In vitro determinants of e-cig aerosol condensate bioavailability and toxicity: Influence of cell culture parameters and utility of a normalized dose metric in e-cig studies

Electronic cigarettes (e-cigs) have a strong foothold in the marketplace as a product to replace tobacco cigarette usage. Despite many researchers investigating the use of e-cigs and possible health issues, there is still controversy concerning how to evaluate and use e-cig condensates. Therefore, to identify factors that influence in vitro e-cig studies, we examined parameters that can impact experimental outcomes. We generated high wattage e-cig aerosol condensate (ECAC) to determine reproducible conditions to evaluate ECAC with respect to cellular survival. Cytotoxicity of ECAC was independent of serum conditions. However, cytotoxicity of ECAC is altered by treatment duration and by physical factors, including cell seeding density and volume of ECAC used. In addition, interactions between ECAC components and cells, as well as the culture vessel surface, diminish the bioavailability of ECAC components in vitro and altered the results obtained. Moreover, the cell seeding density changes reactive oxygen species production in response to ECAC exposure. Our data indicated that normalized ECAC doses (ECAC weight per cell) better reflect the toxicity of ECAC than nominal doses (ECAC percentage). These results provide factors for researchers to consider in the design of in vitro experiments using ECAC.