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Results Establishment of the hypoxic intestinal lumen environment To establish a model closely simulating the physiology and microenvironment of the human colon, we aimed to create a system that incorporates the shear stress conditions and the low-oxygen environment present in the colon, while supporting the growth and maintenance of an intestinal epithelial cell culture. After confirming that the model met the requirements for shear stress (SS) and oxygen levels, the characteristics of the Caco-2 cell layer cultured in the DFC under both aerobic and anaerobic conditions were investigated and compared to static cultures in cell culture inserts. Transcriptomic profiling reveals enterocyte maturation in the Caco-2 DFC model To comprehensively investigate the transcriptional profile of the intestinal epithelium cultured in the system, we conducted transcriptomic analyses on Caco-2 cells cultured for 13 days under both aerobic and anaerobic conditions in the apical channel of the DFC model, and statically under aerobic conditions in cell culture inserts. Upregulation of histone genes, a marker of entry into the S phase of the cell cycle26, may indicate an increase in the number of actively dividing cells in the aerobic DFC, aligning with the observed increase in 3D growth compared to the static model. In the untreated control, both B. fragilis and C. difficile multiplied rapidly during the first three days of the infection (based on CFU/mL in the effluent), demonstrating that the low oxygen levels at the apical cell surface are sufficiently low to support culture of these obligate anaerobes.