title: Integrated 3-dimensional expansion and osteogenic differentiation of murine embryonic stem cells.
abstract: Embryonic stem cell (ESC) culture is fragmented and laborious and involves operator decisions. Most protocols consist of 3 individual steps: maintenance, embryoid body (EB) formation, and differentiation. Integration will assist automation, ultimately aiding scale-up to clinically relevant numbers. These problems were addressed by encapsulating undifferentiated murine ESCs (mESCs) in 1.1% (w/v) low-viscosity alginic acid, 0.1% (v/v) porcine gelatin hydrogel beads (d = 2.3 mm). Six hundred beads containing 10,000 mESCs per bead were cultured in a 50-mL high-aspect-ratio vessel bioreactor. Bioreactor cultures were rotated at 17.5 revolutions per min, cultured in maintenance medium containing leukemia inhibitory factor for 3 days, replaced with EB formation medium for 5 days followed by osteogenic medium containing L-ascorbate-2-phosphate (50 microg/mL), beta-glycerophosphate (10 mM), and dexamethasone (1 microM) for an additional 21 days. After 29 days, 84 times as many cells per bead were observed and mineralized matrix was formed within the alginate beads. Osteogenesis was confirmed using von Kossa, Alizarin Red S staining, alkaline phosphatase activity, immunocytochemistry for osteocalcin, OB-cadherin, collagen type I, reverse transcriptase polymerase chain reaction, microcomputed tomography (micro-computed tomography) and Fourier transform infrared spectroscopic imaging. This simplified, integrated, and potentially scaleable methodology could enable the production of 3-demensional mineralized tissue from ESCs for potential clinical applications.
authors.affiliation: Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College, London, United Kingdom. w.randle@novathera.com
authors.forename: Wesley L
authors.initials: WL
authors.lastname: Randle
authors.forename: Jae Min
authors.initials: JM
authors.lastname: Cha
authors.forename: Yu-Shik
authors.initials: YS
authors.lastname: Hwang
authors.forename: K L Andrew
authors.initials: KL
authors.lastname: Chan
authors.forename: Sergei G
authors.initials: SG
authors.lastname: Kazarian
authors.forename: Julia M
authors.initials: JM
authors.lastname: Polak
authors.forename: Athanasios
authors.initials: A
authors.lastname: Mantalaris
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*