Optimisation of ultrafine particle exposure in an alveolar tri-culture model at the air-liquid interface

Air-liquid interface (ALI) systems have emerged as a physiologically relevant in vitro platform for evaluating the toxicological impact and potential health effects of airborne pollutants. When utilizing collected ultrafine particles (UFPs), application volume and liquid type are critical parameters. Using a smaller volume of liquid for the cell exposure results in a heterogeneous distribution of UFPs across the cell monolayer, whereas application of a sufficient volume optimises even UFP distribution. A buffered solution for UFP administration minimises potential side effects and unravels dose-dependent effects in toxicological endpoints. However, standardised exposure methodologies limit reproducibility and comparability across studies. Therefore, we propose a refined manual exposure technique of suspended airborne pollutants in an adequate exposure volume that bridges the gap between conventional submerged cultures and ALI systems. Our model uses cell culture inserts with A549 epithelial cells, THP-1 macrophages, and EA.hy926 endothelial cells to mimic the in vivo alveolar barrier within the lungs. This approach offers a balance of experimental reproducibility whilst addressing the current challenges of standardisation and feasibility in exposure studies with manual UFP exposure. In conjunction with existing aerosol ALI continuous flow exposure systems, our studies are advancing translational in vitro evaluations, aligning with the 3R principle.