Development of a 48-Well Dynamic Suspension Culture System for Pancreatic Differentiation from Human Embryonic Stem Cells

Background Human pluripotent stem cells (hPSCs) have started to emerge as a potential tool with application in fields of drug discovery, disease modelling and cell therapy. A variety of protocols for culturing and differentiating pluripotent stem cells into pancreatic beta like cells have been published. However, small-scale dynamic suspension culture systems, which could be applied toward systematically optimizing production strategies for cell replacement therapies to accelerate the pace of their discovery and development toward the clinic, are overlooked. Methods Human embryonic stem cell (hESC) line H9 was used to establish the novel 48-well dynamic suspension culture system. The effects of various rotational speeds and culture medium volumes on cell morphology, cell proliferation, cell viability and cell phenotype were evaluated. Effect of cell density on the pancreatic differentiation efficiency from H9 cells in 48-well plates was further investigated. In vitro the function of pancreatic beta like cells was assessed by measuring glucose-stimulated insulin secretion. Main Results A 48-well dynamic suspension culture system for hESC expansion as cell aggregates was developed. With optimized rotational speed and culture medium volume, hESCs maintained normal karyotype, viability and pluripotency. Furthermore, the system can also support the hESC aggregates subsequent differentiation into functional pancreatic beta like cells after optimizing initial cell seeding density. Conclusion A controllable 48-well suspension culture system in microplates for hESCs maintenance, expansion and pancreatic differentiation was developed, which may provide an efficient platform for high-throughput drug screening.