Abstract
A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.