Name: rapid generation of primary murine melanocyte and fibroblast cultures
Uploaded: 2019-06-26T13:00:00.000+00:00
Duration: 6 min 32 s
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 cultures from a single mouse. Refer to the reagent preparation guide in Table 1 for larger scale isolations.
<ol>
	<li>Prepare 1.5 mL of Collagen Solution containing 50 &#181;g/mL collagen in 0.1 M glacial acetic acid.</li>
	<li>In a laminar flow cabinet, coat one well of a 6-well cell culture dish with 8 &#181;g/cm2 (1.44 mL) Collagen Solution. Ensure that the well is completely covered by the Collagen Solution. Incubate the dish at 37 &#176;C for 3 h or at 4 &#176;C overnight. Prior to use, wash the collagen-coated well twice with 1.35 mL of sterile phosphate buffered saline (PBS) (150 &#181;L of PBS per 1 cm2).</li>
	<li>Assemble the tissue chopper in a laminar flow cabinet by carefully placing a chopping disk on the tissue chopper platform and securing a sterile blade to the movable arm.</li>
	<li>Prepare 3 mL of 1x Antibiotic/Antimycotic Solution by diluting 100x antibiotic/antimycotic stock solution in sterile PBS.</li>
	<li>Prepare 3 mL of fresh Skin Digestion Buffer containing 10% fetal bovine serum, 1% penicillin/streptomycin solution, 1% L-glutamine, 10 mg/mL collagenase type I, 0.25% porcine trypsin and 0.02 mg/mL deoxyribonuclease I in RPMI 1640.</li>
	<li>Prepare 6 mL of fresh Melanocyte Media containing 10% fetal bovine serum, 7% horse serum, 1% penicillin/streptomycin solution, 1% L-glutamine, 0.5 mM di-butyryl cyclic AMP (dbcAMP), 20 nM tetradecanoylphorbol 13-acetate (TPA) and 200 pM cholera toxin (CT) in Nutrient Mix F-12 Ham&#39;s media. 
	NOTE: Concentrated dbcAMP, TPA and CT stock solutions can be made, aliquoted and stored for &#62;1 year at -80 &#176;C. Base media lacking these components can be stored for up to 1 month at 4 &#176;C.</li>
	<li>Prepare 4 mL of Fibroblast Media containing 10% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine in Dulbecco&#8217;s Modified Eagle Medium. This media can be stored at 4 &#176;C for up to 1 month.</li>
	<li>Prepare 40 mL of 4% Paraformaldehyde Solution by diluting 16% paraformaldehyde in PBS. Store any excess at 4 &#176;C for 1 month or -20 &#176;C for up to 1 year.</li>
	<li>Prepare a 1% saponin stock solution by mixing 0.5 g of saponin in 50 mL of PBS at 37 &#176;C until the saponin has completely dissolved. Sterilize the solution for long-term storage using a 50 mL syringe fitted with a 0.2 &#181;m PES filter. The resulting saponin stock solution can be kept at 4 &#176;C for 1 month.</li>
	<li>Prepare 200 &#181;L of 0.1% Saponin Solution per mouse by diluting 1% saponin stock solution in PBS containing 3% bovine serum albumin (BSA).</li>
	<li>Prepare 1 mL of Viability Solution by diluting 1 &#181;L of fixable viability dye in 1 mL of PBS.</li>
</ol>
2. Melanocyte and Fibroblast Isolation
<ol>
	<li>Euthanize 0 to 4 day-old male and/or female C57Bl/6J pups by decapitation and remove extremities from the euthanized mice using surgical scissors.</li>
	<li>In a laminar flow cabinet, briefly roll the trunk of each mouse in a sterile Petri dish containing 70% ethanol. Remove the trunk from the ethanol and place it into an empty, sterile Petri dish.</li>
	<li>Using surgical scissors, sterilized in 70% ethanol, make an incision in the skin on the ventral side of the trunk starting from the neck to the tail. Peel the skin from the trunk of the mouse using sterile forceps.</li>
	<li>Place the skin dermis side down in a 6-well dish containing 3 mL of 1x Antibiotic/ Antimycotic Solution and incubate at room temperature for 2-3 min.</li>
	<li>Turn on the tissue chopper with the following settings in place: Slice thickness: 1 &#181;m; Blade force: ~60% of the maximum; Speed: ~50% of the maximum.</li>
	<li>Transfer the skin, dermis side down, to a sterile tissue chopper disk and pass the skin completely through the activated tissue chopper 3 times.</li>
	<li>Transfer the homogenized skin to a sterile, 15 mL conical tube containing 3 mL of Skin Digestion Buffer. Mix the resulting suspension by pipetting up and down 10-15 times with a P1000 micropipette.</li>
	<li>Cap the conical tube and incubate the sample in a 37 &#176;C water bath for 15 min, inverting the tube every 3-5 min.</li>
	<li>Pellet the cells in the skin homogenate by centrifuging the conical tube in a swinging bucket rotor at 750 x g for 5 min at room temperature.</li>
	<li>Using a P1000 micropipette, slowly and completely remove the Skin Digestion Buffer being careful not to disturb the pellet. 
