Development of a Novel Microfluidic Co-culture model to study Organoid Vascularization

Abstract Organoids hold great potential for regenerative medicine and biomedical research. While they are able to recapitulate many structural and functional aspects of their in vivo counterparts, they lack a functional vasculature. In this study, we aimed to coculture human small intestinal organoids (hSIO) with a vasculature in a triple co-culture system with endothelial cells (ECs) and fibroblasts in a microfluidic device. Organoids and micro-vessels favour distinct matrix and medium conditions, which were optimized to sustain growth of all cell types. In addition, we found that organoids exhibit poor survival in monoculture in the microfluidic devices. Interestingly, co-culturing hSIO together with ECs and fibroblasts enhances stemness and survival of hSIO. This effect can be further enhanced by adding the GSK-3β inhibitor CHIR99021 to the medium to enhance Wnt signalling. Direct contact of fibroblasts is not required for either vessel formation or for the effect on stem cell maintenance of organoids, as culturing fibroblasts in side-channels yields larger micro-vessels and better organoid survival. Additionally, we observed that the response of cells to different culture media is strongly dependent on the culture set-up as the second setup allows organoids to be cultured in angiogenic medium. Our results suggest that angiocrine signalling by the vasculature affects stem cell maintenance, which should be further investigated. Our co-culture system offers a platform to study angiocrine signalling in the small intestine, which could in the future be used to unravel different mechanisms behind endothelial mediated intestinal homeostasis and (patho) physiology.