A stratified human colon model reveals SCFA cross-feeding-driven host-microbe dynamics associated with epithelial and immune responses.

Host-microbe interactions are essential for maintaining intestinal homeostasis, yet most in vitro models lack physiological oxygen gradients, mucosal architecture, and reproducibility. We developed the Mucosal Mimetic 3D-Host Aerobic Microbiota Anaerobic Co-culture (Mm 3D-HaeMan) system, a colon-mimetic, oxygen-stratified platform enabling stable co-culture of colonic epithelial cells, human peripheral blood mononuclear cells (hPBMCs), and obligate anaerobes. Oxygen profiling confirmed a stable gradient consistent with human colonic stratification. Using a canonical short-chain fatty acid (SCFA) cross-feeding pair (Anaerostipes caccae and Bifidobacterium bifidum, AcBb consortium), we performed integrated, multilayered analyzes of the host-microbe interface. Co-culture with the SCFA-producing AcBb consortium was associated with enhanced epithelial-mitochondrial respiration, maintenance of transepithelial resistance, tight junction protein expression without barrier disruption, and rebalanced cytokine profiles. Host transcriptomics identified 41 differentially expressed genes (15 upregulated, 26 downregulated) associated with homeostatic-relevant epithelial responses. Simultaneously, bacterial metatranscriptomics revealed 29 differentially expressed gene families; notably, glaK was enriched in bacteria-only cultures; SORD, nuoG, and nuoE were specifically induced during co-culture, indicating host-associated bacterial transcriptional changes. The compartmentalized design permits independent sampling for layer-resolved multi-omics and functional readouts. These findings establish Mm 3D-HaeMan as a physiologically grounded platform for dissecting bidirectional host-microbe responses and immune-associated interactions. This model offers broad applicability for mechanistic research and translational screening of probiotics and microbiome-based therapeutics.