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

This protocol describes a scalable suspension bioreactor workflow for generating three-dimensional pancreatic islet-like organoids from human pluripotent stem cells (hPSCs). The methodology supports the production of approximately 10,000 to 100,000 organoids that mature over 4 to 5 weeks into ?-like and ?-like endocrine cells capable of glucose-responsive insulin and glucagon secretion. The protocol details hPSC adaptation to 3D suspension culture, passaging, cryopreservation, differentiation media preparation, and a six-stage directed differentiation process. Quality control methods, including morphology assessment, flow cytometry, cell counts, and functional glucose-stimulated hormone secretion assays, are included. The workflow is designed for both research-scale and larger-scale manufacturing applications using stirred suspension bioreactors to improve consistency and scalability of stem cell-derived islet production.