We gratefully acknowledge funding to SNT from the US National Institute of Health (K99/R00 HL1431149; R01HL157803; R01DK134590; R24OD034189), the National Science Foundation under Grant No. EEC 1941543, the Claflin Distinguished Scholar Award on behalf of the MGH Executive Committee on Research, and the Polsky Family Award for Leaders in Surgery.&#160;We acknowledge research funding to AAO from the Hassenfeld Family Foundation, the MGH Executive Committee&#160;on Research, and the MGH Center for Diversity and Inclusion.&#160;We acknowledge research funding to GO from the Sarnoff Cardiovascular Research Foundation.

This study was conducted in accordance with the Institutional Animal Care and Use Committee (IACUC), Massachusetts General Hospital, and Jove&#39;s animal guidelines. Hearts (170 - 250 g) were harvested from Yorkshire pigs (30 - 35 kg, age 3-4 months, mixed sex) using a model of donation after brain death and perfused retrogradely (Langendorff) for 6 h prior to loading. All grafts were exposed to a cold ischemia time of approximately 1h during instrumentation.
1. System design
<ol>
	<li>Check that the system is composed of an organ chamber (11.046 inch x 7.595 inch x 3.095 inch), two reservoirs (4 L and 1 L), one double-jacketed oxygenator, and a Windkessel bag (WB). Check that both reservoirs contain a built-in defoamer, which eliminates frothing.</li>
	<li>Check that the components are securely connected with silicone tubing, with the smaller reservoir, the WB, and the organ chamber being in two different configurations (Figure 1A) depending on the perfusion modality (i.e., Langendorff versus loading).
	<ol>
		<li>Connect the base of the organ chamber to the top of the large (venous) reservoir using a 3/8 inch tubing. Place the large reservoir below the organ chamber to allow gravity to circulate the perfusate from the chamber to the reservoir.</li>
		<li>Connect the bottom of the large reservoir to the inflow port of the pump head using 3/8 inch tubing.</li>
		<li>Connect the outflow port of the pump head to a 3/8 inch tube fitted with a 3/8 inch to &#188; inch reducer and &#188; inch tubing on the other end. Connect the &#188; inch to the inflow port of the oxygenator.</li>
		<li>Fit the outflow port of the oxygenator with a &#188; inch tubing with a Y connector at the end. Fit both ends of the Y connector with &#188; inch tubing.</li>
		<li>Sequence 1 - Perfusate flow pattern during Langendorff perfusion (blue dashed line in Figure 1A)
		<ol>
			<li>Fit the first end of the Y connector with a &#188; inch to 3/8 inch expander with an embedded luer connection. Connect the 3/8 inch tubing to the first port at the bottom of the WB. Attach a three-way luer valve to the luer connection on the expander and utilize it for Adenosine drip delivery.</li>
		</ol>
		</li>
		<li>Sequence 2 - Perfusate Flow pattern during loaded perfusion (orange dashed line in Figure 1A)
		<ol>
			<li>Attach the second end of the Y connector to the top of the smaller (loading) reservoir. Attach a Hoffman clamp to the tubing and use it for reservoir filling control.</li>
			<li>Place an overflow line from the top of the loading reservoir to the larger reservoir.</li>
			<li>Fit the bottom of the loading reservoir with an &#188; inch tube and connect to a port at the base of the organ chamber. Add a three-way luer valve halfway through the tubing length for Adenosine delivery and a one-way valve right before reaching the organ chamber.</li>
			<li>Attach the other end of the organ chamber port to a right-angle cannula.</li>
		</ol>
		</li>
		<li>Place the Windkessel bag (WB) directly above the organ chamber. Fit the second overflow port at the bottom of the WB with a 3/8 inch tubing and connect to any inflow port of the venous reservoir. Attach a Hoffman clamp and completely close it to stop fluid through this port during Langendorff and adjust during loaded mode to modulate aortic pressures.</li>
		<li>Connect a three-way luer valve to the overflow port at the top of the WB and use an &#188; inch of tubing to connect the WB to any inflow port in the venous reservoir. Attach a Hoffman clamp to the &#188; inch tubing. Keep the clamp completely closed during Langendorff perfusion and adjust during loading mode to modulate aortic pressures.</li>
