Natural Killer Cell Proliferation Is Dependent on Human Serum and Markedly Increased Utilizing an Enriched Supplemented Basal Medium

We studied the effects of basal medium and human serum on the ex vivo expansion of CD56⁺/CD3natural killer cells (NK). We demonstrated that sorted NK cultured for 18 days with 10% human AB serum without accessory cells in a 2:1 DMEM/F12 basal medium expanded significantly greater (6.4 ± 0.9-fold, n = 14) than when cultured in standard RPMI 1640 basal medium (3.7 ± 0.67-fold, n = 16; p = 0.019). Supplementation of the DMEM/F12 mixture with 2-mercaptoethanol (2ME), ethanolamine, L-ascorbic acid, and sodium selenite significantly augmented NK proliferation (16.3 ± 2.5-fold, n = 11; p < 0.001) compared with DMEM/F12 without supplements. NK growth kinetics demonstrated that both the growth rate and the duration of exponential growth were increased by medium supplements. Addition of 2ME-containing supplements to cultures of NK with accessory cells augments NK proliferation, recruits NK progenitors, and has a serum-sparing effect. For the large-scale expansion of NK, we demonstrate a greater than 30-fold increase (n = 7) in the number of activated natural killer cells (ANK) derived from CD5/CD8-depleted PBMNC when cultured for 35 days in supplemented DMEM/F12 versus RPMI 1640 basal medium (197.6 ± 95.0-fold versus 6.3 ± 2.1-fold, respectively). Serum-free media (AIM-V and X-VIVO 10) were unable to support NK proliferation. These data suggest that utilizing 2:1 DMEM/F12 + 2ME-containing supplements instead of standard RPMI 1640 as a basal medium can increase the proliferation of cytotoxic NK while reducing the culture interval and the amount of serum needed. Replacing RPMI 1640 with supplemented DMEM/F12 may markedly increase the efficiency of large-scale ex vivo NK expansion necessary for clinical immunotherapy trials.