Testing of Aerosols for Lung Toxicity by In-Vitro Studies at the Air-Liquid Interface for up to 24 Hours

The state of the art of studying the health effects of aerosols in vitro is based on submerged exposure of collected particulate matter, suspended in culture medium. However, this method neglects the gas phase including their interactions with particles and cells. It may change the properties of the investigated particles and does not represent the actual process in the human lung. Exposure at the Air-Liquid Interface (ALI) avoids these disadvantages, but requires a comprehensive system to guarantee reproducible conditions. Therefore, KIT and VITROCELL Systems developed a fully automated ALI exposure station. The exposure station offers a complete measurement system for parallel exposure of up to 24 human lung cell cultures towards gases, nanoparticles and complex mixtures such as combustion aerosols. The aerosol flow, temperature, and humidity are adjusted to the conditions resembling the human lung. An internal negative control using humidified synthetic air is also implemented and the particle dose per time can be increased by electrostatic particle deposition. The particle mass per area deposited by diffusional as well as by electrostatic mechanism is monitored online using a quartz crystal microbalance. Additionally, a new tool to reproducibly expose sample grids for transmission electron microscopy was developed and applied. Image evaluation of TEM images delivers dose information with respect to the spatial distribution and the agglomeration state of the deposited particles. Applications of the ALI exposure station are environmental atmospheres and technical emission sources like marine diesel engines or wood combustion. Long-term stability of A549 lung cells was examined for exposure times up to 24 hours by exposing A549 cell cultures towards clean air as well as towards airborne titanium dioxide and copper oxide nanoparticles. Dose measurement data and biological responses as viability (AlamarBlue assay), cytotoxicity (LDH release), and release of cytokines during long-term exposure are reported.