Expansion and Differentiation of NK Cells

Hematopoietic stem cells, isolated from fresh umbilical cord blood (Anthony Nolan, United Kingdom), were expanded and differentiated into GTA002 NK cells as previously described (Spanholtz et al., 2010 (link)). In short, CD34⁺ cells were seeded at a concentration of 10,000 cells/ml in a six-well tissue culture–treated plate (Corning Incorporated) in Glycostem Basal Growth Medium (GBGM^®, FertiPro) supplemented with 10% human serum (Sanquin, Netherlands), 25 ng/ml of TPO, IL-7, Flt-3L, and SCF (Cellgenix), 1 ng/ml GM-CSF, 0.05 ng/ml Il-6 (Cellgenix), and 0.25 ng/ml Neupogen (G-CSF; Amgen BV). After 9 days, TPO was replaced by 20 ng/ml IL-15 (Cellgenix). After 14 days of expansion, the differentiation phase was initiated by adding differentiation medium consisting of GBGM supplemented with 2% human serum, 20 ng/ml of IL-7, IL-15, and SCF, 1 ng/ml GM-CSF, 0.05 ng/ml Il-6, 0.25 ng/ml Neupogen, and 1000 U/ml Proleukin (IL-2; Novartis). The cells were cultured until day 28 and cryopreserved until further use. For usage in cytotoxicity assays, day 28 GTA002 cells were thawed and cultured for 1 week in differentiation medium.