Hanging drop: An in vitro air toxic exposure model using human lung cells in 2D and 3D structures

► Validated for the first time a hanging drop (HD) air toxic exposure method for inhalation exposures effect studies. ► Applied a four compartment mass balance model to calculate cellular exposure levels. ► Compared A549 2-dimentional (2D) cell culture and 3D cell cultures benzene sensitivity. ► Carried out up to 20 days in vitro exposure studies. ► Validated A549 cell hanging drop toluene and xylene exposure EC50 value with published static apical air exposure method. Using benzene as a candidate air toxicant and A549 cells as an in vitro cell model, we have developed and validated a hanging drop (HD) air exposure system that mimics an air liquid interface exposure to the lung for periods of 1h to over 20 days. Dose response curves were highly reproducible for 2D cultures but more variable for 3D cultures. By comparing the HD exposure method with other classically used air exposure systems, we found that the HD exposure method is more sensitive, more reliable and cheaper to run than medium diffusion methods and the CULTEX® system. The concentration causing 50% of reduction of cell viability (EC50) for benzene, toluene, p-xylene, m-xylene and o-xylene to A549 cells for 1h exposure in the HD system were similar to previous in vitro static air exposure. Not only cell viability could be assessed but also sub lethal biological endpoints such as DNA damage and interleukin expressions. An advantage of the HD exposure system is that bioavailability and cell concentrations can be derived from published physicochemical properties using a four compartment mass balance model. The modelled cellular effect concentrations EC50cell for 1h exposure were very similar for benzene, toluene and three xylenes and ranged from 5 to 15mmol/kgdry weight, which corresponds to the intracellular concentration of narcotic chemicals in many aquatic species, confirming the high sensitivity of this exposure method.