Protocol to generate stem cell-derived alpha cells in 3D suspension culture

Pancreatic alpha cells represent a key islet cell type that acts in tandem with pancreatic beta cells to regulate blood glucose levels. Here, we present a protocol to generate stem cell-derived alpha (SC-α) cells using chemically defined small-molecule and growth factor induction in a scalable 3D suspension culture. We describe steps for embryonic stem cell culture and SC-α differentiation. We then detail procedures for quality control and the assessment of SC-α cell function. For complete details on the use and execution of this protocol, please refer to Peterson et al.¹ and Shrestha et al.².