ABSTRACT
Flexible electronics implanted during tissue formation enable chronic studies of tissue-wide electrophysiology. Here, we integrate tissue-like stretchable electronics during organogenesis of human stem cell-derived pancreatic islets, stably tracing single-cell extracellular spike bursting dynamics over months of functional maturation. Adapting spike sorting methods from neural studies reveals maturation-dependent electrical patterns of α and ß-like (SC-α and ß) cells, and their stimulus-coupled dynamics. We identified two major electrical states for both SC-α and ß cells, distinguished by their glucose threshold for action potential firing. We find that improved hormone stimulation capacity during extended culture reflects increasing numbers of SC-α/ß cells in low basal firing states, linked to energy and hormone metabolism gene upregulation. Continuous recording during further maturation by entrainment to daily feeding cycles reveals that circadian islet-level hormone secretion rhythms reflect sustained and coordinate oscillation of cell-level SC-α and ß electrical activities. We find that this correlates with cell-cell communication and exocytic network induction, indicating a role for circadian rhythms in coordinating system-level stimulus-coupled responses. Cyborg islets thus reveal principles of electrical maturation that will be useful to build fully functional in vitro islets for research and therapeutic applications.
ABSTRACT
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.
