Expansion of Primary NK Cells from Peripheral Blood and Cord Blood Hematopoietic Stem Cells

Peripheral blood mononuclear cells (PBMCs), procured from the peripheral blood of healthy donors, were segregated using density gradient centrifugation facilitated by Ficoll-Hypaque (1.077 g/mL). To facilitate PB-NK cell expansion, thawed PBMCs were combined with 2 × 10⁵ K562-mbIL21-feeder cells at a proportion of 1 × 10⁵ cells per ml in StemSpan™ SFEM II medium (Stemcell Technologies). The medium was enriched with 1% L-glutamine (Invitrogen), 100 ng/ml hIL-2 (Peprotech), 20 ng/ml hIL-7 (Peprotech), 20 ng/ml hIL-15 (Peprotech), and 50 μg/ml ascorbic acid (Sigma) [20 (link)]. Both the cytokines and ascorbic acid were freshly added prior to use.
Cord blood CD34⁺ HSPCs were isolated using the CD34 MicroBead Kit (Miltenyi Biotec) [21 (link)]. The enriched HSPC population comprised over 90% CD34⁺ cells. These HSPCs were introduced at 5 × 10⁵ cells per ml into serum-free StemSpan™ SFEM II medium (Stemcell Technologies). The medium was enriched with 1% L-glutamine (Invitrogen), 100 ng/ml hSCF (Peprotech), 100 ng/ml hFlt3-L (Peprotech), 100 ng/ml hTPO (Peprotech), 50 ng/ml hIL-6 (Peprotech), 750 nM SR1 (Sigma), and 50 nM UM171 (Sigma). To maintain optimal cell density, fresh medium was administered every two days before initiating NK cell differentiation, thus ensuring a cell density range of 5 × 10⁵ to 1 × 10⁶ cells per ml.