A Primary Murine Tri-Culture Gut-on-Chip for the Analysis of Host–Microbe Interactions

Animal experiments remain widely used to investigate intestinal physiology and host–microbe interactions despite ethical concerns and limited experimental control. Here, we established and characterised a murine gut-on-chip platform based entirely on primary cells. The microfluidic system integrates murine intestinal epithelial, stromal, and endothelial cells in a spatially organised tri-culture configuration under continuous perfusion. Stable compartmentalisation of the different cell populations was achieved, and cellular organisation was confirmed by immunocytochemical analysis. To evaluate biological responsiveness, the epithelial compartment was exposed to Escherichia coli Nissle 1917. Bacterial exposure was associated with altered epithelial junctional organisation, as indicated by changes in ZO-1 localisation. Gene expression analyses detected markers associated with proliferation, stemness, differentiation, nutrient transport, and signalling pathways. Comparison of tri-culture and epithelial-only configurations suggested differences in epithelial gene expression profiles; however, none of the analysed genes showed statistically significant differences between groups. Together, these findings demonstrate the feasibility of combining primary murine epithelial, stromal, and endothelial cells within a controlled microfluidic environment. The presented platform provides a multicellular in vitro system for investigating host–microbe interactions and epithelial responses under defined experimental conditions.