Human-derived colonoid monolayers as a physiologically relevant model for the study of intestinal inflammation

The increasing incidence of inflammatory bowel diseases highlights the need for preclinical models that mimic the inflamed human intestinal epithelium. Human-derived colonoid monolayers (CDMs) have emerged as promising tools due to their physiological relevance and responsiveness to inflammatory stimuli. This study aimed to characterize the morphological, functional, and molecular responses of CDMs to an inflammatory environment induced by TNFα and IFNγ. Colonic crypts were isolated from healthy donors and cultured in Matrigel® with IntestiCult™ growth medium to generate cystic colonoids. These were dissociated and seeded as monolayers onto inserts. After differentiation, the monolayers were exposed to 25 ng/mL TNFα/IFNγ for 24 hours. Barrier function was assessed by transepithelial electrical resistance (TEER), while cytotoxicity was assessed by the LDH assay. Inflammatory responses were measured by IL-8 and CCL20 secretion, and levels of epithelial and tight junction markers (VILLIN1, CLAUDIN-1, CLAUDIN-2, and MUC2) were analyzed by RT-qPCR. CDMs developed robust epithelial barriers (TEER: 484±58 Ωcm²) and showed a polarized morphology under basal conditions. Cytokine exposure significantly decreased TEER (from 492.4±62.7 to 389.5±23.6 Ωcm², p<0.05) and increased cytotoxicity (~40%). IL-8 and CCL20 release increased significantly from 24.7±21,35 to 342.8±248.1 pg/µg (p=0.01) and from 0.54±0.16 to 6,07±1,58 pg/µg (p<0.001), respectively, indicating a remarkable inflammatory response. RT-qPCR revealed downregulation of CLAUDIN-1 and CLAUDIN-2, highlighting barrier dysfunction. These results demonstrate the ability of CDMs to recapitulate key aspects of intestinal inflammation and support their use as an advanced patient-derived platform for translational intestinal research.