For this purpose, in this study we exposed primary normal human bronchial epithelial (NHBE) cells directly to vapor of two different e-cigarette liquids (0% and 2.4% nicotine). The cells were cultivated on semi-permeable membranes of cell culture inserts and are kept air-lifted in the exposure device, a CULTEX ® RFC compact module. The cell surface is exposed to the surrounding atmosphere and the nutrient supply is realized from the basal side of the cells. We exposed the cultures to e-liquid vapor from 200 puffs and analyzed cell viability and the oxidative stress level in the cells 24 h post exposure. Furthermore, we exposed cells to vapor of the carrier substances glycerol and propylene glycol using the same puff profile and puff numbers. Clean air exposed cells and cells left air-lifted in the incubator were used as positive controls as well as cells exposed to mainstream smoke of 10 cigarettes as negative controls.

Although these investigations have suggested cytotoxic effects of several e-liquids, the significance of the results is limited, since the cell types used in those studies are not in direct contact with e-cigarette liquid in the human body. The primary target organ of e-cigarette vapor is the respiratory tract and therefore, lung-derived cell cultures should be the in vitro model of choice.

Only few studies are available about the health effects of e-cigarettes, which can be divided into chemical, clinical and toxicological studies. In chemical studies, the liquids are analyzed for their ingredients, but mostly only toxicants known from cigarette smoke are investigated, and the search for other unknown, possible toxic compounds is often not included. In clinical trials, users are examined after short-term usage of e-cigarettes with regard to respiratory function and cardiovascular system responses. However, long-term studies indicating possible developments of clinically evident diseases are missing. Toxicological studies comprise a few animal experiments about the effects of inhaled glycerol or propylene glycol and somestudies using established cell lines [ 9 ]. These studies comprise exposure of human embryonal stem cells, human pulmonary fibroblasts and murine neuronal stem cells to e-cigarettes liquids as well as exposure of fibroblasts and cardiomyoblasts to e-cigarette vapor extracts [ 10 13 ].

Electronic cigarette liquids generally consist of a mixture of glycerol and propylene glycol (both used as carrier substances), flavorings and optionally, variable concentrations of nicotine. Although all ingredients, with exception of nicotine, should be approved as food additives, their harmlessness is not proven, since their approval is not necessarily meaningful for their inhalational use. The butterscotch flavor diacetyl for example, which is safe to eat but leads to a severe lung condition known as “popcorn lung” or bronchiolitis obliterans when inhaled, has been found in e-cigarette liquids [ 8 ]. Besides that, the effects of the heated ingredients are unknown.

However, the regulation and control of e-cigarette liquids is neither standardized nor adequate to ensure sufficient quality and safety for the user. Former studies have shown that the labeling and the actual content can differ significantly, for example with regard to nicotine concentrations deviating from the declaration or nicotine-presence in nicotine-free labeled liquids. Furthermore, toxic substances such as nitrosamines and diethylene glycol as well as pharmacological components like rimonabant and aminotadalafil have been found in e-cigarette liquids [ 5 7 ].

In the early 2000s, electronic cigarettes were introduced as an aid for smoking cessation and replacement and in the last few years, the consumption has risen significantly. In 2012, about 288 different e-cigarette brands were available online, whereas this number increased to 466 in January 2014 [ 1 ]. Studies have shown, that the usage of e-cigarettes can help smokers not intending to quit to significantly reduce their cigarette consumption or even stop entirely [ 2 4 ].

2. Materials & Methods

2.1. E-Liquids & Cigarettes The tested refill e-liquids were purchased from Johnsons Creek (Hartland, WI, USA). The e-liquids with the flavor “Tennessee Cured” are propylene glycol based and have nicotine concentrations of 0.0% and 2.4% (24 mg/mL). The ingredients of the liquid are listed on the bottle and are as followed: USP grade propylene glycol, USP grade vegetable glycerol, deionized water, natural flavors, artificial flavors, USP grade nicotine (not in 0.0%), USP grade citric acid (as a preservative). Propylene glycol and glycerol were obtained from Alfa Aesar (Karlsruhe, Germany), with a purity of 99.5%. For cigarette smoke exposure, K3R4F research cigarettes (University of Kentucky, Lexington, KY, USA) with a standard cellulose acetate filter tip were used.

