Modelling biochemical gradients in vitro to control cell compartmentalization in a microengineered 3D model of the intestinal epithelium

Abstract: Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they formed and sensed by the epithelium is still not fully understood. Here we present a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and biomolecular gradients of the ISC niche (v). Biochemical gradients are formed through the hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of precise spatiotemporally controlled gradients, we employ light-sheet fluorescence microscopy and in-silico modelling. The ISC niche biochemical gradients applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.Table of contents (ToC): Biomolecular gradients are fundamental in intestinal epithelial tissue organization and function. The microengineered platform developed, with well-controlled spatiotemporal gradients of intestinal stem cell niche factors, 3D architecture and organoid-derived cells retrieves an in vivo-like cell compartmentalization. The system is easily incorporated in standard cell culture inserts where one can systematically test factors critical for intestinal tissue dynamics.