Quantitative analysis of melanin content in a three-dimensional melanoma cell culture

Soobin Chung,Gippeum J Lim,Ji Youn Lee

doi:10.1038/s41598-018-37055-y

Abstract

Reliable measurement of the amount of melanin produced by melanocytes is essential to study various skin disorders and to evaluate the efficacy of candidate reagents for such disorders or for whitening purposes. Conventional melanin quantification methods are based on absorption spectroscopy, which measures the melanin from lysed cells grown on two-dimensional (2D) surfaces. The 2D culture environment is intrinsically different from in vivo systems though, and therefore cells often lose their original phenotypes. Melanocytes in particular lose their ability to synthesize melanin, thereby requiring melanogenesis stimulators such as alpha-melanocyte stimulating hormone (α-MSH) to promote melanin synthesis. In this study, we compared melanin synthesis in B16 murine melanoma cells grown in 2D and three-dimensional culture environments. B16 cells instantly formed an aggregate in a hanging-drop culture, and synthesized melanin efficiently without treatment of α-MSH. We were able to measure the melanin secreted from a single melanocyte aggregate, indicating that our method enables non-invasive long-term monitoring of melanin synthesis and secretion in a high-throughput format. We successfully tested the developed platform by quantifying the depigmenting effects of arbutin and kojic acid.

Highlights

In vitro assays to measure the amount of melanin, a natural pigment and the primary determinant of skin color, are essential in many studies of skin disorders and for whitening purposes
Before exploring the melanin production by melanocytes in a 3D culture, we investigated the melanogenesis characteristics of our cell lines in a conventional 2D monolayer culture
B16 cells are melanoma cell lines derived from C57BL/6 mice[22], which produce melanin and display metastatic behaviors

Summary

Introduction

In vitro assays to measure the amount of melanin, a natural pigment and the primary determinant of skin color, are essential in many studies of skin disorders and for whitening purposes. We developed an in vitro assay for melanin measurement by combining a 3D cell culture with non-invasive absorption measurement in a well-plate format. While the level of extracellular melanin in control melanocytes without α-MSH treatment was negligible, the amount of measured intracellular melanin was substantial (several picograms per cell).

Results

Conclusion