An in vitro platform with self-sustaining trans-epithelial oxygen gradient to model intestinal barrier function modulation

In vitro models of the small intestine frequently neglect the role of physiological oxygen tension in drug absorption studies. To meet the throughput demands of preclinical drug development while maintaining compatibility with standard permeability assays, we engineered a new technology, termed Gradient-on-Platform. This system enables the establishment of physiologically relevant oxygen gradients across intestinal epithelium in vitro models by leveraging Caco-2 cells metabolic oxygen consumption. In this work, we demonstrated that exposure to physiomimetic oxygen conditions modulates epithelial barrier function, mitigating the excessively tight Caco-2 phenotype typically observed under conventional aerobic cultures, which leads to underestimation of drug bioavailability in vitro. Indeed, while complete hypoxia disrupts epithelial barriers, oxygen gradients drive Caco-2 TEER toward values more consistent with ex vivo measurements and result in a 3-fold increase in paracellular permeability compared to fully aerobic controls. The incorporation of physiologically relevant oxygen gradients into a scalable, assay-compatible platform could represent a pivotal step toward improving the predictive accuracy of in vitro preclinical drug absorption assays.