Evaluating the Impact of E-Liquids on Small Extracellular Vesicles Released from Three-Dimensional Cell Culture of Human Lung Cells

Couchman, Alex

Abstract

Small Extracellular Vesicles (sEVs), membrane-bound vesicles used in cellular communication can be used as a source of biomarkers for lung damage and disease, to minimise need for invasive test procedures. E-cigarette use has grown in recent years, but little is known about how e-liquids impact on sEVs, and therefore how they might cause dysregulation in sEV cargo. Previous studies using two-dimensional cultures of human lung cells have identified that sEVs may be impacted by the addition of e-liquids.
This study used a three-dimensional culture protocol for human lung cells, to more accurately model a physiological environment, compared to a two-dimensional monolayer of cells.
Cultures were treated with flavoured e-liquids, with and without nicotine, and the sEVs released were isolated using size exclusion chromatography. These vesicles were characterised using transmission electron microscopy, nanoparticle tracking analysis, fluorescence nanoparticle tracking analysis, and western blotting techniques to confirm morphology and protein marker presence. Total RNA was isolated, and expression of target microRNA was evaluated using qPCR.
Characterisation techniques confirmed that this project was successful in isolating sEVs from three-dimensional cultures, with vesicles exhibiting the correct “cup-shaped” morphology, 50-200 nm size range, and CD9 and CD63 tetraspanin protein markers to be classified. sEVs derived from e-liquid treated cultures did not exhibit significant differences in size or concentration compared to untreated cultures. Total RNA was successfully isolated from all samples, and expression of microRNAs miR-410-5p and miR-21-5p in sEVs derived from A549 cultures showed variation between treated and untreated samples. This indicates that the e-liquid treatments had an impact on microRNA expression in sEVs, as suggested by previous literature.
The results of this study demonstrate that e-liquids did have an impact on the cargo of sEVs in the microRNA tested, and therefore that sEVs derived from three-dimensional cell culture could be a useful tool in establishing the impacts of e-liquids on potential biomarkers for lung disease.

Citation

Couchman, A. (2024). Evaluating the Impact of E-Liquids on Small Extracellular Vesicles Released from Three-Dimensional Cell Culture of Human Lung Cells. (Thesis). University of Salford

• Thesis Type: Thesis
• Deposit Date: May 13, 2024
• Publicly Available Date: Jul 1, 2024
• Award Date: May 31, 2024