This work was supported by funding from the Department of Pathology and Laboratory Medicine, University of Cincinnati, College of Medicine, to Dr. Onur Kanisicak; a grant from the National Institutes of Health (R01HL148598) to Dr. Onur Kanisicak.&#160;Dr. Onur Kanisicak is supported by the American Heart Association Career Development Award (18CDA34110117). Dr. Perwez Alam is supported by the American Heart Association postdoctoral grant (AHA_20POST35200267). Dr Malina J. Ivey is supported by an NIH T32 grant (HL 125204-06A1).

All experiments should be performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institute of Health (NIH). All protocols displayed in the video were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati, College of Medicine.
1. Preparation before heart extraction from adult mice (and rats)
<ol>
	<li>Prepare the corresponding perfusion, enzyme, and stop solutions, according to the recipes given in Table 1 and Table 2, for rat and mouse isolation, respectively. Filter the solutions through a 0.22 &#181;m filter to remove any contamination or undissolved particles.</li>
	<li>Clean and sterilize the surgical instruments by soaking them in 70% alcohol for 15 min, and subsequently wash with double distilled water. Leave the tools to air dry on a clean paper towel.</li>
	<li>Pre-heat the water bath to 37 &#186;C. Check the water level in the water bath to ensure an uninterrupted circulation of warm water through the perfusion apparatus during the procedure.</li>
	<li>Clean the perfusion apparatus by running with 70% alcohol for 5 min. Repeat. 
	NOTE: Increase the flow rate, which helps to clean the tubing.</li>
	<li>After the alcohol flowthrough, clean the remnants of alcohol by running double distilled water for 10 min.</li>
	<li>Set up 3 medium-size disposable polystyrene weighing dishes to clean the heart after excision. Fill the dishes with 20 mL of myocyte buffer.</li>
	<li>Add 2-3 drops of heparin to each dish with the help of Pasteur pipette and mix well by pipetting multiple times.</li>
	<li>Take a 10 mL syringe with a blunt needle cannula. 
	NOTE: A blunt needle cannula can be prepared by cutting the tip of a clean, sterile needle. A 21 G needle and 14 G needle were used to make the cannula for the mouse and rat, respectively.</li>
	<li>Fill the syringe with perfusion buffer (Table 1). Remove any air bubbles from the syringe and place it in the third dish at an angled position. Secure it with tape. 
	NOTE: For rats, Solution A (Table 2) was used instead of myocyte perfusion buffer.</li>
	<li>Prepare a loose knot with a surgical suture and place it around the needle.</li>
	<li>Inject the mouse with heparin (100 USP units/mouse) by intraperitoneal injection, 20 min before the anesthesia injection.</li>
	<li>Circulate the myocyte perfusion buffer (Table 1) through the perfusion apparatus. Decrease the flow rate to 3 mL/min. 
	NOTE: For rats, Solution A (Table 2) was used instead of myocyte perfusion buffer.</li>
	<li>After 5 min, replace the perfusion buffer with the enzyme solution (Table 1). Saturate the enzyme solution with oxygen during the process. Use a flow rate of 3 mL/min. 
	NOTE: For rat CM isolation, use solution E (Table 2) instead.</li>
	<li>Anesthetize the mouse by intraperitoneal injection of anesthesia. Use the appropriate dose of anesthesia, recommended by IACUC, for the terminal surgery.</li>
	<li>Confirm sufficient depth of anesthesia by the lack of a response to a toe pinch. Then, put the mouse on the surgical platform.</li>
</ol>
2. Extraction of heart from adult mice (and rats)
<ol>
	<li>Sterilize the skin by wiping with 70% alcohol.</li>
	<li>Carefully open the chest.</li>
	<li>Excise the heart. Avoid non-heart tissues during excision.</li>
	<li>Put the heart on the first dish, filled with perfusion buffer (Table 1). 
	NOTE: Use solution A for the rat (Table 2).</li>
	<li>Clear the blood from the heart by gently squeezing.</li>
	<li>Transfer the heart to the second dish.</li>
	<li>Clean the blood from the heart and remove non-cardiac tissues. Subsequently, transfer the heart to the third dish.</li>
	<li>Trim off lungs and other surrounding tissue and cannulate via the ascending aorta. This procedure should be performed as rapidly as possible (&#60;5 min) to improve CM quality and quantity.</li>
</ol>
3. Digestion of the heart
<ol>
	<li>Digestion of the mouse heart 
	NOTE: All the solutions/buffers which circulate through the mouse heart should be oxygenated throughout the procedure.
