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In-vitro culture method of NK (natural killer) cells

A technology of NK cells and in vitro culture, applied in animal cells, vertebrate cells, blood/immune system cells, etc., to increase safety, improve killing activity, and promote growth and proliferation

Inactive Publication Date: 2014-03-12
TIANJIN MEDICAL UNIV CANCER HOSPITAL
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention provides a method for in vitro culture of NK cells in order to solve the problem of large-scale culture of NK cells in the prior art

Method used

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  • In-vitro culture method of NK (natural killer) cells
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  • In-vitro culture method of NK (natural killer) cells

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Embodiment Construction

[0018] The present invention will be described in detail below in conjunction with the drawings and embodiments.

[0019] An in vitro culture method of NK cells, which is carried out according to the following steps:

[0020] a. Dilute Herceptin with PBS to a concentration of 0.85-1.05mg / ml, take 910μl; dilute human immunoglobulin with PBS to a concentration of 1-2mg / ml, take 400μl, combine and then make up to 20ml with PBS, and evenly spread the culture The bottom of the bottle, sealed and placed in a refrigerator at 4°C overnight, take it out the next day, pour out the liquid, and wash twice with PBS;

[0021] b. Take 4-10ml of peripheral enriched blood, perform density gradient centrifugation by Ficoll method, centrifugation speed is 350×g, centrifugation time is 15-20 minutes, aspirate mononuclear cells in the middle interface layer, wash 3 times with PBS, add Serum-free medium, adjust the cell concentration to 1.0×10 6 / ml-3.0×10 6 / ml; then add the cytokine IL-2 to a final con...

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Abstract

The invention discloses an in-vitro culture method of NK (natural killer) cells and belongs to culture of human cells. The in-vitro culture method disclosed by the invention comprises the following steps: merging herceptin diluted by PBS (phosphate buffered saline) and human immunoglobulin diluted by the PBS, then uniformly and fully spreading at the bottom of a culture bottle and standing overnight; additionally taking peripheral blood, performing density gradient centrifugation, sucking a single nuclear cell, adding into a serum-free culture medium, and adjusting the concentration of the cells to 1.0*10<6> / ml-3.0*10<6> / ml; and then adding cell factors IL-2 and IL-15, adding into the culture bottle coated by the herceptin and culturing in an incubator. Therefore, on the basis of ensuring the amplification multiple of various cell subgroups, the growth and the proliferation of the NK cells are promoted, the killing activity of lymphocytes is enhanced, the serum-free culture medium can replace a serum-containing complete culture medium, the number of obtained culture products is equivalent to the activity of the cells, the in-vitro large-scale culture of the NK cells is realized, the in-vitro culture method is used for clinical biological treatment of the NK cells, and the safety in clinical application can be increased by using the in-vitro culture method.

Description

Technical field [0001] The invention relates to the culture of human cells, in particular to an in vitro culture method of NK cells. Background technique [0002] Natural killer cell (NK) is an important immune cell of the body, and it is the body's first line of defense against tumors and viral infections. It is not only related to anti-tumor, anti-viral infection and immune regulation, but also participates in some cases. The occurrence of hypersensitivity reactions and autoimmune diseases. In the process of killing target cells, NK cells can non-specifically recognize target cells, and are not restricted by the major histocompatibility complex (MHC), and have rapid killing and lysis effects on a variety of tumor cells. However, the number of NK cells in peripheral blood of normal people is relatively small, accounting for only 10% to 20% of peripheral blood lymphocytes. Therefore, obtaining high-quality and high-purity NK cells has become one of the most critical issues for ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 任秀宝于津浦
Owner TIANJIN MEDICAL UNIV CANCER HOSPITAL
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