title: Evaluation of effect during cell isolation process in alkaline comet assay using epidermal skin cells.
abstract: The aim of the present study was to evaluate the effect of the cell isolation process in the alkaline comet assay using epidermal skin cells. When we explored the cell isolation method for the alkaline comet assay using the 3-dimensional (3D) human epidermal skin model, we found that DNA damage and cytotoxicity were induced during the cell isolation process. In particular, trypsin 5 min treatment with ethylene diamine tetraacetic acid (EDTA) showed about 5 times %DNA in the tail value compared to without EDTA treatment. In general, EDTA is commonly used for cell isolation, but it is known to induce genotoxicity due to secondary effects. We therefore evaluated the effect of EDTA and pH in the alkaline comet assay on a monolayer culture of rat keratinocytes. As a result, there was a significant increase of %DNA in tail values by treatment with 0.1 w/v% EDTA for 60 min; however, there was no difference in the %DNA in tail values between 0.1 w/v% EDTA/PBS(-) (pH 6.8) and 0.1 w/v% EDTA/PBS(-)  (pH 7.4). These data imply that there is a need to control the EDTA conditions for cell isolation in the epidermal skin cells.
authors.affiliation: Hatano Research Institute, Food and Drug Safety Center (FDSC), Kanagawa, Japan. toyoizumi.t@fdsc.or.jp
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Toyoizumi
authors.forename: Mika
authors.initials: M
authors.lastname: Watanabe
authors.forename: Hajime
authors.initials: H
authors.lastname: Sui
authors.forename: Yuzuki
authors.initials: Y
authors.lastname: Nakagawa
authors.forename: Ryo
authors.initials: R
authors.lastname: Ohta
authors.forename: Kohji
authors.initials: K
authors.lastname: Yamakage
mesh_terms: D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D020552:Comet Assay; D004249:DNA Damage; D004492:Edetic Acid / Q000633:toxicity*; D000078404:Epidermal Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D014357:Trypsin
chemical_list: D004492:Edetic Acid; D014357:Trypsin