An artificial mucus-integrated in vitro model enables stable live probiotic-host co-culture and recapitulates dynamic...

In vitro models for host-microbe interaction often have limited physiological relevance due to the absence of a protective mucus layer, reliance on non-viable bacteria, and short co-culture durations. Here, we present the first scalable and biocompatible in vitro model integrating an artificial mucus barrier that enables stable 48-hour co-culture of live Lactiplantibacillus plantarum OLL2712 with Caco-2 cells at exposure ratios up to 1,000. This model maintained epithelial viability, supported bacterial proliferation, and enabled dynamic host responses. Notably, during co-culture, while hBD-2 gene expression was observed under both with-mucus and without-mucus conditions, protein secretion occurred only in the presence of mucus, reaching approximately a 100-fold higher level than previously reported. This finding underscores the essential role of the mucus barrier in preserving epithelial function and maintaining the downstream host response. The model recapitulates a complete host-microbe feedback loop involving microbial stimulation, antimicrobial peptide (hBD-2) secretion, and subsequent bacterial suppression, thereby linking epithelial defense activation to microbial regulation. It provides a reproducible, physiologically relevant, and animal-free platform for probiotic screening and mechanistic studies of mucus-associated intestinal disorders such as inflammatory bowel disease.