Modeling Disease-Relevant Bile Duct Morphogenesis Defects in a Tunable In Vitro System

cells) or subjected to pharmacological Notch inhibition (CB-103) recapitulate key features of Alagille syndrome, including reduced tube width, impaired folding, loss of lumen continuity, and defective branching. Quantitative analyses revealed a significant reduction in folded epithelial area and complete loss of intermicropattern bridge formation under Notch inhibition, whereas coculture with HUVECs favors lumen expansion and connectivity in control but not in Notch-deprived cells. Epidermal growth factor (EGF) enhanced folding and partially restored proliferation defects induced by CB-103 but failed to rescue branching or tube expansion, highlighting the nonredundant, complementary roles of mitogenic versus morphogenetic cues. Time-lapse imaging revealed that Notch activity is required for the sequential folding-expansion cycle underlying the formation of size-controlled tubes, whereas EGF primarily modulates proliferation within formed folds. This platform allows quantitative, side-by-side comparison of genetic, pharmacological, and microenvironmental perturbations on bile duct morphogenesis. By combining spatial control, a tunable microenvironment, and high-resolution morphometric analysis, it provides a versatile tool for studying developmental defects, dissecting pathway-specific contributions, and exploring regenerative strategies for biliary disorders.