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NK cell culture composition and culture method

A technology for NK cells and cell culture, applied in the field of cell culture, can solve the problems of low killing activity and low purity of NK cells, and achieve the effects of avoiding pollution, improving killing activity and simple culture process.

Active Publication Date: 2016-02-03
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method of stimulating NK cell expansion with simple cell growth factors has low purity and low killing activity of NK cells.

Method used

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  • NK cell culture composition and culture method
  • NK cell culture composition and culture method
  • NK cell culture composition and culture method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Expansion and culture of embodiment 1 NK cells

[0066] 1. Preparation of antigen-presenting cells:

[0067] The 4-1BBL / PCR4TOPO plasmid was amplified by PCR, and then inserted into the NheI-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct the 4-1BBL / pSBSO transposon.

[0068] K562 cells were co-transfected with 4-1BBL / pSBSO transposon and transposase SB11, and sorted by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).

[0069] 4-1BBL-K562 cells were co-transfected with IL-15-Fc(CoOp)-pSBSO and transposase SB11, and sorted by flow cytometry to establish 4-1BBL-mIL-15-K562 artificial antigen-presenting cells.

[0070] The established 4-1BBL-mIL-15-K562 artificial antigen-presenting cells were irradiated with 100Gy radiation.

[0071] 2. Isolation of peripheral blood mononuclear cells

[0072] Collect 40-100mL of peripheral blood, mix it with normal saline at a volume ratio of 1:1, and slowly add the mix...

Embodiment 2

[0076] Expansion and cultivation of embodiment 2 NK cells

[0077] 1. Preparation of antigen-presenting cells:

[0078] The 4-1BBL / PCR4TOPO plasmid was amplified by PCR, and then inserted into the NheI-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct the 4-1BBL / pSBSO transposon.

[0079] K562 cells were co-transfected with 4-1BBL / pSBSO transposon and transposase SB11, and sorted by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).

[0080] 4-1BBL-K562 cells were co-transfected with IL-15-Fc(CoOp)-pSBSO and transposase SB11, and sorted by flow cytometry to establish 4-1BBL-mIL-15-K562 artificial antigen-presenting cells.

[0081] The established 4-1BBL-mIL-15-K562 artificial antigen-presenting cells were irradiated with 100Gy radiation.

[0082] 2. Isolation of peripheral blood mononuclear cells

[0083] Collect 40-100mL of peripheral blood, mix it with normal saline at a volume ratio of 1:1, and slowly add the...

Embodiment 3

[0087] Expansion culture of embodiment 3 NK cells

[0088] 1. Preparation of antigen-presenting cells:

[0089] The 4-1BBL / PCR4TOPO plasmid was amplified by PCR, and then inserted into the NheI-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct the 4-1BBL / pSBSO transposon.

[0090] K562 cells were co-transfected with 4-1BBL / pSBSO transposon and transposase SB11, and sorted by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).

[0091] 4-1BBL-K562 cells were co-transfected with IL-15-Fc(CoOp)-pSBSO and transposase SB11, and sorted by flow cytometry to establish 4-1BBL-mIL-15-K562 artificial antigen-presenting cells.

[0092] The established 4-1BBL-mIL-15-K562 artificial antigen-presenting cells were irradiated with 100Gy radiation.

[0093] 2. Isolation of peripheral blood mononuclear cells

[0094] Collect 40-100mL of peripheral blood, mix it with normal saline at a volume ratio of 1:1, and slowly add the mixed b...

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Abstract

The invention relates to the technical field of cell culture, particularly to an NK (natural killer) cell culture composition and a culture method. The composition comprises an artificial antigen presenting cell, IL-2, IL-15, pseudo-ginseng saponin, blood serum and a basal culture medium. The NK cell culture composition can promote mass proliferation of NK cells, improves the killing activity of the NK cells, avoids extrinsic protein and virus pollution, and reduces production cost; the obtained NK cells are relatively high in purity.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to an NK cell culture composition and a culture method thereof. 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 cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 陈海佳王一飞葛啸虎李丽娟王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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