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Culture medium and method for NK cell expansion in vitro

A technology of NK cells and in vitro expansion, applied in the field of immunology, can solve the problems of high cost of isolating NK cells, inability to achieve high activity, high purity and high amplification multiple of NK cells, limited application, etc. Application safety, enhance NK cell activity, induce NK cell proliferation effect

Inactive Publication Date: 2016-05-11
上海润泉生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low content of NK cells in human peripheral blood (accounting for about 5-10% of lymphocytes), the cost of isolating NK cells from peripheral blood is expensive, and the isolated NK cells are generally in an inactivated state, which limits the its clinical application
To achieve the therapeutic effect, the required effective NK cell quantity should be at least 5×10 in a single batch application. 9 One (and the purity reaches more than 80%). It is currently reported that the use of serum-free culture system can not achieve high activity, high purity and high expansion multiple of NK cells through stimulation and culture of IL-2, IL-21 and related combination factors. , its amplification ability can reach up to 300 times, and there are safety risks in clinical application
Scale expansion is achieved through the autologous serum culture system, and a single culture system is obtained, and the number of cells expanded in a single batch is greater than 1×10 10 NK cells with a purity greater than 90% have not been reported

Method used

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  • Culture medium and method for NK cell expansion in vitro
  • Culture medium and method for NK cell expansion in vitro
  • Culture medium and method for NK cell expansion in vitro

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Experimental program
Comparison scheme
Effect test

Embodiment 1K562

[0128] The preparation of embodiment 1K562 cells

[0129] In this embodiment, K562 cells were prepared by the following method:

[0130] (1) Artificially synthesize GM-CSF sequence, IL-21 / 15 sequence, CD4 sequence, CD137L sequence, CD86 sequence, CD19 sequence and CD64 sequence, and combine IL-21 / 15 sequence, CD137L sequence, CD86 sequence, CD19 sequence and The CD64 sequence is inserted in series with the 2A sequence into the eukaryotic expression vector with antibiotic markers;

[0131] (2) Sequence the eukaryotic expression vector obtained in step (1), purify it after it is correct, and co-transfect it with a transposase-containing plasmid into K562 cells, add antibiotics for drug treatment, and obtain positive transfected cells ;

[0132] (3) adding antibiotics to the positively transfected cells obtained in step (2) for drug treatment, sorting by flow cytometry, and obtaining high-purity transfected K562 cells;

[0133] (4) After drug treatment and 5-20 times of flow c...

Embodiment 2

[0138] The preparation of embodiment 2 culture medium

[0139] 166.7 μg of interleukin 2 was fully mixed and dissolved with 1 mL of lymphocyte serum-free medium to obtain a mixed solution, and then 300 μL of the mixed solution was added to 900 mL of lymphocyte serum-free medium to obtain a mixed solution A;

[0140] Add 0.4g serum substitute to 100mL lymphocyte serum-free medium, resuspend and dissolve in a vortex mixer, then add mixture A to obtain a medium for NK cell in vitro expansion, seal it and store it at 2-8°C spare.

Embodiment 3

[0141] The preparation of embodiment 3 medium

[0142] 1.67 mg of interleukin 2 was fully mixed and dissolved with 1 mL of lymphocyte serum-free medium to obtain a mixed solution, and then 300 μL of the mixed solution was added to 900 mL of lymphocyte serum-free medium to obtain a mixed solution B;

[0143] Add 5 g of serum substitute to 100 mL of RPMI1640 medium, resuspend and dissolve in a vortex mixer, then add mixed solution B to obtain a medium for NK cell expansion in vitro, seal it and store it at 2-8°C for later use.

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Abstract

The invention relates to a culture medium and method for NK cell expansion in vitro. The culture medium is prepared from, by volume, 100% of a lymphocyte serum-free culture medium or the mixture of 1-90% of an RPMI1640 culture medium and 10-99% of a lymphocyte serum-free culture medium, a serum substitute and interleukin 2, wherein the concentration of the serum substitute is 0.5-4g / L, and the concentration of the interleukin 2 is 50-500 micrograms / mL. According to the culture medium and the preparation method, explosive expansion of NK cells in a short time can be achieved, the cost is low, the expanded cells are extremely high in destruction, and the requirement for application of three kinds of medical technologies and the requirement for somatic cell treatment medicine quality control are met.

Description

technical field [0001] The invention relates to the field of immunology, in particular to a medium for in vitro expansion of NK cells and a method for in vitro expansion of NK cells. Background technique [0002] Natural killer cells (NK) are important immune cells in the body and play an important role in anti-tumor and anti-viral infection immunity. Since NK cell killing activity has no MHC limit, it is called natural killer cell. The target cells of NK cells are mainly tumor cells, virus-infected cells, some self-organized cells (such as blood cells) and parasites, etc. Therefore, NK cells are an important part of the body's anti-tumor and anti-infection immunity. Due to these characteristics, NK cells have broad application prospects in cellular immunotherapy. NK cells activated in vitro are highly toxic and can be used as therapeutic preparations for immune cells. Human in vitro and animal experiments have confirmed the possibility of its treatment of various cancers...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302
Inventor 刘军唐欢
Owner 上海润泉生物技术有限公司
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