We are grateful to Prof. emeritus Jean-Claude Perriard and Evelyn Perriard (Swiss Federal Institute of Technology, Switzerland) for the introduction into isolation techniques for neonatal rat and mouse cardiomyocytes. We would like to thank Prof. Ju Chen and Prof. Sylvia Evans (UCSD, USA) for their support. Work in the laboratory of EE was funded by an MRC Career Establishment Grant. SL is supported by a K99/R00 pathway to independence award from the NIH/NHLBI (HL107744). TMM was supported by a postdoctoral fellowship from the American Heart Association (11POST7310066).

The following procedure describes a two-day protocol 16,17 for the isolation and culture of neonatal mouse cardiomyocytes. All solutions are sterile or sterile filtered. All tools are sterilized by surface sterilization with 75% ethanol. Except for the initial tissue extraction, all steps are performed in a sterile laminar flow cell culture hood. This protocol is intended for the isolation of neonatal mouse hearts from one-two litter(s) - approximately 5-14 pups, but may be adapted for larger litter sizes and ...

The use of animal models to study cardiac diseases has become standard in cardiovascular research. Closer biochemical characterization of these models (i.e. studying direct responses of cardiac cells to biochemical or biomechanical stimuli) typically requires the isolation of heart tissues or cardiomyocytes. Studies investigating physiological responses of the heart ex vivo (e.g. to acetylcholine 40, or in ischemia-reperfusion scenarios 41) generally utilize langendorff-pe...

