Name: Isolating Primary Melanocyte-like Cells from the Mouse Heart (Video) | JoVE Journal
Uploaded: 2014-09-29T13:00:00.000+00:00
Duration: 7 min 38 s
Description: Learn to isolate cardiac melanocyte-like cells from mouse hearts using enzymatic digestion and mechanical dissociation. This method enables patch studies and microarray analysis for investigating atrial arrhythmia mechanisms.

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5 minutes

This work was supported by National Institutes of Health award R01 HL105734 to V.V.P. M.D.L. is supported, in part, by National Institutes of Health Research Career Award K08 HL094748. V.V.P. is an Innovative Researcher of the American Heart Association supported by grant 11IRG900384.

1. Preparing the Necessary Solutions and Equipment
<ol>
	<li>First, sterilize the surgical instruments (straight shape forceps, curved shape forceps, straight shape scissors, and curved shape scissors) by autoclaving them for 15 min. at 121 &#176;C. Prepare 2 sets of instruments, where one set is used for dissecting out the hearts and the other set will be used for mincing up the tissue.</li>
	<li>Dissolve 1 packet of MCDB153 culture media in 900 ml of sterilized, filtered water and stir slowly until the powder has dissolved. Add 1.2 g sodium bicarbonate, or 15.7 ml of sodium bicarbonate solution [7.5% w/v], for each liter of final volume of medium being prepared and stir until dissolved. Adjust the pH to 7.2 by adding either 1 N HCl or 1 N NaOH, then pour the media through a 0.2 &#956;m bottle top filter under sterile conditions in a tissue culture hood (biosafety cabinet). Then collect the media and store it at 4 &#176;C.</li>
	<li>Prepare the supplements to add to the culture media (Materials List). The supplements should be added to the media just before the media is needed for plating the cells.</li>
	<li>Prepare DPBS with 10% FBS plus antibiotics (penicillin and streptomycin).</li>
	<li>Next, prepare a 0.25% trypsin solution by adding 0.25 g of trypsin (Bovine pancreas, activity &#62; 2,500 USP units/mg, ultrapure; USB) to 100 ml of DPBS plus antibiotics (penicillin and streptomycin).</li>
	<li>Other required equipment needed to complete this procedure include a stereomicroscope, Petri-dishes (10-cm), 50-ml conical tubes, 40-&#956;m cell strainers, DPBS plus antibiotics, 60-mm culture dishes and 35-mm culture dishes.</li>
</ol>
2. Isolating Hearts from Neonatal Mouse Pups
<ol>
	<li>First, obtain P0 to P2 neonatal mouse pups (n=6 to 8). It is recommended to use pups generated by breeding Dct-Cre homozygous females with males that are heterozygous for either the R26R:EYFP allele or the Z/EG allele to label the melanocyte-like cells. Both R26R:EYFP and Z/EG mice are available from Jackson Laboratories (stock numbers 006148 and 003920, respectively).</li>
	<li>Next, euthanize the neonatal mouse pups, wipe their chests with an alcohol swab and then place the pups in a 10-cm Petri&#160;dish with DPBS.</li>
	<li>Cut the skin over the chest and sternum with sterilized forceps and scissors and then open the chest carefully under a stereomicroscope. The hearts of neonatal mouse pups are very small so be careful to make sure to remove the entire heart with the atria attached.</li>
	<li>Wash the hearts in DPBS and transfer them to a new 10-cm Petri&#160;dish.</li>
</ol>
3. Enzymatic Digestion
<ol>
	<li>Bring the Petri-dish with the excised hearts to a tissue culture hood (biosafety cabinet) and then follow the steps below using sterile techniques in the hood.</li>
	<li>First, wash the hearts three times in sterile DPBS plus antibiotics.</li>
	<li>Place the hearts (n=6-8) in a 60-mm culture dish and add 6-8 ml of 0.5% trypsin solution to the dish.</li>
	<li>Next, cut the hearts into small pieces (less than 1-mm in size) and incubate them at 37 &#176;C for 30 min in a 5% CO2 atmosphere.</li>
	<li>After the incubation period is complete, pour the resulting solution of cardiac tissue and trypsin from the dish into a sterile 50-ml conical tube.</li>
	<li>Then, using a 10-ml sterile pipette, quickly but gently, triturate the solution in the 50-ml conical tube 30-50 times.</li>
	<li>Filter the resulting solution through a 40-&#956;m cell strainer on top of a new, clean 50-ml conical tube to collect the filtrate.</li>
	<li>Next, centrifuge the 50-ml tube at 240 x g in a table-top centrifuge for 5 min at room temperature. Then, gently aspirate off the supernatant and resuspend the remaining pellet in 6-8 ml of sterile DPBS. Repeat this step 2 more times. This step pellets the atrial myocytes and cardiac melanocytes but does not bring down the fibroblasts since the fibroblasts are smaller and will remain in the supernatant.</li>
</ol>
4. Plating Cardiac Melanocyte-like Cells
<ol>
	<li>Following the third rinse after centrifugation, carefully drain or aspirate off the wash solution (DPBS) and resuspend the pellet in 6 ml of MCDB culture media to which the supplements have been added.</li>
	<li>Now, draw the resuspended solution into a sterile transfer pipette and place the cells in a 35-mm dish, or in a well of a 6-well plate, and pool the isolated cardiac cells from 3 neonatal mice in one 35-mm dish or well.</li>
	<li>Incubate the dishes overnight at 37 &#176;C in a 5% CO2 atmosphere, aspirate off the culture media along with any unattached cells and then rinse the plates once with DPBS. Then add fresh media to the plates and replace the media every 2 days. Cells isolated from neonatal or embryonic hearts can be maintained in culture for up to 3 weeks, whereas cells isolated from adult hearts can be maintained in culture for up to 10 days.</li>
</ol>
5. Isolating Cardiac Melanocyte-like Cells from Adult Mouse Heart
<ol>
	<li>First, remove the hearts from mature mice (3-4 weeks-old, n=3) through a mid-line sternotomy after administering heparin 100 U intraperitoneal to the mice and then euthanizing them. It is recommended to use pentobarbital to euthanize mice for this procedure, but this drug is not available in all locations.</li>
	<li>Next, wash the excised hearts in sterile DPBS containing antibiotics in a tissue culture hood (biosafety cabinet). Squeeze the hearts slightly with curved dissecting forceps to remove any remaining blood from them and then rinse the hearts in DPBS one more time.</li>
	<li>Remove the atria and atrioventricular annulus from the hearts (these structures contain the melanocyte-like cells) and place the excised tissue specimens in a 35-mm dish.</li>
	<li>Cut the hearts into small pieces using curved scissors and forceps and then add 6-8 ml of 0.5% trypsin solution to the dish. Then incubate the dishes at 37 &#176;C for 45-60 min.</li>
	<li>Now, follow the steps from the procedure above starting from step 3.6 onwards.</li>
</ol>

