Abstract
Abstract Background The intestinal epithelium is protected by a vital mucus layer which is essential for maintaining gut homeostasis and regulating host-microbial interactions. However, studying this mucus layer in vitro has been challenging due to the lack of physiologically relevant models. Methods We hypothesized that culturing human adult intestinal stem cells (ISCs) under flow conditions on a biomimetic scaffold could better recapitulate in vivo epithelial differentiation and mucus production. To test this, we developed a hydrogel-integrated millifluidic chamber, where ISCs were cultured on decellularized and methacrylated porcine small intestinal submucosa (dSIS-MA) under both static and dynamic (laminar flow) conditions. RNA bulk sequencing was used later to explore gene expression profiles across early and late time points to assess the impact of dSIS-MA and fluid flow on ISC differentiation and function over time. Results Our results reveal significant changes in pathways related to epithelial lineage commitment and intestinal barrier function on dSIS-MA compared to tissue culture plastic and in dynamic compared to static conditions, highlighting the critical influence of matrix structure and fluid flow. Conclusion In summary, we have successfully established a model that mimics in vivo conditions and reveals that surface and shear stress strongly impact intestinal epithelial cell development and function. This advanced in vitro approach enhances our understanding of molecular processes underlying epithelial cell behavior in conditions closely resembling the gut environment, with applications in gut health and disease research.
Published Version
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