Towards an in-vitro multi-cellular human airways model to study E-cigarette effects Vasanthi Bathrinarayanan, P.1 Marshall, L.J.2 Brown, J.E.P.2 Leslie, L.J.1 1School of Engineering and Applied Science, Aston University, Birmingham. B4 7ET. UK; 2 School of Health and Life Sciences, Aston University, Birmingham. B4 7ET. UK. email: vasanthp@aston.ac.uk Introduction Co-culture model vs. Rodent model Despite being poor representations of human airways, innumerable animals especially rodents have been employed in cigarette smoking related studies. The rate of lung development and maturation, mucin production, ciliation of epithelial cells, positioning of bronchial glands are quite different between rodents and human1. Additionally, enforced nasal delivery of cigarette smoke in rodents is distinctly different from a human inhaling cigarette smoke through their mouth. The advent of E-cigarettes (EC) could herald further rodent models with data from in-vivo experiments already increasingly published2,3. Relevant cell types Physiological exposure Better repeatability Assess cell types in isolation Cheaper Quicker Cigarette smoke/ECV Aims of the study Species /individual variation Non-physiological exposure (nose breathers) Nicotine/tar absorption through skin Inter-strain variability Expensive Time-consuming Sacrifice of animals The main aim of the current study was to study EC effects using two novel aspects: An in-vitro multi-cellular human airways model as an effective replacement to the animal models An in-house built smoking machine delivering whole cigarette smoke (WCS) or EC vapour (ECV) following ISO human smoking patterns Materials and Methods Cigarette smoke/ECV 1. In-vitro multi-cellular human airways model: Human pulmonary fibroblasts (HPF) and bronchial epithelial cells (CALU3) were co-cultured at air-liquid interface (ALI) for 11-14 days on permeable snapwell (SW) supports. Post 11-14 days, the cells start producing in-vivo physiologically relevant features such as tight junction formation, cilia production and mucin producing goblet cells. Figure 3. Comparison between our in-vitro methodology and the outdated animal model used in smoking studies. The main advantage the co-culture model is the direct exposure of relevant human upper respiratory tract cell types to WCS or ECV in a physiologically relevant fashion mimicking human smoking behaviour. Additionally, the cell response can be analysed immediately as opposed to the time-consuming animal models. Results Growth medium Pulmonary fibroblasts Bronchial epithelial cells Goblet cells Cilia Mucin 1 Strawberry ECV impacts cell viability only at higher exposure times Figure 1. Diagrammatic representation of the multi-cellular co-culture human airways model. 2. In-house built smoking machine: The smoking machine was constructed using diaphragm pumps, solenoid valves and flow meters. A typical 1 minute cycle consisted of air being delivered to the co-culture model for 58s at a rate of 0.150 L/min, representing normal breathing between puffs (green line). For 2s, Cigarette or EC was puffed at a rate of 1L/min (Red line). The process was carried out for 7 min according to ISO 3308 standards. Figure 4 (A). The co-culture models were exposed to air (control), WCS and Strawberry ECV for 7 min using the smoking machine and cell viability was analysed after 24h using standard XTT assay. While WCS causes a significant decrease in cell viability after 7 min exposure, ECV causes a significant decrease in cell viability only at higher exposure times (6h) (B). N=3, each bar represents Mean±SD from 3 SW’s. ****= p<0.0001 compared to air control. Inlet tube Flow meter Solenoid valve Pumps 58s (Air) 2s (EC) V1 P1 V3 F1 F2 F3 V2 P2 Power supply EC holder Vent Gas diffusion system Perspex blocks Human airways model 2 Conclusions and future work The in-vitro multi-cellular human airways model offers better human in-vivo physiological relevance compared to the rodent models. Model Validation: WCS caused a decrease in cell viability to less than 70% that of the control and hence proved cytotoxic. This result was comparable to the data obtained from animal models4, thus validating the human airways model and the smoking machine. ECV was cytotoxic only after extended period of exposure (6h). Short term exposure (<2h) did not have any influence on the cell viability. EC exposure method and duration can influence EC data Exposure method: A smoking machine which can deliver ECV closely mimicking human smoking/vaping fashion Exposure duration: Currently there are no EC standard testing methods which states a specific EC exposure method and time. We propose that a standard testing protocol for EC safety testing which involves multiple human airway cells cultured at ALI and exposed to EC in a physiologically relevant fashion be should be developed as soon as possible. Future works include investigating expression of pro and anti-apoptotic genes, reactive oxygen species generation post ECV exposure at different time-points. References Figure 2. Schematic illustration of working of in-house built smoking machine. The main purpose of the smoking machine was to mimic human smoking pattern as per ISO standards. The flow rates and puff parameters were computer controlled and hence it can be operated under different regimes, thus adding flexibility to the system. Wright et al (2008). Am J Physiol Lung Cell Mol Physiol. 295(1): L1-L15 McGrath-Morrow et al (2015). PLoS ONE 10 (2)L: e0118344 Sussan et al (2015). PLoS ONE 10 (2)L: e0116831