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NK (natural killer) cell culture medium and culture method of NK cell

A technology of NK cells and culture methods, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve problems such as unsafety, inapplicability, introduction of mycoplasma, viruses, etc.

Active Publication Date: 2015-09-09
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the above method can stimulate the efficient expansion of NK cells, the composition of animal serum is complex, and mycoplasma and viruses may be brought in during the sampling process, which will cause uncertainty and unsafety for NK cell culture; K562 itself, as a tumor cell, is used in clinical There are great doubts in terms of safety, and it is not suitable for clinical NK cell therapy

Method used

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  • NK (natural killer) cell culture medium and culture method of NK cell
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  • NK (natural killer) cell culture medium and culture method of NK cell

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preparation example Construction

[0049] In the present invention, the preparation method of the NK cell culture medium preferably comprises the following steps:

[0050] 83-93 parts by weight of serum-free basal medium, 4-6 parts by weight of plasma, 0.5-1.5 parts by weight of interleukin-2, 0.5-1.5 parts by weight of anti-CD3 monoclonal antibody, and 1-5 parts by weight of insulin-like growth factor 1 The weight part is mixed with 1-3 weight parts of Lycium barbarum polysaccharide to obtain NK cell culture medium.

[0051] In the present invention, the sources and dosages of the serum-free basal medium, plasma, interleukin-2, anti-CD3 monoclonal antibody, insulin-like growth factor 1, and Lycium barbarum polysaccharide are the same as those of the serum-free basal culture described in the above technical scheme. The sources and dosages of the base, plasma, interleukin-2, anti-CD3 monoclonal antibody, insulin-like growth factor 1 and Lycium barbarum polysaccharide are the same, and will not be repeated here. ...

Embodiment 1

[0075] NK cell isolation procedure:

[0076] 1. Separation of PBMC:

[0077] 1) Take 40mL of peripheral blood, transfer it to a 50mL centrifuge tube, and centrifuge at 800g for 10min;

[0078] 2) Collect the upper layer of plasma, dilute the lower layer of cells with twice the volume of normal saline, and gently pipette to mix evenly;

[0079] 3) Take another new 50mL centrifuge tube, add Ficoll separation solution (purchased from Shanghai Sanjin Biotechnology Co., Ltd.) according to 1 / 2 of the volume of the diluted blood, and then slowly add the diluted blood to the surface of the Ficoll liquid; The plasma was centrifuged at 3000g for 10min, filtered and used for later use.

[0080] 4) Set the special brake button Brake of the centrifuge to zero, centrifuge at 700g for 20 minutes, and carefully absorb the buffy coat cells with a Pasteur pipette after centrifugation, and collect them in a centrifuge tube;

[0081] 5) Add 1640 medium (purchased from Shanghai Yubo Biotechnolo...

Embodiment 2

[0092] Plasma, IL-2, OKT-3, IGF-1 and Lycium barbarum polysaccharide were added to the serum-free RPMI medium one by one, the plasma, IL-2, OKT-3, IGF-1, Lycium barbarum polysaccharide and RPMI medium The mass ratio is 5:1:1:3:2:88, shake them well and evenly to obtain NK cell culture medium; according to 5×10 5 Inoculate NK cells in a culture flask at a density of / mL, add the complete medium prepared by the above formula, record the growth situation and detect the killing effect on K562 after two weeks of cultivation, the results are shown in image 3 and Figure 4 , image 3 The proliferation curve of NK cells cultured for the NK cell culture medium prepared in Example 2 of the present invention; Figure 4 It is a graph showing the killing activity of NK cells cultured in Example 2 of the present invention to K562.

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Abstract

The invention provides a NK (natural killer) cell culture medium and a culture method of a NK cell, wherein the NK (natural killer) cell culture medium comprises the following components in parts by weight: 83 to 93 parts of serum-free basal culture medium; 4 to 6 parts of plasma; 0.5 to 1.5 parts of interleukin-2; 0.5 to 1.5 parts of anti-CD3 monoclonal antibody; 1 to 5 parts of insulin-like growth factor 1; 1 to 3 parts of lycium barbarum polysaccharide. According to the NK (natural killer) cell culture medium provided by the invention, the safety is better, and the NK cell cultured by the culture medium with the components can quickly and stably proliferate, and has better killing activity to tumor cell. Experimental results show that, after using the NK cell culture medium provided by the invention for culturing the NK cell for two weeks, the proliferation times soars almost fiftyfold; the killing activity to K562 reaches 91 percent at effector-target ratio being 40: 1.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a culture medium for NK cells and a method for culturing NK cells. Background technique [0002] Natural killer cells (NK) are important immune cells in the body, not only related to anti-tumor, anti-virus infection and immune regulation, but also involved in the occurrence of hypersensitivity and autoimmune diseases in some cases. [0003] NK cells are the body's first line of natural defense against infection and tumors, and are important immune cells of the body. Activated NK cells can synthesize and secrete a variety of cytokines, play a role in regulating immunity and hematopoiesis and directly killing target cells. At present, NK cells have been widely used in the clinical treatment of tumor patients. At the same time, NK cell therapy also has certain curative effects on viral or bacterial infections, immune regulation-related diseases, and anti-aging. [0004] At present, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 葛啸虎陈海佳王一飞曾维杰王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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