Automated Closed-System Expansion of Pluripotent Stem Cell Aggregates in a Rocking-Motion Bioreactor

Pluripotent stem cell suspension aggregates have proven to be an efficient and phenotypically stable means for expansion and directed differentiation. Bioreactor systems with automation of perfusion, fluidization, and gas exchange are essential for scaling up pluripotent stem cell cultures. Since stem cell pluripotency and differentiation are affected by both chemical and physical signals, we investigated a low-shear method for the expansion of cells in a rocking-motion bioreactor. The rocking motion drives continual mixing and aeration, and the single-use disposable bioreactors avoid issues around contamination during seeding, medium exchange, passage, and cell harvest. Serial passaging from a 150 mL to a 1 L scale was demonstrated, achieving cell densities of up to 4 million cells/mL. In an average of 13 experiments, pluripotent stem cell aggregates expanded 5.7-fold (with maximal 9.5-fold expansion) and maintained 97% viability over 4 days in a rocking bioreactor culture. In seven experiments with improved culture conditions, the average expansion was 6.8-fold. Maintenance of pluripotency was confirmed by differentiation to all three germ layers and surface marker expression, and the expanded aggregates maintained a stable normal karyotype. The automation associated with the rocking platform bioreactor required no user intervention during the 4-day culture, providing hands-off expansion of pluripotent stem cells.