Engineered Decellularized Matrix Hydrogels with Crypt–Villus Topography for Forming Functional Intestinal Epithelium

Abstract Creating a functional intestine model remains challenging owing to the complex topography of the intestinal crypt/villus structure. Here, an inverse molding biofabrication technique is presented to address this challenge. A sacrificial hydrogel mold (SHM) with negative crypt/villus features is first fabricated using digital light processing (DLP) 3D printing of poly(ethylene glycol)–norbornene–tyramine (PEGNB‐T) thiol–norbornene hydrogels. Next, decellularized small intestine submucosa–norbornene (dSIS–NB) solution is cast and photopolymerized over the SHM, also via efficient thiol–norbornene photoclick reaction using PEG‐tetrathiol as the crosslinker. The hydrogel construct is placed in a buffer solution to induce autonomous and rapid dissolution of the SHM, creating dSIS–NB hydrogels with the positive crypt/villus structure. Intestinal epithelial cells seeded on the dSIS–NB crypt/villus matrices form a confluent monolayer within 3 days and display correct intestinal polarity. Through transepithelial electrical resistance (TEER) measurements and macromolecular transport studies, the new inverse‐molded dSIS–NB crypt/villus model further demonstrates the selective and drug‐responsive barrier functions. Finally, the unique biofabrication technique is leveraged to create an intestinal disease model carrying regions of both healthy crypts–villi structure and flattened epithelium with hindered macromolecular transport.