	NOTE: A portion of whole skin can be saved at this stage and used as a control for Step 3: Confirmation of Cellular Purity. Strain these cells through a 70 &#956;m cell strainer and then begin at step 3.5 for further processing.</li>
	<li>Thoroughly resuspend the cell pellet in 1 mL of Melanocyte Media by pipetting up and down 15-20 times with a P1000 micropipette. Add the resulting cell solution dropwise to an uncoated well of a 6-well dish containing 1 mL of Melanocyte Media.</li>
	<li>Place the plated skin homogenate in a tissue culture incubator set at 37 &#176;C and 5% CO2. Allow the cultures to incubate for 40 min. 
	NOTE: During this time, some fibroblasts in the skin homogenate will adhere to the uncoated dish while the melanocytes and keratinocytes remain in suspension.</li>
	<li>Transfer the culture supernatant from the uncoated dish to one well of a pre-washed, collagen-coated 6-well dish. Add G418 to the media such that the final concentration is 100 ng/mL.</li>
	<li>Add 2 mL of Fibroblast Media to one well of the uncoated dish, now containing adherent fibroblasts.</li>
	<li>Incubate both cultures overnight in a tissue culture incubator set at 37 &#176;C and 5% CO2.</li>
	<li>Separately aspirate the media and any debris from each culture, 16-24 h after plating. Wash each dish twice with 1 mL of sterile PBS, and then add 2 mL of fresh Melanocyte Media plus 100 ng/mL G418 to the melanocyte culture and 2 mL of fresh Fibroblast Media to the fibroblast culture.</li>
	<li>After the melanocyte cultures have been treated with G418 for 48 h, wash the cells twice with 1 mL of sterile PBS and add 2 mL of fresh Melanocyte Media without G418 to the culture. 
	NOTE: As fibroblasts in the melanocyte culture continue to die off post-G418 treatment, wash the dish with sterile PBS to remove dead cells and add fresh Melanocyte Media. Melanocyte and fibroblast cultures should be passaged when they reach 70-80% confluency.</li>
</ol>
3. Confirmation of Cellular Purity
<ol>
	<li>Aspirate the media from each culture and carefully wash each dish with 1 mL of sterile PBS.</li>
	<li>Add 0.7 mL of 0.25% trypsin to each culture and incubate the cultures in trypsin at 37 &#176;C and 5% CO2 for 1 min.</li>
	<li>Dislodge the cells by pipetting the trypsin against the bottom of the dish using a P1000 micropipette.</li>
	<li>Add 0.7 mL of the appropriate media to each trypsinized culture and transfer the cell solution into a 1.5 mL microcentrifuge tube.</li>
	<li>Pellet the cells by centrifugation at 750 x g for 2 min at room temperature. Carefully remove and discard the supernatant using a P1000 micropipette. 
	NOTE: Steps 3.1-3.5 can be used to passage melanocyte and fibroblast cultures. The resulting pellet should be resuspended in the appropriate media and placed in a new, uncoated (fibroblasts) or pre-washed, collagen-coated (melanocytes) dish.</li>
	<li>Resuspend the cell pellet in 0.5 mL of ice-cold PBS.</li>
	<li>Enumerate and transfer 0.5 million cells to a pre-chilled 1.5 mL microcentrifuge tube.</li>
	<li>Repeat step 3.5 and then resuspend the cells in 100 &#181;L of Viability Solution. Incubate the cell suspension for 30 min at 4 &#176;C in the dark.</li>
	<li>Repeat steps 3.5-3.6 twice.</li>
	<li>Repeat step 3.5, then fix the cells by resuspending the pellet in 100 &#181;L of ice cold 4% Paraformaldehyde Solution. Incubate the suspension for 15 min at room temperature in the dark.</li>
	<li>Repeat step 3.5 twice, each time resuspending the pellet in 0.5 mL of 3% BSA in 1x PBS.</li>
	<li>Repeat step 3.5, then resuspend the cell pellet in 100 &#181;L of 0.1% Saponin Solution. Incubate the suspension for 15 min at room temperature in the dark.</li>
	<li>Repeat step 3.5, then resuspend the cell pellet in 100 &#181;L of 0.1% Saponin Solution containing 0.5 &#181;g of rabbit anti-gp100 antibody, 0.5 &#181;g of rabbit anti-Fibroblast-specific protein 1 (FSP1) antibody and 0.025 &#181;g of mouse anti-Cytokeratin 14 (K14) antibody. Incubate the suspension for 1 h at room temperature in the dark. 
	NOTE: While the K14 antibody was purchased pre-conjugated to Alexa Fluor 647, the gp100 and FSP1 antibodies were conjugated to CF 555 and CF 488, respectively. The staining of control populations should also commence at this step. Control cell populations should be isolated from culture and processed as described in steps 3.5-3.12.</li>
	<li>Repeat step 3.11 twice, to remove any unbound antibody.</li>
	<li>Repeat step 3.5, then resuspend the cell pellet in 200-400 &#181;L of 3% BSA in 1x PBS.</li>
	<li>Pass the cell solution through a 40 &#181;m cell strainer into a 5 mL polystyrene round-bottom tube and analyze the strained cells by flow cytometry.</li>
</ol>

