Feeder-free ex vivo expansion of cord blood-derived natural killer cells for enhanced proliferation and functional maturation

Umbilical cord blood (CB)-derived natural killer (NK(CB)) cells are a promising immunotherapeutic modality due to their cytotoxicity, allogeneic compatibility, and reduced immunogenicity relative to peripheral blood NK cells (NK(PB)). Although several commercial culture media have been optimized for NK(PB) expansion, their suitability for NK(CB) cells remains insufficiently characterized. This study compares the expansion efficiency, phenotypic shifts, functional activation, and cost-effectiveness of NK(CB) cells expanded ex vivo under feeder-free static culture conditions with IL-2-supplemented culture media. CB-derived CD56⁺ cells isolated by magnetic-activated cell sorting were cultured in six candidate culture media, after which CTS™ NK-Xpander™ (NKX) and NK MACS® (NKM) with 1% (NKM1) or 2% (NKM2) supplement were selected for further evaluation. NKX achieved the highest average fold expansion (18.8 ± 7.0), followed by NKM2 (16.8 ± 7.3) and NKM1 (14.3 ± 3.5). All conditions increased cytotoxicity and CD107a degranulation, with NKX consistently yielding stronger functional responses. Phenotypic analysis revealed a shift toward a CD56brightCD16⁻ profile, particularly in NKM cultures, accompanied by cytokine-associated CD16 downregulation. Cost analysis identified NKX and NKM1 as the most cost-efficient conditions, whereas NKM2 doubled manufacturing costs due to higher supplement requirements. In conclusion, NKX and NKM media effectively support feeder-free ex vivo expansion of NK(CB) cells with high viability and robust functional activation. Phenotypic changes, donor-dependent variability, and CD16 downregulation should be considered when designing clinically translatable manufacturing strategies. These findings are essential to enhance scalability, reduce manufacturing costs, and advance NK(CB) cells as an accessible and effective immunotherapy, facilitating their integration into clinical-grade manufacturing and scalable bioprocessing pipelines.