The intestine contains the largest microbial community in the human body, the gut microbiome. Increasing evidence suggests that it plays a crucial role in maintaining overall health. However, while many studies have found a correlation between certain diseases and changes in the microbiome, the impact of different microbial compositions on the gut and the mechanisms by which they contribute to disease are not well understood. Traditional pre-clinical models, such as cell culture or animal models, are limited in their ability to mimic the complexity of human physiology. New mechanistic models, such as organ-on-a-chip, are being developed to address this issue. These models provide a more accurate representation of human physiology and could help bridge the gap between clinical and pre-clinical studies. Gut-on-chip models allow researchers to better understand the underlying mechanisms of disease and the effect of different microbial compositions on the gut. They can help to move the field from correlation to causation and accelerate the development of new treatments for diseases associated with changes in the gut microbiome. This review will discuss current and future perspectives of gut-on-chip models to study host-microbial interactions.

Enterotoxins are a type of toxins that primarily affect the intestines. Understanding their harmful effects is essential for food safety and medical research. Current methods lack high-throughput, robust, and translatable models capable of characterizing toxin-specific epithelial damage. Pressing concerns regarding enterotoxin contamination of foods and emerging interest in clinical applications of enterotoxins emphasize the need for new platforms. Here, we demonstrate how Caco-2 tubules can be used to study the effect of enterotoxins on the human intestinal epithelium, reflecting toxins’ distinct pathogenic mechanisms. After exposure of the model to toxins nigericin, ochratoxin A, patulin and melittin, we observed dose-dependent reductions in barrier permeability as measured by TEER, which were detected with higher sensitivity than previous studies using conventional models. Combination of LDH release assays and DRAQ7 staining allowed comprehensive evaluation of toxin cytotoxicity, which was only observed after exposure to melittin and ochratoxin A. Furthermore, the study of actin cytoskeleton allowed to assess toxin-induced changes in cell morphology, which were only caused by nigericin. Altogether, our study highlights the potential of our Caco-2 tubular model in becoming a multi-parametric and high-throughput tool to bridge the gap between current enterotoxin research and translatable in vivo models of the human intestinal epithelium.

Candida albicans is an opportunistic pathogenic yeast commonly found in the gastrointestinal tract of healthy humans. Under certain conditions, it can become invasive and cause life-threatening systemic infections. One mechanism used by C.albicans to breach the epithelial barrier is the secretion of candidalysin, a cytolytic peptide toxin. Candidalysin damages epithelial membranes and activates the innate immune response, making it key to C.albicans’ pathogenicity and a promising therapeutic target. Although candidalysin mediates C. albicans translocation through intestinal layers, its impact on epithelial responses is not fully understood. This study aims to characterize this response and develop scalable, quantitative methodologies to assess candidalysin’s toxicological effects using gut-on-chip models. We used the OrganoPlate^® platform to expose Caco-2 tubules to candidalysin and evaluated their response with trans-epithelial electrical resistance (TEER), protein detection, and immunostaining. We then validated our findings in a proof-of-concept experiment using human intestinal organoid tubules. Candidalysin impaired barrier integrity, induced actin remodeling, and increased cell permeability. It also induced the release of LDH, cytokines, and the antimicrobial peptide LL37, suggesting cellular damage, inflammation, and antimicrobial activity. This study strengthens our understanding of candidalysin’s role in C. albicans pathogenesis and suggests new therapeutic strategies targeting this toxin. Moreover, patient-derived organoids show promise for capturing patient heterogeneity and developing personalized treatments.

