Isolating Primary Melanocyte-like Cells from the Mouse Heart

Summary

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. 

Abstract

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.

Introduction

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 'cardiac melanocytes' 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.

Protocol

1. Preparing the Necessary Solutions and Equipment

1. 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 °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.
2. 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 μm bottle top filter under sterile conditions in a tissue culture hood (biosafety cabinet). Then collect the media and store it at 4 °C.
3. 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.
4. Prepare DPBS with 10% FBS plus antibiotics (penicillin and streptomycin).
5. Next, prepare a 0.25% trypsin solution by adding 0.25 g of trypsin (Bovine pancreas, activity > 2,500 USP units/mg, ultrapure; USB) to 100 ml of DPBS plus antibiotics (penicillin and streptomycin).
6. Other required equipment needed to complete this procedure include a stereomicroscope, Petri-dishes (10-cm), 50-ml conical tubes, 40-μm cell strainers, DPBS plus antibiotics, 60-mm culture dishes and 35-mm culture dishes.

2. Isolating Hearts from Neonatal Mouse Pups

1. 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).
2. Next, euthanize the neonatal mouse pups, wipe their chests with an alcohol swab and then place the pups in a 10-cm Petri dish with DPBS.
3. 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.
4. Wash the hearts in DPBS and transfer them to a new 10-cm Petri dish.

3. Enzymatic Digestion

1. 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.
2. First, wash the hearts three times in sterile DPBS plus antibiotics.
3. 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.
4. Next, cut the hearts into small pieces (less than 1-mm in size) and incubate them at 37 °C for 30 min in a 5% CO₂ atmosphere.
5. 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.
6. Then, using a 10-ml sterile pipette, quickly but gently, triturate the solution in the 50-ml conical tube 30-50 times.
7. Filter the resulting solution through a 40-μm cell strainer on top of a new, clean 50-ml conical tube to collect the filtrate.
8. 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.

4. Plating Cardiac Melanocyte-like Cells

1. 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.
2. 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.
3. Incubate the dishes overnight at 37 °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.

5. Isolating Cardiac Melanocyte-like Cells from Adult Mouse Heart

1. 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.
2. 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.
3. 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.
4. 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 °C for 45-60 min.
5. Now, follow the steps from the procedure above starting from step 3.6 onwards.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MCDB 153	Sigma-Aldrich	M7403	Dissolve in water
DPBS, no calcium no magnesium	Invitrogen	14190136	Stock conc.: 1x Working conc.: 1x
Penicillin-streptomycin	Invitrogen	15140163	Stock conc.: 100x Working conc.: 1x
Fetal Bovine Serum	Life Technology	Certified	Working conc.: 4%
TPA	Sigma-Aldrich	P1585	Dissolve in DMSO Stock conc.: 3.2 μM Working conc.: 32 nM
Transferrin	Sigma-Aldrich	T8158	Dissolve in DPBS Stock conc.: 0.1 mg/ml Working conc.: 1 μg/ml
Dibutyryl cyclic AMP	Sigma-Aldrich	D0627	Dissolve in DPBS Stock conc.: 0.5 mM Working conc.: 5 μM
Insulin	Sigma-Aldrich	I6634	Dissolve in 10 mM acetic acid Stock conc.: 1 mg/ml Working conc.: 5 μg/ml
Basic FGF	BD Biosciences	354060	Dissolve in water Stock conc.: 4 ng/μl Working conc.: 4 ng/ml