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Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method

A NK cell and cryopreservation technology, applied in the field of cell engineering, can solve the problems of unclear NK cell expansion and NK cell survival cannot last for more than one week, and achieve the effect of improving NK expansion and high NK cell expansion efficiency.

Active Publication Date: 2016-08-10
杭州朔溪生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the prior art, cell viability is still an issue, and NK cell survival after resuscitation cannot last more than a week
At the same time, it remains unclear whether the recovered NK cells can further expand

Method used

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  • Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method
  • Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method
  • Method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and application of method

Examples

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Embodiment 1

[0033] Example 1 This example discloses a method for efficiently expanding cryopreserved NK cells, and the specific steps are as follows:

[0034] 1. Culture of human cell lines

[0035] Wild-type K562 and Raji cell lines were obtained from the American Type Culture Collection (ATCC). And these cells were placed in RPMI-1640 medium (GIBCO) containing 10% fetal bovine serum (GIBCO), and placed in 37 ° C, 5% CO 2 cultured in an incubator.

[0036] 2. Plasmid construction

[0037] pFastBac1 (Invitrogen, Carlsbad, CA) was used as the plasmid backbone for the expression of ZFNs and fusion genes encoding mbil15, 4-1BBL and mbIL21 with the cytomegalovirus (CMV) promoter. Among them, the specific construction of pFastBac-ZFN has been reported before (Tay, Tan et al.2013).

[0038] For the construction of the pFB-fusion gene host, the DNA fragment encoding the fusion gene was synthesized by AIT Biotechnology for the first time according to the sequence gene reported in the prior ar...

experiment example

[0059] 1. Phenotype detection experiment of expanded NK cells

[0060] A broad array of expanded NK cell adhesion molecules and receptors was tested. Most highly expressed receptors and adhesion molecules, with a few exceptions - activating receptors, NKp44, and inhibitory receptors, surface CD158a, h, CD158i and CD158e1 / e2 ( Figure 5 ).

[0061] The expression of adhesion molecules after resuscitation of NK cells cultured for 7 days and the expression of adhesion molecules and receptors after re-stimulation with K562-mbIL15-41BBL-mbIL21 for 1 week were also compared. The surface expression of the inhibitory receptors, CD158a, h and CD158e1 / e2, and the activating receptors, NKG2D and NKp44, was significantly increased. It was shown that the expression of the surface marker CD16 is downregulated by cryopreservation of NK cells (Sakamoto, Ishikawa et al. 2015). Consistent with these results, there was a significant decrease (up to 86%) in the expression of the surface marker...

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Abstract

The invention belongs to the field of cell engineering, in particular to a method for high-efficient expansion and cryopreservation of NK (Natural Killer) cells and an application of the method. According to the invention, fusion genes are subjected to gene recombination to an AAVS1 (Adeno-Associated Virus Integration Site 1) transposon through a plasmid, and the freezing / thawing step is introduced to the NK cell expansion step taking a modified K562 cell line as a basis in the intermediate stage of cell expansion; finally, the NK cells are expanded and saved. A K562-mbIL15-41BBL-mbIL21 cell line developed by the integration technology of site specific genes is used, and the activity, purity and cytotoxicity of the NK cells are high to realize high expansion efficiency of the NK cells to improve NK expansion. The method for the high-efficient expansion and cryopreservation of the NK (Natural Killer) cells and the application of the method for the high-efficient expansion and cryopreservation of the NK (Natural Killer) cells disclosed by the invention have great market prospects and economic values.

Description

technical field [0001] The invention belongs to the field of cell engineering, and in particular relates to a method for efficiently expanding cryopreserved NK cells and an application thereof. Background technique [0002] Human natural killer (NK) cells are composed of lymphocytes of the innate immune system that express CD56 and lack CD3 (CD-CD56+). NK cells have a broad spectrum of tumor-killing functions that can induce ligand binding through cytotoxic granules such as perforin and granzymes, such as death receptor interaction, Fas-Fas ligand binding and TNF-related apoptosis and other mechanisms to exert anti-tumor effects. NK cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the membrane receptor FcRγIII (CD16). Target recognition and activation of NK cells works through a balance of inhibition and activation of NK cell surface receptors and associated ligands expressed on target cells, which allows an immediate response to pathogen inva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0783C12N13/00A01N1/02
CPCC12N5/0646C12N13/00C07K14/54C07K14/5443C07K14/70578C12N15/85C12N2502/30C12N2501/2302Y02A50/30
Inventor 卓朗王晖王柯
Owner 杭州朔溪生物医药有限公司
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