UCB collection at delivery was approved (see “Compliance with ethical standards”). HPC-NK cells were generated as described [11 (link)] with the following minor modifications. Cells were cultured for 5–7 weeks in 6-well tissue culture plates (Corning, 3506), using NK MACS Basal medium and supplement (NK MACS, Miltenyi Biotec, 130–114-429) complemented with 10% human serum (HS, Sanquin) during expansion (day 0–14) and 2–10% HS during differentiation (from day 14). HPC-NK cells (> 70% CD56+) were used directly or cryopreserved. Cryopreserved HPC-NK cells were thawed and used after 5–7 days of culture in NK MACS containing 10% HS, 50 ng/ml recombinant human (rh)IL-15 (Immunotools, 11340155) and 0.2 ng/ml rhIL-12 (Miltenyi Biotec, 130–096-704). For experiments, HPC-NK cells were resuspended in Iscove's Modified Dulbecco's Medium (IMDM, Gibco, 21980–032) supplemented with 10% fetal calf serum (FCS, Integro, 5–45900 or Corning, 35–079-CV) (IMDM10), except assays with primary AML samples (10% HS), proliferation assays, and some serial killing experiments in microwells (NK MACS medium + 10% HS or FCS, respectively).
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