P0054 Human colonoid-derived monolayers and Caco-2: comparative analysis of inflammatory responses

Abstract Background Given the increasing incidence of inflammatory bowel diseases, there is a growing need for reliable preclinical models that replicate the inflamed intestinal epithelium. Immortalized Caco-2 cells are widely considered the standard in vitro model for intestinal studies, but human-derived colonoid monolayers (CDMs) could be proposed as a powerful alternative to more accurately mimic the pathophysiological features of intestinal inflammation. The present study aims to compare the functional and morphological responses induced by inflammatory cytokines TNFα and IFNγ on CDMs and Caco-2. Methods Human colonic crypts from biopsies of 5 healthy volunteers were embedded in Matrigel® and cultured for 5-7 days. IntestiCult™ OGM was used to expand cystic-like colonoids, and single cells were seeded onto coated 24-well inserts. Upon reaching confluence, the medium was switched to IntestiCult™ ODM. Caco-2 cells were cultured for 21 days on 24-well inserts. Differentiated CDMs and Caco-2 monolayers were exposed to 25 ng/mL TNFα-IFNγ for 24 hours. Cell differentiation was assessed by immunocytochemistry (ICC), and barrier integrity was evaluated by transepithelial electrical resistance (TEER) measurement. Cytotoxicity was assessed through the LDH assay. IL-8 and CCL20 release were quantified. Results Both models showed increasing TEER over the growth period, indicating the gradual development of epithelial barriers, up to 484±58 Ω*cm2 for CDMs and 700±184 Ω*cm2 for Caco-2. ICC staining for epithelial markers villin and MUC2 confirmed polarization and differentiation under basal conditions in both models, cytokines reduced the expression of epithelial markers. Inflammation reduced the barrier integrity of the monolayers (approximately 18%), although more uniformly in Caco-2 than in CDMs. IL-8 and CCL20 release was augmented by cytokines in both models, (for IL-8 of about 20 times in CDM and 2 times in Caco-2). As regards cell viability, CDMs (40% cytotoxicity) showed higher sensitivity to cytokines than Caco-2 (22% cytotoxicity). Conclusion Exposure to an inflammatory environment altered cell morphology and impaired epithelial barrier integrity in both CDMs and Caco-2 monolayers. These findings highlight the potential of CDMs as an advanced model for studying personalized responses in intestinal inflammation.