A human intestinal epithelial-mesenchyme-immune triple culture system for disease modelling.

Introduction: The intestinal mucosa plays a vital role in nutrient absorption, drug metabolism, and pathogen defence. Advances in single-cell technologies have highlighted the specialised roles of various cell types that execute these diverse functions. Beyond intestinal epithelial cells, fibroblasts regulate the extracellular matrix (ECM) and modulate pro-inflammatory signalling, while antigen-presenting cells (macrophages and dendritic cells) maintain intestinal homeostasis and immune responses. However, the incorporation of such cellular complexity within existing Methods: We developed a human intestinal co-culture model that incrementally mimics the mucosal cellular environment, comprising intestinal epithelial cells, intestinal fibroblasts, and antigen-presenting cells. The model incorporated co-cultures of both adult and foetal cells to facilitate studies of intestinal development, barrier function, inflammation, and viral infections. Results: We successfully established an advanced multi-cellular intestinal co-culture model that recapitulates key features of the native mucosal environment. This model demonstrated the capacity for ECM deposition, Paneth cell differentiation, and functional immune interactions. Notably, the co-culture system effectively modelled immune responses during viral infections, demonstrating the utility of incorporating multiple cell types and developmental stages. Discussion: Our co-culture model improves the physiological relevance of in vitro studies by incorporating multi-cellular complexity previously lacking in existing systems. This advancement enables the exploration of epithelial-mesenchymal-immune crosstalk in intestinal health and disease, providing a powerful platform for studying intestinal development, barrier dysfunction, and infection-related pathology.