title: Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors.
abstract: : The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only 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 × 106cells per milliliter 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 supports maintenance 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 efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture.
  
  
  
  Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Kropp
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Kempf
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Caroline
authors.initials: C
authors.lastname: Halloin
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Robles-Diaz
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Annika
authors.initials: A
authors.lastname: Franke
authors.affiliation: Institute of Technical Chemistry, Gottfried-Wilhelm-Leibniz University Hannover, Hannover, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheper
authors.affiliation: Eppendorf AG Bioprocess Center, Juelich, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Kinast
authors.affiliation: Natural and Medical Science Institute (NMI) at the University of Tuebingen, Reutlingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Knorpp
authors.affiliation: Natural and Medical Science Institute (NMI) at the University of Tuebingen, Reutlingen, Germany.
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Joos
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Haverich
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Martin
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany zweigerdt.robert@mh-hannover.de.
authors.forename: Robert
authors.initials: R
authors.lastname: Zweigerdt
authors.affiliation: Department of Cardiac, Thoracic, Transplantation, and Vascular Surgery, Leibniz Research Laboratories for Biotechnology and Artificial Organs, Hannover, Germany REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Olmer
mesh_terms: D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology*
keywords: Human pluripotent stem cell expansion; Induced pluripotent stem cells; Metabolism; Perfusion; Single-use stirred tank bioreactors; Three-dimensional suspension culture