Establishment of a 3D multi-layered in vitro model of inflammatory bowel disease.

Crohn's Disease and Ulcerative Colitis, the main types of Inflammatory Bowel Disease (IBD), are life-threatening gastrointestinal disorders with no definitive cure. The establishment of biorelevant in vitro models that closely recapitulate the IBD microenvironment is of utmost importance to validate newly developed IBD therapies. To address the existing flaws in the current representation of the IBD microenvironment, we propose a novel three-dimensional (3D) in vitro model comprising a multi-layered gastrointestinal tissue with functional immune responses under inflammatory conditions. The multi-layered architecture consists of a lamina propria-like hydrogel with human intestinal fibroblasts (HIF), supporting an epithelial layer composed of Caco-2 and HT29-MTX cells, along with an endothelial layer surrogating the absorptive capillary network. A collagen-alginate composite matrix was optimized for the lamina propria-like hydrogel, preserving HIF metabolic activity and morphology over time. To achieve immune competence, pre-differentiated THP-1-derived macrophages were incorporated into the epithelial barrier. Inflammation was induced through the optimization of an inflammatory cocktail consisting of E. coli O111:B4 lipopolysaccharide combined with a specialized cytokine array (tumor necrosis factor-α, interferon-γ, and interleukin-1β). This inflammation-inducing stimulus led to a significant upregulation of pro-inflammatory cytokines commonly associated with IBD onset, including CCL20, IL-6, CXCL9 and CXCL10. Altogether, this 3D in vitro model has the potential to accelerate the drug development pipeline by providing reliable permeability and efficacy outputs for emerging therapies, reducing unnecessary animal experiments. Moreover, it offers a valuable in vitro platform for studying IBD pathophysiology and cell interplay dynamics.