In-vitro expansion method for NK (Natural Killer) cells

Abstract

The invention discloses an in-vitro expansion method for NK (Natural Killer) cells. The in-vitro expansion method comprises the following steps: (1) preparing a reagent A which comprises main components of a 50 ng / ml anti-CD3 monoclonal antibody and a 50 ng / ml anti-CD16 monoclonal antibody, a reagent B which comprises a main component of 300 IU / ml IL-2, and a reagent C which comprises main components of 20 ng / ml IL-12 and 20 ng / ml IL-15, and preparing 40 ml of a lymphocyte separation liquid as a reagent D; (2) enveloping the reagent A in a T-175 culture flask, and leaving to stand overnight at 4 DEG C; (3) treating peripheral blood so as to obtain cells and serum; (4) suspending the cells, adding the reagent D, performing centrifugal collection on PBMC (Peripheral Blood Mononuclear Cell); (5) suspending the PBMC in an X-VIVO15 culture medium, adding the reagent B, the reagent C and autologous serum, and performing incubation; (6) replenishing the culture medium, the reagent B, the reagent C and the autologous serum on a third day, a sixth day, a ninth day and a twelfth day respectively, and collecting cells on a fourteenth day. The in-vitro expansion method has the beneficial effects that as the anti-CD3 monoclonal antibody and the anti-CD16 monoclonal antibody are inoculated in advance, and the cells are screened firstly, the expansion times and the purity of the NK cells in in-vitro culture are improved.

Description

technical field [0001] The invention relates to a cell expansion method, in particular to an NK cell expansion method in vitro, and belongs to the field of biotechnology. Background technique [0002] NK cells are a group of cell subpopulations whose phenotype is CD3-, CD56+. Their killing activity is not limited by MHC and does not depend on antibodies. They are called natural killer cells. NK cells usually contain a large amount of perforin and granzyme B. When the target cells are activated, NK cells attack the target cells by releasing perforin and granzyme B. The content of NK cells in human blood is extremely low (5%-10% of mononuclear cells), so their clinical application is greatly limited and they need to be expanded in vitro. [0003] At present, most of the NK cell culture systems stimulate the growth of cells by adding IL-2, IL-15, IL-18 and other interferons. Purity is also not up to the mark. [0004] In addition, culturing by feeder cells has good results i...

AI Technical Summary

Problems solved by technology

[0003] At present, most of the NK cell culture systems stimulate the growth of cells by adding IL-2, IL-15, IL-18 and other interferons. Purity is not enough

[0004] In addition, culturing by feeder cells has good results in terms of quantity and purity, but feeding itself has certain risks, so it cannot be the best choice

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