Evaluating the biological effects of electronic cigarettes using a novel in-house designed aerosol delivery system and an in-vitro co-culture model of the human airways

Although electronic cigarettes (ECs) have been widely promoted as safer alternatives to tobacco cigarettes, limited scientific data is currently available on their possible health effects. The current study aims to investigate the potential effects of ECs using a novel in-house designed cigarette/EC aerosol delivery system and physiologically relevant 2D and 3D in-vitro human airway models. Submerged cultures of BEAS 2B and CALU 3 bronchial epithelial cells were used to investigate the effects of nicotine and its oxidative metabolite cotinine. Results demonstrated that neither nicotine nor cotinine had any significant impact on the bronchial epithelial cell viability or IL-6/IL-8 pro-inflammatory mediators’ production. Further, treatment of submerged cultures of a number of airways related cell types to extracts of commercially available ECs of different nicotine strength, flavourings and brands showed that while the differing nicotine strengths had no impact on the cell viability, flavourings significantly influenced cell viability, with strawberry and cherry flavoured ECs demonstrating the highest cytotoxicity. Moreover, same flavours from different brands produced different effects on cells. Finally, a co-culture human airways model consisting of CALU 3 bronchial epithelial cells and MRC-5 pulmonary fibroblasts cultured at air-liquid-interface were treated to whole cigarette smoke (WCS) and EC vapour (ECV) at different exposure times (7 m, 1 h, 2 h, 3 h, 4.5 h and 6 h) as per the ISO:3308 standard smoking regime using an in-house designed bespoke, automated aerosol delivery system. Results demonstrated that while WCS caused a significant reduction in cell viability post 7 min exposure, ECV produced cytotoxic effects only at exposure times ≥ 3 h. A significant increase in oxidative stress and IL-6/IL-8 production was observed post 3 h ECV treatment, both of which are hallmark characteristics of airway inflammatory conditions like chronic obstructive pulmonary disorder. Overall, results from the current study suggest that ECs have the potential to cause substantial airways damage, and as yet, cannot be regarded as safe alternatives to tobacco cigarettes. Importantly, a standardised testing method is urgently required in order to elucidate on the long-term health effects of ECs