2.2. Cell Isolation & Cultivation Normal human bronchial epithelial (NHBE) cells were isolated from ring-shaped bronchus samples of two different donors, and the obtained cells were named NHBE48 and NHBE33. The samples were received from a male patient (age 69) with a lung adenocarcinoma in the right upper lobe (NHBE33) and from of a 75-year old cancer patient during a wedge resection of the upper lobe (NHBE48). Both samples were obtained from the Bielefeld Evangelical Hospital (Bielefeld, Germany). In accordance with the Declaration of Helsinki, both subjects gave their informed consent to the research use of the removed lung tissue samples. Upon arrival in our laboratory, the bronchus samples were incubated for 24 h at 4 °C on a rocking platform in incubation medium (MEM medium containing dithiothreitol (0.5 mg/mL), DNase (10 µg/mL) and antibiotics (40 µg/mL tobramycin, 50 µg/mL vancomycin, 50 µg/mL ceftazidime, 2.5 µg/mL amphotericin B, 50 U/mL penicillin/streptomycin)). Afterwards, the ring shaped samples were transferred into a PBS-containing Petri dish, opened longitudinally, isolated from residual parenchyma and cut into smaller pieces of approximately 8 × 5 mm. The bronchus pieces were then placed into cryovials, containing DMEM with 10% FCS and 10% DMSO and frozen to −80 °C. After storage at −80 °C overnight, the vials were moved to a liquid nitrogen tank and stored until needed. For cell isolation, the samples were thawed in a water bath at 37 °C, transferred into Petri dishes and rinsed with PBS after removal of the freezing medium. Incubation medium containing 0.1% protease XIV was added and the samples were incubated for 1–2 h at 4 °C on a rocking platform. Afterwards, bronchial epithelial cells were isolated by thoroughly scraping the luminal surface of the bronchus pieces with a scalpel. The cell suspensions were homogenized, pipetted into centrifugation tubes and centrifuged for 10 min at 170× g . The resulting cell pellets were resuspended in 4.5 mL–9 mL AEGM medium. The cell suspensions of each sample were then equally divided to two collagen IV coated wells of a 6-well plate to grow in culture. After the first passage, NHBE cells were cultivated in collagen IV coated culture flasks using AEGM Medium. After reaching 80%–90% confluence, the cells were seeded on collagen IV coated cell culture inserts (seeding density: 2.1 × 105/cm2). The cells were cultivated under submerged conditions and supplied with AEGM medium for 1 day before the apical medium was removed and the cells were transferred to the exposure module. The exposure experiments were performed with cells of passages 2–4. This procedure was identical for cells of both donors. MEM medium was obtained from Lonza (Basel, Switzerland); PBS, penicillin/streptomycin, DMEM from Biochrom, (Cambridge, UK) and AEGM medium from Promocell (Heidelberg, Germany). All other cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

2.3. Exposure All exposure experiments were performed in a CULTEX® RFS compact module (Cultex Laboratories GmbH, Hannover, Germany), exposing six cell culture inserts at a time in each experimental run. For e-cigarette vapor experiments, a Reevo Mini-S (In-Smoke, Winnenden, Germany) was used, equipped with a 3.3 V/900 mAh battery and a vaporizer with a resistance of 2.2 Ohm. The e-cigarette was connected to the piston pump of a smoking robot and 200 puffs were taken with a puff volume of 35 mL, a puff duration of 2 s and a blow-out time of 7 s. The smoking robot was operated in asynchronous mode, meaning that puffs were taken successively, so that the cells were surrounded by vapor during the whole exposure time. For a better distribution, the e-liquid vapor was diluted with synthetic air (1 L/min) before sucked into the CULTEX® RFS compact via a vacuum pump with a flow rate of 5 mL/min/insert. The exposures to pure glycerol and propylene glycol were performed using the same puff parameters and puff numbers. For mainstream smoke exposure, 10 K3R4F cigarettes were smoked by the smoking robot using the same parameters as described for the e-cigarette. Each cigarette was puffed six times. The freshly generated main stream smoke was equally diluted with synthetic air (1 L/min) and also entered the CULTEX® RFS compact with a rate of 5 mL/min/insert. The clean air exposure (clean air control) was performed for 30 min with the flow rates described above. The flow rates were controlled by mass flow controllers (IQ+ Flow and EL-Flow Select, Bronckhorst, Ruurlo, The Netherlands). The exhaust air was directed back to the fume hood. As a second control, cell cultures were used that remained air-lifted in the incubator for the exposure time (incubator control).

2.4. Analysis The analyses were done 24 h after the exposure to allow the cells to respond to the exposure. In order to analyze cell viability and oxidative stress in the same cell, two cell-based assays were combined. The oxidative stress was analyzed first, using the ROS-Glo™ H 2 O 2 Assay (Promega, Madison, WI, USA). Afterwards, cell viability was measured with the CellTiter-Blue® Assay (Promega). For the ROS-Glo™ H 2 O 2 Assay, AEGM medium (200 µL) and H 2 O 2 substrate solution (50 µL) were added on the surface of the cells. After 3 h incubation at 37 °C/5% CO 2 , 75 µL of the solution was transferred into a white 96 well plate. Detection solution (75 µL) was added and after 20 min incubation at room temperature, the relative luminescence was measured. In order to measure the cell viability in the same cell culture insert, the remaining medium was removed from the cells, and 300 µL AEGM medium as well as 60 µL CellTiter-Blue® solution were added. The cultures were then incubated for 2 h at 37 °C/5% CO 2 . Afterwards, 100 mL of the solution were pipetted into a black 96 well plate to measure the fluorescence at 544 Ex /590 Em nm.