Perfluorocarbon-liquid interface culture method enhances mucus production and allows evaluation of drug permeability...

Pulmonary drug administration is a promising route for systemic delivery of therapeutics. Airway epithelial cells cultured under air-liquid interface (ALI) conditions are commonly used to evaluate drug permeation across differentiated epithelium. However, ALI culture presents limitations in reproducibility and reliability due to challenges in maintaining adequate moisture at the air-cell interface. In this study, we investigated a novel in vitro culture system in which the air compartment of ALI is replaced with Fluorinert FC-40, a liquid perfluorocarbon that serves as an oxygen carrier, creating a Fluorinert-liquid interface (FLI). Transepithelial electrical resistance values and paracellular permeability in Calu-3 and 16HBe14o- cells were comparable between ALI and FLI, suggesting that FLI supports differentiation similarly to ALI. Furthermore, mucin expressions were elevated under ALI and FLI relative to submerged culture, with FLI showing higher expression. Morphological analysis revealed that FLI culture produced a thicker and more uniform mucus layer than ALI. Drug permeability studies using rose bengal revealed improved epithelial barrier function under FLI. These findings indicate that FLI is a viable alternative to ALI, offering enhanced mucus production and epithelial barrier function. Thus, the FLI culture system provides a physiologically relevant in vitro model for pulmonary drug permeability studies.