	<ol>
		<li>Carefully remove the cannulating needle from the syringe and connect it to the Langendorff perfusion apparatus.</li>
		<li>Avoid any air bubbles going into the heart, which may affect the flow-through of the enzyme solution and digestion.</li>
		<li>Move the water jacket up to provide a homogenous environment to the heart. Allow the enzyme solution to flow through the heart at a speed of 2-3 mL/min. 
		NOTE: The speed of the passing solution is critical and has a significant impact on the outcome of CM quantity and quality.</li>
		<li>Let the enzyme solution flow through the heart for 2 min.</li>
		<li>At 2 min, add 40 &#181;L of 100 &#181;M CaCl2 solution to the enzyme solution and mix. Let the enzyme solution pass through the heart for another 10-15 min. 
		NOTE: Once the flow becomes smooth, the heart becomes brownish and soft, indicating the even distribution of the collagenase enzyme and proper digestion of heart.</li>
	</ol>
	</li>
	<li>Digestion of the rat heart 
	NOTE: All the solutions/buffers which circulate through the rat heart should be oxygenated throughout the procedure.
	<ol>
		<li>Carefully remove the cannulating needle from the syringe and connect it to the Langendorff perfusion apparatus.</li>
		<li>Avoid any air bubbles going into the heart, which may affect the flow-through of the enzyme solution and digestion.</li>
		<li>Move the water jacket up to provide a homogenous environment to the heart.</li>
		<li>Start the flow of solution A at a speed of 2-3 mL/min for 3-5 min to remove any blood from the heart. 
		NOTE: The speed of the passing solution is critical and has a significant impact on the outcome of CM quality.</li>
		<li>Once the blood is cleared from the heart, switch from solution A to solution E (Table 2). Titrate solution E with CaCl2 as indicated below for a final concentration of 0.1 mM in solution:
		<ol>
			<li>After 10 min of perfusion, add 12.5 &#181;L of 0.1 M CaCl2.</li>
			<li>After 15 min of perfusion, add 25 &#181;L of 0.1 M CaCl2.</li>
		</ol>
		</li>
		<li>Let the enzyme solution pass through the heart for another 30-40 min, until the flow becomes rapid and the heart is pliable.</li>
	</ol>
	</li>
</ol>
4. Preparation of CM single-cell suspension
<ol>
	<li>Preparation of the CM single-cell suspension (mouse)
	<ol>
		<li>Remove the heart from the Langendorff perfusion system. Move it to a 60 mm Petri dish filled with 5 mL of enzyme solution and transfer the dish to a biosafety hood. 
		NOTE: Ensure sufficient heart digestion before removing the heart from the Langendorff perfusion system. The digestion time depends on the enzyme activity, the flow of solution, and the size of the heart.</li>
		<li>Perform any further mechanical disaggregation in a biosafety hood to ensure sterility and avoid contamination.</li>
		<li>Carefully remove the atria (1/4th of the basal heart portion) and extra fat tissues.</li>
		<li>Mince the heart with forceps into fine pieces.</li>
		<li>Take a sterile Pasteur pipette and cut its tip at a 45&#186; angle.</li>
		<li>Use this Pasteur pipette to dispense the heart tissue in a single-cell suspension by gentle pipetting. Optimal digestion provides a suspension of single-cell cardiomyocytes. 
		NOTE: Remove the narrow, bottom portion of the transfer pipet to reduce CM damage due to mechanical searing.</li>
		<li>Then, add 5 mL of stop solution to stop the enzyme activity, which avoids the possibility of overdigestion.</li>
		<li>Take a fresh 50 mL conical and place a sterile 100 &#181;m cell strainer on it.</li>
		<li>Pass the cardiomyocyte suspension through the cell strainer to remove the tissue chunk. Wash the Petri dish and strainer with another 5 mL of stop solution (Table 1) to collect any cardiomyocytes that remain attached to the strainer.</li>
	</ol>
	</li>
	<li>Preparation of the CM single-cell suspension (rat)
	<ol>
		<li>Remove the heart from the Langendorff perfusion system. Move the heart to a 100 mm Petri dish filled with 5 mL of solution A and move the dish to a biosafety hood. 
		NOTE: Ensure sufficient heart digestion before removing the heart from the Langendorff perfusion system. The digestion time depends on the enzyme activity, the flow of solution, and the size of heart.</li>
		<li>Perform any further mechanical disaggregation in a biosafety hood to ensure sterility and avoid contamination.</li>
		<li>Carefully remove the atria (1/4th of the basal heart portion) and extra fat tissues.</li>
