Transition of Natural Killer cells expansion platform from static flasks to stirred tank bioreactor

Abstract

NK cells cultivation in static conditions has been established and studied many years ago. Since then, many different protocols have appeared and have been widely used to generate cell populations intended for therapeutic and scientific use. Common methods that utilize static suspension culture have limited scalability, making commercialization of such cell therapies challenging. The focus of this work lay in the investigation of the dynamic cultivation of NK-92 in stirred tank bioreactor and shake flasks compared to static cultivation in T-flasks.To be able to evaluate the production of NK-92 cells and generate stable data, a complete laboratory set-up was thoroughly tested and implemented. The scale-up from shake flask based on power input to volume unit was found to be suitable. Consequently, an experimental bioreactor set-up was then designed and integrated. This included inoculum procedure, stirring and aeration, monitoring of permittivity, dissolved oxygen (DO) concentration, O2/CO2 concentrations in off-gas, pH, as well as a detailed off-line sampling plan. A tailored sampling plan comprised of an assessment of viable cell concentration (VCC), viability and cytotoxicity using flow cytometry and enzyme-based concentration measurements of extracellular D-glucose, L-lactate, L-glutamine, L-glutamate, ammonium, and pyruvate was presented.We were able to transform the static NK-92 cells cultivation to dynamic conditions in shake flasks to stirred tank bioreactor. Nine days of batch cultivation resulted in high viable cell concentration in all three conditions. Briefly, the fold expansion of 9.96 ± 0.33, 8.97 ± 0.60, and 6.64 ± 0.09 was achieved in T-flasks, shake flasks, and bioreactor respectively. No significant differences in specific uptake/production rates during the growth phase in all three conditions were detected. Despite slightly lower fold expansion in shake flasks and significantly lower fold expansion in bioreactor, the cells from all three conditions demonstrated equally high cytotoxicity against K562-GFP reporter cells at an E:T ratio of 1, highlighting one of the major critical quality attributes of these cells. Lastly, in this work we underlined the dynamics of specific uptake/production rates of D-glucose, L-lactate and pyruvate as the key molecules in oxidative glycolysis as well as specific uptake/production rates of L-glutamine, L-glutamate and ammonium ions as a bridge between amino acid metabolism and TCA-cycle.