Isolation, Culture, and Transfection of Melanocytes

Abstract

Located in the basal epidermis and hair follicles, melanocytes of the integument are responsible for its coloration through production of melanin pigments. Melanin is produced in lysosomal‐like organelles called melanosomes. In humans, this skin pigmentation acts as an ultraviolet radiation filter. Abnormalities in the division of melanocytes are quite common, with potentially oncogenic growth usually followed by cell senescence producing benign naevi (moles), or occasionally melanoma. Therefore, melanocytes are a useful model for studying melanoma, as well as pigmentation and organelle transport and the diseases affecting these mechanisms. This chapter focuses on the isolation, culture, and transfection of human and murine melanocytes. The first basic protocol describes the primary culture of melanocytes from human skin and the maintenance of growing cultures. The second basic protocol details the subculture and preparation of mouse keratinocyte feeder cells. The primary culture of melanocytes from mouse skin is described in the third basic protocol, and, lastly, the fourth basic protocol outlines a technique for transfecting melanocytes and melanoma cells. Curr. Protoc. Cell Biol. 63:1.8.1‐1.8.20. © 2014 by John Wiley & Sons, Inc.

Melanocytic nevi and melanoma: unraveling a complex relationship

“…For this, reason a keratinocyte feeder layer is often used in the culture of melanocytes. 198 The specific signals that underlie this phenomenon and whether or not proximity to keratinocytes in the epidermis has a role in the process of maturation in nevi is not known.…”

Mesenchymal stem cells promote human melanocytes proliferation and resistance to apoptosis through PTEN pathway in vitiligo

“…Pure primary melanocytes were generated from foreskin obtained from male circumcision patients (n = 40) aged 20-25 years old. The foreskin specimens were washed with 1% penicillin-streptomycin antibiotic-supplemented DPBS and digested with a 0.25% Dispase II solution at 4°C for 16-18 h. The epidermis and dermis were separated, and the epidermis was digested with 0.25% trypsin for 10-15 min and incubated with M254 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (Gibco) and 1% Human Melanocyte Growth Supplement-2 (Gibco) at 37°C in a 5% CO 2 atmosphere [25]. Keratinocytes were obtained with the same protocol and incubated with KSFM (Gibco) and serum-free medium.…”

Transcriptional co-activator regulates melanocyte differentiation and oncogenesis by integrating cAMP and MAPK/ERK pathways

“…Primary cells were isolated from whole epidermises of 2 days old WT, CRTC1 KO, CRTC2 KO and CRTC3 KO pups and cultured in differentiation media containing TPA (P1585, Sigma) and cholera toxin (C8052, Sigma), following previously described detailed protocol 59 . For skin melanoblast quantification, WT and CRTC3 KO mice were crossed with mice carrying iDCT:GFP transgene system.…”

“…To account for differences in total amounts of cells isolated from each animal, quantification was expressed as percentage of GFP positive cells per epidermis. In experiments where XB2 feeder keratinocytes were used, cells were obtained from Wellcome Trust Functional Genomics Cell Bank and prepared using mitomycin c treatment (M4287, Sigma), as previously described 59 . In some experiments, catalase (LS001896, Worthington Biochemical) and an antioxidant supplement (A1345, Sigma) were added to the primary cultures in the attempt to improve survival and differentiation of CRTC3 KO melanoblasts.…”