Mucin hydrogel fibers enable oxygen-limited co-culture of Bifidobacterium bifidum and intestinal epithelial cells.

The intestinal mucus layer forms the interface between the gut microbiota and the epithelium, and in vitro platforms are needed to study host-microbe interactions at this interface. However, such platforms must reconcile conflicting conditions: obligately anaerobic bacteria require a low-oxygen environment, whereas epithelial cells require conditions compatible with barrier maintenance. In addition, simple co-culture systems lack a glycan-presenting mucus matrix. Here, we established a co-culture platform in a conventional Transwell format without complex microfluidic devices by integrating a mucin matrix with a simple oil-based method for restricting oxygen influx. Using polyethylene oxide (PEO) as a transient spinning aid and removing it after glutaraldehyde crosslinking, we fabricated a three-dimensional porous hydrogel fiber composed predominantly of porcine gastric mucin (PGM). The fiber retained the examined mucin-derived O-linked glycan epitope on its surface. Bifidobacterium bifidum adhered to and proliferated on the fiber, forming a biofilm-like structure and infiltrating the porous network. In nutrient-restricted medium, bacterial growth occurred only in the presence of the fiber; this result demonstrates growth associated with the presence of the fiber but not O-glycan-specific utilization. A mineral oil overlay restricted oxygen influx and, together with oxygen consumption by Caco-2 cells, lowered the dissolved O2 concentration at the sensor position immediately beneath the epithelial layer to approximately 0.1 mL/L (approximately 2% of air saturation). Combining methylcellulose, the mucin hydrogel fiber, and mineral oil enabled co-culture while maintaining both epithelial barrier function, as assessed by transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) staining, and B. bifidum viability, as assessed by reculture. This mucin-predominant, glycan-presenting hydrogel fiber provides an accessible, device-free platform for studying the intestinal host-microbe interface.