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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Antegrade perfusion of the mouse heart
NOTE: The plastic transfer pipette used for sucking the heart should be soft and not be sharply tapered towards the tip. Choose a small vascular clamp with serration. The recommended instruments are listed in the Table of Materials.
<ol>
	<li>Excision of the mouse heart and clamping the aorta 
	NOTE: The adult mice (&#62;8 weeks old) should be euthanized by an overdose of sodium pentobarbital (&#62;300 mg/kg, intraperitoneal [i.p.] injection) with heparin (8000 unit/kg).
	<ol>
		<li>Excise the mouse heart quickly by sucking.
		<ol>
			<li>Open the thoracic cavity quickly to expose the heart. Cut the plastic transfer pipette, the tip of which is approximately the same size as, or slightly smaller than, the exposed heart (usually at a site approximately 1 cm from the 0.5-mL mark towards the tip, but it depends on the heart size).</li>
			<li>Suck the heart into the pipette, raise the pipette to create enough space to insert scissors, and excise the heart with curved scissors from the dorsal side, avoiding damaging the atria.</li>
			<li>Immediately transfer the excised heart to the 30-mL glass beaker containing ice-chilled CIB-EGTA to stop the contraction. This procedure usually takes &#60;1 min.</li>
		</ol>
		</li>
		<li>Cleaning around the aorta
		<ol>
			<li>Transfer the heart to a 35-mm culture dish filled with ice-chilled CIB-EGTA and remove the lung and other visible tissues, and then transfer the roughly cleaned heart to the heart stand filled with chilled CIB-EGTA and place it with the apex down.</li>
			<li>Under the stereoscopic microscope, remove the fat and connective tissues to clean around the aorta. If the length of the cut aorta is too long including the brachiocephalic artery, the left common carotid artery, or the left subclavian artery, cut off the aorta just under the brachiocephalic artery to shorten it in order to proceed to the next step. This procedure usually takes approximately 4 min</li>
		</ol>
		</li>
		<li>Clamping the aorta and placing the clamped heart on the perfusion plate
		<ol>
			<li>Insert the injection needle and start perfusion to discharge blood
			<ol>
				<li>Under the microscope, place the heart in the heart stand. The operator should face the anterior surface of the heart, pick up the end of the aorta with tweezers, and clamp the aorta near the atria with a small vascular clamp while gently pushing down on the aorta a bit.</li>
				<li>Place the clamped heart on the perfusion plate with the anterior side up, and then cover it with a few drops of CIB-EGTA to keep it from drying out. This procedure usually takes &#60;20 s.</li>
			</ol>
			</li>
			<li>Antegrade perfusion of the heart 
			NOTE: First, perfuse the heart with CIB-EGTA to discharge blood and prevent clotting.
			<ol>
				<li>Insert the injection needle and start perfusion to discharge blood
				<ol>
					<li>Set the 20-mL syringe filled with prewarmed CIB-EGTA connected to the flexible extension tube and a marked injection needle on the infusion pump. Start the pump at a slow rate of 0.5 mL/min to carefully fill the needle and tube with CIB-EGTA and be sure to prevent any air from entering the tube.</li>
					<li>Place the injection needle on the perfusion plate with the shorter side of the diagonal shape in front. Slide the needle towards the apex of the heart until it is touching it, and then carefully insert the needle near the apex of the left ventricle into the ventricular chamber without twisting. Do not detach the needle from the plate while performing insertion.</li>
					<li>Watch the red mark to estimate the depth of the needle insertion. When the needle insertion is completed, blood flowing from the coronary artery should start to be discharged.</li>
					<li>Fix the injection needle on the plate with tape, and increase the pump speed to 1 mL/min. This procedure usually takes approximately 30 s. If the heart is perfused successfully, the flow of the CIB-EGTA in the capillary just under the epicardium can be seen under the microscope.</li>
				</ol>
				</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>Extension tube&amp;nbsp;</td><td>Top, Japan&amp;nbsp;</td><td>X1-50</td><td>Connect with syringe and injection needle for antegrade perfusion.</td></tr><tr><td>Heater mat&amp;nbsp;</td><td>Natsume Seisakusho, Japan&amp;nbsp;</td><td>KN-475-3-40&amp;nbsp;</td><td>Equipment to warm the perfusion plate.</td></tr><tr><td>Infusion pump&amp;nbsp;</td><td>TERUMO, Japan</td><td>&amp;nbsp;TE-311</td><td>&amp;nbsp;Infusion syringe pump for antegrade perfusion.</td></tr><tr><td>Injection needle (27 gauge)</td><td>&amp;nbsp;TERUMO, Japan&amp;nbsp;</td><td>NN-2719S&amp;nbsp;</td><td>Needle for insertion into the left ventricle.</td></tr><tr><td>Insulin (from bovine pancrease)&amp;nbsp;</td><td>Sigma-Aldrich, USA&amp;nbsp;</td><td>I5500&amp;nbsp;</td><td>Dissolve in 0.1 M HCl.</td></tr><tr><td>Plastic syringe (20 mL)&amp;nbsp;</td><td>TERUMO, Japan</td><td>&amp;nbsp;SS-20ES&amp;nbsp;</td><td>Use for infusion of CIB-EGTA.</td></tr><tr><td>Vascular clamp&amp;nbsp;</td><td>Karl Hammacher GmbH, Germany&amp;nbsp;</td><td>HSE 004-35&amp;nbsp;</td><td>Small straight vascular clamp used for clamping aorta.</td></tr><tr><td>All other reagents</td><td>&amp;nbsp;Nacalai Tesque, Japan</td><td></td><td></td></tr></tbody></table>

