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Method for stably amplifying high-purity and high-cytotoxic-activity NK cells in vitro

A NK cell, high-purity technology, used in cell culture active agents, animal cells, vertebrate cells, etc., can solve the problems of weak killing function, low NK cell purity, unstable culture system, etc., and achieve high cytotoxic activity, The effect of reducing the number of

Inactive Publication Date: 2019-11-29
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

[0006] In order to solve the deficiencies existing in the prior art, and solve the shortcomings of the existing NK cell culture technology, such as cumbersome, low NK cell purity, weak killing function, and unstable culture system, the present invention provides a serum-free, feeder-free NK cell in vitro expansion The expansion method can stably and massively expand NK cells with high purity and high cytotoxic activity under in vitro culture conditions, so as to effectively provide NK cells that meet the needs of clinical applications.

Method used

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  • Method for stably amplifying high-purity and high-cytotoxic-activity NK cells in vitro
  • Method for stably amplifying high-purity and high-cytotoxic-activity NK cells in vitro
  • Method for stably amplifying high-purity and high-cytotoxic-activity NK cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 In vitro expansion of NK cells-isolation of monocytes

[0039] 1. Isolation of Monocytes

[0040]1.1 Under the normal working condition of the biological safety cabinet, add Ficoll-Paque Plus lymphocyte separation medium to 50ml sterile centrifuge tubes;

[0041] 1.2 Dilute the patient's peripheral whole blood and DPBS evenly in proportion and slowly inject it into the upper layer of the lymphocyte separation liquid in the centrifuge tube;

[0042] 1.3 20°C, 400×g, centrifuge for 30 minutes;

[0043] 1.4 Use a sterile pipette to draw the plasma layer in the separation tube, put the plasma into a 50ml sterile centrifuge tube, inactivate it in a water bath at 56°C for 30 minutes, centrifuge at 800g×10 minutes, and transfer the upper layer of plasma to a new 50ml sterile centrifuge tube middle;

[0044] 1.5 Pipette the isolated mononuclear cell layer (PBMCs) into a 50ml sterile centrifuge tube;

[0045] 1.6 Add DPBS, mix well, centrifuge at 20°C, 400×g for 10...

Embodiment 2

[0047] Example 2 In vitro expansion of NK cells-CD3 + T cell depletion

[0048] 2.1 According to the counting result of step 1.7, take out 5×10 5 Cells were placed in a 1.5ml EP tube for phenotype detection before sorting. The remaining cells were centrifuged at 20°C and 400×g for 10 minutes, washed once more, and the supernatant was discarded. 1 mM EDTA in DPBS (0.5% HSA, 1 mM EDTA, DPBS) to adjust the cell concentration to 1 × 10 8 cells / ml, transfer to 5ml sterile flow tube, add Easysep at 150μl / ml sample TM Human CD3Positive Selection Cocktail II in Human CD3Positive Selection KitII, mix well, and place at room temperature for 3min;

[0049] 2.2 will Easysep TM Mix RapidSphereTM50100 in Human CD3Positive Selection Kit II, add 90 μl / ml sample to the flow tube in step 2.1, mix well, and place at room temperature for 3 minutes;

[0050] 2.3 Use 0.5% HSA, 1mM EDTA, DPBS to make up the volume of the cell suspension in the 5ml sterile flow tube in step 2.2 to 2.5ml, and ...

Embodiment 3

[0052] Example 3 NK cell expansion in vitro-NK cell culture

[0053] 3.1 According to the counting result in step 2.4, take out 5×10 5 Cells were placed in 1.5ml EP tubes for phenotypic detection after sorting. The remaining cell suspension was supplemented with L500 medium to make up the remaining cell volume to 14ml, mixed well, and centrifuged at 20°C, 400×g for 5min;

[0054] 3.2 Discard the supernatant, centrifuge at 20°C, 400×g for 5 minutes;

[0055] 3.3 Aspirate the supernatant and use SuperCulture containing 10% autologous serum TM L500 (L500) medium to adjust the cell concentration to 1 × 10 6 cells / ml, add IL-2 to make the final concentration 1500IU / ml, and add cytokines (the final concentration of IL-15, Il-12 and IL-21 is 200ng / ml) and OK432 (the final concentration is 2ng / ml) ml), mix well.

[0056] 3.4 Place cells at 37°C, saturated humidity, 5.0% CO 2 cultured in an incubator.

[0057] 3.5 On the 4th day of culture, the cells were blown evenly, and 20 μl ...

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Abstract

The invention discloses a method for stably amplifying high-purity and high-cytotoxic-activity NK cells in vitro, and particularly relates to a method for stably amplifying a large number of the high-purity and high-cytotoxic-activity NK cells in vitro by autologous or allogeneic peripheral blood mononuclear cells, which comprises the following steps: (1) separating mononuclear cells; (2) removingCD3<+> T lymphocytes in the mononuclear cells through immune sorting; (3) adding a serum-free cell culture medium for NK cell culture and cell factors IL-15, IL-12, IL-21, IL-2 and OK432, carrying out in-vitro culture for 2-5 days, and completely changing a solution; and (4) changing the NK cell solution; and (5) harvesting the NK cells. According to the invention, a large number of the high-purity and high-cell-activity NK cells are stably amplified under the in-vitro culture condition, meanwhile, the number of T cells is reduced, and the requirement for clinical application is satisfied.

Description

Technical field: [0001] The invention relates to the field of immune cell therapy, in particular to a method for stabilizing autologous or allogeneic peripheral blood mononuclear cells in vitro, and massively expanding NK cells with high purity and high cytotoxic activity. Background technique: [0002] Cellular immunotherapy is one of the most promising tumor treatment methods at present. It can kill tumor cells through in vitro expansion or transformation and then infuse into patients, or activate the immune system of the body to enhance the autoimmune function of tumor patients to resist tumors. At present, NK cell immunotherapy has received more and more attention. NK cells account for 5-15% of human peripheral blood lymphocytes, and their phenotype is generally defined as CD3 - CD56 + , NK cells can be further subdivided into two main subgroups: CD56 with immune regulation function high CD16 - cells and CD56 with cytotoxic activity dim CD16 + cell. NK cells play ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/2321C12N2501/20
Inventor 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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