Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Summary

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

Abstract

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

Introduction

The goal of this study was to develop a simple and effective protocol to isolate melanocytes from the epidermis of adult skin for biological research and clinical applications. Melanocytes, which are located in the basal epidermis of the skin and in hair follicles, play an important role in pigmentation of the skin and hair by producing melanin¹. The resulting skin pigmentation from epidermal melanocytes acts as an ultraviolet radiation filter that reduces/prevents DNA damage to underlying cells in the skin². The abnormal proliferation of melanocytes in the skin is quite common, such as in the formation of benign n....

Protocol

The use of adult foreskin tissues in this protocol has been approved by the Human Research Ethics Committee (No.2015120401, date: May 12, 2015).

NOTE: Perform all the following procedures in a sterile environment to prevent contaminations of cells and cultures.

1. Preparations

1. Gather fresh adult foreskin tissues from circumcision surgeries in 15 mL tubes containing 10 mL of phosphate buffer saline (PBS) and store in 4 °C.
   NOTE: Separate the tissues as detailed below within 24 h after their excision.
2. Prepare reagents and culture medium.
   1. Prepare 3% P/S (penicillin/strep....

Results

Figure 1 shows a schematic diagram comparing the conventional and the new methods. The procedure for tissue preparation and digestion with the new method is the same as the procedure for the conventional method, the only difference being that the isolated cells are resuspended in 10 mL of TIVA medium in the presence of 10 µM Y-27632. Two days after seeding, replace the media with normal TIVA medium without Y-27632. The conventional method of isolating primary melanocytes from skin tissues us.......

Discussion

The protocol described here was based on a recent publication¹⁶. Some attention should be paid to the following critical steps to achieve the best results with the new method. First, successful separation of the epidermis and the dermis is crucial. Cut the adult foreskin tissues into 3-4 mm wide strips using a scalpel blade to make the dispase work more thoroughly and easily to separate the epidermis from the dermis. Second, when separating those two layers, contamination of the dermis should be a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa fluor-594 donkey anti-mouse IgG	Thermo Fisher Scientific	A21203	For Immunofluorescence
Alexa fluor-594 donkey anti-rabbit IgG	Thermo Fisher Scientific	R37119	For Immunofluorescence
Cell Culture Dish	Eppendorf	30702115	For cell culture
Cell Strainer	Corning incorporated	431792	Cell filtration
15 mL Centrifuge Tube	KIRGEN	171003	For cell centrifuge
50 mL Centrifuge Tube	KIRGEN	171003	For cell centrifuge
CO2 Incubator	Thermo Scientific	51026333	For cell incubating
Constant Temperature Shaker	Shanghai Boxun	150036	For water bath
DAPI	Abcam	ab104139	For Immunofluorescence
Dispase	Gibco	17105-041	For melanocyte isolation
DMEM	Thermo Scientific	C11995500	Component of neutralization medium
Fetal Bovine Serum	Biological Industries	04-001-1AC5	Component of neutralization medium
Fluorescence microscope	Olympus	5E44316	For Immunofluorescence
Forskolin	MCE	HY-15371	Induce pigmentation
Glutamine	Thermo Fisher Scientific	25030081	melanocyte culture medium
Ham's F12	Thermo Scientific	11330032	melanocyte culture medium
Inverted microscope	Olympus	5C42258	For cell microscopic observation
3-isobutyl-1-methyl xanthine (IBMX)	Sigma	17018	melanocyte culture medium
mouse anti-human MITF	Abcam	ab12039	For Immunofluorescence
Na3VO4	Sigma	S6508	melanocytes culture medium
N6,2'-O-dibutyryladeno-sine 3',5'-cyclic monophosphate (dbcAMP)	Sigma	D0627	melanocyte culture medium
12-O-tetradecanoyl phorbol-13-acetate (TPA)	Sigma	79346	melanocyte culture medium
Penicillin Streptomycin	Thermo Scientific	15140-122	Antibiotics
rabbit anti-human Ki-67	Abcam	ab15580	For Immunofluorescence
Phosphate buffered solution	Solarbio Life Science	P1020-500	Washing solution
Sorvall ST 16R Centrifuge	Thermo Scientific	75004380	Cell centrifugation
TC20TM automated cell counter	Bio-Rad	1450102	Automatic cell counting
0.05% Trypsin	Life Technologies	25300-062	For melanocyte dissociation
Y-27632	Sigma	Y0503	For melanocyte isolation