antegrade perfusion model: an ex vivo technique to perfuse isolated mouse heart

Source: Omatsu-Kanbe, M. et al., <a href="https://www.jove.com/v/61866">An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice.</a> J. Vis. Exp. (2021).
This video describes an antegrade perfusion technique of an isolated mouse heart during which a perfusate is injected through the left ventricle, and eventually, it perfuses the entire myocardium. This method can be used to isolate cardiomyocytes from murine models and generate decellularized hearts for further downstream applications.

Antegrade Perfusion Model: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

Source: Omatsu-Kanbe, M. et al., An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice. J. Vis. Exp. (2021).
This video describes an ...

Begin by placing an anesthetized mouse in the supine position. Dissect the thoracic cavity to visualize the heart. Use a pipettor fitted with a wide bore tip to apply suction pressure and aspirate the heart from the mouse&#8217;s body into the tip. Excise the heart at the aorta to harvest it from the mouse&#39;s body.
Immerse the isolated heart into a beaker containing a nutrient-rich buffer supplemented with an anticoagulant agent. The anticoagulant in the buffer forms a complex with calcium present in the cardiomyocytes - the heart muscle cells, ceasing contraction of cardiac muscles and preventing blood coagulation.
Next, place the heart apex-side down on a heart stand kept in a Petri dish containing suitable buffer. Clamp the aorta above the atria and transfer the clamped heart to a perfusion plate.
Subsequently, take a syringe-needle assembly containing an appropriate perfusion buffer. Insert the needle into the left ventricle piercing the ventricular chamber.
With the aorta clamped, intraventricular injection pressurizes aortic valves to open. Consequently, the buffer flushes blood antegradely through the coronary arteries, thoroughly perfusing heart muscles. Finally, the buffer effuses through the pulmonary veins. This provides a successfully perfused heart for downstream applications.

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1.2K Views. Source: Omatsu-Kanbe, M. et al., An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice. J. Vis. Exp. (2021). This video describes an antegrade perfusion technique of an isolated mouse heart during which a perfusate is injected through the left ventricle, and eventually, it perfuses the entire myocardium. This method can be used to isolate cardiomyocytes from murine models and generate decellularized hearts for further downstream applications. 

Video: Antegrade Perfusion Model: An Ex Vivo Technique to Perfuse Isolated Mouse Heart - Concept

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

JoVE Journal

Bioengineering

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Medicine

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 ...

Antegrade Perfusion Model: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

Transcript

Antegrade Perfusion Model: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

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