A Bioengineered Platform for Enhanced Observability of Patterned Gastrointestinal Organoid Monolayers with Bilateral Access

Studying the physiology and pathology of gastrointestinal (GI) tissues requires tools that can accurately mimic their complex architecture and functionality in vitro. Organoids have emerged as one such promising tool, though their closed structures with poorly accessible lumen and limited observability makes readouts challenging. In this study, we introduce a bioengineered organoid platform that generates bilaterally accessible 3D tissue models, allowing independent manipulation of both the apical and basal sides of patterned epithelial monolayers. We successfully constructed gastric, small intestinal, caecal, and colonic epithelial models that faithfully reproduce tissue-respective geometries and exhibit high physiological relevance, evidenced by the regionalization of stem cells and transcriptional resemblance to real epithelia. The gained observability allowed single-cell tracking over time and studies into the motility of cells in immersion and air-liquid interface cultures. Additionally, this model recapitulated Trichuris muris infection of the caecum epithelium, allowing the first live imaging of syncytial tunnel formation. Overall, this platform offers accessible organoids with improved observability, making it a valuable tool for investigating the dynamics of GI epithelial cells and their interactions with pathogens.