The In Vitro Wound-Scratch Assay: Applications, Technical Advances, and Limitations in Wound Healing Research

The wound-scratch assay is a widely used in vitro model for studying collective cell migration, a fundamental process contributing to wound closure and re-epithelialisation. Owing to its simplicity, low cost, and adaptability, it has become a foundational tool for early-stage wound-healing research and therapeutic screening. The assay involves generating a defined gap in a confluent cell monolayer and monitoring gap closure over time as a surrogate readout of repair. This narrative review examined 199 published studies, identifying 73 relevant to wound healing. A technical hierarchy of wound creation methods was identified across three main categories: mechanical approaches (e.g., pipette tips and cell scrapers), accessible but prone to operator-dependent variability; semi-automated systems (e.g., inserts and wound maker devices), which improve reproducibility; and fully-automated robotic platforms offering high precision and high-throughput capability. While these advances enhance technical consistency, they do not overcome the assay's fundamental biological constraints. Importantly, gap closure in the wound-scratch assay primarily reflects planar collective cell migration and does not recapitulate the integrated inflammatory, vascular, metabolic, and extracellular matrix-dependent processes that govern wound repair in vivo. Consequently, bioactive compounds acting through antioxidant, anti-inflammatory, angiogenic, or matrix-modulating pathways may have their therapeutic potential underestimated or misclassified when assessed using migration-only readouts. Preliminary in-house (unpublished) data are presented to illustrate this limitation, demonstrating modest migration effects for compounds with established wound-healing activity in vivo. Despite these limitations, the wound-scratch assay remains a valuable first-line, hypothesis-generating tool when interpreted appropriately, with future utility dependent on integration with adapted models and complementary assays for translation.