[Development of a method for isolation, culture and proliferation of mouse NK cells].
OBJECTIVE
To establish a method to isolate, purify, and culture mouse nature killer (NK)-cells in vitro.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yang
Zhi-gang
ZG
Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China. yangzhigangcn@yahoo.com.cn
Zeng
Yao-ying
YY
He
Xian-hui
XH
Wang
Tong
T
Jiang
Xun
X
Tang
De-shen
DS
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008863:Microspheres