[Development of a method for isolation, culture and proliferation of mouse NK cells].

To establish a method to isolate, purify, and culture mouse nature killer (NK)-cells in vitro.NK-cells were isolated from splenic mononuclear cells (MNC) by a two-step adherence system and magnetic microbeads actived cell sorting (MACS), then these cells were cultivated with feeder cells and IL-2 in RPMI 1640 medium for 5, 10, 15, and 20 days. The enriched cells were counted and stained with anti-mouse CD3-FITC and anti-mouse NK1.1-PE. The purity of the NK-cells was determined by flow cytometry and the cytotoxicity to YAC-1 targets was detected by MTT assay.In the two-step adherence system, the enriched cells cultivated for 5, 10, 15, and 20 days were 0.5 x 10(7), 1.4 x 10(7), 2.6 x 10(7), and 3.0 x 10(7) respectively, and the percent of CD3(-) NK1.1(+) cells was 18.36%, 43.44%, 55.68%, and 60.03% respectively. The cytotoxicity to YAC-1 targets was significantly higher than that of splenic MNC(P<0.01), and increased from 41.93% up to 54.38%, 66.54%, 79.38%, and 83.86% respectively at 25:1 of effector:target ratio. After NK-cells were purified by MACS, the purity reached 93.60%ls, the enriched cells was 1.5 x 10(6), but proliferated to only 1.9 x 10(6) 20 days later.NK-cells isolated by using the two step adherence system proliferate abundantly, and the purity reaches 55%-60%. The cytotoxicity to YAC-1 targets is about 80% at 25:1 of effector:target ratio.