<ol><li>Hornyak, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitf+dosage+as+a+primary+determinant+of+melanocyte+survival+after+ultraviolet+irradiation%5BTitle%5D)+AND+%22Pigment+Cell+Melanoma+Res%22%5BJournal%5D)">Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.</a> Pigment Cell Melanoma Res. 22, 307-318 (2009).</li>
<li>Yonetani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+expansion+of+immature+melanoblasts+and+their+ability+to+repopulate+melanocyte+stem+cells+in+the+hair+follicle%5BTitle%5D)+AND+%22J.+Invest.+Dermatol%22%5BJournal%5D)">In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.</a> J. Invest. Dermatol. 128, 408-420 (2008).</li>
<li>Levin, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Melanocyte-like+cells+in+the+heart+and+pulmonary+veins+contribute+to+atrial+arrhythmia+triggers%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.</a> J. Clin. Invest. 119, 3420-3436 (2009).</li>
<li>Patel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+insights+in+the+cellular+basis+of+atrial+fibrillation%5BTitle%5D)+AND+%22Expert+Rev.+Cardiovasc.+Ther%22%5BJournal%5D)">Novel insights in the cellular basis of atrial fibrillation.</a> Expert Rev. Cardiovasc. Ther. 8, 907-915 (2010).</li>
<li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+neurons+and+melanocytes+induced+from+immortal+lines+of+postnatal+neural+crest-like+stem+cells%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.</a> FASEB J. 23, 3179-3192 (2009).</li>
<li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((P16Ink4a+in+melanocyte+senescence+and+differentiation%5BTitle%5D)+AND+%22J.+Natl.+Cancer.+Inst%22%5BJournal%5D)">P16Ink4a in melanocyte senescence and differentiation.</a> J. Natl. Cancer. Inst. 94, 446-454 (2002).</li>
<li>Cook, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+melanoblasts+in+culture%3A+Expression+of+BRN2+and+synergistic+regulation+by+fibroblast+growth+factor-2%2C+stem+cell+factor%2C+and+endothelin-3%5BTitle%5D)+AND+%22J.+Invest.+Dermatol%22%5BJournal%5D)">Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.</a> J. Invest. Dermatol. 121, 1150-1159 (2002).</li>
<li>Fang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem Cells. 24, 1668-1677 (2006).</li>
<li>Brito, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timeline+and+distribution+of+melanocyte+precursors+in+the+mouse+heart%5BTitle%5D)+AND+%22Pigment+Cell+Melanoma+Res%22%5BJournal%5D)">Timeline and distribution of melanocyte precursors in the mouse heart.</a> Pigment Cell Melanoma Res. 21, 464-470 (2008).</li>
<li>Yajima, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolation+of+heart+melanocytes+is+specified+and+the+level+of+pigmentation+in+the+heart+may+correlate+with+coat+color%5BTitle%5D)+AND+%22Pigment+Cell+Melanoma+Res%22%5BJournal%5D)">The isolation of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color.</a> Pigment Cell Melanoma Res. 21, 471-476 (2008).</li>
</ol>

We have nothing to disclose.