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The authors have no conflicts of interest to disclose.

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 consistent chopping force and granularity of a mechanical tissue chopper along with selective media and plating techniques. By reducing processing complexity and hands-on time, this procedure also enables researchers to conduct large-scale experiments efficiently.
We recommend several steps to enhance the yield and consistency using this protocol. First, prior to step 2.4, we advise using curved forceps to scrape away any adipose tissue from the dermal side of the skin. This process will reduce the fat cake formed after centrifugation. Next, proper mechanical dissociation of the skin is critical to the success of this protocol and can be enhanced by rotating the homogenizer disk ~60&#176; after each pass of the blade. Another key step is to ensure that all of the Skin Digest Buffer is removed in step 2.10. Failure to do so will hinder cell adhesion to the dish and significantly decrease yield. Because the pellet is not firmly affixed to the bottom of the 15 mL conical tube in step 2.10, the cells can be easily aspirated if the supernatant is not slowly removed. If thorough removal of the Skin Digestion Buffer is a challenge, we suggest washing the pellet in 2-3 mL of Melanocyte Media and then repeating steps 2.9-2.10 prior to plating. Finally, when resuspending the cell pellet in step 2.11, it is important to pipet vigorously in order to break up any clumps and ensure that the cells are evenly distributed on the surface of the cell culture dish. Debris and dead cells will be observed in the culture during the first few days after isolation. These dead cells may hinder the growth of the culture and should be removed by washing the dish with PBS and then adding fresh media.
This protocol outlines a method to generate primary melanocyte and fibroblast cultures from a single mouse. However, the procedure can be effectively adjusted to accommodate larger cohorts of mice (see Table 1). For larger cohorts, combining skin homogenate from 4-5 pups results in a 30-40% confluent 10 cm dish of melanocytes within 4-5 days of isolation. Several techniques can be used to improve the efficiency of this protocol when working with multiple animals. First, we typically euthanize the animals in sets of four, processing two animals at a time. We have found that homogenized skin from two pups can be combined at steps 2.7-2.8 by increasing the volume of Skin Digest Buffer to 6 mL. To save time, we begin isolating skin from the next pair of pups while the first set is undergoing homogenization and digestion. This approach ensures that the cell populations are isolated soon after euthanasia and reduces the processing time required for multiple animals. When working with older mice (i.e., postnatal days 2-4), we have found that passing the skin a fourth time through the active tissue chopper (step 2.6) enhances skin homogenization and cellular yield. Using this technique, we see no age-dependent difference in the melanocyte or fibroblast yield.
This protocol details how to simultaneously generate fibroblast and melanocyte cultures from the same skin sample. We have observed that keratinocytes in the initial melanocyte culture remain adhered to the dish when short-term trypsinization is performed along with the forceful dislodgement of melanocytes (see steps 3.2-3.3). While we have not optimized propagation methods for these remaining cells, we hypothesize that continued culture of such adherent populations in keratinocyte media supplemented with 100 ng/mL G418 could result in an enriched cell population.

