Expansion of NK Cells using K562 Feeder Cells

NK cells were expanded for 14 days using K562-mbIL21-41BBL feeder cells as described previously [58 (link)]. On day 0, 5 million mononuclear cells, separated from a buffy coat with Ficoll-Paque gradient centrifugation, were suspended in 40 ml RPMI-1640 with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L- glutamine, and 100 U/mL penicillin with 100 mg/mL streptomycin (PS) (referred to as R10), supplemented with 10 ng/ml recombinant human IL-2 (R&D Systems, 202-IL-050) together with 10 million irradiated (100 Gy) K562-mbIL21-41BBL feeder cells. After 7 days of culture and two passages, additional feeder cells were added in a 1:1 ratio. After 14 days of culture, NK cells were purified using an NK Cell Isolation Kit (Miltenyi) and frozen. Before the scRNAseq co-culture experiments, NK cells were thawed and cultured in R10 supplemented with 10 ng/ml recombinant human IL-2 (R&D Systems, 202-IL-050) 3 days before the experiments.