Method for efficient in-vitro amplification of peripheral blood NK cells

Abstract

The invention relates to the technical field of cell amplification culture and discloses a method for efficient in-vitro amplification of peripheral blood NK cells. The method comprises the followingsteps: 1) performing anticoagulation on whole blood by heparin sodium, and diluting by PBS; 2) spreading the diluted anticoagulant whole blood on the liquid level of a lymphocyte separation liquid; 3)centrifuging, absorbing a white film layer, blowing, beating and uniformly mixing the white film layer, centrifuging, discarding the supernatant and repeatedly washing; 4) removing CD3+ lymphocytes,dyeing peripheral blood monocyte antibodies and sorting the cells; and 5) diluting the CD3+ lymphocytes, adding IL-2, IL-15, IL-21 and L-7 and performing amplification. The CD3+ cells are removed before amplification, so that the cellular immunogenicity is greatly reduced; meanwhile, four specific cell factors are used according to the specific proportion to activate and amplify the NK cells, so that the amplification multiple of the NK cells is guaranteed and the safety of the NK cells during treatment is greatly improved.

Description

technical field [0001] The invention relates to the technical field of cell expansion and culture, in particular to a method for efficiently expanding peripheral blood NK cells in vitro. Background technique [0002] NK cells, also known as natural killer cells, are large granular lymphocytes, derived from bone marrow, accounting for 5% to 15% of peripheral blood lymphocytes, and are important immune cells. Scientists first identified NK cells in 1975, which can directly kill tumor cells in the absence of T and B cells. Unlike cytotoxic CD8+ T cells, NK cells can directly kill MHC-negative tumor cells without pre-sensitization when killing tumor cells, which makes NK cells widely used in adoptive cell immunotherapy. [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...

AI Technical Summary

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 large-scale expansion of NK cells in vitro. At present, after in vitro expansion and culture, NK cells can only be expanded several times or more than ten times, and the purity is not ideal, which cannot meet the practical application.

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