Name: rapid generation of primary murine melanocyte and fibroblast cultures
Uploaded: 2019-06-26T13:00:00
Description: Watch this Scientific Journal Video about Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures at JoVE.com

The authors thank the Damon Runyon Foundation (Innovation Award #38-16 to C.E.B.) and Pelotonia (B.M.M.) for financial support. We are grateful to C. Haines and C. Wormsbaecher who provided comments to improve the manuscript text. This work benefitted from the Ohio State Comprehensive Cancer Center&#8217;s Analytical Cytometry Shared Resource which is supported by NIH P30 CA016058.

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&#8217;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 cult...

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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...

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 production1,2,3. 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 follicles4. Here, we detail a simple procedure that enables the concurrent generation of primary melanocyte and fibroblast cultures from murine skin.
Melanocytes are pigment-producing cells found in many locations throughout the human body, including the basal epidermis, iris, cochlea, brain and hair follicles5. The primary function of melanocytes is to generate and secrete melanin-containing vesicles called melanosomes5,6. Melanosomes contain two major classes of melanin: brown/black eumelanin and yellow/red pheomelanin6,7. Biochemical processes within the melanocyte regulate the relative abundance of each melanin species and help to determine hair, skin and eye color8,9. Melanin also serves to absorb UVR and protect sun-exposed tissues from mutagenesis10.
Melanocyte dysfunction can cause pigmentary defects and increase skin cancer susceptibility. For example, the hyper-pigmented skin patches characteristic of melasma are the result of focal melanin overproduction, whereas germline genetic mutations which compromise genes involved in melanin synthesis lead to albinism11,12. Intimate knowledge of melanocyte biology is required to develop strategies that will correct such pigmentary defects and ultimately improve the psychosocial well-being of individuals afflicted with these diseases. Deficits in melanin production and/or the preferential synthesis of pheomelanin are also associated with increased skin cancer risk10. This risk is believed to result from reduced UVR protection6,13. Thus, methods to enhance or restore eumelanin production in melanocytes may reduce the incidence of skin cancer in these populations.
Mesenchymal fibroblasts establish the connective tissue and structural support for all organs of the body, including the dermal layer of the skin14. Excretion of proteins such as collagen, elastin, laminin and fibronectin enable fibroblasts to form the extracellular matrix (ECM) that is essential for tissue integrity1,14. Fibroblasts also play essential roles in processes such as wound healing, inflammation, angiogenesis and cancer formation/progression1,15,16.
Similar to melanocytes, defects in fibroblast activation and function can promote tumorigenesis and disease. For example, inappropriate fibroblast activation commonly leads to the formation of fibrosis, resulting from the enhanced deposition of excess ECM components into the surrounding tissue. As fibroblasts maintain much of the body&#8217;s structural integrity, fibrosis promotes diseases that affect numerous tissues and organs, including idiopathic pulmonary fibrosis, systemic sclerosis, liver cirrhosis and cardiac fibrosis15. Fibroblasts also play a critical role in cancer16. Cancer associated fibroblasts (CAFs) are the most abundant non-malignant cells in the microenvironment of many tumors. CAFs have been shown to promote tumor proliferation, progression and therapeutic resistance by modulating tissue stiffness, local cytokine production and immune function16.
Primary cell cultures provide researchers with genetically tractable models to identify and mitigate melanocyte and fibroblast defects that lead to disease. However, current methods to establish melanocyte cultures are time consuming and technically challenging. The need for trypsinization and delicate separation of the epidermis and dermis contributes to variability in experimental yield and makes it difficult to perform large-scale experiments. Furthermore, protocols to simultaneously isolate melanocytes and fibroblasts from whole skin are currently lacking in the field.
We have developed a method to reduce the processing steps, variability and time required to establish melanocyte and fibroblast cultures from the same whole skin sample. Using a mechanical homogenization method followed by a brief digestion, our strategy significantly decreases the amount of hands-on time required to isolate primary melanocytes while enabling concurrent fibroblast isolation. The increased speed, efficiency and consistency of this protocol, in combination with the ability to isolate melanocytes from 0-4 day old mice, provides researchers the flexibility to perform a wider array of experiments than previously possible.

