Improving the Culture of Human Skin Explants for Use in Preclinical Testing of Wound Healing Treatments

Background: Cultured human skin explants provide preclinical models to investigate drug delivery and the efficacy of topical treatments for wound healing. However, different culture conditions may affect cell viability, proliferation, and even wound healing. Since animal-derived supplements can influence the investigation of human physiological responses, this study evaluated the effects of non-animal supplements on the ex vivo wound healing process to improve the use of this model for preclinical drug efficacy tests. Methods: In in vitro scratch assays using HaCaT cells and fibroblasts, for media supplemented with normal human serum (NHS), oxygen carriers (OCs) had a positive impact on cell migration, supporting the further evaluation in ex vivo skin culture models. Human skin explants with standardized superficial wounds were cultured in four supplemented media: (i) Dulbecco's Modified Eagle Medium High Glucose (DMEM) with fetal calf serum (FCS), (ii) DMEM with NHS and OC, (iii) CnT-Prime^TM with NHS and OC, and (iv) EpiLife™ with NHS and an OC. Results: During the 12-day culture, we observed re-epithelialization in all groups with the exception of EpiLife + NHS + OC (with no Ca⁺⁺ supplement). For these samples, starting from day 6, we noticed a loosening of the dermal-epidermal junction and disruption of the upper epidermal layer. Furthermore, an immunohistochemical analysis of extracellular matrix components and remodeling factors, including type I and III collagen, transforming growth factor-β2, and matrix metalloproteinase-9, provided insights into tissue repair dynamics. Conclusions: NHS plus OC is comparable to FCS supplementation and represents a more physiological and ethical alternative.