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.

The contribution of efficient production of monozygotic twins to beef cattle breeding

“…A study assessing the effectiveness of dislodging cells using enzymes reported that trypsin can stimulate DNA synthesis in lymphocyte cells and that this increase in stimulation was observed when the cells were exposed to proteases for more than 1 min [ 100 ]. Another study reported that treatment using Trypsin-EDTA induced DNA damage during cell isolation, in particular via the action of EDTA [ 101 ]. Accordingly, it was suggested that Trypsin-EDTA may affect the developmental competence of blastomeres, following exposure for even a few minutes.…”