Aims: Perfluorocarbons (PFCs) are inert, oxygen-rich fluids with applications in neonatal and adult liquid ventilation and potential for innovations related to hypoxic environments, such as under water and for space. Despite promising clinical applications, a laboratory model to comprehensively test PFC implications and predict human airway responses is lacking. We hypothesise that an organotypic airway epithelial cell (AEC) model is needed assess PFC impacts and provide predictive outcomes to inform the in vivo scenario that conventional submerged cultures cannot replicate. This study evaluated PFC exposure in two in vitro systems: a traditional submerged model using 16HBE14o- cells and an air-liquid interface (ALI) model using hSABCi and primary nasal AECs, cultured on transwells to mimic the human airway epithelium. Methods: PFC exposures were conducted at 2, 8, and 24 hours (submerged model) and extended to 72 hours (ALI model). We tracked gross morphological changes via microscopy, quantified apoptosis and autophagy markers through protein biochemistry, and assessed epithelial permeability using trans-epithelial electrical resistance (TEER) and tight junction protein abundance. Statistical analyses included at least three biological replicates per condition. Results: In the submerged 16HBE14o- model, PFC exposure led to significant apoptotic changes by 24 hours, with marked autophagic disruption. Nutrient deprivation, confirmed by starvation experiments, was a key driver of cytotoxicity due to media/PFC phase separation. Conversely, hSABCi cells in the ALI model remained viable, with no apoptosis or autophagic disruption over the exposure periods (P > 0.05 vs. control). Similarly, primary nasal AECs showed consistent viability and stability in autophagic and apoptotic markers, indicating a more accurate representation of in vivo conditions. TEER measurements and tight junction protein levels in the ALI model suggested PFC did not compromise epithelial integrity (P > 0.05 vs. control). PBS exposure, included as a liquid control, underscored baseline sensitivity in the primary nAEC model, evidenced by SQSTM1 upregulation and pronounced barrier dysfunction over 24-72 hours (P < 0.05-0.001). These findings underscore the unique biocompatibility of PFC in maintaining cellular integrity, in contrast to the disruptive effects observed with PBS. Conclusion: This is the first study to describe PFC exposure in an organotypic airway model. Results indicate that the ALI model more accurately preserves airway epithelial integrity during PFC exposure than submerged models, which are limited by nutrient depletion effects. The findings support the use of ALI cultures to replicate the human airway architecture for evaluating PFC's biological effects and offer a platform for preclinical applications in respiratory medicine. This organotypic approach may inform future therapeutic and hypoxia-related interventions and contributes significantly to the field by providing a viable model for understanding PFC interactions with airway epithelial cells. We are now trialling PFC emulsions that also contain azithromycin and steroid therapies for extended therapeutic applications.
