Three-dimensional multicellular layer structure: an advanced in vitro model for studying inflammatory bowel diseases

Inflammatory Bowel Disease (IBD) is a group of complex and debilitating gastrointestinal disorders characterised by chronic inflammation of the intestinal mucosa. This condition is multifactorial, involving genetic predispositions, environmental influences, dysregulated immune responses, and impaired epithelial barrier function. Researchers have already proposed the use of Extracellular Vesicles (EVs) as a therapeutic approach for inflammatory diseases. EVs are lipid bilayer-delimited nanoparticles secreted by cells that possess anti-inflammatory and pro-regenerative properties. We aim to develop a three-dimensional (3D) multilayer structure (MLS) model that mimics the physiological complexity of the intestinal mucosa, serving as a ready-to-use primary platform for drug testing. MLS, built with Caco-2 cells and BJ fibroblasts, were stimulated with pro-inflammatory cytokines to replicate IBD-like conditions, and within this model, the anti-inflammatory potential of EVs was further investigated. In the MLS, fibroblasts and Caco-2 cells integrated with one another, demonstrating that Caco-2 cells require fibroblasts for assembly. The results obtained from RT-qPCR analysis displayed a significant enhancement in the expression of both pro-inflammatory and anti-inflammatory genes. Specifically, EVs led to an increase in the expression of the IL-10 anti-inflammatory gene and leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5 +) stemness gene, suggesting a possible regulatory role of EVs in reducing inflammation and promoting a more balanced immune response. MLSs provide a valuable tool to study the intricate interactions between EVs, epithelial cells, and stromal components, more accurately mimicking native tissue architecture than traditional 2D cultures. The findings contribute to the burgeoning field of 3D models as tools for regenerative medicine applications.