		<li>Connect the third outflow port on the WB to the aortic port on the organ chamber through the 3/8 inch tubing, with an interruption in the lower 3/4 of the length for a temperature probe.</li>
	</ol>
	</li>
</ol>
2. Perfusate system preparation
<ol>
	<li>Prepare a base perfusate composed of 0.96% Krebs-Henseleit Buffer, 9.915 mM Dextran, 25 mM sodium bicarbonate, 1.054 mM bovine serum albumin, 1% Pen Strep, 0.13% insulin, 0.02% hydrocortisone, 0.5% heparin, and 2.75 mM calcium chloride. Bring the volume to 4 L using distilled water.</li>
	<li>Perfusion system setup
	<ol>
		<li>Rinse all tubing, system components, and reservoirs with distilled water and reconnect in the correct sequence (Figure 1A).</li>
		<li>Place the Windkessel bag between two acrylic plates and tighten it with the screw-spring setup (Supplementary Figure 1).</li>
		<li>Attach a three-way luer valve to the luer connection of the aortic port and the atrial port of the organ chamber (Figure 1B). Connect two pressure sensors to the organ box, one at the valve connected to the aortic port at the top of the organ chamber, and one at the valve connected to the atrial port at the bottom of the organ chamber.</li>
		<li>Calibrate the pressure sensors by opening them to air (0 mmHg pressure) and setting this reading to 0 in the recording device.</li>
		<li>Connect two flow sensors to the system tubing. Connect the sensor measuring aortic flow to the 3/8 inch tubing connecting the WB with the aortic port. Connect the sensor measuring atrial flow to the tubing connecting the loading reservoir to the atrial port.</li>
		<li>Connect a temperature probe to the 3/8 inch tubing connecting the WB to the aortic port. Connect a de-airing tube to the third port of the valve attached to the aortic port and keep it open at all times. Connect the other end of the line to any inflow of the venous reservoir.</li>
		<li>Connect the heat exchanger to the oxygenator and set it to 38 &#176;C. Connect the 100% oxygen line to the gas inflow port on the oxygenator. Turn oxygen on to 0.5 L/min.</li>
	</ol>
	</li>
	<li>System priming
	<ol>
		<li>Add 2 L of perfusate to the system using the organ chamber. Turn on the pulsatile pump and allow the perfusate to circulate (skipping the loading reservoir) until the perfusate is oxygenated to a minimum pO2 of 400 mmHg and the temperature has reached ~35 &#176;C.</li>
		<li>Allow the perfusate to circulate and fill all the system components to remove air. Massage the tubes containing any remnant air bubbles to remove them. Then, remove any air trapped in the aortic port by increasing the pressure by partially occluding the fluid outflow and increasing the pump flow rate. This increase in pressure will force any air through the purge/de-airing tube.</li>
		<li>Allow the perfusate to circulate until it reaches the desired temperature (37 &#730;C, monitored continuously via a temperature probe), conduct an initial assessment of the biochemical parameters to ensure correct ion concentration (Table 1) and adequate oxygenation. 
		NOTE: Read the ion and pH levels after the solution has been brought up to temperature (37 &#176;C) and has been properly oxygenated.</li>
	</ol>
	</li>
</ol>
3. Cardiac graft procurement
<ol>
	<li>Sedate animals with an intramuscular injection of atropine (0.04 mg/kg), telazol (4.4 mg/kg), and xylazine (2.2 mg/kg).</li>
	<li>Once sedated, transfer the animals into the operating room and obtain venous access using an IV line placed in either ear.</li>
	<li>Administer a bolus of Propofol (0.16-0.33 mg/kg) through the IV line and test noxious stimuli 3 min after injection. If no reflexes are present, intubate the animal and maintain anesthesia through continuous isoflurane (3%-5%) inhalation and intravenous fentanyl (5-20 &#181;g/kg/h) as needed.</li>
	<li>Once intubated, place a pressure cuff in either of the front limbs and connect an EKG sensor on the apex of the lower lip for oxygen saturation monitoring.</li>
	<li>Initiate a saline drip and administer it through the ear IV line. Place a surgical drape over the ventral aspect of the animal.</li>
