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NK cell culture medium and NK cell culture method

A technology of NK cells and culturing methods, applied in the field of NK cell culture medium and NK cell culture, can solve the problem that the immune activity of NK cells is not very high, and achieve the effects of enhancing lethality, reducing interference and improving activity

Inactive Publication Date: 2016-12-07
浙江译美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Therefore, in order to obtain a larger number of NK cells in the prior art, researchers have carried out large-scale reproduction through in vitro culture. However, because the in vitro environment is not exactly the same as the environment in the human body, the NK cells Immunity is not very high

Method used

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  • NK cell culture medium and NK cell culture method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] NK cell culture medium:

[0039] Serum-free basal medium 94g, plasma 7g, anti-CD3 monoclonal antibody 0.6g, insulin-like growth factor 1 2g, PBS buffer 20g, squid ink 1g, safflower polysaccharide 2g, allicin 2.5g, alfalfa polysaccharide 1g and cinnamon bark Aldehyde 2g;

[0040] NK cell culture:

[0041] S1. Collect 10ml of peripheral blood from a healthy person, and dilute the peripheral blood with 20ml of diluent to obtain diluted blood;

[0042] S2. Add 10ml Ficoll to centrifuge tube 1, and slowly add diluted blood along the inclined tube wall;

[0043] S3. Put the centrifuge tube 1 into a constant temperature room at 18°C ​​and centrifuge at a speed of 1500r / min for 30min;

[0044] S4, gently absorb the PBMC layer in the centrifuge tube one of S3 with a straw and move it into the centrifuge tube two;

[0045] S5. Add a sufficient amount of diluent to the second centrifuge tube for washing, and centrifuge at 1500r / min for 5min to remove the supernatant to obtain ...

Embodiment 2

[0049] NK cell culture medium:

[0050] Serum-free basal medium 98g, plasma 10g, anti-CD3 monoclonal antibody 1.6g, insulin-like growth factor 1 4g, PBS buffer 30g, squid ink 4g, safflower polysaccharide 6g, allicin 4.5g, alfalfa polysaccharide 3g and cinnamon bark Aldehyde 4g;

[0051] NK cell culture:

[0052] S1. Collect 10ml of peripheral blood from a healthy person, and dilute the peripheral blood with 30ml of diluent to obtain diluted blood;

[0053]S2. Add 7.5ml Ficoll to centrifuge tube 1, and slowly add diluted blood along the inclined tube wall;

[0054] S3. Put the centrifuge tube 1 into a constant temperature room at 18°C ​​and centrifuge at a speed of 1800r / min for 45min;

[0055] S4, gently absorb the PBMC layer in the centrifuge tube one of S3 with a straw and move it into the centrifuge tube two;

[0056] S5. Add a sufficient amount of diluent to the second centrifuge tube for washing, and centrifuge at 1800r / min for 7min to remove the supernatant to obtain...

Embodiment 3

[0060] NK cell culture medium:

[0061] Serum-free basal medium 96g, plasma 8.5g, anti-CD3 monoclonal antibody 1g, insulin-like growth factor 1 3g, PBS buffer 25g, squid ink 2.5g, safflower polysaccharide 4g, allicin 3.5g, alfalfa polysaccharide 2g and Cinnamaldehyde 3g;

[0062] NK cell culture:

[0063] S1. Collect 10ml of peripheral blood from a healthy person, and dilute the peripheral blood with 15ml of diluent to obtain diluted blood;

[0064] S2. Add 5ml Ficoll to centrifuge tube 1, and slowly add diluted blood along the inclined tube wall;

[0065] S3. Put the centrifuge tube 1 into a constant temperature room at 18°C ​​and centrifuge at 1670r / min for 37min;

[0066] S4, gently absorb the PBMC layer in the centrifuge tube one of S3 with a straw and move it into the centrifuge tube two;

[0067] S5. Add a sufficient amount of diluent to the second centrifuge tube for washing, and centrifuge at 1650r / min for 6min to remove the supernatant to obtain clean PBMC cells; ...

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Abstract

An NK cell culture medium is prepared from, by weight, 94-98 parts of serum-free basic culture medium, 7-10 parts of plasma, 0.6-1.6 parts of anti-CD3 monoclonal antibody, 2-4 parts of first para-insulin growth factor, 20-30 parts of PBS buffer solution, 1-4 parts of squid ink, 2-6 parts of flos carthami polysaccharide, 2.5-4.5 parts of allicin, 1-3 parts of alfalfa polysaccharide and 2-4 parts of cinnamaldehyde. Serum-free basic culture medium, plasma, anti-CD3 monoclonal antibody and first para-insulin growth factor together form a human body marrow environment required by NK cell growth. Flos carthami polysaccharide, allicin, alfalfa polysaccharide and cinnamaldehyde can improve activity of the NK cells easily, and accordingly killing capacity of the NK cells is enhanced. Squid ink contains IL-2 which can induce activation and differentiation of the NK cells, and accordingly multiplication efficiency of the NK cells is easily improved. The activated NK cells can synthesize and secrete various cytokines, and accordingly tumor cell differentiation and multiplication are directly inhibited.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a culture medium for NK cells and a method for culturing NK cells. Background technique [0002] Natural killer cells (NK) are important immune cells in the body, not only related to anti-tumor, anti-virus infection and immune regulation, but also involved in the occurrence of hypersensitivity and autoimmune diseases in some cases. Maturation depends on the bone marrow and thymus microenvironment. The function of NK cells is mainly realized in the following ways. NK cell natural killing activity: NK cells recognize target cells (some tumor cells, virus-infected cells, some self-organized cells, parasites, etc.), and then NK cells release perforin and cytotoxic factors, and activated NK cells release TNF factors , so as to achieve the possibility of lysing and killing target cells. NK cytotoxicity (ADCC effect), mainly due to the FcγR Ⅲ A on the surface of NK cells, which mainly bin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/84C12N2500/90C12N2501/06C12N2501/105C12N2501/515C12N2501/90C12N2501/999
Inventor 陈继冰吴振化
Owner 浙江译美生物科技有限公司
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