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Updated: Aug 5, 2025
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Published on: May 17, 2021
Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers
Dean S Rosenthal1, Li-Wei Kuo1, Sarah L Seagrave1
1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University School of Medicine, Washington, DC 20057, USA.
Abstract:
The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2β1 integrin, which is present only on basal and stem cells. The presence of β1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers.
Insights
This study introduces a new high-throughput Skin Immuno-CometChip assay to detect DNA damage in skin cells. The assay accurately predicts genotoxicity using 2D and 3D skin models.
Area of Science:
- Dermatology
- Toxicology
- Molecular Biology
Background:
- Topical genotoxic agents target skin basal and stem cells, potentially causing mutations.
- Accurate genotoxicity testing requires relevant skin models, as many compounds remain unevaluated.
- DNA damage in these cells can lead to tumor suppressor or oncogene mutations.
Purpose of the Study:
- To develop and validate a high-throughput assay for detecting DNA damage in epidermal stem and basal cells.
- To assess the genotoxicity of various compounds using a novel skin model.
- To establish a reliable method for predicting chemical genotoxicity in skin.
Main Methods:
- Development of the Skin Immuno-CometChip assay utilizing microwells and collagen binding to isolate basal/stem cells.
- Application of the assay to 2D epidermal cell cultures and 3D skin equivalents.
- Detection of DNA damage via immunofluorescent labeling, alkaline comet assay, and automated image analysis.
Main Results:
- The Skin Immuno-CometChip assay successfully captured and analyzed DNA damage in single basal cells.
- Automated quantification of DNA damage was achieved with improved comet detection software.
- The assay demonstrated accurate prediction of genotoxicity in both 2D and 3D skin models.
Conclusions:
- The Skin Immuno-CometChip assay is a validated high-throughput method for assessing skin genotoxicity.
- 3D skin cultures and 2D keratinocyte monolayers can be reliably used to predict chemical genotoxicity.
- This assay provides a crucial tool for evaluating the safety of topical agents.
