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

CN105861435AInactive Publication Date: 2016-08-17NANJING HWATAO BIOPHARM CO LTD
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2016-08-17
Estimated Expiration
Not applicable · inactive patent

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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.
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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,...

Examples

Embodiment 1

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

[0056] 1. Using the in vitro expansion method of natural killer cells according to the embodiment of the present invention to carry out the isolation of peripheral blood mononuclear cells (PBMC) and the cultivation of NK cells (example)

[0057] 100ml of peripheral blood was collected by hand, and the collected blood sample was transferred to a 50ml centrifuge tube. 700g, centrifuge for 10min, absorb the upper layer of plasma, inactivate at 56°C, and keep it ready for cultivation.

[0058] Dilute the lower layer of blood with 0.9% normal saline to the original volume, and mix well. The diluted blood was equally divided into 5 parts, slowly added to the centrifuge tubes added with lymphocyte separation solution, 900g, and centrifuged for 20min. Aspirate the milky white mononuclear cell layer at the interface of the separation solution, centrifuge and wash twice and count. Resuspend PBM...