Scalable generation of 3D pancreatic islet organoids from human pluripotent stem cells in suspension bioreactors

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 β-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)