The invention relates to the technical field of
cell culture, and provides an NK
cell in-vitro amplification culture method which comprises the following steps: S1, separating to obtain a mononuclear
cell of
peripheral blood, resuspending the mononuclear cell by using a serum-free NK cell basic culture medium, and then inoculating the mononuclear cell to an NK
cell activation culture medium for incubation; s2, culturing to the third day, supplementing the NK cell to activate the culture medium for amplification, then supplementing the NK cell to activate the culture medium once
every other day, and culturing to the 19th day to obtain high-purity NK cells; the NK
cell activation culture medium is a serum-free NK cell basal culture medium which contains IL-2 (
Interleukin-2), IL-12 (
Interleukin-12), IL-18 (
Interleukin-18), IL-21 (Interleukin-21), a CD16
antibody, a CD56
antibody, 4-1BBL (4-1BBL),
nicotinamide,
sodium diethyldithiocarbamate, polyinosinic acid and
plasma. The method is simple and convenient to operate and short in culture period, the amplification efficiency reaches up to 1050 times within 19 days, and the NK cells are high in purity and accord with
clinical treatment standards.