Engineering a reproducible 3D wound model with defined macrophage polarization and partial-thickness mechanical injury

<title>Abstract</title> <p> Despite the acknowledged importance of macrophages in regenerative processes, the field lacks a standardized and reproducible <italic>in-vitro</italic> skin wound model incorporating macrophages, fibroblasts and keratinocytes. To address this, a 3D immunocompetent <italic>in-vitro</italic> wound model based on commercially available cells was established. For the dermal layer, adult dermal fibroblasts (NHDF) and monocytes (THP-1) were embedded in rat-tail collagen, where macrophages were polarized towards M1- and M2-like phenotypes. M1-polarized macrophages exhibited a pro-inflammatory signature, whereas M2-like cells showed reduced pro-inflammatory signature. However, anti-inflammatory mediators were not markedly elevated in models with M2. Human keratinocytes (HaCaT) were used to establish an epidermal layer and integrated onto the dermal layer using a newly developed “Slide-Tight-Inside” method. Using this method a reproducible thin epidermal layer can be generated. A novel static wounding device enabled the generation of standardized partial-thickness injuries penetrating both epidermal and dermal layer while preserving a residual dermal tissue, causing a destructive tissue damage resembling clinical wounds. Models could be co-cultured and observed for up to 21 days and seven days post-wounding. This model provides phenotypic macrophage polarization, standardized wounding and epidermal-dermal architecture in a single platform supporting its use for investigating wound repair mechanism and evaluating drug interventions in a reproducible <italic>in-vitro</italic> setting. </p>