Evaluation of a high-throughput in vitro endothelial cell migration assay for the assessment of nicotine and tobacco delivery products

• Published in: Toxicology letters 2020-11, Vol.334, p.110-116
• Main Authors: Bishop, Emma, Breheny, Damien, Hewitt, Katherine, Taylor, Mark, Jaunky, Tomasz, Camacho, Oscar M., Thorne, David, Gaça, Marianna
• Format: Article
• Language: English
• Subjects: Aerosols; Cell Migration Assays; Cell Movement - drug effects; Electronic Nicotine Delivery Systems; Endothelium; Endothelium, Vascular - drug effects; High-Throughput Screening Assays; Human Umbilical Vein Endothelial Cells; Humans; In vitro; Migration; Nicotine - toxicity; Particulate Matter - toxicity; Tobacco heating; Tobacco Products - toxicity
• ISSN: 0378-4274; 1879-3169; 1879-3169
• DOI: 10.1016/j.toxlet.2020.07.011

[Display omitted] •Assessment of THP and 3R4F AqE in HUVECs using cytotoxicity and migration assays.•Advancement of the scratch wound assay from a 24-well format to 96-well format.•Change in data analysis matric from migration rate to relative wound density (RWD).•3R4F response was comparable to historical HUVEC migration and cytotoxicity data.•THP extract (even at 100 % AqE) had no effect on HUVEC viability or migration. Endothelial cell migration is a critical process in the maintenance of healthy blood vessels. Impaired endothelial migration is reportedly associated with the development of cardiovascular diseases. Here, we report on the development of a 96-well in vitro endothelial migration assay for the purpose of comparative toxicological assessment of a novel THP relative to cigarette smoke, to be able to rapidly inform regulatory decision making. Uniform scratches were induced in confluent human umbilical vein endothelial cells using the 96-pin wound maker and exposed to 3R4F cigarette or THP aqueous extracts (AqE). Endothelial migration was recorded over 24 h, and the rate of wound closure calculated using mean relative wound density rather than migration rate as previously reported. This self-normalising parameter accounts for starting wound size, by comparing the density of the scratch to the outer region at each time-point. Furthermore, wound width acceptance criteria was defined to further increase the sensitivity of the assay. 3R4F and THP AqE samples were tested at comparable nicotine concentrations. 3R4F showed significant cytotoxicity and inhibition of wound healing whereas THP AqE did not show any response in either endpoint. This 96-well endothelial migration assay was suitably sensitive to distinguish combustible cigarette and THP test articles.