Anatomy of a bioengineered human pigmented skin equivalent to provide fundamental insights into skin tone melanin dynamics

Full-thickness skin equivalents provide a platform for preclinical screening, streamlining the clinical trial process and reducing the need for animal testing while also providing a tool capable of fundamental insights into skin biology. Understanding the specific role of melanin dynamics across various skin tones is essential not only to better understand its function in photoprotection but is also better representative of a diverse population. Although pigmented skin equivalents (PSEs) have been reported in the literature, they rarely recapitulate the structural location of melanin within native keratinocytes, which is pivotal to its photoprotective role. This is due in part to the reliance of existing technologies on exogenous or animal-derived extracellular matrix (ECM) constituents or the complete lack of a dermal compartment. In this study, we describe the development of novel PSEs representative of skin pigmentation phenotypes in vitro, which comprise fibroblast-secreted endogenous ECM and a differentiated, well-organised epidermis that resembles diverse skin tones. We demonstrate that these skin tones display morphological differences at a gross, histological and ultrastructural level. We then utilised the system to provide fundamental insights into the processes of melanogenesis, melanin transfer from melanocytes to keratinocytes, supranuclear cap formation and melanosome organisation within the epidermis. Quantification of melanosome dynamics allowed for comparison to native tissue and among skin tones, providing a detailed comparison among experimental conditions. This innovative technology enables a wide range of applications, such as studying pigmentation mechanisms in skin responses to external stimuli, disease modelling and drug testing involving the interactions between the epidermis and dermis.