Validation of the in vitro comet assay in conjunction with an air‐liquid interface exposure of cigarette smoke in human lung epithelial cells

Currently, most in vitro testing for genotoxicity caused by cigarette smoke is performed using submerged non-human cell cultures. In an effort to improve the cell culture model, we used human lung epithelial cells and grew them on microwell fittings, which induces a more organotypical differentiation. In an effort to improve the exposure model, we then exposed the cells to cigarette smoke in the in vitro single cell gel electrophoresis assay (comet assay) using a novel air-liquid interface exposure system, which better simulates the exposure in inhalation studies. The exposure system (Vitrocell®) consists of a smoking robot, a dilution system, and an exposure chamber with 24 MicroWell fittings for higher throughput. Two human lung epithelial cell lines, BEAS-2B and A549, were exposed to different flows of freshly generated, diluted mainstream smoke from the reference cigarette 3R4F. A clear dose-dependent increase in DNA-damage, expressed as tail intensity, was obtained in all experiments for both cell lines (p ≤ 0.001), with A549 cells demonstrating a higher resistance to genotoxic insults than BEAS-2B cells. Reproducibility and repeatability were acceptable, with a relative standard deviation of approximately 25%. The study shows that the in vitro comet assay in combination with the Vitrocell 24® air-liquid exposure system is able to detect cigarette-smoke-induced DNA damage in a reproducible and repeatable manner with a high throughput.