Epithelial model-dependent host-microbe interactions in human intestinal models under anaerobic Transwell co-culture

The intestinal epithelium and gut microbiota exist at a dynamic aerobic-anaerobic interface that is difficult to reproduce in vitro because epithelial cells require oxygen, whereas many gut bacteria are oxygen-sensitive. Here, we used an accessible anaerobic Transwell co-culture framework to systematically compare epithelial model-dependent host-microbe interaction outcomes, rather than positioning the system as a fundamentally new device. Using this platform, we compared the widely used tumor-derived Caco-2 cells with human intestinal epithelial cells (hIECs) derived from pluripotent stem cells to evaluate how epithelial cell origin influences microbial growth, metabolite dynamics, and host responses. Across multiple representative bacterial species, epithelial model choice strongly affected co-culture outcomes. When interpreted relative to each model's corresponding medium-only control, hIEC-based cultures tended to better support the growth or persistence of Lactiplantibacillus plantarum, Bifidobacterium longum, and Akkermansia muciniphila, whereas Caco-2-based cultures showed stronger growth responses for Escherichia coli and Clostridioides difficile. Distinct differences in short-chain fatty acid production and compartmental distribution further suggested that metabolite readouts depend on epithelial model context. Microbial co-culture increased expression of intestinal transcription factors, stem cell-associated genes, and ZO-1 in Caco-2 cells, whereas hIECs exhibited distinct transcriptional responses. Notably, under the defined hIEC culture conditions, exposure to the opportunistic pathogen E. coli was associated with increased expression of epithelial markers related to differentiation, lineage specification, and maturation, whereas Caco-2 cells showed increased cell death. Together, these findings suggest that epithelial cell origin is an important factor shaping in vitro host-microbe interaction outcomes and demonstrate that side-by-side comparison under an anaerobic Transwell co-culture framework can reveal model-dependent responses that may be obscured in single-model studies.