Method for amplifying NK cells through in-vitro cultivation

A technology for NK cells and in vitro culture, applied in the field of in vitro culture and expansion of NK cells, can solve the problems of low NK cell expansion, complicated technical operations, and low purity, and achieve simple and easy technical operations, strong cytotoxicity, and The effect of excellent performance

Inactive Publication Date: 2015-12-23
SHANGHAI CLAISON BIOTECH
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Problems solved by technology

[0007] The main purpose of the present invention is to solve the problems of low NK cell amplification factor, low purity, complicated technical operation and high cost in the prior art

Method used

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  • Method for amplifying NK cells through in-vitro cultivation
  • Method for amplifying NK cells through in-vitro cultivation
  • Method for amplifying NK cells through in-vitro cultivation

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Embodiment 1

[0025] 1. Isolation of Peripheral Blood Mononuclear Cells (PBMC) and Culture of NK Cells

[0026] The patient's peripheral blood mononuclear cells (PBMC) were collected by a blood cell separator, the collected blood sample was transferred to a centrifuge tube, centrifuged at 700g for 10min, and the upper layer of plasma was absorbed for later use; the blood sample was restored to the original volume with 0.9% normal saline, mixed Mix well; slowly add the diluted blood to the Ficoll separation medium, centrifuge at 900g for 20min; absorb the milky white mononuclear cell layer at the separation interface, wash and centrifuge twice with 0.9% normal saline; resuspend the PBMC with serum-free medium, and adjust the cells Concentration to 2.0×10 6 / mL.

[0027] Add 0.02KE / mL group A streptococcus preparation, 1000U / mL IL-2 and 5% autologous serum, and culture in a 37°C incubator. Add NK serum-free medium containing 1000 U / mL IL-2 every 3 to 4 days according to the counting results...

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Abstract

The invention relates to a method for amplifying NK cells through in-vitro cultivation, in particular to a method for greatly amplifying the NK cells from autologous peripheral blood mononuclear cells through in-vitro cultivation. The method includes the steps of activating the NK cells with group-A streptococcus preparations as the irritant and cultivating the NK cells through the combined common stimulation of cell factors IL-2 so that the obtained NK cells can have the advantages of being large in number, high in purity and cytotoxicity and the like and can meet the clinical requirements. Specifically, the autologous peripheral blood mononuclear cells are inoculated into a serum-free medium to be cultivated, and the group-A streptococcus preparations and the cell factors IL-2 are added for inducing activation and proliferation. The method is easy, convenient and rapid to operate; the NK cells can be activated and amplified only through the group-A streptococcus preparations and the cell factors IL-2; the amplification times, flow phenotype and cytotoxicity of the NK cells are ensured, the cultivation cost of the NK cells is greatly reduced, and use and popularization are simple and easy.

Description

[technical field] [0001] The invention relates to a method for cultivating and expanding NK cells in large quantities in vitro with autologous peripheral blood mononuclear cells. [Background technique] [0002] In 1975, when scientists were studying the killing characteristics of T cells on target cells, they identified for the first time a group of cells that could directly kill tumor cells in the absence of T and B cells. Because this group of cells has no MHC restriction on the recognition of target cells, and the killing activity does not require prior immune sensitization, it is named Natural Killer cells (Nature Killer cells, NKs). NKs belong to the lymphocyte lineage and are the main bearers of the body's innate immunity and the core regulatory cells of acquired cellular immunity. They play an important role in tumor immunity, anti-viral infection and removal of non-self cells. Due to the extremely low content of NKs in human peripheral blood and the low distribution...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 张燕程双凤张赞
Owner SHANGHAI CLAISON BIOTECH
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