	<li>Administer a bolus of fentanyl citrate (5 &#181;g/kg) intravenously in preparation for the sternotomy.</li>
	<li>Make a vertical incision between the sternal notch and xiphoid with a 10-blade scalpel (~25 cm). Following the incision, utilize an electrocautery to divide the subcutaneous fat and fascia until reaching the sternum.</li>
	<li>Once exposed, separate the adherent pericardium from the sternum using blunt finger dissection at the caudal aspect of the sternum. To do so, place your finger on the dorsal aspect of the sternum and separate any adherent tissue between the sternum and viscera.</li>
	<li>Insert a chisel at the caudal aspect of the sternum. While applying skyward force to the chisel, use a mallet to advance the chisel through the sternal bone.</li>
	<li>After the median sternotomy is complete, determined by the complete separation of the sternum (from xiphoid to sternal notch), place a sternal retractor and open it until full exposure.</li>
	<li>Incise the pericardium cranially with Metzenbaum scissors until the aorta and pulmonary artery are visible.</li>
	<li>Place two purse string sutures (4-0 Prolene) on the aorta and secure with a tourniquet snare. Take care that the sutures go through the media layer of the aorta but not through the lumen. Superficial sutures will fail to keep the aortic root cannula in place, and deep sutures will cause the aorta to bleed.</li>
	<li>Administer a bolus of heparin (100 U/kg) through the ear IV and allow it to circulate for 3 min.</li>
	<li>Insert a 9F aortic root cannula perpendicular to the aorta in between the purse string sutures and secure by gently tightening the tourniquet snare. Ensure no leakage around the cannulation site. Allow the cardioplegia line of the aortic root cannula to be de-aired by letting blood flow from the aorta.</li>
	<li>Connect a bag of cardioplegic solution to the luer lock connection. Pressurize the cardioplegia bag using a pressure bag to result in 80 mmHg during flushing. Connect a peristaltic pump to the second line of the aortic root cannula and use it to extract 1 L of blood directly from the aorta.</li>
	<li>Separate red blood cells using a blood salvage device (red blood cells are centrifuged and washed with saline). Add red blood cells to the perfusion system after it has reached the desired temperature.</li>
	<li>Once enough blood is collected, utilize a trans-thoracic cross to clamp the aorta and open the cardioplegia line to flush the organ.</li>
	<li>Immediately after cross-clamp and flush initiation, vent the heart via a 5 cm incision on the left atrial appendage and by completely severing the inferior vena cava. Add ice to the thoracic cavity at this time to help reduce the temperature of the organ.</li>
	<li>After successful venting, severe or tie off the superior vena cava to stop warm blood from the head from reaching the cooled heart.</li>
	<li>Once the heart has been flushed with 1 L of cardioplegia solution, divide the great vessels, including the aorta, main pulmonary artery, superior and inferior vena-cavae, and bilateral pulmonary veins. This completes the cardiac explant. Remove the heart from the cavity. 
	NOTE: Maintain as much length as possible of the great vessels attached to the graft. Cut the pulmonary veins as close to the lungs as possible.</li>
	<li>Wrap the organ in a laparotomy sponge and place it on ice.</li>
</ol>
4. Graft preparation
<ol>
	<li>Cut the branches of the aortic arch to create one outflow tract. Insert the aortic cannula through this tract and secure with a zip tie and 4-0 silk suture (Figure 2).</li>
	<li>Place a bipolar pacing wire on the posterior wall of the right ventricle. Cut the pulmonary veins to form a single inflow tract into the left atrium.</li>
	<li>Create two purse-stringed 4-0 proline sutures through the perimeter of the left atrium tract. Secure the sutures with tourniquet snares and leave untied until loading.</li>
	<li>Close the left atrium appendage using a simple continuous suture (4-0 Prolene). Record the initial heart weight.</li>
</ol>
5. Cardiac graft revival
<ol>
	<li>Clamp the 3/8 inch tubing right before the aortic port to stop perfusate flow. Position the heart with the posterior wall facing the operator. Angle the organ chamber at roughly 20&#176;. 