		<li>Mince the heart with forceps into fine pieces.</li>
		<li>Take a sterile Pasteur pipette and cut its tip at a 45&#186; angle.</li>
		<li>Use this Pasteur pipette to dispense the heart tissue in single-cell suspension by gentle pipetting. Optimal digestion provides a suspension of single-cell cardiomyocytes. 
		NOTE: Remove the narrow, bottom portion of the transfer pipet to reduce CM damage due to mechanical searing.</li>
		<li>Add 5 mL of solution B (Table 2) to the CM suspension and pass the solution through a 100 &#181;m cell strainer to remove any remaining pieces of fat or other non-digested tissue.</li>
		<li>Collect filtrate in a fresh 50 mL conical vial.</li>
		<li>Use another 5 mL of solution B to wash the Petri dish and any remaining CM through the cell strainer.</li>
	</ol>
	</li>
</ol>
5. Removal of non-CMs
<ol>
	<li>Removal of non-CMs from the adult single-cell suspension (mouse)
	<ol>
		<li>Centrifuge the cell suspension at 20 x g for 3 min.</li>
		<li>Discard the supernatant and resuspend the cells in 10 mL of stop solution (Table 1). 
		NOTE: Increasing the concentration of BSA in the stop solution will increase its viscosity and reduce the sedimentation rate of non-CMs.</li>
		<li>Resuspend the CMs by gentle inversion of the tube 3-5 times.</li>
		<li>At 3 min intervals, add 10 &#181;L of 100 &#181;M CaCl2 solution and mix. Repeat thrice.</li>
		<li>After the fourth addition, centrifuge the cardiomyocyte suspension at 20 x g for 3 min.</li>
		<li>Discard the supernatant.</li>
		<li>Resuspend CMs in pre-warmed (37 &#186;C), adult mouse CM plating media (Table 3).</li>
	</ol>
	</li>
	<li>Removal of non-CMs from the adult single-cell suspensions (rat)
	<ol>
		<li>Pellet cells at 20 x g for 3 min at 25 &#186;C.</li>
		<li>Discard the supernatant and resuspend the cells into 25 mL of solution B (Table 2).</li>
		<li>Mix the cells by gentle inversion and place it in a tube stand to allow the CM to settle under gravity.</li>
		<li>Carefully discard the supernatant.</li>
		<li>Resuspend the cells in fresh 25 mL of solution B and titrate it to 1.0 mM CaCl2 by stepwise addition of 50 &#181;L, 75 &#181;L, and 100 &#181;L of 0.1 M CaCl2 at 3-5 min intervals. 
		NOTE: A higher concentration of BSA in solution B could be used at this step. Increasing the concentration of BSA in solution B increases the viscosity and thus, reduces the sedimentation rate of non-CMs.</li>
		<li>Pellet cells at 20 x g for 3 min at 25 &#186;C, aspirate the supernatant, and add the desired volume of adult rat cell culture media (Table 4).</li>
		<li>Seed CMs on a laminin coated plate. 
		NOTE: Pre-plating of CMs on a non-coated culture plate can be used to minimize the contamination of non-CM cells.</li>
	</ol>
	</li>
</ol>
6. Adult CM plating
<ol>
	<li>Pre-plating
	<ol>
		<li>Resuspend the cardiomyocytes into culture media.</li>
		<li>Pre-plate the cells into a 60 mm or 100 mm dish for mouse or rat CM, respectively.</li>
		<li>Incubate CMs for 2 h in an incubator supplemented with 5% CO2 at 37 &#186;C.</li>
		<li>During the pre-plate incubation, coat the cell culture plates with laminin (10 &#181;g/mL in PBS) for long term CM culture.</li>
	</ol>
	</li>
	<li>After 2 h of pre-plating, collect CMs in a 50 mL conical vial.</li>
	<li>Collect the cells from the dish and re-plate them into a laminin coated 24 well culture plate for transfection experiments.</li>
	<li>Culture cells in an incubator at 37 &#186;C supplemented with 5% with CO2. 4-6 hours is sufficient to adhere to the cardiomyocytes with the surface, and thus, transfection can be performed 6 hours post-plating. 
	NOTE: The plating medium containing FBS is commonly used and shows better compatibility with proliferation studies. However, a serum-free medium can be used for experiments where secretory factors are being analyzed.</li>
</ol>
7. Transfection
<ol>
	<li>Incubate the CMs to the cell culture plates coated with laminin for 4-6 hours.</li>
	<li>Four hours after CM seeding on the laminin coated plate, transfect cells with siRNAs (50 nM) of interest using a transfection reagent (e.g., Lipofectamine RNAiMAX) according to the manufacturer&#8217;s protocol.</li>
	<li>Change media 24 hours following transfection and every 24 hours after that for up to 20 days.</li>
	<li>After 20 days, fix cells with 4% PFA in PBS for downstream applications, including immunocytochemistry for cardiac-specific markers such as Troponin to ensure live cardiomyocytes were successfully cultured long-term.</li>
</ol>

