Human natural killer cell expansion in vitro using mitomycin-C treatment as an alternative to irradiation of modified K562 feeder cells.

Abstract Introduction Current methods for in vitro expansion of Natural Killer (NK) cells require feeder cell irradiation, which is not readily available in many biomedical research or clinical facilities. Here we evaluated mitomycin-C (MMC) treatment of feeder cells as an alternative to irradiation for the purpose of expanding NK cells in human peripheral blood mononuclear cell (PBMC) cultures. Methods Using PBMCs from 4 healthy blood donors and genetically modified K562 feeder cells (K562-mb21-41BBL cells) that preferentially stimulate NK cells, we evaluated the efficacy of MMC treatment in limiting the proliferation of the feeder cells at a range of doses (0 ug/mL, 2 ug/mL, 10 ug/mL, 20 ug/mL, 30 ug/mL, and 200 ug/mL) with 3-hour incubation. We used 7AAD dye and CFSE proliferation dye to measure viability and cell proliferation, respectively. We then compared the percentages of NK cells (CD3neg, CD56pos) in PBMCs cultured for 14 days in the presence of MMC-treated feeder cells or irradiated feeder cells using multiparametric flow cytometry. Results We found that 10 ug/mL dose of MMC for 3-hrs achieved a significantly higher percentage of viable cells compared to higher doses (p<0.05, ANOVA), and also significantly limited proliferation compared to untreated feeder cells. Furthermore we found no significant difference in NK cell expansion by 10 ug/mL MMC treated feeder cells and 100 grays irradiated feeder cells. Discussion In conclusion, we found that 10 ug/mL mitomycin-C treatment of modified K562 feeder cells was optimal for limiting their proliferation for NK cell expansion that was equivalent to using irradiated feeder cells.