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Method for co-culturing, inducing and amplifying gamma delta T cells and NK cells

A technology of NK cells and co-cultivation, applied in the direction of cell culture active agents, biochemical equipment and methods, animal cells, etc., can solve the problems of high cost, achieve the effects of simplifying the treatment process, strong toxicity/activity, and reducing culture costs

Inactive Publication Date: 2019-12-13
安徽瑞达健康产业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] In the existing research, most of the single immune cells or killer cells are used for cell therapy or combined therapy in clinical treatment. bring more economic pressure

Method used

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  • Method for co-culturing, inducing and amplifying gamma delta T cells and NK cells
  • Method for co-culturing, inducing and amplifying gamma delta T cells and NK cells
  • Method for co-culturing, inducing and amplifying gamma delta T cells and NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Obtaining of γδT-NK cells

[0045] Procurement of γδT-NK cells Mononuclear cells (PBMCs) were isolated from peripheral blood and γδT-NK cells were expanded:

[0046] (1) Turn on the biological safety cabinet 30 minutes before use;

[0047] (2) Take out D-PBS from the refrigerator before use, and let it stand at room temperature for 30 minutes;

[0048] (3) Transfer 30ml of peripheral blood samples (heparin anticoagulant) to two sterile 50ml centrifuge tubes, 15ml in each tube, then add 22.5ml of sterile D-PBS to each tube, invert the centrifuge tube repeatedly, and mix thoroughly;

[0049] (4) Take another two 50ml sterile centrifuge tubes, add 15ml Ficoll-Paque Plus solution respectively, and then slowly add 24ml blood diluted in step 3 (drawn from the two sterile tubes in step 3) to form Layering, 20°C, 400×g, centrifuge for 30 minutes;

[0050] (5) Put the two 50ml centrifuge tubes in step 4 into a biological safety cabinet, then use a 10ml pipette to su...

Embodiment 2

[0063] Example 2 Obtaining of γδT-NK cells

[0064] Procurement of γδT-NK cells Mononuclear cells (PBMCs) were isolated from peripheral blood and γδT-NK cells were expanded:

[0065] (1) Turn on the biological safety cabinet 30 minutes before use;

[0066] (2) Take out D-PBS from the refrigerator before use, and let it stand at room temperature for 30 minutes;

[0067] (3) Transfer 30ml of peripheral blood samples (heparin anticoagulant) to two sterile 50ml centrifuge tubes, 15ml in each tube, then add 22.5ml of sterile D-PBS to each tube, invert the centrifuge tube repeatedly, and mix thoroughly;

[0068] (4) Take another two 50ml sterile centrifuge tubes, add 15ml Ficoll-Paque Plus solution respectively, and then slowly add 24ml blood diluted in step 3 (drawn from the two sterile tubes in step 3) to form Layering, 20°C, 400×g, centrifuge for 30 minutes;

[0069] (5) Put the two 50ml centrifuge tubes in step 4 into a biological safety cabinet, then use a 10ml pipette to su...

Embodiment 3

[0082] Example 3 Effect of γδT-NK cell mixture on SK-BR-3 cells

[0083] Use 5μM CFSE to stain SK-BR-3 cells, mix NK cells or γδT cells or γδT-NK cells or PBS and SK-BR-3 cells at a ratio of 20:1, incubate at 37°C for 4h, add 1 μg / ml PI dye, CFSE+PI double-positive cells are dead cells, such as Figure 5 As shown, the mixture of γδT cells and NK cells can significantly enhance the killing activity of cancer cells, almost 100% killing SK-BR-3 cells.

[0084]It can be concluded from the above examples that γδT cells and NK cells can be co-induced and expanded through the above-mentioned co-culture mode, and the two kinds of cells can promote the growth and expansion of each other, and the expansion amount can fully meet the clinical needs, simplifying the induction of single cells. The expansion culture mode, co-inducing and expanding the cultured cells, saves a lot of economic and time costs, and the co-cultured cells have better killing effect on SK-BR-3 cells than single cel...

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Abstract

The invention discloses a method for co-culturing, inducing and amplifying gamma delta T cells and NK cells. The gamma delta T cells and the NK cells are obtained in a co-culture manner; peripheral blood is collected to separate and screen mononuclear cells and stimulate and induce the mononuclear cells to induce the gamma delta T cells and the NK cells, the gamma delta T cells and the NK cells are subjected to activation amplification culture to amplify a gamma delta T cell and NK cell composite cell substance having characteristics of large number, high amplification multiple, strong cytotoxicity and the like so as to form the co-culture cell composition through compounding, and the co-culture cell composition has good clinical application value. The co-culture induced amplification method improves the cell culture efficiency, greatly reduces the culture cost, reduces the difficulty of a culture technology, simplifies the clinical treatment process, reduces the treatment cost, and simplifies the treatment means.

Description

Technical field: [0001] The invention relates to a method for inducing and expanding cells, in particular to a method for inducing and expanding by co-culture of γδT cells and NK cells, and the application of the γδT-NK cell composite cells obtained by the method on cancer cell SK-BR-3. Background technique: [0002] γδT cells are immune cells that can not only kill cancer cells, tumor stem cells, but also recognize cancer antigens. [0003] γδ T cells are T cells that perform innate immune functions, and their TCR consists of γ and δ chains. Such T cells are mainly distributed in mucosa and subcutaneous tissues such as intestinal respiratory tract and urogenital tract, and only account for 0.5%-1% of CD3+ T cells in peripheral blood. Its TCR lacks diversity and can directly recognize some complete polypeptide antigens. The types of antigens recognized by γδT cells are limited: ① HSP; ② lipid antigens extracted from CD1 molecules on the surface of infected cells; ③ certain...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17A61P35/00C12N5/0636C12N5/0646C12N2500/30C12N2501/2302C12N2501/2315C12N2501/2321C12N2501/51C12N2501/515A61K2300/00
Inventor 邢永梅邓蒙蒙程箫刘丹吴疆王保如
Owner 安徽瑞达健康产业有限公司
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