<table border="0" cellpadding="0" cellspacing="0" style="width:860px;" width="859">
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			<td height="21" style="height:21px;width:360px;">0.2 &#181;m PES sterile syringe filter</td>
			<td style="width:183px;">VWR</td>
			<td style="width:151px;">28145-501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">10 cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:151px;">430167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">40 &#181;m cell strainer</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">22363547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">5 mL polystyrene round-bottom tubes</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">352008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">6-well cell culture dish</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">SIAL0516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">70 &#181;m cell strainer</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Antibiotic Antimycotic Solution (100x)</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">A5955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bovine Serum Albumin</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">BP9706-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CF 488A Mix-n-Stain Antibody Labeling Kit</td>
			<td>Biotium</td>
			<td>92273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CF 555 Mix-n-Stain Antibody Labeling Kit</td>
			<td>Biotium</td>
			<td>92274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cholera Toxin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">C8052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Collagen from rat tail</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">C7661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Collagenase Type I</td>
			<td style="width:183px;">Worthington Biochemicals</td>
			<td style="width:151px;">LS004156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Corning Penicillin/Streptomycin Solution</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">30-002-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cytokeratin 14 Antibody Alexa Fluor 647</td>
			<td style="width:183px;">Novus Biologicals</td>
			<td style="width:151px;">NBP2-34403AF647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Deoxyribonuclease I</td>
			<td style="width:183px;">Worthington Biochemicals</td>
			<td style="width:151px;">LS002058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Di-butyryl cyclic AMP</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">D0627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:151px;">12800-082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">eBioscience Fixable Viability Dye eFluor 780</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:151px;">65-0865-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ethanol, 200 proof</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">22032601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Fetal Bovine Serum</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">12306C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">FSP1/S100A4 antibody</td>
			<td style="width:183px;">Millipore Sigma</td>
			<td style="width:151px;">07-2274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">G418 Disulfide</td>
			<td style="width:183px;">P212121</td>
			<td style="width:151px;">LGB-418-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Glacial Acetic Acid</td>
			<td style="width:183px;">VWR</td>
			<td style="width:151px;">VWRV0714</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Horse Serum</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">26050088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HyClone L-Glutamine</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:151px;">SH3003402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">McIlwain Tissue Chopper</td>
			<td style="width:183px;">Ted Pella</td>
			<td style="width:151px;">10180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Melanoma gp100 antibody</td>
			<td style="width:183px;">Abcam</td>
			<td style="width:151px;">ab137078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Nutrient Mix F-12 Ham&#39;s Media</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">N6760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phorbol 12-Myristate 13-acetate</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Pierce 16% Formaldehyde</td>
			<td style="width:183px;">Thermo Fisher</td>
			<td style="width:151px;">28908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Porcine Trypsin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">85450C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">RPMI 1640 media</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Saponin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:151px;">S-7900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tissue Chopper Blade</td>
			<td style="width:183px;">Ted Pella</td>
			<td style="width:151px;">121-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tissue Chopper Plastic Disk</td>
			<td style="width:183px;">Ted Pella</td>
			<td style="width:151px;">10180-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Trypsin</td>
			<td style="width:183px;">VWR</td>
			<td style="width:151px;">VWRL0154-0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid generation of primary murine melanocyte and fibroblast cultures

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.

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...

Watch this Scientific Journal Video about Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures at JoVE.com

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

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.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

