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

A technology of natural killer cells and in vitro expansion, applied in animal cells, vertebrate cells, cell culture active agents, etc., can solve the problems of low NK cell content, large demand, and restrictions on the application of NK cells, achieving obvious effects, strong killing effect

Inactive Publication Date: 2016-08-17
NANJING HWATAO BIOPHARM CO LTD
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  • Abstract
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  • Application Information

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Problems solved by technology

[0003] However, due to the extremely low content of NK cells in human peripheral blood and the low distribution frequency (<10%) in tumor tissues, they only account for 5%-10% of mononuclear cells, which greatly limits the use of NK cells as adoptive immune cells. clinical application
For many years, people have been trying to achieve a large amount of NK cell expansion in vitro, but the current conventional technology is mainly to add a combinat

Method used

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

Examples

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[0054] Example one

[0055] 1. Isolation of peripheral blood mononuclear cells (PBMC) and NK cell culture

[0056] 1. Use the in vitro expansion method of natural killer cells according to the embodiments of the present invention to separate peripheral blood mononuclear cells (PBMC) and culture NK cells (Example)

[0057] Collect 100ml of peripheral blood by hand collection, and transfer the collected blood sample to a 50ml centrifuge tube. 700g, centrifugal separation for 10 minutes, aspirate the upper layer of plasma, inactivate it at 56°C, and use it for later use.

[0058] Dilute the lower layer of blood to its original volume with 0.9% saline and mix well. Divide the diluted blood into 5 equal parts, slowly add them to the centrifuge tube with the lymphocyte separation solution, and centrifuge at 900g for 20min. Aspirate the milky white mononuclear cell layer at the interface of the separation liquid, centrifuge and wash twice and count. Resuspend PBMC in serum-free medium and...

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Abstract

The invention discloses an in-vitro amplification method of natural killer cells (NK). The in-vitro amplification includes the steps of S1, culturing mononuclear cells separated from peripheral blood with a serum-free medium while adding in IFN-gamma to stimulate for a first preset time; S2, adding class-I cell factors in the serum-free medium for stimulation induction after the first preset time; S3, adding in class-II cell factors in the serum-free medium for induced amplification after culturing for a second preset time, and then performing cell fluid replacement and culturing for a third preset time; and S4, collecting the cells. According to embodiments of the invention, the natural killer cells cultured by the in-vitro amplification method have high killing action and high significant effect to target cells.

Description

technical field [0001] The invention relates to the technical field of cell culture, and more specifically, relates to an in vitro expansion method of natural killer cells. Background technique [0002] Natural killer cells (NK) are cytotoxic lymphocytes belonging to the lymphocyte lineage that contain perforin and granzyme granules. Its recognition of target cells is not restricted by MHC, it can directly kill tumor cells without prior sensitization, and can also secrete cytokines to regulate the functions of other immune cells. It is the main bearer of the body's natural immunity and the core of acquired cellular immunity. Regulatory cells play an important role in tumor immunity, anti-viral infection and removal of non-self cells. Studies have proved that natural killer cells are not only an important component of the natural immune system, but also have some characteristics of acquired immune cells. In the immune system, NK cells respond faster than T cells or B cells,...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/84C12N2500/90C12N2501/2301C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/24C12N2501/515
Inventor 齐来俊姚逸江
Owner NANJING HWATAO BIOPHARM CO LTD
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