The earliest reports for the successful dissociation and culture of rodent heart cells dates back to the 1960's 1,2. Even then, Harary and Farley noticed that cultured cardiomyocytes "may provide a unique system for the study of the requirements of the periodic contractility [, and may] provide a means of determining the contribution of various metabolic pathways for the [beating] process". Although Harary and Farley isolated and cultured cardiomyocytes from young rats, and the original protocol has been adapted and modified by many scientists over the years, the general isolation and culturing procedure has not greatly changed. However, better enzymes 3, standardized solutions 4,5, and the addition of the reversible channel and myosin ATPase inhibitor BDM to protect cells during the isolation procedure 6-9 has significantly improved cell-yield and viability.
Adult vs. neonatal cardiomyocytes
Cardiomyocytes isolated and cultured from neonatal mice or rats have several advantages over cultures of adult cardiomyocytes. Foremost, the isolation procedure for neonatal mouse or rat hearts is easier and less costly, when compared to the isolation of cardiomyocytes from adult mouse or rat 10. Neonatal cardiomyocytes are far less sensitive to reintroduction into a calcium-containing medium after dissociation, greatly increasing cell-yield. Another big advantage is that neonatal mouse cardiomyocytes undergo a more rapid dedifferentiation - redifferentiation cycle that typically results in spontaneously beating cells 20 hr after plating, while adult cardiomyocytes typically require pacing to induce contraction. Neonatal cardiomyocytes are also more readily transfectable with liposomal transfection methods, whereas adult cardiomyocytes require viral vectors for successful delivery of transgenic DNA. In contrast to neonatal cardiomyocytes, culture of adult rodent cardiomyocytes 11-13 allows for investigations of myofibrillar degradation and eventual reestablishment of the contractile apparatus. These characteristic morphological changes in adult cardiomyocytes occur over periods of 1-2 weeks. The dedifferentiation - redifferentiation cycle is accompanied by reexpression of the fetal gene program, thereby mimicking pathological changes observed in human cardiomyopathies 14. Another advantage of adult rat cardiomyocytes over the culture of neonatal cardiomyocytes is the ability to culture these cells for long periods of time.
Rat vs. mouse cardiomyocytes
The isolation and culture of rat neonatal cardiomyocytes has some benefits over that of mouse neonatal cardiomyocytes, including higher yields of viable cells and increased transfection rates. However, the wide usage of genetically modified mouse models for cardiac diseases (e.g. the muscle lim protein knockout mouse as model for dilated cardiomyopathy 15) has led to the adaptation of the isolation procedure for cardiomyocytes derived from neonatal mice. Although the protocols used to isolate neonatal rat and mouse cardiomyocytes are nearly identical, greater care must be taken in the selection of an appropriate enzyme mix for the latter. Indeed, neonatal mouse cardiomyocytes are generally more susceptible to overdigestion, resulting in a reduced cell-yield and viability. Moreover, the plating density should be adjusted, because cardiomyocytes derived from neonatal mice are somewhat smaller compared to cells derived from neonatal rat hearts.
With many uses for the investigation of morphological, electrophysiological, biochemical, cell-biological and biomechanical parameters as well as for the process of myofibrillogenesis, cultured neonatal cardiomyocytes have become one of the most versatile systems for the study of cardiac cell functions in vitro. The first step to a successful assay however, depends on an easy and reliable methodology to isolate neonatal mouse cardiomyocytes. Our protocol draws its methodology from many sources and was optimized for reproducibility and robustness. We discuss factors that influence cardiomyocyte-yield and viability, and provide a variety of options for the optimization of isolation and culture conditions.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>BDM (2,3-Butanedione monoxime) 6-8</td>
			<td>Sigma</td>
			<td>B-0753</td>
			<td>prepare 0.2M stock solution in HBSS (without Ca2+, Mg2+), filter sterilize, can be kept at 4 &deg;C up to 6 months; Caution: Prolonged usage of BDM other than during isolation procedure may result in non-beating cells, decreased cell viability and/or significantly altered gene-expression during cardiomyocyte culture46,47.</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td>can be substituted with collagenase type II from Worthington</td>
		</tr>
		<tr>
			<td>Collagenase type II3</td>
			<td>Worthington</td>
			<td>CLS-2</td>
			<td>substitute for Collagenase/Dispase mix from Roche</td>
		</tr>
		<tr>
			<td>1x trypsin solution (0.25%) with EDTA</td>
			<td>e.g. cellgro</td>
			<td>25-053-CI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1x Penicillin/ Streptomycin solution with EDTA in HBSS</td>
			<td>e.g. cellgro</td>
			<td>30-002-Cl</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1x PBS (without Ca2+, Mg2+)</td>
			<td>e,g, cellgro</td>
			<td>21-040-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS (Hank's balanced salt solution; without Ca2+, Mg2+) 4</td>
			<td>e.g. cellgro</td>