Cardiac melanocytes are present in anatomic locations within the heart that commonly give rise to atrial arrhythmia triggers. These regions include the pulmonary veins, the atrioventricular annulus, the posterior left atrium and the foramen ovale (Figure 4). To understand the role these cells play in arrhythmogenesis, we develop techniques to isolate them and study their physiology. However, we recognized that improving this technique to yield healthier and larger quantities of cardiac melanocytes would ...

We recently identified a novel cell population in the heart of mice and humans that express melanin-synthesis enzymes and morphologically resemble cutaneous melanocytes. We called these cells &#39;cardiac melanocytes&#39; and found they are present in anatomic locations from which atrial arrhythmia triggers often originate in humans. We also found cardiac melanocytes contribute to atrial arrhythmias in mice with germline deletion of Dct. Cardiac melanocytes are sparsely distributed throughout the mouse atrium where they comprise less than 0.1% of all atrial cells. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from the mouse heart using Cre-inducible melanocyte specific markers. Our initial procedure was developed from those used to isolate neonatal murine cardiomyocytes and yielded very small numbers of cells suitable for patch clamp recordings or PCR analysis. However, to improve the health and viability of cardiac melanocytes, for more in-depth electrophysiological analysis or biochemical studies, we developed a refined procedure from those designed to isolate cutaneous melanocytes. The procedure described and demonstrated in this review has the advantage of producing healthier cardiac melanocytes that can be plated as a pure population or with cardiomyocytes.

<table><tbody><tr><td>MCDB 153</td><td>Sigma-Aldrich</td><td>M7403</td><td>Dissolve in water</td></tr><tr><td>DPBS, no calcium no magnesium</td><td>Invitrogen</td><td>14190136</td><td>Stock conc.: 1x&lt;br/&gt; Working conc.: 1x</td></tr><tr><td>Penicillin-streptomycin </td><td>Invitrogen</td><td>15140163</td><td>Stock conc.: 100x&lt;br/&gt; Working conc.: 1x</td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technology</td><td>Certified</td><td>Working conc.: 4%</td></tr><tr><td>TPA</td><td>Sigma-Aldrich</td><td>P1585</td><td>Dissolve in DMSO&lt;br/&gt;Stock conc.: 3.2 &amp;mu;M&lt;br/&gt;Working conc.: 32 nM</td></tr><tr><td>Transferrin</td><td>Sigma-Aldrich</td><td>T8158</td><td>Dissolve in DPBS&lt;br/&gt;Stock conc.: 0.1 mg/ml&lt;br/&gt;Working conc.: 1 &amp;mu;g/ml</td></tr><tr><td>Dibutyryl cyclic AMP</td><td>Sigma-Aldrich</td><td>D0627</td><td>Dissolve in DPBS&lt;br/&gt;Stock conc.: 0.5 mM&lt;br/&gt;Working conc.: 5 &amp;mu;M</td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td>Dissolve in 10 mM acetic acid&lt;br/&gt;Stock conc.: 1 mg/ml&lt;br/&gt;Working conc.: 5 &amp;mu;g/ml</td></tr><tr><td>Basic FGF</td><td>BD Biosciences</td><td>354060</td><td>Dissolve in water&lt;br/&gt;Stock conc.: 4 ng/&amp;mu;l&lt;br/&gt;Working conc.: 4 ng/ml</td></tr></tbody></table>

isolating primary melanocyte-like cells from the mouse heart

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

The protocol described in this article is an improved technique compared to the one we previously published for isolating cardiac melanocytes from the murine heart. This procedure employs the use of fluorescent markers driven by melanin synthesis enzyme-specific Cre-recombinase to label cardiac melanocyte-like cells. It is important to use a marker to label cardiac melanocytes when working with freshly isolated embryonic or neonatal cells, since the morphological differences that distinguish cardiac melanocytes from atri...

