RESUMO
We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and ß-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células , Diferenciação Celular , Ilhotas Pancreáticas , Organoides , Células-Tronco Pluripotentes , Humanos , Organoides/citologia , Organoides/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Técnicas de Cultura de Células/métodos , Criopreservação/métodosRESUMO
Here, we present a protocol for producing 3D pancreatic-like organoids from human pluripotent stem cells in suspension bioreactors. We describe scalable techniques for generating 10,000-100,000 organoids that further mature in 4-5 weeks into α- and ß-like cells with glucose-responsive insulin and glucagon release. We detail procedures for culturing, passaging, and cryopreserving stem cells as suspended clusters and specify growth media and differentiation factors for differentiation. Finally, we discuss functional assays for research applications. For complete details on the use and execution of this protocol, please refer to Alvarez-Dominguez et al.1.
