[Method for expansion in vitro of CD3-CD56+CD16+NK cells highly purified from human peripheral blood]

The aim of this study was to establish an efficient method for expansion in vitro of natural killer (NK) cells highly purified from human peripheral blood. The CD3-CD56+CD16+ NK cells purified by the negative sorting method of MACS (magnetic microbeads activated cells sorting) were expanded with the different combinations of IL-2, SCF, IL-15 in SCGM (stem cell growth medium) supplemented with 10% human AB serum for 18 days. Cultures were fed with fresh medium and cytokines every 3 days. The sum of cells was counted for evaluating the efficiency of expansion. Then the purity of the CD3-CD56+CD16+ NK cells were determined by flow cytometry and the cytotoxicity to K562 targets was detected by CCK-8 assay in the end. Furthermore, the same way was used to explore the relationship between the efficiency of expansion, cytotoxicity to K562 targets of NK cells and the dose of IL-2. The results showed that after peripheral blood mononuclear cells (PBMNC) were purified by the negative sorting method of MACS, the purity of CD3-CD56+CD16+ NK cells increased from (12.70±2.66)% to (93.03±1.72)%. The CD3-CD56+CD16+ NK cells purified by MACS were expanded with the different combinations of IL-2, SCF, IL-15 in SCGM supplemented with 10% human AB serum for 18 days. The expanding multiple of IL-2/IL-15/SCF group was significantly higher than other groups (p<0.05). The purity of NK cells in the groups with cytokines was not significantly lower than that before expansion (p>0.05). The cytotoxicity of the groups with cytokines was significantly higher than that before expansion. Especially, the cytotoxicity (%) of NK cells in IL-2/IL-15 group and IL-2/IL-15/SCF group was more than 90%. The expanding multiples of low-dose group, medium-dose group and high-dose group were significantly higher than that of zero-dose group (p<0.05), but no significant difference was found between themselves (p>0.05). The cytotoxicity of the groups with IL-2 was significantly higher than that before expansion. Cytotoxicity to K562 cells in high-dose group was significantly higher than that in others (p<0.05); there was no significant difference between low-dose group and medium-dose group (p>0.05). It is concluded that cytokines in the 4 groups were efficient for expansion and the cytotoxicity of highly purified NK cells in vitro. IL-2/SCF/IL-15 combination is the most efficient one among different combinations, and enhanced significantly the cytotoxicity of NK cells against K562 targets. The efficiency of expansion and the cytotoxicity in vitro of NK cells are not related with the dose of IL-2, when IL-2<1,000 U/ml. It is indicated that IL-2 of high-dose (≥1,000 U/ml) may enhance the cytotoxicity of NK cells in vitro more efficiently.