Development of an in vitro three-layered skin wound healing model for pre-clinical testing

Abstract Skin wound healing involves many cell types in a stepwise process of tissue regeneration. Reepithelialization is an essential characteristic of successful healing. In tissue engineering, mimicking the complex process of injury repair in vitro is challenging and requires the development of advanced skin models. In this study, a simple and reproducible method for wounding three-layered skin models on membranes with different pore sizes (0.4, 1, 3 μm) was established. The model allows the investigation of reepithelialization processes in a more complex environment. Hemalaun-eosin (HE) and 3-[4,5-dimethylthiazole-2-yl]-2,5- diphenyltetrazolium bromide (MTT) staining proved sufficient removal of the epidermis directly after wounding. An increasing pore size of the culture membrane delayed the reepithelialization time. Transepithelial electrical resistance (TEER) measurements provided non-invasive monitoring of reepithelialization, showing increasing values 4 days after wounding for the skin models on 0.4 and 1 μm membranes but not for those on 3 μm membranes. Cytokine quantification of interleukin (IL)-6 and IL-8 complemented the TEER results with increasing levels directly after wounding for all skin models. This skin wounding model could be used to simulate different wounding scenarios and test wound matrix materials. Furthermore, it could be adapted by adding immune cells to resemble the in vivo setup more closely.