	NOTE: This position of the graft perfusion is chosen to augment passive drainage and is consistent with previously published data demonstrating significant improvement in function compared to hanging26.</li>
	<li>Place the aortic cannula at a 90&#176; angle from the aortic port and unclamp the aortic line slowly. De-air the aortic cannula by the progressive flow of perfusate into it.</li>
	<li>Slowly decrease the angle in the aortic cannula until it is in line with the aortic port, and fully connected to the aortic line.</li>
	<li>Once fully connected, intermittently gently massage the heart to prevent distention due to left ventricular filling. During this period, vent the left ventricle through the open left atrium.</li>
	<li>Concomitantly, monitor the aortic pressures and maintain within the acceptable range (30 - 40 mmHg).</li>
	<li>Initiate data acquisition and start the adenosine drip at a rate of 333 uL/min 
	NOTE: Adenosine (2 mg/mL) is added to the perfusion protocol to mimic the current conditions of clinical perfusion. However, it is important to point out that the associated vasodilation may exacerbate unwanted organ edema30,31,32.</li>
	<li>Connect the pacing wires to the pacing box and set it to 60 bpm as backup pacing.</li>
	<li>If fibrillation is present, defibrillate the heart using paddles with 30 J. Deliver as many shocks as needed until achieving rhythmic contractions.</li>
	<li>Once an organized rhythm is present (paced or intrinsic), discontinue the manual venting and place EKG leads directly on the heart using hook needles.</li>
	<li>Tighten the screws on the Windkessel bag until the waveform displayed from the aortic pressure sensor mimics a sine wave. Perfuse heart in Langendorff configuration for 6 h as described previously33.</li>
</ol>
6. Cardiac graft loading
<ol>
	<li>Connect the right-angle cannula to the atrial port of the organ chamber. Once connected, clamp the cannula and allow the fluid into the loading reservoir by releasing the Hoffman clamp in the line between the oxygenator and the loading reservoir.</li>
	<li>Fill the loading reservoir until the pressure reaches 15 - 20 mmHg. Increase the output on the pump to maintain both aortic and atrial pressure.</li>
	<li>Insert half of the right-angle metal tip into the left atrium with the tip pointing toward the appendage. This placement most consistently promotes Mitral Valve competence.</li>
	<li>Insert the pressure sensor inside the left ventricle for left ventricular pressure recording. Release the clamp on the cannula and allow the left atrium to fill.</li>
	<li>Once de-aired, use the previously placed sutures and tourniquet snares to completely close the opening of the left atrium. Adjust the cannula and snares as needed to minimize fluid leakage.</li>
	<li>After securing the cannula in the left atrium, stop retrograde perfusion to the aorta completely by clamping the line from the oxygenator to the WB.</li>
	<li>Move the adenosine drip from the line leading to the WB, to the line between the loading reservoir and the atrial port.</li>
</ol>
7. End of perfusion
<ol>
	<li>Record biochemical readings, heart rate, aortic/atrial flow, and pressures every 30 min for the duration of the experiment (10 h). Obtain biochemical readings from the atrial line for inflow measurement and directly from the pulmonary artery for outflow readings.</li>
	<li>At the end of perfusion, stop the data acquisition, remove the heart, and dispose of it.</li>
	<li>Mobilize the non-fixed parts of the system to a large sink, disassemble, rinse, and clean with copious amounts of water.</li>
	<li>Once no remnants of blood are visible in the components, re-assemble the system. Add large quantities of water to the system through the organ chamber and roughly 100 mL of liquid alkaline detergent to circulate with water.</li>
	<li>Once the detergent has been mixed well with the water, clamp the system lines to maintain all reservoirs filled with the detergent solution.</li>
	<li>Disconnect the oxygenator from the rest of the system and rinse once more with water to remove all soap.</li>
	<li>Once the soap is completely removed, blow dry the oxygenator by blasting air through the fluid input at the highest rate possible.</li>
</ol>

Enhanced Cardiac Assessment in Ex Vivo Perfusion with Left Ventricular Loading

DV is an employee and Founder of VentriFlo, Inc., Pelham, NH, and has patent applications relevant to this study. For more information see https://ventriflo.com/patents/.&#160;SNT has patent applications relevant to this study and serves on the Scientific Advisory Board for Sylvatica Biotech Inc., a company focused on developing organ preservation technology. All competing interests are managed by the MGH and Partners HealthCare in accordance with their conflict-of-interest policies.&#160;AR and AAO receive research funding from Paragonix Technologies Inc.

Normothermic machine perfusion is a powerful modality for organ preservation and assessment that has greatly impacted the field of cardiac transplantation by expanding the donor pool of adult hearts36. This expansion is the result of the ability to currently utilize a small pool of hearts previously considered unsuitable for transplantation. Normothermic machine perfusion preserves cardiac grafts in a beating state, offering the opportunity for both functional and metabolic assessment. However, de...

