1. 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

2. 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?

3. 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

4. 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)

5. 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

6. 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

7. 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

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

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

10. 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

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

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

13. 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< compared to control

14. 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.

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

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

17. 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.

18. 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.

19. Thank you!