Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors

Abstract

© AlphaMed Press 2016. Theroutine applicationofhumanpluripotent stemcells (hPSCs)andtheirderivatives inbiomedicineanddrug discoverywill require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cellonly aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D] ) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85×10 6 cells permilliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supportsmaintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells’ energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs’ energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficientGMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture.