This work was supported by National Heart, Lung, and Blood Institute Grant HL-36573. We thank Mr. David Sowa for editing this manuscript.

All research on mice was done according to procedures and guidelines of the National Institutes of Health, and the protocols were approved by the Institutional Animal Care and Use Committee of the University of Toledo, College of Medicine and Life Sciences.
1. Perfusion System Preparation
<ol>
	<li>The day before isolation, fill the entire perfusion system (including reservoirs) with a solution of fast alkaline residue-free detergent. Allow the solution to soak into the system for a few hours&#160;or overnight.</li>
	<li>The next morning, adjust the thermocycler temperature to 37 &#176;C and allow the solution to warm up. Remove the detergent solution and flush the system thoroughly with sterilized distilled water. Do not forget all the side branches.</li>
	<li>Fill the system with perfusion buffer via a constant peristaltic pump and draw any air bubbles out from the aorta chamber (bubble trap) with a side-mounted syringe to protect the heart from coronary occlusion. The flow rate of the peristaltic pump should be checked routinely.</li>
</ol>
2. Preparation of Buffers, Culture Media, and Dishes
<ol>
	<li>Perfusion Buffer: Make 500 ml of 1x&#160;perfusion buffer prior to use. This calcium-free buffer contains 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-HEPES, 12 mM NaHCO3, 10 mM KHCO3, 0.032 mM phenol red, 30 mM taurine, 10 mM BDM, and 5.5 mM glucose. Adjust the pH to 7.0 with 2 M HCl and filter through a 0.2 &#956;m&#160;filter, store at 4&#160;&#176;C. 
	Note: a)&#160;The perfusion solution should be prepared on the same day when it is used. In some cases, it is acceptable to store the solution for less than 3 days at 4 &#176;C. b)&#160;This buffer has a high potassium concentration up to 15.3 mM, which can keep the cardiomyocytes quiescent, reduce ATP consumption and increase Ca2+-tolerant ability.</li>
	<li>Digestion Buffer (300 U/ml, 25-50 ml/heart): prepare just prior to perfusion. Weigh 15,000 U of total activity of type II collagenase, dissolve in 50 ml perfusion buffer in culture hood. Add 25 &#956;l of 100 mM CaCl2 (stock solution, filter sterilized) to make the final calcium concentration 50 &#956;M. Warm the buffer to 37 &#176;C when ready for isolation. Unlike trypsin, collagenase works best in the presence of 50-100 &#956;M Ca2+.</li>
	<li>Stop Buffer (50 ml/heart): Operate in culture hood. Mix 45 ml of perfusion buffer with 5 ml sterile FBS.
	<ol>
		<li>Ca2+ solution I (12.5 &#956;M Ca2+): Add 2.5 &#956;l of 100 mM CaCl2 to 20 ml of stop buffer and mix.</li>
		<li>Ca2+ solution II (100 &#956;M Ca2+): Add 10 &#956;l of 100 mM CaCl2 to 10 ml of stop buffer and mix.</li>
		<li>Ca2+ solution III (400 &#956;M Ca2+): Add 40 &#956;l of 100 mM CaCl2 to 10 ml of stop buffer and mix.</li>
		<li>Ca2+ solution IV (900 &#956;M Ca2+): Add 90 &#956;l of 100 mM CaCl2 to 10 ml of stop buffer and mix.</li>
	</ol>
	</li>
	<li>Plating medium (50 ml/heart): Transfer 43 ml of MEM containing 2 mM L-glutamine and 1.26 mM CaCl2 into a sterile 50-ml conical tube in culture hood and add 5 ml of FBS, 1 ml of BDM stock solution (500 mM, filter sterilized), and 0.5 ml penicillin/streptomycin (10,000 U/ml). Mix and equilibrate for 2-3 hr&#160;prior to myocyte isolation in 2% CO2, 37 &#176;C incubator with the caps loose. Right before myocyte plating, add 0.5 ml of 200 mM ATP (pH 7.2, stock solution, filter sterilized) into the medium.</li>
	<li>Culture medium (50 ml/heart): Transfer 48 ml of MEM containing 2 mM L-glutamine and 1.26 mM CaCl2 into a sterile 50-ml conical tube in culture hood and add 1 ml of BDM stock solution (500 mM, filter sterilized), and 0.5 ml penicillin/streptomycin (10,000 U/ml). Mix and equilibrate in 2% CO2, 37 &#176;C incubator prior to myocyte isolation. Prior to use, add 0.5 ml of 100 mg/ml BSA (filter sterilized) into the medium. 
