Natural killer (NK) cells are a promising avenue for cancer immunotherapy, but efficient clinical-scale production remains challenging. Here, we present an optimized protocol for the activation and expansion of NK cells (NKAE) and its validation under clinical-grade conditions. NK cells were expanded from either total peripheral blood mononuclear cells (PBMCs) or CD45RA+ subsets using irradiated K562 feeder cells expressing IL15-41BBL or IL21-41BBL, with culture media including RPMI-1640, SCGM, NK MACS, and TexMACS. Expansion rate, purity, activation markers, cytotoxic function, and transcriptomic changes were evaluated. Clinical-grade NKAE cells were produced using the CliniMACS Prodigy system. NK MACS and TexMACS media provided superior NK cell purity and minimal T cell contamination. While NKAE generation from CD45RA+ cells was feasible, PBMCs offered higher cell yield and purity. The greatest expansion and purity were achieved with PBMCs stimulated by K562mbIL21-41BBL, though activation status and cytotoxicity were similar across all conditions. Transcriptomic profiling revealed marked differences between resting NK cells and NKAE cells expanded with either IL15- or IL21-expressing feeders. All clinical-grade NKAE preparations met regulatory quality criteria. Our findings demonstrate that GMP-compliant NK cells suitable for immunotherapy can be efficiently expanded using different starting cell populations and feeder strategies, supporting their use in clinical applications.
