Engineering a Multilayered Skin Equivalent: The Importance of Endogenous Extracellular Matrix and Model Flexibility for Nuanced and Accurate Skin Studies.

Human skin equivalents (HSEs) are a valuable tool for both academic and industrial laboratories to further the understanding of skin physiology in a range of health and disease situations. There have been many advances in the development of HSEs that successfully recapitulate the structure of human skin in vitro; however a main limitation is variability due to the use of complex protocols and exogenous extracellular matrix (ECM) proteins. Previously, we have developed a robust, reproducible, and unique full thickness HSE, using a consistent scaffold, commercially available cells and defined low-serum media. Alvetex scaffold technology allows fibroblasts to produce their own endogenous ECM proteins, alleviating the need for exogenous collagen and supporting epidermal differentiation and stratification. This model is highly flexible, with the potential to use other cell populations for more complex HSEs, alter the culture regime to experimental needs, and perform preclinical testing using the same tools as in vivo studies. Our full thickness skin equivalent is generated using a detailed step-by-step protocol, which sequentially forms the multi-layered structure of human skin in vitro and is detailed below.