	Note: Using a 2% CO2 incubator facilitates maintaining the culture medium pH at 6.9-7.0, which helps the myoctyes maintain their rod-shaped morphology up to 24 hr.</li>
	<li>Laminin (1-2 &#956;g/cm2)-coated dishes or plates: Transfer 200 &#956;l of 1 mg/ml natural mouse laminin to 20 ml of sterilized PBS (w/o calcium and magnesium) and mix. Add 1 ml to 35-mm dishes, 2.5 ml to 60-mm dishes, or 5 ml to 100-mm dishes, shake to evenly cover the surfaces, and place in 2% CO2, 37 &#176;C incubator until myocyte plating. 
	Note: If the dishes or plates are not used on the day coated, they can be sealed with Parafilm and stored at 4&#176;C overnight&#160;(Slow Coating). Sealing is required to prevent evaporation and contamination. Coated plates can be kept at 4 &#176;C for up to 1 week.</li>
</ol>
3. Mouse Heart Removal and Cannulation
<ol>
	<li>Soak the scissors and forceps in 70% ethanol for 30 min and let them dry.</li>
	<li>Weigh the mouse to the nearest 0.1 gram. Record its body weight, strain, sex, and date of birth.</li>
	<li>Inject 200 &#956;l heparin (100 IU/mouse) i.p. 10 min prior to anesthesia to prevent coagulation of blood in the coronary arteries. Anesthetize the mouse with 200 mg/kg body weight of ketamine and 10 mg/kg body weight of xylazine by i.p. injection and wait for 5-10 min&#160;until the mouse stops responding to tail/toe pinches.</li>
	<li>Secure the mouse in the supine position by gently fixing the forepaws and hindpaws to a pinnable work surface (i.e. styrofoam) on an animal surgery tray or table bench near the perfusion system.</li>
	<li>Wipe the chest and abdomen with 70% ethanol. Make a midline skin incision from mid abdomen to the diaphragm with a surgical scissor.</li>
	<li>Cut the diaphragm with another surgical scissor and hold the sternum with curved serrated forceps. Cut bilaterally and retroflect thoracic cage to expose the heart.</li>
	<li>Lift the heart slightly using fine curved serrated and atraumatic forceps and dissect the heart out of the thoracic cavity as close as possible to the dorsal thoracic wall.</li>
	<li>Transfer the heart to a 100-mm dish containing cold perfusion buffer. Carefully trim away the connective tissues such as lungs, thymus, and bronchi.</li>
	<li>Identify the aorta and its cranial branches, which are usually hidden by thymus and fat pad. Cut the aorta below its first branch13, grasp the aortic wall, lift the heart, and slightly slip it on to the perfusion fluid-filled aortic cannula (1.0 mm OD stainless steel cannula with blunt/flat tip and notches 1 and 2 mm above the tip; or a 22 G reusable feeding needle) using two ultra fine-tip forceps. 
	Note: a)&#160;The perfusion fluid should be dripping during heart cannulation to prevent gas bubbles from entering the coronary arteries; b)&#160;Keep the tip of the cannula just above the aortic valve.</li>
	<li>Clamp the aorta to the cannula and ligate the aorta to the cannula with 6-0 surgical silk. The whole cannulation should take less than 120 sec.</li>
</ol>
4. Heart Perfusion and Digestion
<ol>
	<li>Perfuse the heart with calcium-free perfusion buffer at flow rate of 4 ml/min about 4-5 min until the effluents become clear, then switch to digestion buffer containing 50 &#956;M CaCl2 and perfuse for 3.5-20 min depending on mouse strain, perfusion pressure and collagenase activity. If applicable, increase the aorta pressure to 70-80 mmHg when digesting. When necessary, enzyme perfusate can be recirculated for digestion. Typically, 300 U/ml enzyme buffer will digest a heart in 10-12 min at a flow rate of 4 ml/min; If applying afterload pressure at 70 mmHg, 300 U/ml will take only 3.5-5.5 min at a flow rate of 4 ml/min.</li>
	<li>Stop digestion when the heart becomes slightly pale and flaccid. It should be spongy when gently pinched.</li>
</ol>
5. Cells dissociation and Calcium reintroduction
<ol>
	<li>Pull the aorta from the cannula with 70% ethanol-soaked forceps and put the heart into a sterile 60-mm dish containing 2.5 ml digestion buffer. Move into the culture hood using sterile supplies, staying mindful of sterile techniques for this and follow-up steps.</li>
	<li>Remove aorta, atria, and great vessels with fine surgical scissors. Gently tease the ventricle into 10-12 small pieces with two fine-tip forceps.</li>
	<li>Gently pipette the heart pieces and cells up and down ~10x&#160;with a 10-ml transfer pipette and then transfer to a 15-ml polypropylene conical tube. Rinse the dish with 7.5 ml calcium solution I containing 12.5 &#956;M CaCl2 and transfer this into the tube, resulting in a final volume of 10 ml.</li>
	<li>Gently pipette the cell suspension up and down using a fine-tip transfer pipette to disperse the large pieces of heart tissue.</li>
	<li>Then centrifuge for 3 min at 20 x g to separate out small non-myocyte cells such as endothelial cells and fibroblasts.</li>
	<li>Aspirate off the supernatant and resuspend the myocyte pellet in 10 ml calcium solution I with a supplement of 100 &#956;l of 200 mM ATP (pH 7.2, stock solution, filter sterilized). Leave the tube in the hood for 3-5 min.</li>
	<li>Transfer duplicate 10 &#956;l aliquots to a hemacytometer to count rod-shaped and round myocytes. Calculate the total number of myocytes and calculate the percentage of rod-shaped myocytes.</li>
	<li>Centrifuge the myocytes for 3 min at 20 x g. Remove the supernatant and resuspend the pellets in 10 ml calcium solution II containing 100 &#956;M CaCl2. Disperse the myocytes with the fine-tip transfer pipette by pipetting up and down.</li>
	<li>Repeat the above step (5.8) using calcium solution III (400 &#956;M CaCl2) and IV (900 &#956;M CaCl2), respectively.</li>
	<li>For mouse myocyte primary culture, resuspend the final myocyte pellet in 5 ml of plating medium. Count the total number of myocytes and calculate the percentage of rod-shaped myocytes.</li>
</ol>
6. Cell Culture
<ol>
	<li>Adjust the concentration of rod-shaped myocytes to 25,000 rod-shaped myocytes/ml in a 50-ml tube. Gently pipette the myocytes using a 10-ml pipette to suspend them well. Avoid cell sedimentation in the pipette and tube to ensure equal cell density for each dish or plate.</li>
	<li>After aspirating off the laminin coating solution, plate the myocytes into those dishes or plates. Gently slide the dishes or plates forward and backward and side-to-side in a cross-like pattern three to four times on the surface of the culture hood.</li>
	<li>Place the dishes or plates in a 2% CO2 incubator at 37 &#176;C. Incubate for 1-3 hr to allow myocyte attachment at a rate of about 80%.</li>
	<li>After attachment, gently aspirate off the medium containing unattached myocytes and cell debris. Gently add culture medium to the sides of the dishes or plates. Perform this medium change one by one. Immediately return the dishes or plates to the incubator.</li>
	<li>After O/N culture, the myocytes can be changed to the same medium without BDM before studies of short term cell signaling. Keep the BDM in long term culture for hypertrophic or apoptosis assays. 
	Note: BDM (2,3-butanedione monoxime) can inhibit the myosin-ATPase and prevent contraction. Its inhibition of myosin-based contraction is readily reversible. It is reported that BDM may have some potential side effects, such as acting as a non-specific phosphatase, increasing calcium release from SR, and alteration of free calcium concentration and cellular electrical properties, so before the treatment, the BDM should be removed. Moreover, in some cases, a specific myosin II inhibitor Blebbistatin could be an alternative drug to inhibit cardiomyocyte contraction8,9.&#160; However, in our cases, BDM is better than Blebbistatin for the sake of the quality and quantity of the cardiomyocytes.</li>
</ol>

<ol>
	<li>Louch, W. E., Sheehan, K. A., Wolska, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> J. Mol. Cell. 51, 288-298 (2011).</li><li>Gross, D. R., Gross, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolated heart preparations, problems, and pitfalls. In: Animal Models in Cardiovascular Research. , 109-130 (2009).</li><li>Schl&#252;ter, K. D., Piper, H. M., Dheim, S., Mohr, F. W., Delmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+ventricular+cardiomyocytes.">Isolation and culture of adult ventricular cardiomyocytes.</a> Practical Methods in Cardiovascular Research. , 557-567 (2005).</li><li>O&#39;Connell, T. D., Ni, Y. G., Lin, K. M., Han, H. P., Yan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+cardiac+myocytes+for+signaling+studies.">Isolation and culture of adult cardiac myocytes for signaling studies.</a> AfCS research reports. 1 (5), 1-9 (2003).</li><li>O&#39;Connell, T. D., Rodrigo, M. C., Simpon, P. C., Vivanco, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+mouse+cardiac+myocytes.">Isolation and culture of adult mouse cardiac myocytes.</a> Methods in Molecular Biology. 35, 271-296 (2007).</li><li>Nilolskaya, A., Sharma, V., Sigg, D. C., Laizzo, P. S., Xiao, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+models+and+methods.">Cell culture models and methods.</a> Cardiac Electrophysiology Methods and Models. , 213-235 (2010).</li><li>Merx, M. W., Schrader, M., Dhein, S., Mohr, F. W., Delmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+working+heart.">The working heart.</a> Practical Methods in Cardiovascular Research. , 173-189 (2005).</li><li>Kabaeva, Z., Zhao, M., Michele, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+extends+culture+life+of+adult+mouse+cardiac+myocytes+and+allows+efficient+and+stable+transgene+expression.">Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 1667-1674 (2008).</li><li>Lou, Q., Li, W., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+dynamic+instability+and+wavelength+in+arrhythmia+maintenance+as+revealed+by+panoramic+imaging+with+blebbistatin+vs.+2,3-butanedione+monoxime.">The role of dynamic instability and wavelength in arrhythmia maintenance as revealed by panoramic imaging with blebbistatin vs. 2,3-butanedione monoxime.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 262-269 (2012).</li><li>Liu, C. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+Na/K-ATPase+potentiates+Marinobufagenin-induced+cardiac+dysfunction+and+myocyte+apoptosis.">Reduction of Na/K-ATPase potentiates Marinobufagenin-induced cardiac dysfunction and myocyte apoptosis.</a> J. Biol. Chem. 287 (20), 16390-16398 (2012).</li><li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+roles+of+the+cardiac+Na+/Ca2+-exchanger+in+ouabain-induced+inotropy,+cell+signaling,+and+hypertrophy.">Different roles of the cardiac Na+/Ca2+-exchanger in ouabain-induced inotropy, cell signaling, and hypertrophy.</a> Am. J. Physiol. Heart Circ. Physiol. 304, 427-435 (2013).</li><li>Liu, L. J., Zhao, X. C., Pierre, S. V., Askari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+PI3K-AKT+signaling+pathway+with+digitalis-induced+hypertrophy+of+cardiac+myocytes.">Association of PI3K-AKT signaling pathway with digitalis-induced hypertrophy of cardiac myocytes.</a> Am. J. Physiol. Cell Physiol. 293, 1489-1497 (2007).</li><li>Flynn, J. M., Santana, L. F., Melov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Cell+Transcriptional+Profiling+of+Adult+Mouse+Cardiomyocytes.">Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes.</a> J. Vis. Exp. (58), (2011).</li></ol>

The authors declare that they have no competing financial interests.

For the best preparation, the most critical steps are: 1) promptly hooking up the mouse heart to the cannula after excision; 2) appropriate heart perfusion. Also, note that water quality, perfusion temperature, pH of buffer, sterilization, chemical purity, and clean, non-contaminated tubing and chambers are also important factors. 18.2 m&#937; molecular biology-grade H2O is highly recommended for buffer preparation. The pH of 200 mM ATP stock solution should be adjusted to 7.2, a step which is easily missed.

It is e...

Cardiomyocytes are not proliferative. There are some atrial cardiomyocyte cell lines, like HL-1 and AT-1 cells derived from mouse atrial tumors; however, there are no adult ventricular cardiomyocyte cell lines available for research. Primary cell cultures of adult mouse cardiomyocytes provide a powerful model for heart research at the cellular and molecular levels. To date, they have been used extensively for biochemical, physiological, and pharmacological research1. Additionally, the frequent use of genetically modified mice has necessitated effective methods of cardiomyocyte isolation. Pure culture allows for conditions free from interaction with other organs and the systemic circulation, such as through endogenous neurohormonal and hormone-like factors2,3. However, successful isolation of cardiomyocytes can be challenging.
The protocol we introduce herein is based on our experiences with adult rat cardiomyocytes and the method described by O&#39;Connell et al4,5. Especially in 20075, they described detailed techniques from buffer preparations to cell culture and functional assay. Since mouse myocytes are highly susceptible to contracture compared to the rat cardiomyocytes, the buffers or media used for the perfusion, digestion, Ca2+ toleration, plating and culture in the mouse protocol are supplemented with a nonspecific excitation-contraction coupling inhibitor, 2,3-Butanedione monoxime (BDM) to inhibit their spontaneous contraction, hence the viability and yield of rod-shaped myocytes improve significantly. In the protocol introduced here, the myocytes are separated in a high potassium buffer from the isolated heart by modified Langendorff perfusion with type II collagenase. Collagenase II effectively breaks down the intercellular matrix and releases cells. The perfusion solution also keeps cellular metabolism at a low level6. In addition, the isolated heart system from Harvard Apparatus we used here is well-designed for precise temperature and constant pressure control7. This approach provides highly reproducible preparations and uniform populations of single cell type, which can be used in overnight&#160;culture for measuring signaling proteins or 2-3 day culture for cardiac hypertrophic assays.

<table border="0" cellpadding="0" cellspacing="0" width="774">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="41" rowspan="2" style="height:41px;width:257px;">Isolated heart system for small rodent</td>
			<td style="width:131px;">Harvard Apparatus</td>
			<td style="width:99px;">IHSR-mouse 73-4019</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Aortic Cannula, OD 1.0 mm</td>
			<td style="width:131px;">Harvard Apparatus</td>
			<td style="width:99px;">73-2816</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Dumont #4 Forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11294-00</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Straight sharp serrated scissors</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">14070-12</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Serrated Graefe forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11050-10</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Curved, serrated Graefe forceps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">11052-10</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Straight Mini Serrefines clamps</td>
			<td style="width:131px;">Fine science tools</td>
			<td style="width:99px;">18054-28</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">MEM</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:99px;">11575-032</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Collagenase II</td>
			<td style="width:131px;">Worthington</td>
			<td style="width:99px;">4176</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mucosal universal detergent</td>
			<td style="width:131px;">Sigma</td>
			<td style="width:99px;">Z637181</td>
			<td>Mucasol is a fast alkaline residue-free detergent.</td>
		</tr>
	</tbody>
</table>

isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

1. Successful Isolation Quantification
Two criteria are used to quantify the success of the isolation: first, the total number of cardiomyocytes isolated, and second, the ratio of rod-shaped calcium-tolerant to round non-tolerant myocytes. Generally this protocol takes around 75-90 min&#160;from heart removal to myocyte plating and yields around 1 million rod-shaped cardiomyocytes (70-90% of total myocytes harvested) from one adult mouse heart. This may vary with mouse body weight and strain. Typ...

Watch this Scientific Journal Video about Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy at JoVE.com

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

isolation-culture-adult-mouse-cardiomyocytes-for-cell-signaling-vitro

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47.4K Aufrufe.  University of Toledo College of Medicine and Life Sciences.  Wir beschreiben eine zuverlässige Methode zur Isolierung von adulten Herzmuskelzellen der Maus. Dieses Protokoll liefert ein konsistentes Ergebnis für die Kultur von funktionellen adulten Kardiomyozyten aus einer Vielzahl von gentechnisch veränderten Mäusen. 

Serie: Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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.

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 ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...