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><tbody><tr><td>0.2 &amp;micro;m PES sterile syringe filter</td><td>VWR</td><td>28145-501</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>5 mL polystyrene round-bottom tubes</td><td>Fisher Scientific</td><td>352008</td><td></td></tr><tr><td>6-well cell culture dish</td><td>Sigma-Aldrich</td><td>SIAL0516</td><td></td></tr><tr><td>70 &amp;micro;m cell strainer</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>Antibiotic Antimycotic Solution (100x)</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP9706-100</td><td></td></tr><tr><td>CF 488A Mix-n-Stain Antibody Labeling Kit</td><td>Biotium</td><td>92273</td><td></td></tr><tr><td>CF 555 Mix-n-Stain Antibody Labeling Kit</td><td>Biotium</td><td>92274</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma-Aldrich</td><td>C8052</td><td></td></tr><tr><td>Collagen from rat tail</td><td>Sigma-Aldrich</td><td>C7661</td><td></td></tr><tr><td>Collagenase Type I</td><td>Worthington Biochemicals</td><td>LS004156</td><td></td></tr><tr><td>Corning Penicillin/Streptomycin Solution</td><td>Fisher Scientific</td><td>30-002-CL</td><td></td></tr><tr><td>Cytokeratin 14 Antibody Alexa Fluor 647</td><td>Novus Biologicals</td><td>NBP2-34403AF647</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Worthington Biochemicals</td><td>LS002058</td><td></td></tr><tr><td>Di-butyryl cyclic AMP</td><td>Sigma-Aldrich</td><td>D0627</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco</td><td>12800-082</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>eBioscience Fixable Viability Dye eFluor 780</td><td>Thermo Fisher</td><td>65-0865-14</td><td></td></tr><tr><td>Ethanol, 200 proof</td><td>Fisher Scientific</td><td>22032601</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>12306C</td><td></td></tr><tr><td>FSP1/S100A4 antibody</td><td>Millipore Sigma</td><td>07-2274</td><td></td></tr><tr><td>G418 Disulfide</td><td>P212121</td><td>LGB-418-1</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>VWR</td><td>VWRV0714</td><td></td></tr><tr><td>Horse Serum</td><td>Fisher Scientific</td><td>26050088</td><td></td></tr><tr><td>HyClone L-Glutamine</td><td>Fisher Scientific</td><td>SH3003402</td><td></td></tr><tr><td>McIlwain Tissue Chopper</td><td>Ted Pella</td><td>10180</td><td></td></tr><tr><td>Melanoma gp100 antibody</td><td>Abcam</td><td>ab137078</td><td></td></tr><tr><td>Nutrient Mix F-12 Ham&#039;s Media</td><td>Sigma-Aldrich</td><td>N6760</td><td></td></tr><tr><td>Phorbol 12-Myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>Pierce 16% Formaldehyde</td><td>Thermo Fisher</td><td>28908</td><td></td></tr><tr><td>Porcine Trypsin</td><td>Sigma-Aldrich</td><td>85450C</td><td></td></tr><tr><td>RPMI 1640 media</td><td>Sigma-Aldrich</td><td>R8758</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>S-7900</td><td></td></tr><tr><td>Tissue Chopper Blade</td><td>Ted Pella</td><td>121-6</td><td></td></tr><tr><td>Tissue Chopper Plastic Disk</td><td>Ted Pella</td><td>10180-01</td><td></td></tr><tr><td>Trypsin</td><td>VWR</td><td>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 transferred to an uncoated well of a 6-well dish. The high density of cells caused the media to appear cloudy. However, after 40 min of incubation, larger cell and tissue conglomerates were observed in the media and adherent fibroblasts could be seen with an inverted light microscope (Figure 1).
The non-adherent cells from the fibroblast culture were transferred to one well of a collagen-coated 6-well dish in order to isolate melanocytes. Addition of G418 killed any remaining fibroblasts in the homogenate, leaving numerous dead cells floating in the media for the next 5 days (Figure 2A-C). After 4-5 days of growth, the plated melanocytes began to take on a stereotypical dendritic phenotype with melanocytic granules (Figure 2C). This phenotype persisted after passaging of the culture (Figure 2D). While clusters of contaminating keratinocytes were initially observed in the melanocyte cultures (Figure 2A), these populations were lost upon subsequent passaging.
Flow cytometric analyses were used to confirm the purity of the resulting primary melanocyte and fibroblast cultures. C5N mouse keratinocytes17, day 10 primary melanocyte cultures and day 6 primary fibroblast cultures were stained with: anti-gp100 (differentiated melanocytes), anti-Cytokeratin 14 (K14; keratinocytes) and anti-Fibroblast-specific protein 1 (FSP1; fibroblasts) (Figure 3). In these cells, gp100 staining was specific to the primary melanocytes, while FSP1 positivity was only observed in fibroblasts (Figure 3A,B). Expression of K14 was limited to C5N keratinocytes (Figure 3C). &#160;Additional analyses were performed to determine the purity of our melanocyte cultures 1, 2, 3, 4 and 10 days after isolation (Figure 4). Combined fibroblast and keratinocyte contamination never exceeded 25% of the total cells in culture (Figure 4B,C). Nevertheless, dramatic increases in gp100 were not observed until the fourth day in culture (Figure 4A). We attribute this observation to the reduced expression of gp100 in melanocyte precursors (i.e., melanoblasts), many of which appear fully differentiate by day 10 in culture (Figure 4A). In summary, these data demonstrate that our rapid isolation protocol simultaneously produces cultures enriched for melanocytes and fibroblasts.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Step #</td>
