An investigation into E-cigarette cytotoxicity in-vitro using a novel 3D differentiated co-culture model of human airways

Abstract

Currently there is a lack of consensus on the possible adverse health effects of E-cigarettes (ECs). Important factors including cell model employed and exposure method determine the physiological relevance of EC studies. The present study aimed to evaluate EC cytotoxicity using a physiologically relevant in-vitro multicellular model of human airways. Human bronchial epithelial cells (CALU-3) and pulmonary fibroblasts (MRC-5) were co-cultured at air-liquid-interface for 11-14 days post which they were exposed to whole cigarette smoke (WCS) or EC vapour (ECV) at standard ISO-3308 regime for 7 m using a bespoke aerosol delivery system. ECV effects were further investigated at higher exposure times (1 h-6 h). Results showed that while WCS significantly reduced cell viability after 7 m, ECV decreased cell viability only at exposure times higher than 3 h. Furthermore, ECV caused elevated IL-6 and IL-8 production despite reduced cell viability. ECV exposure also produced a marked increase in oxidative stress. Finally, WCS but not ECV exposure induced caspase 3/7 activation, suggesting a caspase independent death of ECV exposed cells. Overall, our results indicate that prolonged ECV exposure (≥3 h) has a significant impact on pro-inflammatory mediators' production, oxidative stress and cell viability but not caspase 3/7 activity.

Mechanisms of the lipopolysaccharide‑induced inflammatory response in alveolar epithelial cell/macrophage co‑culture

“…However, increased levels of the proinflammatory cytokine, TNF-α, were observed following 6, 24 and 48 h of LPS exposure, and increased levels of IL-1β, IL-6 and IL-8 were observed across all time intervals from 6 to 48 h, which indicated that these cytokines may be important factors for maintaining persistent inflammation. Some co-culture models of chronic inflammatory have been studied for more than 7 days (19,28); in the current study, EpCs and MPs viability following LPS treatment was monitored for 7 days; however, the A549 (EpCs) cells began to die after 72 h (the OD value was quite low), whereas THP-1 cells (MPs) grew well from 0 to 7 days (data not shown). In a previous study, the inflammatory status of airway epithelial cells and MPs was determined at 24 and 48 h (29), thus in the present study, the inflammatory effects were observed until up to 48 h of co-culture.…”

A lower impact of an acute exposure to electronic cigarette aerosols than to cigarette smoke in human organotypic buccal and small airway cultures was demonstrated using systems toxicology assessment

“…Numerous studies have evaluated the potential toxicity of ECs in vitro [10–14]; however, a large number of in vitro studies examined the effects of the EL that were applied directly on cell cultures but not the effects of exposure to the vapor/aerosol [15–22]. Although such studies may provide insights into the potential toxicity of the EL, they do not reflect the real exposure to EC; in reality, the human respiratory system is exposed to the EC aerosols and not to the EL.…”

A Summary of In Vitro and In Vivo Studies Evaluating the Impact of E-Cigarette Exposure on Living Organisms and the Environment

“…Similarly, there is a lack of consensus on the possible harmful effects of e-cigarette vapor on a 3D differentiated co-culture model of human airways. The study, conducted on a multicellular model of human cells comprising human bronchial epithelial cells (CALU-3) and pulmonary fibroblasts (MRC-5), indicate that exposure to e-liquid ingredients including PG:VG, 16 mg/mL nicotine and proprietary flavors significantly impact cell viability, proinflammatory mediator production and oxidative stress [118]. In turn, a study on EpiAirway tissue, a human airway culture derived from primary human tracheal/bronchial epithelial cells, demonstrated similar cell viability to untreated controls following exposure to commercially available e-cigarette aerosols, with and without menthol flavoring, containing 4.5% and 3% nicotine by volume [119].…”