Waterpipe tobacco smoke (WTS) bubbles through water before being inhaled and is a complex mixture of particulate gases formed by the heating of tobacco, flavourings and humectants, usually using an external charcoal piece. To date, there are no validated methods to test the toxicity in an in vitro setting. So far, only a limited number of publications have investigated the effects of WTS on cell cultures, and even less have used 3D human airway epithelia combined with a standardized waterpipe smoking machine. There is a clear need to develop methods to ensure that data is obtained in a repeatable and reproducible manner. Following the puffing regime set in ISO Standard 22486, this study aimed at investigating the optimal system settings of an air-liquid interface (ALI) exposure system (Vitrocell Shisha testing device combined with VC1) to achieve dose delivery with minimal run-to-run variation. This was achieved by using a trapping DMSO solution in the exposure chambers which was analyzed by fluorescence. Subsequently, nicotine was quantified in the same trapping solution.

The method was first validated using reference 3R4F cigarettes smoked according to the Health Canada Intense regime. By simply changing the smoke dilution, a clear dose-response in fluorescence was observed. Waterpipe smoke was then analyzed but did not produce significant delivery with the dilution parameters used for 3R4F. Therefore, puff exhaust time, number of smoking runs, and set-up of the dilution bar were modified to try to achieve a similar deposition as for 3R4F.

This preliminary work is a prerequisite for the interpretation and comparison of future experiments using this system. The standardization of this set-up will identify the parameters required to reach biological significance and extrapolate in vitro results on 3D cell cultures to in vivo models by measuring endpoints such as cell viability, trans-epithelial electrical resistance (TEER) and cilia beating frequency (CBF).