MBRA 3.0: integrating the mucus environment for advanced high-throughput in vitro intestinal microbiome modeling.

The colonic mucus layer is a dynamic barrier that plays central roles in intestinal health, and recent studies highlight that it harbors a distinct and functionally critical microbial community. However, most in vitro gut models fail to recapitulate this mucosal niche, limiting mechanistic investigation of microbiota-mucus interactions. Here, we developed the MBRA 3.0 system, a next-generation chemostat engineered to integrate mucus-coated carriers and enable high-throughput dissection of spatial microbiome dynamics. Using fecal microbiota from eight human donors, we report that mucus addition does not impact total bacterial density but selectively shapes microbial community structure, metabolic output, and pro-inflammatory potential in a donor-dependent manner. Notably, MBRA 3.0 resolves stable, compositionally distinct mucus-associated and luminal communities, mirroring in vivo spatial heterogeneity. Integration of this mucosal niche also modulates short-chain fatty acid (SCFA) profiles and inflammatory signatures, highlighting the relevance of the spatial context for intestinal microbiota research. Hence, MBRA 3.0 offers a scalable and customizable platform to model mucus-microbiota interactions, advancing our understanding of gut ecology and supporting translational discovery in gastrointestinal health and disease.