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

Abstract

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.

Fucoxanthin Holds Potential to Become a Drug Adjuvant in Breast Cancer Treatment: Evidence from 2D and 3D Cell Cultures

“…Regardless of the inconclusive mechanistic data reported in 2D, the most promising combination was explored in a more complex cell model—3D MCAs. Although many studies reported the effects of Fx in 2D cultures, in 3D cultures, as far as we investigated, the literature is limited to four studies [ 53 , 70 , 71 , 72 ]. Only de la Mare and collaborators reported Fx effects in a BC cell line cultured in 3D [ 53 ].…”

A Novel and Effective Method for Human Primary Skin Melanocytes and Metastatic Melanoma Cell Isolation

“…They also displayed the ability to form spheroids after repetitive long-term trypsinization [ 22 ]. It is noteworthy that melanocytes cultured in spheroids have higher viability and functional activity than melanocytes cultured in monolayers [ 3 , 22 ]. Furthermore, melanocytes isolated from the spheroids possess morphological features of melanocyte stem cells, as they displayed high proliferative potential [ 10 , 22 ].…”

“…The production of melanin may depend on the culture conditions (such as 2D culture or medium type) and its duration [ 23 ]. It has been reported that melanocytes lose the ability to synthesize melanin in a monolayer culture [ 3 ]. However, we observed that melanocytes and melanoma cells retained the ability to produce melanin, even when growing in a monolayer culture.…”

“…Melanocyte dysfunction can result in the loss of pigmentation, and appearance of hypo- or hyperpigmented spots and may lead to the development of malignant melanoma [ 3 ]. According to WHO, 324,635 new cases of melanoma and 57,043 deaths related to this cancer were registered in 2020 around the world [ 4 ].…”

The Duo of Osteogenic and Angiogenic Differentiation in ADSC-Derived Spheroids

“…It is now widely accepted that 3D culturing of cells in the form of spheroids or organoids is a “bridge” between monolayer cultures and native tissues ( Pampaloni et al, 2007 ). In 3D culture, cells acquire emergent functionality via multilevel interactions through cell junctions, microvesicle exchange, and extracellular matrix (ECM), which altogether provides the regulation of their collective behavior as well as restoration of initial cellular phenotype and functional activity ( Repin et al, 2014 ; Zurina et al, 2018 , 2020 ). Indeed, it was shown that such arrangements within spheroids as intercellular junctions and cell–matrix interactions result in the appearance of a local microenvironment, which facilitates proper cellular differentiation ( Lee et al, 2016 ; Muller et al, 2019 ).…”