Human Melanocyte-Derived Spheroids: A Precise Test System for Drug Screening and a Multicellular Unit for Tissue Engineering

Pigmentation is the result of melanin synthesis, which takes place in melanocytes, and its further distribution. A dysregulation in melanocytes' functionality can result in the loss of pigmentation, the appearance of pigment spots and melanoma development. Tissue engineering and the screening of new skin-lightening drugs require the development of simple and reproduciblein vitromodels with maintained functional activity. The aim of the study was to obtain and characterize spheroids from normal human melanocytes as a three-dimensional multicellular structure and as a test system for skin-lightening drug screening. Melanocytes are known to lose their ability to synthesize melanin in monolayer culture. When transferred under non-adhesive conditions in agarose multi-well plates, melanocytes aggregated and formed spheroids. As a result, the amount of melanin elevated almost two times within seven days. MelanoDerm (TM) (MatTek) skin equivalents were used as a comparison system. Cells in spheroids expressed transcription factors that regulate melanogenesis: MITF and Sox10, the marker of developed melanosomes-gp100, as well as tyrosinase (TYR)-the melanogenesis enzyme and melanocortin receptor 1 (MC1R)-the main receptor regulating melanin synthesis. Expression was maintained during 3D culturing. Thus, it can be stated that spheroids maintain melanocytes' functional activity compared to that in the multi-layered MelanoDerm (TM) skin equivalents. Culturing both spheroids and MelanoDerm (TM) for seven days in the presence of the skin-lightening agent fucoxanthin resulted in a more significant lowering of melanin levels in spheroids. Significant down-regulation of gp100, MITF, and Sox10 transcription factors, as well as 10-fold down-regulation ofTYRexpression, was observed in spheroids by day 7 in the presence of fucoxanthin, thus inhibiting the maturation of melanosomes and the synthesis of melanin. MelanoDerm (TM) samples were characterized by significant down-regulation of only MITF, Sox10 indicating that spheroids formed a more sensitive system allowed for quantitative assays. Collectively, these data illustrate that normal melanocytes can assemble themselves into spheroids-the viable structures that are able to accumulate melanin and maintain the initial functional activity of melanocytes. These spheroids can be used as a more affordable and easy-to-use test system than commercial skin equivalents for drug screening.