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

The overall goal of this procedure is to isolate viable cardiac melanocyte like cells from the mouse heart. This is accomplished by first harvesting hearts from euthanized mice. The second step is to mince the hearts and dissociate the cells using enzymatic digestion and mechanical agitation.Next, the slurry of cells is centrifuged and the pellet containing the cardiomyocytes and melanocyte like cells is resus suspended. The final step is to plate and culture. The cells ultimately isolated cardiac melanocyte like cells can be used for patch studies or microarray analysis in response to pathophysiological stressor to assess cellular excitability or the status of the transcriptome.The main advantage of this technique over existing method designed to isolate and culture the cardiomyocytes, is that it results in the isolation of many wearable cardiac melanocytes like cells. Well, we first had the idea for this method when we discovered a new cell population, the mouse heart, and we wanted to investigate its physiology. To begin, prepare two sets of sterilized surgical instruments, one for dissecting out the hearts and another for mincing up the tissue.Next, prepare the media supplements, but wait to add them to the culture media until just before plating the cells. After preparing the culture media filter to sterilize the solution while working in a tissue culture hood, store the media at four degrees Celsius. Prepare the DNA's one solution by adding 0.05 grams of DNA's, one to 100 milliliters of DPBS with 10%FBS plus antibiotics.Next, prepare a 0.5%tryin solution by adding 0.5 grams of trypsin to 100 milliliters of DPBS plus antibiotics. Lastly, gather together other equipment needed to complete the procedure, including Petri dishes, 50 milliliter conical tubes, 40 micron cell strainers, DPBS plus antibiotics, 60 millimeter and 35 millimeter culture dishes. After euthanizing P zero to P two neonatal mouse pups, wipe their chest with alcohol and place them in a 10 centimeter P two dish With DPBS working under a stereo microscope, carefully open the chest cavity and remove the entire heart with the atria attached.Wash the hearts in DPBS and transfer them to a new 10 centimeter Petri dish. Move the Petri dish with the excised hearts into a tissue culture hood and follow sterile technique for the following steps. First, wash the hearts three times in sterile DPBS plus antibiotics.Place the hearts in a 60 millimeter culture dish and add six to eight milliliters of 0.5%trips in solution. Next, cut the hearts into small pieces and incubate them at 37 degrees Celsius for 30 minutes. After the incubation is complete, pull the resulting solution from the dish into a sterile 10 milliliter conical tube.Using a 10 milliliter sterile pipette quickly but gently rerate the solution 30 to 50 times, filter the resulting suspension through a 40 micron cell strainer into a new clean 50 milliliter conical tube. After centrifugation, gently aspirate off the supinate and resuspend the remaining pellet in six to eight milliliters of sterile DPBS. Repeat this step two more times.Following the third rinse after centrifugation, carefully drain or aspirate off the wash solution and resuspend the pellet in six milliliters of supplemented culture media. Next, draw the resuspended solution into a sterile transfer pipette and place the cells in a 35 millimeter dish. Pull the isolated cardiac cells from three neonatal mice after an overnight incubation, aspirate off the culture media along with any unattached cells, and then rinse the plates once with DPBS, add fresh media to the plates and replace the media.Every two days, cells isolated from neonatal or embryonic hearts can be maintained in culture for up to three weeks, whereas cells isolated from adult hearts can be maintained in culture for up to 10 days. To isolate melanocyte like cells from an adult mouse heart, first, wash the excised hearts in sterile DPBS containing antibiotics in a tissue culture hood. Squeeze the heart slightly with curved dissecting forceps to remove any remaining blood, and then rinse the heart in DPBS one more time.Next, remove the atria and atrial ventricular annulus from the hearts and place the excise tissue specimens in a 35 millimeter dish. Cut the hearts into small pieces using curved scissors and forceps. Add six to eight milliliters of 0.5%trips in solution to the dish and incubate a 37 degrees Celsius for 45 to 60 minutes.After the incubation, repeat the steps demonstrated earlier to obtain a suspension of adult cardiac cells and platers before. In this example, the white arrow identifies the cardiac melanocyte in both panels. Note that without the aid of the fluorescent marker, the cardiac melanocyte is difficult to identify and distinguish from the atrial myocytes attached to the dish, the light blue arrowhead point to atrial cells that are not well attached to the dish shown here, a healthy cardiac melanocytes isolated from a neonatal mouse heart.They have developed extensive dendritic processes and closely resemble cutaneous melanocytes. Once mastered, this technique can be done in a few hours if it's performed properly. After watching this video, you should have a good understanding of how to isolate viable melanocyte cells from the heart.That'll be suitable for physiologic and molecular analysis.

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Learn to isolate cardiac melanocyte-like cells from mouse hearts using enzymatic digestion and mechanical dissociation. This method enables patch studies and microarray analysis for investigating atrial arrhythmia mechanisms.

Isolating Primary Melanocyte-like Cells from the Mouse Heart

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