Aerosolized fluorescent tracers provide insight into particle deposition and cellular uptake at the air-liquid interface
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Aerosols present unique challenges for in vitro toxicity evaluation with New and Alternative Methods (NAMs). They can be difficult to generate and transport in a reproducible manner and require complex air-liquid interface (ALI) exposure systems to mimic realistic inhalation exposures. Deposition and cellular uptake following in vitro ALI exposures are rarely reported which prevents dose-response modeling and comparison across exposure systems. A possible alternative is to re-submerge ALI cell cultures and utilize a direct-dosing method which simplifies exposure methods for high-throughput screening. However, re-submersion of primary human bronchial epithelial cells (pHBEC) grown at the ALI disrupts airway epithelial barrier integrity and changes physicochemical properties of particles which may affect cellular uptake and toxicity. To assess possible dosimetric differences between exposure methods, we compared cellular uptake and basolateral translocation of two fluorescent tracers (fluorescein and rhodamine) that were delivered as liquid aerosols at ALI or by direct-dosing in a human bronchial epithelial cell line (16HBE) and pHBECs. We found that the delivery method altered cellular uptake, basolateral transport, and mucus retention of fluorescent tracers. In 16HBEs, cellular uptake of rhodamine and basolateral transport of fluorescein reached a maximum at ALI and decreased as direct-dosing volumes increased. We also observed volume-dependent changes in mucus retention of rhodamine in pHBECs. Furthermore, these tracers reveal that cell-free matrices may not accurately estimate cell deposition in ALI exposure systems. Together, these data highlight the need for improved analytical assays to support inhalation NAMs and suggest that direct-dosing studies may not be equivalent to ALI exposures. [Abstract does not reflect views or policies of the U.S. EPA.]