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

Abstract

We studied the effects of basal medium and human serum on the ex vivo expansion of CD56+/CD3- natural 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.(ABSTRACT TRUNCATED AT 250 WORDS)

NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions

“…In a search for suitable conditions that would reproducibly generate high numbers of functional NK cells from early progenitor cells, we isolated CD34 + CD38 + cells from cord blood, bone marrow and mobilized PB and cultured them under different conditions combining cytokines and stromal cells. As previously reported [21], we found that a mixture of 10 % human serum and 5 % FCS was optimal for the proliferation and maturation of NK cells (data not shown). No significant proliferation was observed in the absence of SCF which was therefore systematically added.…”

Isolation and characterization of canine natural killer cells

“…CD5-depleted leukocytes were cultured for 14 days (9 samples) or for 21-25 days (4 samples) days in media containing 60% DMEM, 30% HAMS F-12 + 2 mM L-glutamine, 10% heat-inactivated human serum, ß-mercaptoethanol ethanolamine, sodium selenite, and ascorbic acid, as described (Pierson et al, 1995). Samples for the 14 day culture were divided into 3 treatment groups, each containing 1,000 IU/mL of IL-2, 10 ng/mL of IL-15, or 1,000 IU/mL IL-2 + 10 ng/mL IL-15.…”

Human natural killer cells with polyclonal lectin and immunoglobulinlike receptors develop from single hematopoietic stem cells with preferential expression of NKG2A and KIR2DL2/L3/S2

“…The DMEM/F12-based medium was developed to maximize NK cell growth 50 and is supplemented with 24 M 2-mercaptoethanol, 50 M ethanolamine, 20 mg/L ascorbic acid, 50 g/L sodium selenite (Na 2 SeO 3 ), 100 U/mL penicillin, and 100 U/mL streptomycin (Gibco). Twenty percent heat-inactivated human AB serum (North American Biologicals, Miami, FL) was used at culture initiation that was reduced to 10% for subsequent media changes.…”