (final concentration 5 μg / mL);

[0088] ② Coating: add the coating solution prepared above into a T175 cell culture flask, lay it flat, mix well, and coat overnight at 4°C in the dark;

[0089] (2) Induction culture of peripheral blood mononuclear cells (PBMC):

[0090] ①Blood collection: collect 50mL of peripheral blood;

[0091] ②Preparation of autologous plasma: Centrifuge the peripheral blood collected above at 700 g at room temperature for 20 minutes, the upper layer is the plasma layer, and the lower layer is the white blood cell and red blood cell layer;

[0092] Gently aspirate the ...

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Abstract

The invention relates to in-vitro culture of immune cells and in particular relates to in-vitro induction culture of CIK cells. The preparation method disclosed by the invention comprises the following steps: coating a cell culture bottle by fibronectin and a CD3 monoclonal antibody; preparing autologous plasma; acquiring peripheral blood mononuclear cells PBMC; inducing the PBMC by IL-2, IFN-gamma and the autologous plasma; and performing multiplication culture. The CIK cells prepared by the preparation method disclosed by the invention are great in quantity of CIK cells, high in cell viability, high in ratio of CD3+CD56+effector cells and high in cytolytic activity, can meet clinical requirements and have high anti-tumor abilities.

Description

technical field [0001] The present invention relates to in vitro culture of immune cells, and more specifically relates to in vitro induced culture of CIK cells. Background technique [0002] CIK (cytokine induced killer) cells, that is, cytokine-induced killer cells, are a type of heterogeneous mononuclear cells with CD3+CD56+ cells as the main effector cells obtained after co-culture stimulation with CD3 monoclonal antibody and various cytokines A cell population that simultaneously expresses NK cell (natural killer cell) surface markers (CD56 molecules) and T cell surface markers (CD3 molecules), so it is also called NKT cells (natural killer T cells); it also has strong anti-tumor activity of T cells , and the non-MHC (major histocompatibility antigen)-restricted tumor killing characteristics of NK cells. CIK cells have the characteristics of high anti-tumor activity, broad anti-tumor spectrum, low toxicity to normal tissues, and highly expandable in vitro, etc., and ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2500/90C12N2501/2302C12N2501/24C12N2501/515C12N2533/52
Inventor 吴琨陈汉森徐丽婉徐家科
Owner 暨赛国际再生医学科技有限公司
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