This protocol describes a rapid and simple method to generate primary melanocyte and fibroblast cultures from mice. Because this technique does not require that the epidermis and dermis are separated, it can be performed quickly and consistently with minimal training. This method can be used to study cutaneous melanocyte and fibroblast biology.Experiments in these cultures can provide insights relevant to cancer, wound healing, and pigmentation defects. Before beginning, make sure to prepare all of the necessary reagents and check the temperature of the water bath. If you plan to process multiple samples, refer to the reagent scaling guide in Table One.Demonstrating this procedure will be Brandon Murphy, a graduate student from my laboratory. In a laminar flow cabinet, briefly roll the trunk of each euthanized mouse in a sterile Petri dish containing 70%ethanol. Remove the mouse from the ethanol and place it into an empty sterile Petri dish.Next, sterilize surgical scissors in 70%ethanol. Use these scissors to make an incision in the skin on the ventral side of the trunk, starting from the neck and continuing to the tail. Using sterile forceps, peel the skin away from the trunk of the mouse, and remove excess adipose tissue from the dermal side of the skin.Place the skin, dermis side down, into a six well dish containing three milliliters of 1X antibiotic antimycotic solution. Incubate at room temperature for two to three minutes. Then, turn on the tissue chopper and adjust the settings to those shown here.Be sure to exercise caution when installing the tissue chopper blade and when operating the machine. Transfer the skin, dermis side down, to a sterile tissue chopper dish and pass the skin completely through the tissue chopper three times. After this, transfer the homogenized skin to a sterile 15 milliliter conical tube containing three milliliters of skin digestion buffer.Using a P1000 micro-pipette, mix the resulting suspension by pipetting up and down vigorously 10 to 15 times. Cap the conical tube and incubate the sample in a water bath at 37 degrees Celsius for 15 minutes, making sure to invert the tube every three to five minutes. Next, centrifuge the tube in a swinging bucket rotor at 750 times G at room temperature for five minutes to pellet the cells in the skin homogenate.Use a P1000 micro-pipette to slowly and completely remove the skin digestion buffer, being careful not to disturb the pellet. Be sure to aspirate off all the skin digest buffer. If you are having trouble, wash the cell pellet with two to three mils of melanocyte media and repeat centrifugation and remove excess skin digest buffer.Then thoroughly resuspend the cell pellet in one milliliter of melanocyte media by pipetting up and down 15 to 20 times with a P1000 pipette. Transfer the resulting cell solution drop wise to an uncoated well in a six well dish that contains one milliliter of melanocyte media. Incubate the plated skin homogenate in a tissue culture incubator at 37 degrees Celsius with 5%carbon dioxide for 40 minutes.After this, transfer the culture supernatant from the uncoated dish to one well of a pre-washed collagen coated six well dish. Add G-418 to the media, such that the final concentration is 100 nanograms per milliliter. Add two milliliters of fibroblast media to one well of the uncoated dish now containing adherent fibroblasts.Incubate both cultures overnight in a tissue culture incubator at 37 degrees Celsius with 5%carbon dioxide. After 16 to 24 hours of incubation, separately aspirate the media and any debris from each culture. Wash each dish twice using one milliliter of PBS for each wash.Then, add two milliliters of fresh melanocyte media to the melanocyte culture and add two milliliters of fresh fibroblast media to the fibroblast culture. In this study, fibroblast and melanocyte cultures are simultaneously generated from the same skin sample. When the homogenized skin is first plated, the media appears cloudy.However, after incubation larger cell conglomerates and adherent cell fibroblasts become visible in the dish. Non-adherent cells from the culture are transferred to a collagen coated dish and treated with G-418. Fibroblasts killed by G-418 are seen floating in the melanocyte culture medium for the next five days.After four to five days in culture, the plated melanocytes begin to take on a dendritic phenotype with melanocytic granules. This phenotype persists upon passaging, while clusters of contaminating keratinocytes are lost. The purity of the resulting melanocyte and fibroblast cultures is confirmed using flow cytometry.Expression of the melanocyte marker GP100 is specific to the melanocyte cultures, while the fibroblast marker, FSP1, is found solely in the primary fibroblasts. Control C59 keratinocyte cells are the only cultures that stain positive for the keratinocyte marker, K14. Additional analyses are performed to determine how long it takes to establish enriched melanocyte cultures.GP100 positive cells begin to appear on day four and peak after 10 days in culture. This procedure can be used to rapidly generate melanocyte and fibroblast cultures for a variety of in vitro and high throughput omic assays.

Research

JoVE Journal

Cancer Research

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