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 reproducible in vitro models 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 of TYR expression, 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 Zurina et al.Melanocyte-Derived Spheroids as a Test System 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.
Функциональная активность меланоцитов обусловливает защитные свойства кожи против воздействия ультрафиолета, вызывающего фотостарение. Однако изучение меланоцитов in vivo затруднено тем, что необходимо использовать животные модели и трудоемкие методы анализа, а культивирование клеток в монослойной культуре in vitro сопряжено с потерей тканеспецифичных маркеров клеток. Данная работа посвящена получению и изучению сфероидов из меланоцитов, так как 3D культивирование меланоцитов в виде сфероидов может сохранить их фенотип и функциональность. Исследование проводили на первичной культуре меланоцитов кожи человека. Клетки культивировали в монослое в полной ростовой среде до 4 пассажа. Далее клетки помещали на агарозные планшеты с микролунками в посевной плотности 3,3 х 10 кл./мл. Анализ полученных сфероидов производили с помощью фотометрии, иммуноцитохимии и ПЦР в реальном времени. Было показано, что, при культивировании в монослое, к 4 пассажу снижалось количество синтезируемого меланина. Тогда как в 3D условиях меланоциты формировали компактные сфероиды, внутри которых в процессе культивирования не только сохранялся синтез, но и накапливался меланин. Была выявлена экспрессия специфических генов TYR и MCR1, и увеличивался синтез белков, участвующих в меланогенезе - gp100 и MITF. Таким образом, в данном исследовании было показано, что меланоциты in vitro способны формировать длительно живущие, жизнеспособные 3D структуры - сфероиды, с сохранением фенотипа и синтеза тканеспецифичных маркеров. Поэтому данные сфероиды могут быть успешно использованы как тест-системы для оценки эффективности препаратов, направленных на регуляцию уровня пигментации кожи. The functional activity of melanocytes determines protective properties of the skin against the effect of ultraviolet radiation, which causes photo-aging. However, studying melanocytes in vivo and in an in vitro monolayer culture is difficult because animal models and laborious analytical methods are required, and the culturing is associated with loss of tissue-specific cell markers. Since 3D cultivation of melanocytes in the form of spheroids can preserve their phenotype and functionality, this work focused on obtaining and studying melanocyte spheroids. The study was conducted using a primary culture of human skin melanocytes. Cells were cultured in a monolayer to the fourth passage. Then these cells were placed in agarose plates with microwells at the cell suspension concentration of 3.3 х 10 in vitro . These features make the melanocyte spheroids a convenient test-system for studying toxicity and efficiency of drugs targeted at regulation of skin pigmentation.
Обзор включает данные анализа современной литературы по вопросам происхождения и дифференцировки клеток-предшественников меланоцитов в эмбриогенезе и взрослом состоянии, их локализации и роли в пигментации кожи и волос. Особое внимание в обзоре уделено рассмотрению факторов, участвующих или влияющих на процессы нормального меланогенеза и патологических нарушений пигментации, обусловленных влиянием генетических и эпигенетических факторов, а также процессов старения клеток. Идентификация и понимание процессов меланогенеза, а также механизмов изменения функциональной активности меланоцитов кожи человека облегчит понимание патогенеза нарушений пигментации и позволит разрабатывать новые высокоэффективные препараты для профилактики и терапии заболеваний, коррекции возрастных изменений, а также препаратов, снижающих риск развития рака кожи. Melanocytes represent an important type of human skin cells. They synthesize the pigment melanin, which determines skin pigmentation and provideds protection from ultraviolet radiation and other external factors. Regulation of pigmentation involves many factors essential for development, regeneration, and aging of melanocytes and their precursors as well as the factors involved in synthesis of melanin, formation, transport and distribution of melanosomes and melanocyte-specific transcription factors that control the expression and function of all these genes. This review focused on origination and differentiation of melanocyte progenitor cells in embryogenesis and adulthood, their localization and role in skin and hair pigmentation. Particular attention was paid to the factors involved in or affecting processes of normal melanogenesis, pigmentation abnormalities due to genetic and epigenetic factors, and the processes of cell aging. The authors underlined that melanogenesis and the pigment packing and transportation to epithelial cells are complex and multifactorial processes determined by many external and internal factors, such as performance of genes, enzymes, structural proteins, and effects of hormones and medicines. Age-related changes in cells and the body as a whole are serious factors of pigmentation disorders. Despite numerous studies, information about possibilities of influencing the processes of aging or pathological disorders of skin pigmentation is extremely scarce. Identification and understanding processes involved in melanogenesis and mechanisms of changes in the functioning of human skin melanocytes will facilitate understanding the pathogenesis of pigmentation disorders and help developing new, highly effective drugs for prevention and treatment of diseases or age-related changes, specifically for prevention or reducing the risk of skin cancer.
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