Homogeneous shear distribution improves NK-92 cell cytotoxicity in a clinically relevant 2 L membrane-stirred bioreactor.

The immortalized NK-92 cell line is widely used to study natural killer (NK) cell biology and develop immunotherapies. NK-92 cells exhibit strong cytotoxic activity against tumor and virus-infected cells and are often used as a functional surrogate for primary NK cells. Beyond research applications, NK-92 cells are currently being evaluated in clinical trials as an allogeneic, off-the-shelf cell therapy. NK cells are typically cultured in static systems, which limits scalability. For clinical and commercial applications, large-scale cell expansion requires scalable platforms, such as bioreactors. However, conventional bubble-aerated bioreactors generate shear stress and foam, which can impair proliferation during prolonged culture and compromise cell quality. To address these limitations, a membrane-based stirring and aeration system was compared to a conventional pitched-blade impeller with microsparger aeration in 2 L stirred-tank bioreactors. NK-92 cells were expanded from static pre-cultures into shake flasks and subsequently inoculated into each bioreactor with continuous feeding to maintain ideal nutrient supply. Both systems supported comparable growth, viability, and metabolic profiles. Cells showed comparable growth, viability and metabolism profile in both systems. However, cells expanded in the membrane-based system exhibited markedly higher cytotoxicity and cytotoxic capacity. Computational fluid dynamic simulations of both systems suggest that this observation is likely attributable to the more homogeneous shear distribution in the membrane-stirred setup compared to the pitched-blade configuration. Overall, this work presents a novel cultivation method to produce highly cytotoxic NK-92 cells in a well scalable stirred-tank bioreactor platform for allogeneic off-the-shelf cell therapy.