Context &Objective: Part of the digestive system, the small intestine is an organ with multi-scale curvatures, such as microstructures, tissue folds and the tubular wall. The microstructures shaping the intestinal epithelium and called crypt-villus units are repetitive and compartmentalized structures that display high aspect ratios and dense spatial packing. On these curved surfaces, highly organized stem cells (crypts) and differentiated cell compartments (villus) reside in concave and convex regions, respectively. Deformed by a combination of muscular and external movements, this complex architecture increases surface area and promotes nutriment absorption. However, it is affected in case of severe pathologies that are not yet fully understood and still require effective treatments.

Reproducing this microarchitecture in vitro is essential for developing physiologically relevant models of intestinal biology but remains technically challenging due to fabrication limitations. Conventional methods such as photolithography and standard 3D printing are insufficient to replicate the high aspect ratio, smoothly contoured structures found in vivo. We thus aim to use an alternative method to replicate the complex crypt-villus architecture of the intestinal epithelium in vitro to investigate the physiopathology of the small intestine.

Methods: Micromilling was employed to fabricate moulds that accurately replicate the full 3D geometry of crypt-villus units. These moulds were used to cast microstructured hydrogels with smooth surfaces and high aspect ratios. Primary mouse intestinal epithelial cells were grown onto the hydrogel scaffolds. To mimic physiological motions, dynamic and cycling mechanical actuation was introduced with magnetic membranes.

Results: The microstructured hydrogels supported the formation of polarized epithelial monolayers that self-organize along the scaffold curvature, with distinct proliferative and differentiated regions. Additionally, the ability to apply in vivo-like folding through magnetic actuation provides a means to study the impact of mechanical forces on epithelial organization.

Conclusion: While static curvature alone is sufficient to recreate the spatial organization of intestinal cell types in crypt-villus units, in vivo this curvature is highly dynamic. Integrating dynamic folding introduces a powerful platform to investigate how physiologically relevant mechanical stimuli influence intestinal tissue architecture and function.