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There is a critical need to establish a protocol for adult cardiomyocyte isolation and long-term culture to perform cell-specific mechanistic studies. There are only a few reports discussing adult CM isolation protocols, and even fewer of them are used for long-term culture of adult mice CM15,16,17. It is been shown that the adult rat CM has a higher tolerance to in vitro culture than the adult mice CM10<...

Cardiac diseases are one of the leading causes of death worldwide. Almost all types of cardiac injury result in a significant loss of adult cardiomyocytes (CMs). Adult mammalian hearts are unable to repair their cardiac injury due to the senescent nature of the adult CM1. Thus, any insult to the adult mammalian heart results in a permanent loss of CMs, leading to reduced cardiac function and heart failure. Unlike adult mammals, small animals like zebrafish and newt hearts can regenerate their cardiac injury through existing CM proliferation2,3,4. A worldwide effort is ongoing to find a novel therapeutic intervention for cardiac injury via both proliferative and non-proliferative approaches. In past decades, various types of genetic mouse models have been developed to study cardiac injury and repair. However, using in vivo animal models continues to be an expensive approach with the additional complexity to decipher a cell-autonomous mechanism from secondary effects. Besides, in vivo systems are challenging to analyze CM specific effects of a pharmacological intervention that induces cardioprotective signaling from the CM.
Moreover, the long-term culture of adult CMs is necessary to perform CM proliferation analyses. CM proliferation assays require a minimum of 4-5 days for cells to be induced into the cell cycle and to obtain accurate data after that. Additionally, studies that utilize isolated CMs for electrophysiological studies, drug screening, toxicity studies, and Ca++ homeostasis studies are all in need of an improved culture system5,6,7. Furthermore, recent studies show the cardioprotective significance of cytokines secreted from CMs (cardiokines)8,9. In order to investigate the therapeutic role and molecular mechanism of these cardiokines during heart repair and regeneration, a prolonged culture is required.
Adult rat CMs are robust enough for single-cell isolation and long-term culture in an in vitro system10,11,12. However, adult mouse CMs are of great interest for in vitro assays, due to the availability of a variety of genetically modified mouse models, which allows for the design and execution of various innovative analyses that are not possible with rat CM13. In contrast to adult rat CM isolation, it is quite challenging to obtain a single-cell suspension from adult mouse hearts, and the long-term culture of adult mouse CMs in culture is even more challenging.
Adult CM isolation from mouse and rat hearts using a Langendorff system has previously been established to study CM function5,14,15. Here, we have described in detail the protocols for adult CM isolation from both rats and mice, as well as a modified long-term culture, transfection, and CM proliferation of isolated cells.

