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Culture method for NK cells

A technology of NK cells and culture methods, applied in the field of NK cell culture, can solve the problems of NK cell culture uncertainty, lack of correct evaluation of cells, and inapplicability of NK cell therapy, etc., and achieve weight loss, low cost, and high specificity sexual effect

Inactive Publication Date: 2017-11-28
BINZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To sum up, the problem existing in the prior art is that at present, the method of simply co-cultivating antigen-presenting cells and NK cells is mainly used to promote the proliferation of NK cells
Although the above method can stimulate the efficient expansion of NK cells, the composition of animal serum is complex, and mycoplasma and viruses may be brought in during the sampling process, which will cause uncertainty and unsafety for NK cell culture; K562 itself, as a tumor cell, is used in clinical There are great doubts about safety, not suitable for clinical NK cell therapy; lack of correct assessment of cultured cells

Method used

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  • Culture method for NK cells

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Effect test

Embodiment 1

[0059] Expansion and culture of embodiment 1 NK cells

[0060] 1. Isolation of peripheral blood mononuclear cells

[0061] Collect 40-100mL of peripheral blood, mix it with normal saline at a volume ratio of 1:1, and slowly add the mixed blood mixture and lymphocyte separation solution to the lymphocyte at a volume ratio of 2:1. The upper layer of the cell separation solution was centrifuged at 3000rpm for 20min;

[0062] Viewed from top to bottom of the centrifuge tube after centrifugation, they are respectively plasma, buffy coat layer (ie, mononuclear cell layer, PBMC), lymphocyte separation medium, and red blood cell layer. The PBMC layer is extracted, and the collected PBMC is washed with normal saline and reconstituted. After suspension, centrifuge at 1500rpm for 10min, repeat washing twice, and collect PBMC;

[0063] 2. Expansion and culture of NK cells

[0064] The collected PBMC and the above-prepared antigen-presenting cells were mixed and cultured at a ratio of 1...

Embodiment 2

[0066] Expansion culture of NK cells

[0067] 1. Isolation of peripheral blood mononuclear cells

[0068] Collect 40-100mL of peripheral blood, mix it with normal saline at a volume ratio of 1:1, and slowly add the mixed blood mixture and lymphocyte separation solution to the lymphocyte at a volume ratio of 2:1. The upper layer of the cell separation solution was centrifuged at 3000rpm for 20min;

[0069] Viewed from top to bottom of the centrifuge tube after centrifugation, they are respectively plasma, buffy coat layer (ie, mononuclear cell layer, PBMC), lymphocyte separation medium, and red blood cell layer. The PBMC layer is extracted, and the collected PBMC is washed with normal saline and reconstituted. After suspension, centrifuge at 1500rpm for 10min, repeat washing twice, and collect PBMC;

[0070] 2. Expansion and culture of NK cells

[0071] The collected PBMC and the above-prepared antigen-presenting cells were mixed and cultured at a ratio of 1:3, seeded in a c...

Embodiment 3

[0072] Expansion culture of embodiment 3 NK cells

[0073] 1. Isolation of peripheral blood mononuclear cells

[0074] Collect 40-100mL of peripheral blood, mix it with normal saline at a volume ratio of 1:1, and slowly add the mixed blood mixture and lymphocyte separation solution to the lymphocyte at a volume ratio of 2:1. The upper layer of the cell separation solution was centrifuged at 3000rpm for 20min;

[0075] Viewed from top to bottom of the centrifuge tube after centrifugation, they are respectively plasma, buffy coat layer (ie, mononuclear cell layer, PBMC), lymphocyte separation medium, and red blood cell layer. The PBMC layer is extracted, and the collected PBMC is washed with normal saline and reconstituted. After suspension, centrifuge at 1500rpm for 10min, repeat washing twice, and collect PBMC;

[0076] 2. Expansion and culture of NK cells

[0077] The collected PBMC and the antigen-presenting cells prepared above were mixed and cultured at a ratio of 1:2, in...

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Abstract

Belonging to the field of medical technology, the invention discloses a culture method for NK (natural killer) cells. The method includes the steps of: isolating peripheral blood to obtain PBMC cells for standby use; inoculating conventionally cultured K562 cells in a medium; then replacing a serum-free DMEM culture solution for synchronization, then using a basic medium for resuspension, adding IL-2 and IL-15, conducting cell culture, supplementing liquid every 3 days, adding K562 cells till culture for 6-7 or 8 days, and performing culture for 16-18d to obtain NK cells; and then conducting inoculation molding. The method provided by the invention uses autoserum to promote cell proliferation and avoids exogenous protein and virus contamination. The method provided by the invention only needs a low dose of IL-2, and greatly reduces the production cost. The NK cells cultured by the culture method provided by the invention have high purity.

Description

technical field [0001] The invention belongs to the field of medical technology, in particular to a method for cultivating NK cells. Background technique [0002] Cell therapy is a new technology for disease treatment that has emerged in recent years. It refers to the use of the characteristics of certain cells with specific functions, which are obtained by bioengineering methods and / or processed by in vitro expansion and special culture to enhance the ability of these cells. Immunity, killing pathogens and tumor cells, promoting tissue and organ regeneration and body recovery and other therapeutic effects, so as to achieve the purpose of treating diseases. Therapeutic cells include: NK, γδT, CD3AK, DC-CIK, etc., and their efficacy, specificity, overall efficiency, and side effects have gradually improved. [0003] NK cells (natural killer cells), also known as natural killer cells, belong to lymphocyte lineage cells, cytotoxic lymphocytes containing perforin and granzyme g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/09
CPCC12N5/0646C12N2501/2302C12N2501/2315C12N2502/30
Inventor 付强栾希英薛江楠孙雨飞王卓亚
Owner BINZHOU MEDICAL COLLEGE
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