Evaluation of a high throughput multiparallel stirred bioreactor system using an apple cell line.

Cultivating dedifferentiated plant cells in bioreactors offers a continuous, scalable, reproducible, and fully controlled platform for producing valuable secondary metabolites. However, performance is highly dependent on cultivation conditions. In this study, we investigated the impact of different bioreactor configurations on the growth and metabolite production of an apple plant cell suspension culture. Stirred-tank bioreactors (STRs) with different vessel geometries (flat and round-bottom) were evaluated at the multi-liter scale (2-5 L) and compared, for the first time with plant cells, with a high-throughput small volume parallel system (240 mL). Using these mini-bioreactors, we studied the effect of impeller type (Rushton, marine, elephant ear) and pO2 cascade controls on cell growth and triterpene production. Results revealed that triterpene content and biomass yield varied significantly across cultivation systems and that plant cells were highly sensitive to shear stress, with variation coefficient reaching 56.2%-63.5%. Configurations with elephant ear-type impellers seemed to reduce shear stress, supporting stable triterpene accumulation (14.15 mg/g cells DW) and higher biomass (11.8 g DW/L). The high-throughput system closely mirrored multi-liter STR performance, demonstrating consistent and reliable prediction at larger scales. In contrast, wave bioreactors and flask cultures yielded distinct results, emphasizing the importance of continuous monitoring and system-specific optimization. Overall, these findings demonstrate that careful selection of bioreactor design and control strategy is critical for maximizing plant cell growth and metabolite production, and that small-scale high-throughput systems can be effectively used as process development tool for plant cell-based biomanufacturing.