The invention relates to a method for in-vitro amplification of NK cells, and in particular relates to a method for massive in-vitro amplification of NK cells, wherein the method comprises the following steps of: a, inoculating a
peripheral blood mononuclear
cell in a CD3McAb and CD226McAb pre-coated culture
bottle for coculture; b, adding 1L-2 and 1L-18, coculturing for 72hours to stimulate amplification of NK cells; c, transferring the NK cells, K562 cells after lethal treatment and a serum-
free medium containing 1L-2 and 1L-18 in a
cell culture bag for coculture; and d, collecting the NK cells. According to the method for in-vitro amplification of the NK cells, two antibodies CD3McAb and CD226McAb are simultaneously coated, so the
cell factor synthesis and ADCC effect are promoted, and killing
toxicity of the NK cells is remarkably improved; the activation and amplification on the NK cells are achieved just by the 1L-2 and 1L-18 cell factors, so the amplification multiple and
cell toxicity of the NK cells are guaranteed, and the cost of
cell culture is reduced.