title: Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.
abstract: The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks, and the process was optimized using a factorial design approach, involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled, automated and reproducible large-scale bioreactor culture systems.
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Cláudia C
authors.initials: CC
authors.lastname: Miranda
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Tiago G
authors.initials: TG
authors.lastname: Fernandes
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: M Margarida
authors.initials: MM
authors.lastname: Diogo
authors.affiliation: Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: Joaquim M S
authors.initials: JM
authors.lastname: Cabral
mesh_terms: D001549:Benzamides / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004149:Dioxoles / Q000494:pharmacology; D004492:Edetic Acid / Q000737:chemistry; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D000071841:PAX6 Transcription Factor / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015203:Reproducibility of Results; D013535:Suspensions
chemical_list: C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D015415:Biomarkers; D003470:Culture Media; D004149:Dioxoles; C554430:LDN 193189; D050814:Octamer Transcription Factor-3; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; C494173:POU5F1 protein, human; D011720:Pyrazoles; D011743:Pyrimidines; D013535:Suspensions; D004492:Edetic Acid
keywords: Human pluripotent stem cells; Neural commitment; Neural precursor cells; Stirred-tank bioreactors; Suspension aggregates