title: High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations.
abstract: Strategies for the production of pluripotent stem cells (PSCs) rely on serially dissociated adherent or aggregate-based culture, consequently limiting robust scale-up of cell production, on-line control and optimization of culture conditions. We recently developed a method that enables continuous (non-serially dissociated) suspension culture-mediated reprogramming to pluripotency. Herein, we use this method to demonstrate the scalable production of PSCs and early derivatives using acoustic filter technology to enable continuous oxygen-controlled perfusion culture. Cell densities of greater than 1 × 10⁷  cells/mL were achieved after 7 days of expansion at a specific growth rate (µ) of 0.61 ± 0.1 day⁻¹ with a perfusion rate (D) of 5.0 day⁻¹. A twofold increase in maximum cell density (to greater than 2.5 × 10⁷  cells/mL) was achieved when the medium dissolved oxygen was reduced (5% DO). Cell densities and viabilities >80% were maintained for extended production periods during which maintenance of pluripotency was confirmed by stable expression of pluripotency factors (SSEA-1 and Nanog), as well as the capacity to differentiate into all three germ layers. This work establishes a versatile biotechnological platform for the production of pluripotent cells and derivatives in an integrated, scalable and intensified stirred suspension culture.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ricardo P
authors.initials: RP
authors.lastname: Baptista
authors.forename: David A
authors.initials: DA
authors.lastname: Fluri
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D019149:Bioreactors; D001709:Biotechnology; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002454:Cell Differentiation; D002470:Cell Survival; D005947:Glucose / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D016256:Lewis X Antigen / Q000378:metabolism; D051379:Mice; D000071317:Nanog Homeobox Protein; D010100:Oxygen / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; D016256:Lewis X Antigen; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D019344:Lactic Acid; D005947:Glucose; D010100:Oxygen