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NK cell amplification method

A NK cell and cell technology, which is applied to animal cells, vertebrate cells, cell culture active agents, etc., can solve the problem of small amplification multiple, and achieve the effect of simple operation, low cost and good amplification effect.

Pending Publication Date: 2020-05-15
深圳市芙丽嘉科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing method of stimulating NK cell expansion with cytokine combinations can increase the expansion factor relatively small, generally around one or two hundred times, which is difficult to meet the clinical demand for NK cells

Method used

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Examples

Experimental program
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Embodiment 1

[0028] A method for expanding NK cells using peripheral blood mononuclear cells is provided, comprising the following steps:

[0029] 1. Collect peripheral blood: Use heparin sodium anticoagulant tube to collect peripheral blood.

[0030] 2. Plasma preparation:

[0031] ① Mix the peripheral blood by blowing up and down with a pipette, then take a sample for sterility test and cell count;

[0032] ② Balance the peripheral blood in the 4 centrifuge tubes with a Pasteur pipette, and centrifuge at 500×g for 7 minutes;

[0033] ③ After centrifugation, use a pipette to absorb the upper light yellow plasma layer and transfer it to a new centrifuge tube. The remaining liquid is the concentrated blood cells;

[0034] ④Seal the two centrifuge tubes containing plasma with parafilm, mark the sample code, and inactivate in a water bath at 56°C for 30 minutes;

[0035] ⑤ After plasma inactivation is completed, centrifuge at 900×g for 10 minutes;

[0036] ⑥After centrifugation, take the ...

Embodiment 2

[0057] A method for expanding NK cells using peripheral blood mononuclear cells is provided, the difference from Example 1 is that the final concentration of IL-2 added during the culture process is 700IU / mL.

Embodiment 3

[0059] A method for expanding NK cells using peripheral blood mononuclear cells is provided, the difference from Example 1 is that 2% human albumin is added to the serum-free medium instead of autologous plasma.

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Abstract

The invention provides a highly-efficient NK cell amplification method. The amplification method comprises the following steps: (1) carrying out separating so as to obtain mononuclear cells; and (2) adding a serum-free culture medium and an activating factor, and carrying out culturing, wherein the activating factors are IL-2 monoclonal antibody and CD3 monoclonal antibody. According to the NK cell amplification method provided by the embodiments of the invention, in-vitro amplification is directly carried out after mononuclear cells are separated out, and a combination of specific activatingfactors is selected, so extremely highly-efficient NK cell amplification is realized, and the amplification multiple can reach 3000 times or above, and is increased by an order of magnitude compared with the amplification multiple of a conventional amplification method; and the highly-efficient NK cell amplification method has good amplification efficiency and is simple in operation.

Description

technical field [0001] The invention relates to the technical field of NK cell culture, in particular to an NK cell expansion method. Background technique [0002] Natural killer (NK) cells are the most important type of lymphocytes in the third type of lymphocytes without the characteristic surface characteristics of T and B cells, and they are the bridge connecting the innate and specific immune systems. Under normal circumstances, NK cells in the body mostly circulate in the peripheral blood and exist in an unactivated state. NK cells can quickly kill malignant cells and virus-infected cells without sensitization, and exhibit special immunoregulatory and cytotoxic functions. However, the proportion of NK cells in the peripheral blood in vivo is very small, generally no more than 10% of the total number of lymphocytes, and it is difficult to obtain a large number of high-purity NK cells in a short period of time. [0003] For this reason, researchers try to obtain NK cel...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/515C12N2506/11
Inventor 贺光锐苑英姿王慧琴
Owner 深圳市芙丽嘉科技有限公司
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