| Abstract |
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.
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