Supernatant from primary human gut microbiota fermentation as a modulator of intestinal barrier in in vitro models

Abstract The human intestine harbours a complex microbial community that closely interacts with intestinal epithelial cells, the first line of host defence. Although several in vitro models have been developed to study host–microbiota interactions, direct co-culture of gut microbiota and mammalian cells remains technically challenging. Here, we evaluated an in vitro approach for testing human faecal fermentation supernatant on intestinal barrier function using differentiated Caco-2/HT29-MTX monolayers maintained in static cell culture inserts or a dynamic gut-chip model. Fermentation supernatant diluted in HBSS was applied for 4 h, and barrier function was assessed by transepithelial electrical resistance (TEER) in static cultures and by Lucifer Yellow apparent permeability (P app ) in both models. Under the conditions tested, exposure to the fermentation supernatant did not compromise barrier integrity. Lucifer Yellow Papp was significantly lower in cell culture inserts (1.04×10⁻⁷±8.98×10⁻⁹ cm·s⁻¹) than in the dynamic gut-chip (7.96×10⁻⁷±1.82×10⁻⁷ cm·s⁻¹), highlighting differences in epithelial permeability between static and dynamic culture conditions. To enable prolonged exposure studies, different carrier conditions were evaluated. HBSS was unsuitable for 24 h incubation, resulting in reduced cell metabolic activity and TEER, whereas HBSS-HEPES maintained cell viability and barrier function. A TNF-α-induced barrier dysfunction was subsequently established, resulting in a 45% decrease in TEER and a 48% increase in Lucifer Yellow P app compared with the healthy control. Treatment with fermentation supernatant diluted in HBSS-HEPES was associated with a reduction in the increased permeability, with P app values becoming comparable to those of the healthy control condition. restored barrier integrity, demonstrating its capacity to repair impaired intestinal epithelial. Overall, this study establishes a practical in vitro framework for evaluating faecal fermentation-derived products using intestinal epithelial models representing both healthy and barrier-impaired conditions. The findings highlight the importance of selecting appropriate culture platforms, carrier conditions, and exposure periods when assessing the effects of complex microbiota-derived products on intestinal barrier function and provide a basis for the subsequent testing of fermentation samples in cell-based intestinal models.