title: Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems.
abstract: Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.
authors.affiliation: Department of Bioengineering, and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Badenes
authors.affiliation: Department of Bioengineering, and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Tiago G
authors.initials: TG
authors.lastname: Fernandes
authors.affiliation: Department of Bioengineering, and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Cláudia S M
authors.initials: CS
authors.lastname: Cordeiro
authors.affiliation: Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America.
authors.forename: Shayne
authors.initials: S
authors.lastname: Boucher
authors.affiliation: Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Kuninger
authors.affiliation: Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America.
authors.forename: Mohan C
authors.initials: MC
authors.lastname: Vemuri
authors.affiliation: Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America.
authors.forename: Maria Margarida
authors.initials: MM
authors.lastname: Diogo
authors.affiliation: Thermo Fisher Scientific, Cell Biology, Life Sciences Solutions, Frederick, Maryland, United States of America.
authors.forename: Joaquim M S
authors.initials: JM
authors.lastname: Cabral
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D008863:Microspheres; D019096:Vitronectin / Q000494:pharmacology*
chemical_list: D003470:Culture Media; D019096:Vitronectin