Visualizing Scar Development Using SCAD Assay - An <em>Ex-situ</em> Skin Scarring Assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or "SCar-like tissue in A Dish" that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids researchers in understanding fundamental processes and mechanisms of wound repair, directly exploring the effects of modulators on wound healing outcomes.