title: Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors.
abstract: The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 106hiPSCs were expanded into 2 × 109hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.
authors.affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology II, University of Würzburg, Würzburg, Germany.
authors.forename: Chee Keong
authors.identifier: 0000-0003-3803-1200
authors.initials: CK
authors.lastname: Kwok
authors.affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology II, University of Würzburg, Würzburg, Germany.
authors.forename: Yuichiro
authors.identifier: 0000-0002-1885-1896
authors.initials: Y
authors.lastname: Ueda
authors.affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology II, University of Würzburg, Würzburg, Germany.
authors.forename: Asifiqbal
authors.initials: A
authors.lastname: Kadari
authors.affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology II, University of Würzburg, Würzburg, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Günther
authors.affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology II, University of Würzburg, Würzburg, Germany.
authors.forename: Süleyman
authors.initials: S
authors.lastname: Ergün
authors.affiliation: The life science business of Merck KGaA Darmstadt, Germany.
authors.forename: Antoine
authors.initials: A
authors.lastname: Heron
authors.affiliation: EMD Millipore Corporation, Bedford, Massachusetts, USA.
authors.forename: Aletta C
authors.initials: AC
authors.lastname: Schnitzler
authors.affiliation: EMD Millipore Corporation, Bedford, Massachusetts, USA.
authors.forename: Martha
authors.initials: M
authors.lastname: Rook
authors.affiliation: Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell Biology II, University of Würzburg, Würzburg, Germany.|Institute of Molecular Biology & Center for Molecular Biosciences Innsbruck, Genomics, Stem Cell Biology and Regenerative Medicine Leopold-Franzens-University & CMBI, Innsbruck, Innsbruck, Austria.
authors.forename: Frank
authors.identifier: 0000-0002-6489-714X
authors.initials: F
authors.lastname: Edenhofer
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D019149:Bioreactors; D002448:Cell Adhesion; D002449:Cell Aggregation; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D059785:Karyotype; D008297:Male; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D012015:Reference Standards; D013535:Suspensions; D055815:Young Adult
chemical_list: D015415:Biomarkers; D013535:Suspensions
keywords: bioprocessing; human pluripotent stem cells; process optimization; scalable culture system; single-use bioreactors; stirred suspension culture