Isolation and Expansion of Primary Human NK Cells

Primary human NK cells were isolated from peripheral blood mononuclear cells (PBMC) obtained by Ficoll-Paque separation from buffy coats provided by the Finnish Red Cross. NK cells were isolated using a human NK cell isolation kit (Miltenyi Biotec). NK cells from three donors were pooled to achieve enough material for DSRT and RNA sequencing. The purity of NK cells was evaluated by flow cytometry using CD56-PE (clone NCAM16.2) and CD3-APC (clone SK7) staining (BD Biosciences), and the final pool contained >90% CD56⁺CD3⁻ cells in all replicates. Primary bone marrow mononuclear cells (BM MNC) were isolated from bone marrow aspirates from healthy donors using Ficoll-Paque separation. All normal NK cells were cultured in R10 supplemented with 2.5 ng/mL recombinant human IL-2 (Peprotech). BM MNC were cultured in Mononuclear Cell Medium (PromoCell). To obtain actively proliferating normal NK cells, NK cells were expanded using an artificial antigen-presenting cell K562 variant (K562-aAPC) as previously described^{39 (link)}. Briefly, PBMC were isolated from buffy coats by Ficoll-Paque separation and co-cultured with irradiated (100 cGy) K562-aAPCs at a ratio of 1:2 (PBMC:aAPC) in RPMI1640 with 50 IU/mL IL-2 at 200,000 PBMC/mL. Cultures were refreshed with half-volume media changes every two to three days, and re-stimulated with aAPCs at ratio of 1:1 every seven days.