Transcriptomic profiling reveals substrate- and shear stress-dependent maturation of human small intestinal epithelial cells

Primary human intestinal epithelial cells (IECs) require microenvironments that reproduce various in vivo cues to maintain survival, differentiation, and function in vitro. In this study, we investigated how intestinal stem cell (ISC)-derived monolayers respond to biomimetic substrates and shear stress using 3D-printed hydrogels based on bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA) integrated into a custom millifluidic system. Extensive bulk RNA sequencing experiments revealed that, compared with Matrigel-coated tissue culture plastic, dSIS-MA hydrogels supported survival- and differentiation-related signaling, stabilized gene expression over time, and promoted absorptive lineage maturation while reducing crypt-associated signatures. By applying dynamic culture conditions to the hydrogel system, IECs underwent transcriptional remodeling, characterized by activation of metabolic and immune pathways. Longitudinal analysis further indicated that shear stress enhanced metabolic pathway–associated gene expression and promoted differentiation toward absorptive lineages. These findings establish dSIS-MA hydrogels with controlled fluid flow as a biomimetic in vitro model that supports survival, maturation of human IECs and enables transcriptional adaptation to defined biochemical and mechanical cues, supporting future applications in disease modelling, drug testing, and regenerative medicine.