title: Scalable platform for human embryonic stem cell differentiation to cardiomyocytes in suspended microcarrier cultures.
abstract: A scalable platform for human embryonic stem cell (hESC)-derived cardiomyocyte (CM) production can provide a readily available source of CMs for cell therapy, drug screening, and cardiotoxicity tests. We have designed and optimized a scalable platform using microcarrier cultures in serum-free media supplemented with SB203580 mitogen-activated protein kinase-inhibitor. Different microcarriers (DE-53, Cytodex-1 and 3, FACT, and TOSOH-10) were used to investigate the effects of type, size, shape, and microcarrier concentrations on the differentiation efficiency. hESCs propagated on TOSOH-10 (protamine derivatized 10-μm beads) at the concentration of 0.125 mg/mL produced 80% beating aggregates, threefold cell expansion, and 20% of CMs (determined by fluorescence-activated cell sorting for myosin heavy chain and α-actinin expression). The ratio of CM/hESC seeded in this system was 0.62 compared to 0.22 in the embryoid body control cultures. The platform robustness has been tested with HES-3 and H1 cell lines, and its scalability was demonstrated in suspended spinner cultures. However, spinner culture yields dropped to 0.33 CM/hESC probably due to shear stress causing some cell death. Cells dissociated from differentiated aggregates showed positive staining for cardio-specific markers such as α-actinin, myosin heavy and light chain, troponin I, desmin, and emilin-2. Finally, CM functionality was also shown by QT-prolongation (QTempo) assay with/without Astemizole. This study represents a new scalable bioprocessing system for CM production using reagents that can comply with Good Manufacturing Practice.
authors.affiliation: Bioprocessing Technology Institute, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore. marti.lecina@gmail.com
authors.forename: Marti
authors.initials: M
authors.lastname: Lecina
authors.forename: Sherwin
authors.initials: S
authors.lastname: Ting
authors.forename: Andre
authors.initials: A
authors.lastname: Choo
authors.forename: Shaul
authors.initials: S
authors.lastname: Reuveny
authors.forename: Steve
authors.initials: S
authors.lastname: Oh
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004526:Efficiency; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D008863:Microspheres; D055616:Microtechnology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D009994:Osmolar Concentration; D013535:Suspensions; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: D013535:Suspensions