1. 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.
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 days on permeable snapwell (SW) supports. Post 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 Comparison between our in-vitro methodology and the outdated animal model used in smoking studies.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 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< 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
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