Development of an aerosol-compatible cell culture exposure system and its application to quantify cellular uptake of particles at the air-liquid interface
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Inhalation is the one of the three primary modes of chemical exposure; however, significant challenges in developing reproducible exposure systems, dosimetry methods, and appropriate in vitro models have led to its vast underrepresentation in high-throughput screening initiatives. To meet screening demands for a diverse range of inhaled toxicants, we utilized fluorescent tracers and Computational Fluid Dynamics (CFD) modeling to develop an Aerosol-compatible Cell Culture Exposure System (ACCES) which achieves serial dilution of aerosols and volatile compounds at air-liquid interface (ALI). CFD modeling was a valuable tool to optimize operational parameters, while aerosolized fluorescein and rhodamine quantified cell deposition within the ACCES. Both fluorescent tracers revealed that cell-free matrices (filters, cell culture media, etc) may not accurately predict cell deposition at ALI and should be used with caution. Deposition studies in two other commercial systems, the VITROCELL Cloud and MedTec CelTox, confirmed these results. Furthermore, we compared cellular uptake of fluorescent tracers delivered at ALI or by direct liquid application in a human bronchial epithelial cell line (16HBE) and primary human bronchial epithelial cells (pHBEC) given that liquid application of particles to ALI cultures is often proposed as a time- and cost-effective alternative to ALI exposures. 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 translocation of fluorescein reached a maximum at ALI and decreased as direct-dosing volumes increased. The opposite trend was observed in pHBECs: it appeared that higher apical volumes disrupted the mucus barrier and increased basolateral transport and cellular uptake. Together, these data highlight the need for improved analytical assays to quantify deposition and cellular uptake during in vitro inhalation assays and suggest that direct liquid application studies may not be equivalent to ALI exposures. [Abstract does not reflect views or policies of the U.S. EPA.]