Systematically Assessing Natural Compounds’ Wound Healing Potential with Spheroid and Scratch Assays

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.