			<td>21-022-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>e.g. cellgro</td>
			<td>10-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>M-199</td>
			<td>e.g. cellgro</td>
			<td>10-060-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>e.g. cellgro</td>
			<td>35-011-CV</td>
			<td>cell-culture grade</td>
		</tr>
		<tr>
			<td>horse serum</td>
			<td>e.g. cellgro</td>
			<td>35-030-CV</td>
			<td>cell-culture grade</td>
		</tr>
		<tr>
			<td>Leibovitz L-155</td>
			<td>e.g. cellgro</td>
			<td>10-045-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AraC (Cytosine-B-D-arabino-furanoside hydrochloride)</td>
			<td>Sigma</td>
			<td>C-6645</td>
			<td>proliferation inhibitor, prepare 1 mM stock solution in H2O, filter sterilize, store at 4 &deg;C</td>
		</tr>
		<tr>
			<td>phenylephrine</td>
			<td>Sigma</td>
			<td>P-6126</td>
			<td>chronotropic agent, prepare 100 mM stock solution in H2O, filter sterilize, store at 4 &deg;C or -20 &deg;C</td>
		</tr>
		<tr>
			<td>isoproterenol hydrochloride</td>
			<td>Sigma</td>
			<td>I-6501</td>
			<td>chronotropic agent, prepare 1 mM stock solution in H2O, filter sterilize, store at 4 &deg;C or -20 &deg;C</td>
		</tr>
		<tr>
			<td>0.1% collagen solution</td>
			<td>Sigma</td>
			<td>C-8919</td>
			<td>extracellular matrix for coating</td>
		</tr>
		<tr>
			<td>3 mg/ml collagen type 1 solution</td>
			<td>Advanced BioMatrix</td>
			<td>5005-B</td>
			<td>alternative to Sigma collagen solution</td>
		</tr>
		<tr>
			<td>cell strainer</td>
			<td>e.g. Fisherbrand</td>
			<td>22363548</td>
			<td>appropriate filter size:40 &mu;m-100 &mu;m</td>
		</tr>
		<tr>
			<td>syringe filter 0.2 &mu;m</td>
			<td>e.g. Fisherbrand</td>
			<td>09-719C</td>
			<td>for sterile filtration of digestion medium</td>
		</tr>
		<tr>
			<td>straight scissors</td>
			<td>e.g. Fine Sciences Tools</td>
			<td>91460-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>curved scissors</td>
			<td>e.g. Fine Science Tools</td>
			<td>91461-11</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont No. 7 forceps</td>
			<td>e.g. Fine Science Tools</td>
			<td>91197-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>perforated spoon</td>
			<td>e.g. Fine Science Tools</td>
			<td>10370-19</td>
			<td>optional, for transfer of heart tissue</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>e.g. Gibco</td>
			<td>15250-061</td>
			<td>live cell staining</td>
		</tr>
		<tr>
			<td>Neubauer hemocytometer</td>
			<td>e.g. Prosource Scientific</td>
			<td>3500</td>
			<td>alternatively use: disposable hemocytometer C-chip or automated cell counting systems</td>
		</tr>
		<tr>
			<td>50 ml Falcon tubes</td>
			<td>e.g. Fisherbrand</td>
			<td>14-432-23</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml Falcon tubes</td>
			<td>e.g. Fisherbrand</td>
			<td>05-527-90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20 ml syringe</td>
			<td>e.g. BD Medical</td>
			<td>14-820-19</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml serological pipette</td>
			<td>e.g. Falcon</td>
			<td>357551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30 mm cell culture dish</td>
			<td>e.g. Nunc</td>
			<td>153066</td>
			<td>for standard culture of cardiomyocytes</td>
		</tr>
		<tr>
			<td>30 mm cell culture dish, glass bottom</td>
			<td>MatTek</td>
			<td>P35G-0-10-C</td>
			<td>for live cell imaging with inverted microscope</td>
		</tr>
		<tr>
			<td>10 cm cell culture dish</td>
			<td>e.g. Nunc</td>
			<td>172958</td>
			<td>for preplating</td>
		</tr>
		<tr>
			<td>Escort III</td>
			<td>Sigma</td>
			<td>L3037</td>
			<td>for liposomal transfection, alternatively use lipofectamin 2000</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Life Technologies, Invitrogen</td>
			<td>52887</td>
			<td>substitute for Escort III</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Buffers and media: 
			<ul>
				<li>Isolation medium (filter sterilize) 
				20 mM BDM 
				0.0125% trypsin 
				in HBSS4 (without Ca2+, Mg2+)</li>
				<li>Digestion medium (filter sterilize) 
				20 mM BDM 
				1.5 mg/ml Roche Collagenase/Dispase enzyme mix 
				in L15 medium</li>
				<li>Plating medium 
				65% DMEM high glucose 
				19% M-199 
				10% horse serum 
				5% fetal calf serum 
				1% penicillin/streptomycin</li>
				<li>Maintenance medium 
				78% DMEM high glucose 
				17% M-199 
				4% horse serum 
				1% penicillin/streptomycin 
				optional: 
				1 &mu;M AraC 
				1 &mu;M isoproterenol or 0.1 mM phenylephrine</li>
			</ul>
			The plating and maintenance medium can be stored at 4 &deg;C for up to 6 months.</td>
		</tr>
	</tbody>
</table>

isolation and culture of neonatal mouse cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Using this protocol, we isolated hearts from 8 one day old neonatal mice (Figures 1A, 1B, Supplemental Movie S1). After washing and mincing the hearts with scissors (Figures 1C-1F), tissue fragments were predigested in isolation medium over night at 4 &deg;C with gentle agitation. Following predigestion (Figure 1G), we transferred the tissue fragments into freshly made digestion medium, and incubated the tissue fragments for 20 min at 37 &deg;C with gentle agitation. The...

Watch this Scientific Journal Video about Isolation and Culture of Neonatal Mouse Cardiomyocytes at JoVE.com

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...