Abstract
Understanding cellular processes involved in wound healing is very important given that there are diseases, such as diabetes, in which wounds do not heal. To model tissue regeneration, we focus on two cellular processes: cellular proliferation, to replace cells lost to the wound, and cell motility, activated at the wound edges. We address these two processes in separate, drug responsive, in vitro models. The first model is a scaffold-free three-dimensional (3D) spheroid model, in which spheroids grow larger – to a certain extent – with increased time in culture. The second model, the scratch wound assay, is focused on cell motility. In conjunction with collagen staining, it analyzes changes to the coverage of the wound edge and wound bed. Our workflow gives insights into candidate compounds for wound healing as we show using manuka honey (MH) as an example. Spheroids are responsive to oxidative damage by hydrogen peroxide (H2O2) which affects viability but mostly produces disaggregation. Conversely, MH supports spheroid health, shown by size measurements and viability. In two-dimensional scratch wound assays, MH helps close wounds with relative less collagen production and increases the loose cellular coverage adjacent to and within the wound. We use these methods in the undergraduate research laboratory as teaching and standardization tools, and we hope these will be useful in similar settings.
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Abbreviations
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- DMSO:
-
Dimethyl sulfoxide
- EDTA:
-
Ethylenediaminetetraacetic acid
- MRI:
-
Montpellier Ressources Imagerie
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
- PBS:
-
Phosphate buffered saline
- SDS:
-
Sodium dodecyl sulfate
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Acknowledgments
This work has been supported by Montgomery College, SCIR 297 program, and by support from Schoenberg Fellowship to V.V. We are thankful to Lauren Kimlin and to Greta Babakhanova for their help with the ImageJ analyses and for the constant assistance and support of Arifur Rahman, Ya Yu Shao, and Chris Standing, our lab staff.
Contributions
G. V. performed experiments, analyzed data, and reviewed the manuscript; M. A. analyzed data and prepared figures; L. P., S. A., M. V., D. C., W. T., and N. T. performed experiments and analyzed data; and V. V. conceived the studies, performed experiments, analyzed data, and wrote the manuscript. All authors read and approved the manuscript.
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Virador, G. et al. (2022). Systematically Assessing Natural Compounds’ Wound Healing Potential with Spheroid and Scratch Assays. In: Turksen, K. (eds) Cell Biology and Translational Medicine, Volume 17. Advances in Experimental Medicine and Biology(), vol 1401. Springer, Cham. https://doi.org/10.1007/5584_2022_727
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DOI: https://doi.org/10.1007/5584_2022_727
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