Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Summary

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Abstract

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

Introduction

Mammalian skin is a multilayered organ that protects the body from foreign pathogens and ultra violet irradiation (UVR). The skin also plays a critical role in homeostatic processes such as wound healing, temperature regulation and vitamin D production^1,2,3. Mammalian skin consists of three major cell types: melanocytes, fibroblasts and keratinocytes. These cell types populate different layers of the skin, with keratinocytes making up the epidermis, fibroblasts residing in the dermis and melanocytes localizing to the epidermal-dermal junction and hair follicles

Protocol

Obtain approval from your institutional animal ethics committee before commencing this or any other study involving animals. Experiments performed in this protocol were approved by the Ohio State University’s Institutional Animal Care and Use Committee (IACUC, protocol #2012A00000134).

1. Protocol Preparation

NOTE: The following reagent preparation instructions are appropriate for the generation of 9 cm² melanocyte and fibroblast cultures from a single mouse. Refer to the reagent preparation guide in Table 1 for larger scale isolations.

1. Prepare 1.5 mL....

Results

Male and female C57Bl/6J mice were euthanized on postnatal days 0-4 and the truncal skin was subjected to mechanical dissociation as described above. After chopping, the skin formed a viscous slurry lacking any sign of structural tissue. Centrifugation of this slurry resulted in the formation of a large cell pellet at the bottom of the conical tube and a layer of adipose floating on top of the supernatant. This adipose layer was discarded with the supernatant while the remaining cell pellet was resuspended and transferre.......

Discussion

The in vitro culture of primary melanocytes and fibroblasts has led to significant advancements in our understanding of skin biology and disease. This protocol improves upon prior melanocyte isolation methods by reducing the time and technical savvy needed to generate consistent melanocyte cultures while allowing for the simultaneous isolation of skin fibroblasts. A novel, time-saving element of this procedure is that the dermis and epidermis do not need to be separated. Instead, skin cells are isolated using the consist.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µm PES sterile syringe filter	VWR	28145-501
10 cm cell culture dish	Corning	430167
40 µm cell strainer	Fisher Scientific	22363547
5 mL polystyrene round-bottom tubes	Fisher Scientific	352008
6-well cell culture dish	Sigma-Aldrich	SIAL0516
70 µm cell strainer	Fisher Scientific	22363548
Antibiotic Antimycotic Solution (100x)	Sigma-Aldrich	A5955
Bovine Serum Albumin	Fisher Scientific	BP9706-100
CF 488A Mix-n-Stain Antibody Labeling Kit	Biotium	92273
CF 555 Mix-n-Stain Antibody Labeling Kit	Biotium	92274
Cholera Toxin	Sigma-Aldrich	C8052
Collagen from rat tail	Sigma-Aldrich	C7661
Collagenase Type I	Worthington Biochemicals	LS004156
Corning Penicillin/Streptomycin Solution	Fisher Scientific	30-002-CL
Cytokeratin 14 Antibody Alexa Fluor 647	Novus Biologicals	NBP2-34403AF647
Deoxyribonuclease I	Worthington Biochemicals	LS002058
Di-butyryl cyclic AMP	Sigma-Aldrich	D0627
Dulbecco's Modified Eagle Medium	Gibco	12800-082
Dulbecco's Phosphate Buffered Saline	Sigma-Aldrich	D8537
eBioscience Fixable Viability Dye eFluor 780	Thermo Fisher	65-0865-14
Ethanol, 200 proof	Fisher Scientific	22032601
Fetal Bovine Serum	Sigma-Aldrich	12306C
FSP1/S100A4 antibody	Millipore Sigma	07-2274
G418 Disulfide	P212121	LGB-418-1
Glacial Acetic Acid	VWR	VWRV0714
Horse Serum	Fisher Scientific	26050088
HyClone L-Glutamine	Fisher Scientific	SH3003402
McIlwain Tissue Chopper	Ted Pella	10180
Melanoma gp100 antibody	Abcam	ab137078
Nutrient Mix F-12 Ham's Media	Sigma-Aldrich	N6760
Phorbol 12-Myristate 13-acetate	Sigma-Aldrich	P8139
Pierce 16% Formaldehyde	Thermo Fisher	28908
Porcine Trypsin	Sigma-Aldrich	85450C
RPMI 1640 media	Sigma-Aldrich	R8758
Saponin	Sigma-Aldrich	S-7900
Tissue Chopper Blade	Ted Pella	121-6
Tissue Chopper Plastic Disk	Ted Pella	10180-01
Trypsin	VWR	VWRL0154-0100