<table>
	<tbody>
		<tr>
			<td>2,3-Butane Dione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B-0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>APExBIO</td>
			<td>B1387</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>449709</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plate</td>
			<td>Corning Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>Cel-miR-67</td>
			<td>Dharmacon</td>
			<td>CN-001000-01-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type2</td>
			<td>Worthington</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Graduated Transfer Pipettes</td>
			<td>Fisherbrand</td>
			<td>13-711-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable polystyrene weighing dishes</td>
			<td>Sigma-Aldrich</td>
			<td>Z154881-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s medium</td>
			<td>Thermo Scientific</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU</td>
			<td>Life Technologies</td>
			<td>C10337</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Corning</td>
			<td>35-015-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Point High Precision Forceps</td>
			<td>Fisherbrand</td>
			<td>22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G-5400</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Hausser Scientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sagent Pharmaceuticals</td>
			<td>PSLAB-018285-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>High Precision Straight Broad Strong Point Tweezers/Forceps</td>
			<td>Fisherbrand</td>
			<td>12-000-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I0516-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-8281</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>746436</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Life Technologies</td>
			<td>13778-150</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M-2643</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Natural Mouse Laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Henry Schein</td>
			<td>24352</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease XIV</td>
			<td>Sigma-Aldrich</td>
			<td>P5147-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Selenium</td>
			<td>Sigma-Aldrich</td>
			<td>229865+5G</td>
			<td></td>
		</tr>
		<tr>
			<td>siMeis2</td>
			<td>Dharmacon</td>
			<td>s161030</td>
			<td></td>
		</tr>
		<tr>
			<td>siRb1</td>
			<td>Dharmacon</td>
			<td>s128325</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Blunt/SharpDissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>28252</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Very Fine Precision Tip Forceps</td>
			<td>Fisherbrand</td>
			<td>16-100-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma-Aldrich</td>
			<td>T8158-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-smooth, beveled-edge finish scissor</td>
			<td>Fisherbrand</td>
			<td>22-079-747</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Scientific</td>
			<td>3006S</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

The current modified protocol allows efficient isolation and culture of rat and mice CMs in vitro. For rat CM isolation, a total of 3 adult (12-week-old) male Fischer 344 rats were used in the procedure. Figure 1 shows the surgical apparatus and isolation setups that are required in the procedure; each part has been marked and described in the figure legend. Collagenase type 2 was used for digestion, which yields a high quantity of high quality CMs from successful isolation (<strong class="x...

Watch this Scientific Journal Video about Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes at JoVE.com

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

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Research

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Biology

8.9K Views.  University of Cincinnati. Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Video: Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

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 Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

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 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.

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.

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