Preparation method of NK (Natural Killer) cells

A NK cell and cell technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of complex process, uncontrollable factors, high cost, and achieve simple amplification method, strong amplification ability, The effect of high safety

Inactive Publication Date: 2018-04-13
GENESIS STEMCELL REGENERATIVE MEDICINE ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many ways to cultivate and activate NK cells in vitro, such as adding animal serum to the culture system, stimulating NK cells in vitro by using fire-extinguished K562 cells as a feeder layer, or transferring exogenous genes into cells through gene recombination technology or through magnetic stimulation. NK cells are sorted out by bead sorting and expanded in vitro, etc. Although these methods can effectively expand NK cells, the process is complicated, the cost is high, there are many uncontrollable factors, and the safety needs to be verified, so it is not suitable for clinical NK cell therapy

Method used

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  • Preparation method of NK (Natural Killer) cells
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  • Preparation method of NK (Natural Killer) cells

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preparation example Construction

[0027] A method for preparing NK cells, the preparation of NK cells is completed through the steps of culture flask coating, peripheral blood collection, mononuclear cell isolation, inoculation, amplification, cell harvesting and identification; the specific steps are as follows:

[0028] (1) Culture flask coating: 1 mg / ml humanized CD16 monoclonal antibody and 1 mg / ml OKT-3 monoclonal antibody, dilute the antibody to the required concentration with sterile DPBS pH 7.6-8.2, and coat the T75 flask , 10 ml per bottle, overnight at 4°C or pre-coated at 37°C for 1 h, the optimal concentration is 3ug / ml humanized CD16 monoclonal antibody and 2 ug / ml OKT-3 monoclonal antibody;

[0029] (2) Peripheral blood collection: The test items of HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-Treponema pallidum antibody, ALT, and mycoplasma were all negative in the tertiary hospitals. Draw 50-80 ml of peripheral blood by a professional nurse, put it in a 10 ml sterile blood collection...

Embodiment 1

[0042] A method for preparing NK cells, the preparation of NK cells is completed through the steps of culture flask coating, peripheral blood collection, mononuclear cell isolation, inoculation, amplification, cell harvesting and identification; the specific steps are as follows:

[0043] (1) Coating of culture flask: 1 mg / ml humanized CD16 monoclonal antibody and 1 mg / ml OKT-3 monoclonal antibody, diluted with sterile DPBS at pH 7.6 to 30 ug / ml humanized CD16 monoclonal antibody Antibody and 20 ug / ml OKT-3, take 1 ml each into a 15 ml centrifuge tube, then add 8 ml DPBS, mix by pipetting, transfer the coating solution to a T75 bottle, and leave overnight at 4°C

[0044] (2) Peripheral blood collection: The test items of HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-Treponema pallidum antibody, ALT, and mycoplasma were all negative in the tertiary hospitals. 50 ml of peripheral blood was drawn by a professional nurse, put in a 10 ml sterile blood collection tube cont...

Embodiment 2

[0050] A method for preparing NK cells. The preparation of NK cells is completed through the steps of culture flask coating, peripheral blood collection, mononuclear cell isolation, inoculation, amplification, cell harvesting and cell identification; the specific steps are as follows:

[0051] (1) Coating of culture flask: 1 mg / ml humanized CD16 monoclonal antibody and 1 mg / ml OKT-3 monoclonal antibody, diluted with sterile DPBS pH 7.6 to 30 ug / ml humanized CD16 monoclonal antibody and 20 ug / ml OKT-3, take 1 ml each into a 15 ml centrifuge tube, then add 8 ml DPBS, mix by pipetting, transfer the coating solution to a T75 bottle, and coat at 37°C for 1 h;

[0052] (2) Peripheral blood collection: The test items of HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-Treponema pallidum antibody, ALT, and mycoplasma were all negative in the tertiary hospitals. 60 ml of peripheral blood was drawn by a professional nurse, put in a 10 ml sterile blood collection tube containing l...

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Abstract

The invention discloses a preparation method of NK (Natural Killer) cells. The preparation of the NK cells is prepared through the steps of coating a culture bottle, collecting peripheral blood, separating mononuclear cells, inoculating, amplifying and harvesting the cells. A serum free culture medium provided by the invention has clear components and high safety; after the mononuclear cells are cultured and activated by the culture medium, the amplification capability is strong; after being cultured for 15 to 18 days, the mononuclear cells are amplified for 160 to 200 times; after the cells are detected by a flow cytometer, the content of the NK cells of CD3<->CD56<+> is 65 to 70 percent and the content of T cells of an NK sample of CD3<+>CD56<+> is 5 to 15 percent; the content of NK andNKT cells exceeds 75 percent and the killing activity on K526 reaches 85 percent or above when the multiplicity of infection is 20 to 1; furthermore, an amplification method is simple and efficient and large-scale production is facilitated.

Description

technical field [0001] The invention relates to a method for preparing cells, in particular to a method for preparing NK cells. Background technique [0002] The discovery of NK cells has a history of several decades. It belongs to the innate immune system and is the first line of defense given by the human immune system. NK cells account for about 5%-10% of lymphocytes in the blood, and the characteristic indicator is CD3-CD56+, which is a type of lymphocytes without T and B cell characteristic markers, and also exists in peripheral tissues, such as liver and peritoneal cavity , placenta, etc., NK cells circulating in peripheral blood are usually in an immune quiescent state, once activated by cytokines, they will infiltrate into most tissues to attack tumor cells and virus-infected cells. [0003] NK cells are an important part of the innate immune system and an important immunoregulatory cell for the body to resist infection and prevent malignant transformation of cells....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/105C12N2501/2301C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/24C12N2501/90C12N2501/998
Inventor 王军霞谢再东潘春雷吴锋殷鉴强刘定生
Owner GENESIS STEMCELL REGENERATIVE MEDICINE ENG CO LTD
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