Saari, M.; Jahan, F.; Andersson, L.; Syreeni, A.; Vehmaan-Kreula, P.; Koski, J.; Jarvela, E.; Kerkela, E.; Paavilainen, H.; Schenkwein, D.; Yla-Herttuala, S.; Vettenranta, K.; Goos, H.; Korhonen, M.

Natural killer (NK) cells are increasingly recognized as a versatile therapeutic platform, yet their translation is hindered by limited ex vivo proliferation. Feeder cells serve as robust stimulatory component supplying activating signals required to initiate large-scale NK cell expansion. Here, using bench-scale cultures, we evaluated how distinct engineered K562-based feeder cells influence NK cell proliferation, phenotype maintenance, potential for activation, and post-cryopreservation function. Across conditions, feeder-based systems consistently enabled superior, up to 500-fold higher NK cell yield compared to feeder-free system. Variants incorporating membrane-bound costimulatory and cytokine cues yielded the most favorable balance between expansion and functional preservation. Simple adjustments to cryopreservation, including high-density-freezing and centrifuge-free-thawing, further supported NK cell recovery. Together, these findings highlight feeder cells as essential upstream reagents for effective NK cell bioproduction and provide foundational biological insights to guide the rational design and validation of future scalable NK cell manufacturing platforms.