From INFOGEST to Intestine-on-Chip: Fluidic-Equipped Platforms to Assess Digested Food Matrices and Fecal Microbiota Effects on the Intestinal Barrier

One of the major challenges in modern food science and nutrition research is understanding how food components and microbial metabolites influence the intestinal barrier. Traditional two-dimensional monocultures are overly simplistic and lack physiological relevance, and animal models are limited by ethical concerns and high costs. Organ-on-chip technology, therefore, offers an innovative approach to overcoming these limitations by providing dynamic, human-relevant experimental models. The present research project aimed to develop a novel in vitro platform integrating simulated gastrointestinal digestion (INFOGEST) with dynamic intestine-on-chip systems in order to study the biological effects of digested food matrices and microbial metabolites on intestinal epithelial function. A key aspect of the study involved genetically modified rice expressing Apolipoprotein A-I Milano (AIM), a natural ApoA-I variant known for its potent anti-inflammatory and cardioprotective properties. The rice was used as a sustainable, plant-based bioreactor to produce a bioactive protein with therapeutic potential, introducing the concept of edible, plant-derived nutraceuticals. The experimental workflow involved optimizing digestion and storage protocols to enhance the biocompatibility of INFOGEST digesta samples for in vitro intestinal barrier studies while maintaining the samples' biological activity. Initially, a 2D Caco-2 intestinal epithelial model was set up to pre-screen for cytotoxicity and barrier integrity and to determine the most tolerable concentrations for use in more advanced, physiologically relevant intestine-on-chip experiments. Specifically, two intestine-on-chip platforms - the Mimetas OrganoPlate® and the IVTech LiveBox® - were optimized and used to recreate dynamic flow conditions and mechanical stress in order to study the interaction between food matrices, microbiota, and the intestinal epithelium. The analytical approaches included LDH cytotoxicity assays, TEER measurements, Lucifer Yellow and Rhodamine-123 permeability tests, ELISA, Western blotting and gene expression analysis of intestinal barrier and inflammatory markers (ZO-1, OCLN, APOA1, EZR, MDR1 and IL-6). The Mimetas microfluidic device was primarily used to evaluate the effects of digested food matrices, such as apple and cabbage, based on the specific characteristics of each platform. TEER measurements and immunofluorescence analyses demonstrated this platform's suitability for investigating the interaction between food-derived compounds and the intestinal barrier. On the other hand, the IVTech millifluidic system was used to evaluate the impact of microbial metabolites originating from Bacteroides thetaiotaomicron and human faecal microbiota fermentations on epithelial physiology. Within this framework, this study also present a case report on an analysis of the metabolites produced by the human female faecal microbiota when incubated with digested genetically modified AIM rice. The IVTech platform was effective in visualizing the biological response of the intestinal barrier to all faecal metabolite treatments. Overall, the INFOGEST–intestine-on-chip approach successfully reproduced key aspects of human intestinal physiology, enabling complex interactions between diet and microbiota to be investigated. Exposure to AIM-enriched rice digesta and microbial metabolites modulated epithelial barrier function without inducing cytotoxicity. In conclusion, this research establishes an integrated, reproducible and ethical in vitro model for studying food–microbiota–host interactions, as well as for screening functional food components and bioactive compounds. This represents a significant step towards more sustainable, physiologically relevant, and animal-free experimental systems that are consistent with the 3Rs principle and the goals of next-generation food and biomedical research.