A collagen hydrogel-based intestinal model enabling physiological epithelial–immune cell interactions in host–microbe studies†

The human intestine orchestrates complex immune responses to external stimuli, including trillions of gut microbes. Understanding host–microbiota interactions remains challenging due to the complex cellular composition of intestinal tissues. In this study, we present InTRIC (Intestinal model with Tissue-Resident Immune Components), a three-dimensional intestinal model incorporating tissue-resident immune cells within a biomimetic extracellular matrix. We developed a hydrogel system by combining UV-crosslinked methacrylated collagen with a native collagen blend that prevents matrix shrinkage while maintaining physiologically relevant mechanical properties, a fibrillar microstructure and minimal toxicity for cell embedding. Prior to establishing InTRIC, we validated THP-1 viability in the collagen hydrogel (>14 days), verified their differentiation into distinct macrophage phenotypes, and confirmed appropriate gene expression in Caco-2 cells on the collagen substrates. The integrated model comprises THP-1 macrophages embedded within the collagen matrix and Caco-2 cells forming a functional epithelium with intact junctions and physiologically relevant TEER values. The InTRIC platform enables both the assessment of cytokine profiles and the visualization of transepithelial macrophage infiltration in response to commensal (Lacticaseibacillus rhamnosus) and pathogenic (Pseudomonas aeruginosa) bacteria. Notably, P. aeruginosa exposure induced a four-fold increase in macrophage infiltration and elevated IL-8 secretion. Compared to conventional cocultures without collagen, InTRIC exhibited attenuated inflammatory cytokine secretion, suggesting microenvironmental modulation of epithelial–immune communication. Additionally, M2-polarized macrophages showed reduced basal IL-8 secretion but increased responsiveness to bacterial stimulation. This proof-of-concept model demonstrates the importance of incorporating both immune cells and appropriate extracellular matrices in recapitulating intestinal conditions and offers a physiologically relevant test platform for applications in drug discovery, toxicology, and microbiome research.