			<td>Reagent</td>
			<td>1 pup</td>
			<td>5 pups</td>
		</tr>
		<tr>
			<td>1.1</td>
			<td>Collagen Solution</td>
			<td>1.5 mL</td>
			<td>4.5 mL</td>
		</tr>
		<tr>
			<td>1.2</td>
			<td>Plate size</td>
			<td>6-well</td>
			<td>10 cm</td>
		</tr>
		<tr>
			<td>1.4</td>
			<td>Antibiotic / Antimycotic Solution</td>
			<td>3 mL</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>1.5</td>
			<td>Skin Digestion Buffer</td>
			<td>3 mL</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>1.6</td>
			<td>Melanocyte Media</td>
			<td>6 mL</td>
			<td>30 mL</td>
		</tr>
		<tr>
			<td>1.7</td>
			<td>Fibroblast Media</td>
			<td>4 mL</td>
			<td>20 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Reagent preparation guide for different cohort sizes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59468/59468fig1.jpg" /> 
Figure 1: Representative images of primary murine fibroblast cultures. Shown are 20x images of fibroblasts immediately following isolation (A), as well as 24 (B) and 48 (C) h post-isolation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59468/59468fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59468/59468fig2.jpg" /> 
Figure 2: Representative images of primary melanocyte cultures. Shown are 20x images of melanocyte cultures 1 (A), 2 (B) and 4 days (C) post-isolation. Contaminating keratinocytes are indicated by the arrow in &#39;A&#39;. (D) Representative image of primary melanocyte cultures after passaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59468/59468fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59468/59468fig3.jpg" /> 
Figure 3: Flow cytometric assessments of culture purity. Representative histograms showing gp100 (A; differentiated melanocytes), FSP1 (B; fibroblasts) and K14 (C; keratinocytes) positivity in primary melanocytes (day 10), primary fibroblasts (day 6) and C5N keratinocytes. After gating on live cells, 10,000 events were analyzed from each population. Single color controls were used for compensation and the resulting data was visualized with FlowJo software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59468/59468fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59468/59468fig4.jpg" /> 
Figure 4: Time course analysis of melanocyte purity. Representative histograms showing melanocyte purity 1-10 days after initial isolation. Samples were stained, analyzed and graphed as described in Figure 3. Positive and negative control populations were as follows: gp100--human melanocytes (+), primary murine fibroblasts (-); FSP1--primary murine fibroblasts (+), human melanocytes (-); K14--C5N cells (+), human melanocytes (-). The average positivity and standard deviation for at least three distinct melanocyte cultures is shown in the upper right-hand corner of each graph. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59468/59468fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

rapid-generation-of-primary-murine-melanocyte-and-fibroblast-cultures

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.7K Views.  The Ohio State University. Dit protocol beschrijft een snelle en eenvoudige methode om primaire melanocyten- en fibroblastculturen van muizen te genereren. Omdat deze techniek niet vereist dat de opperhuid en dermis worden gescheiden, kan het snel en consistent worden uitgevoerd met minimale training. Deze methode kan worden gebruikt om cutane melanocyten en fibroblast biologie te bestuderen.Experimenten in deze culturen kunnen inzichten bieden die relevant zijn voor kanker, wondgenezing en pigmentatiedefecten. ...

Video: Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Developmental Biology

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Biology

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Medicine

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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

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.

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

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

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