Biomimetic villi-crypt scaffold-on-chip with tunable mechanical properties for intestinal epithelium modeling

The small intestine possesses a complex architecture and microenvironment. Current in vitro three-dimensional models fail to fully replicate the architectural, biophysical and biochemical cues in both healthy and pathological intestine tissues. In this study, we designed and engineered a biomimetic villi-crypt scaffold-on-chip via digital light processing (DLP) 3D-printing. The fabricated villi-crypt scaffold-on-chip model is specifically designed to emulate physiological mechanical properties and enable advanced investigation of intestinal epithelial architecture, cellular functions, and interactions with fluid flow, while also being compatible with downstream proteomic analysis. Using gelatin methacryloyl (GelMA) and poly(ethylene glycol) diacrylate (PEGDA), we fabricated high-fidelity villi and crypt-like structures with tuned mechanical properties and enhanced long-term stability. By optimizing the GelMA-PEGDA composition, we achieved precise microarchitecture with minimal swelling or deformation. Computational fluid dynamic studies demonstrated the consistency of the villi-crypt scaffold-on-chip model with the physiological shear forces observed in the intestinal epithelium. Among the tested formulations, the Villi-(Rigid)-Crypt scaffold exhibits superior structural stability and a more physiologically relevant intestinal-like environment, maintaining its integrity in culture. In contrast, the Villi-(Flexible)-Crypt scaffold presents superior flexibility while still supporting cell growth. Proteomic analysis revealed that the different mechanical properties of the fabricated biomimetic villi-crypt scaffold-on-chip models can modulate cells functions towards barrier formation, epithelial polarization, and metabolic activity, or even expression of mucus-associated and adhesion proteins. These results confirm the model's relevance for in vitro studies of intestinal epithelial function and dynamics, offering a powerful tool for drug screening and modeling.