The intestinal epithelial barrier is crucial for gut homeostasis, with dysfunction linked to inflammatory disorders like inflammatory bowel disease (IBD). While cytokines are established mediators of barrier disruption, the role of lipid signaling remains poorly understood. Using a microfluidic platform, we cultured epithelial tubules and exposed their luminal (apical) side to TNF‐α, IL‐1β, and IFN‐γ (0–200 ng/mL) under serum‐containing or serum‐free conditions. Barrier function was assessed primarily via transepithelial electrical resistance (TEER), with DRAQ7 staining and actin cytoskeletal analysis providing complementary indicators of membrane integrity and structural disruption. In parallel, a targeted liquid chromatography–tandem mass spectrometry approach was used to profile lipid mediators across apical and basolateral compartments. Cytokine exposure significantly impaired barrier integrity, as indicated by reduced TEER, alongside associated cell damage and structural changes reflected by DRAQ7 staining and actin remodeling. The remodeling effect was lower in serum‐free medium. Lipid profiling revealed inflammatory signatures characterized by an increase in prostaglandins in the luminal compartment, particularly under serum‐free conditions. PGF1α increased under both media conditions, whereas the rest of the changes were condition‐specific, with a rise in PGE1, PGE2, and PGD2, among others, particularly under serum‐free conditions. Simultaneously, changes in other eicosanoids, but not in prostaglandins, were detected in the basolateral compartment under serum‐free conditions. This proof‐of‐principle study demonstrates how medium composition significantly influences inflammatory responses and lipid signaling patterns in a physiologically relevant gut‐on‐chip model. Our integrated approach reveals the complex spatial organization of lipid mediators during cytokine‐induced barrier dysfunction and provides a valuable framework for investigating the interplay between inflammation, barrier integrity, and lipid metabolism in intestinal pathophysiology.

Evaluating cellular responses to toxic compounds is essential for assessing the safety and potential hazards of drugs, environmental pollutants, and food contaminants. Traditional in vitro methods often lack the precision and scalability required for comprehensive toxicological assessments. This protocol presents an efficient and scalable approach for quantifying cellular damage and toxicity using organ-on-chip technology, specifically the OrganoPlate® platform. By combining fluorescent probes—DRAQ7 for cell membrane integrity, ActinGreen for cytoskeletal changes, and NucBlue for nuclear counting—with high-throughput image analysis via CellProfiler, this method provides detailed and quantitative insights into cellular damage induced by toxic compounds. The protocol includes step-by-step instructions for staining, image acquisition, and data analysis, as well as troubleshooting guidance. CellProfiler’s open-source nature, flexibility, and automation capabilities enable reproducible, high-throughput workflows, offering significant advantages over traditional manual image analysis. Its ability to assess cytotoxicity in human tissue models makes this protocol a valuable tool for safety testing in drug discovery and environmental toxicology. Furthermore, the approach is highly adaptable, accommodating a variety of cell types and toxic compounds, and is well-suited for rapid screening and risk assessment across diverse research and industrial applications.

Candida albicans is an opportunistic pathogenic yeast commonly found in the gastrointestinal tract, vagina, and mouth of healthy humans. Under certain conditions, it can become invasive, causing mucosal or life-threatening systemic infections. One mechanism used by C . albicans to breach the epithelial barrier is the secretion of candidalysin, a cytolytic peptide toxin. Candidalysin damages epithelial membranes and activates the innate epithelial immune response, making it key to C . albicans’ pathogenicity and a promising therapeutic target. Although candidalysin mediates C. albicans translocation through intestinal layers, its impact on epithelial responses is not fully understood. This study aims to characterize this response and develop scalable, quantitative methodologies to assess candidalysin’s toxicological effects using gut-on-chip models. We used the OrganoPlate®, a microfluidic platform to culture up to 64 perfused, membrane-free intestinal epithelial tubes. We exposed Caco-2 tubes to candidalysin and evaluated their response with trans-epithelial electrical resistance (TEER), protein detection, and immunostaining. We then validated our findings in a proof-of-concept experiment using human intestinal organoid tubules. Candidalysin impaired barrier integrity, as indicated by decreased TEER and increased permeability in a fluorescent dye assay. It also induced actin remodeling and DRAQ7™ dye uptake, a marker of cell permeability. This disruption was associated with the release of LDH, cytokines, and the antimicrobial peptide LL37, suggesting cellular damage, inflammation, and antimicrobial activity. This study strengthens our understanding of candidalysin’s role in C. albicans pathogenesis and suggests new therapeutic strategies targeting this toxin. Moreover, the use of patient-derived organoids shows promise for capturing patient heterogeneity and developing personalized treatments. Graphical