Assessing E-cigarette cytotoxicity using two in-vitro methodologies Pranav Vasanthi Bathrinarayanan, M.Sc PhD Student- Mechanical Engineering, School of Engineering and Applied Science, Aston University, Birmingham, UK Supervised by: Dr. Laura Leslie, School of Engineering and Applied Science Co-supervised by: Dr. James Brown, School of Life and Health Sciences Dr. Lindsay Marshall, School of Life and Health Science

Introduction The impact of E-cigarettes (ECs) on human health cannot be conclusively determined with the current limited available information on their effects. Studies on EC cytotoxicity often report contradictory findings. For Eg: Lerner et al reported that EC induces cytotoxicity and oxidative stress Misra et al reported that EC did not induce any cytotoxic or genotoxic effects How does this contradiction arise?

Obstacles to effective EC research Plethora of E-cigarette types- 1st gen, E-pen, Tank model Variations in composition of E-liquid Inconsistencies in labelling of constituents Source of funding Most importantly… There are no standard testing methods to evaluate E-cigarette safety Pictures of E-cigs

Issues with the research performed so far Lack of relevance with respect to: Models employed (human vs rodent) Cell lines chosen (airway cells vs other cell types) Complexity of model (multiple vs single cell) Exposure method (extracts vs vapour)

Aims of the study The main aims of the current study is to Assess EC cytotoxicity using two in-vitro methodologies Demonstrate the influence of cell model, cell type and exposure method on safety data

Methodologies employed in this study Methods Methodologies employed in this study Exposure method Cell models Whole smoke/EC vapour (ECV) Aqueous extracts Submerged monocultures ALI Co-cultures

Aqueous extract collection Strawberry EC/Cigarette was drawn at a rate of 35mL/2s followed by a break of 28s for a total time of 7 minutes. Flow metre 12V pump Power supply Growth medium EC/Cigarette Cigarette holder Inlet tube Outlet tube

Exposure of submerged monocultures CSE/ECE ANALYSE RESPONSE AFTER 24h Epithelial cells= BEAS 2B or CALU 3 Fibroblasts= Human pulmonary fibroblasts (HPF)

Co-culture human airways model at ALI MUCUS CILIA GOBLET CELLS EPITHELIAL CELLS PULMONARY FIBROBLASTS GROWTH MEDIUM

In-house built smoke/vapour delivery system CO-CULTURE AIRWAYS MODEL V3 F1 F2 F3 FLOW METER SOLENOID VALVE V1 V2 12v PUMPS P.S: Not to scale P1 P2 POWER SUPPLY

Exposure of ALI co-cultures to smoke or vapour Cigarette smoke OR EC vapour from smoking machine ANALYSE RESPONSE AFTER 24h

1. Effect of extracts on submerged monocultures Results 1. Effect of extracts on submerged monocultures

N=3, quadruplicate samples from each repeat N=3, quadruplicate samples from each repeat. Each bar represents Mean±SD. Statistical analysis using One-way ANOVA where ****= p<0.0001 compared to control

Results summary: Submerged cultures Both, ECE and CSE caused a significant decrease in cell viability compared to control. While CSE was cytotoxic (<70%) in all three cell types, ECE was cytotoxic only in BEAS 2B. Not all cells responded similar to the extracts BEAS 2B was found most susceptible to the extracts.

2. Effect of whole smoke/vapour on ALI co-cultures

N=3, where each repeat represents an individual SW N=3, where each repeat represents an individual SW. Each bar represents Mean±SD.

Discussion In both methods, EC appeared less cytotoxic than tobacco cigarettes. Extracts indicated more toxicity than their whole smoke counterparts. This could be due to longer exposure time (24h) of submerged cultures compared to ALI cultures (7 min) Submerged cultures exposure to extracts are quick and easy to repeat but less physiologically relevant. ALI co-cultures exposure to WCS/ECV are expensive and time consuming but are physiologically relevant.

Conclusions Cell model influence Different cells of airways respond differently to EC’s and hence relevant cells are to be chosen. Delivery system influence A system that can deliver EC vapour in a physiologically relevant manner. The proposed EC standard testing method: An in-vitro ALI cell model involving multiple cells that are exposed to EC in a fashion that mimics human vaping as closely as possible.

Thank you!