Orthotopic heart transplantation is the current gold standard of care for end-stage heart failure1. Unfortunately, the field is significantly limited by a severe donor shortage crisis, resulting in only 2,000 heart transplants being performed each year when over 20,000 people would benefit from the lifesaving procedure2. This organ shortage is expected to worsen as the prevalence of heart failure in the United States alone is projected to surpass 8 million individuals by 20303. Steady increases in waitlist survival times - as a result of improved medical management, advances in mechanical circulatory support, and amendments to the UNOS allocation policy - have resulted in a further increase in the number of patients in need of transplantation at any given moment4,5.
Ex vivo machine perfusion or normothermic machine perfusion (NMP) is a preservation modality that has facilitated the expansion of the supply pool by allowing the use of organs donated after circulatory death (DCD) while achieving some extension of preservation times5,6,7,8. Unlike static cold storage, the current gold standard for preservation, NMP maintains organs in a metabolically active state, which creates the opportunity for real-time monitoring and graft assessment, becoming the standard preservation method for DCD grafts8,9. However, NMP devices currently used clinically are restricted to the Langendorff perfusion mode, which lacks quantitative metrics to predict transplantation outcomes and is unable to capture functional parameters6. For instance, lactate accumulation during Langendorff perfusion has been denoted as the best metabolic predictor of post-transplantation outcomes and is currently used in the clinical setting as a proxy for cardiac graft health10. However, even as the best assessment biomarker, it fails to reliably anticipate the need for mechanical circulatory support post-transplantation11,12. Similarly, the predictive capabilities of commonly utilized hemodynamic parameters (i.e., aortic pressure and coronary blood flow) are largely limited by the retrograde nature of current clinically used configurations for heart machine perfusion9.
The development of assessment protocols for accurate and precise determination of cardiac graft health during NMP would have an immense impact in the field beyond improving post-transplantation outcomes. Objective predictive tools would enable the reliable evaluation and likely utilization of marginal or extended criteria organs (i.e., prolonged warm (&#62; 30 min) and cold ischemia times (&#62; 6 h), increased donor age (&#62; 55), other comorbidities, etc.) from both DCD and brain death donors (DBD) that are currently rejected for transplantation due to the stringent selection criteria13. By enabling the use of marginal hearts, NMP could facilitate an increase in the organ supply as it is estimated that successful transplantation of half the hearts that currently go unused would be sufficient to eliminate the heart waitlist within 2-3 years14. Hemodynamic measurements obtained from left ventricular loading during NMP have garnered significant attention within the field due to their potential as objective assessment parameters. Previous studies have demonstrated that these parameters, such as left ventricular pulse pressure, contractility, and relaxation, are more indicative of cardiac graft function than metabolic trends15,16,17.
In effect, efforts have been dedicated to the development and identification of optimal loading methods to maximize assessment accuracy. Through these efforts, other groups have identified the most relevant mode of aortic perfusion during loading, whereby a stronger correlation between hemodynamic parameters and post-transplantation function was seen when implementing a passive afterload (i.e., no retrograde perfusion to the aorta during loading) when compared with pump-supported afterload (i.e., retrograde perfusion to the aorta during loading)18. This indicates that assisted coronary perfusion likely masks functional deficiencies. Previous studies have successfully incorporated passive afterloads into perfusion setups by implementing systems that mimic the Windkessel effect18,19,20. The Windkessel effect aids in dampening the fluctuation in blood pressure, maintaining continuous blood flow to the tissues and improving coronary perfusion. This protocol achieves the Windkessel-based passive afterload using a modified intravenous (IV) bag enclosed in two spring-loaded plates, where coronary perfusion is exclusively dependent on heart ejection (patent pending).
The use of passive left atrium (LA) pressurization (i.e., gravity-dependent pressurization) during loading, although common practice in small animal heart perfusions, is rarely utilized in the loading of large hearts21,22,23. Instead, the large majority of methods reported in the literature rely on secondary pumps for LA pressurization18,24,25,26,27,28. The pressurization of the LA through a gravity-dependent reservoir, rather than by pump, significantly simplifies the implementation of loading protocols. The use of gravity provides a fixed and constant pressure source, which greatly decreases the need for complicated control systems to achieve and maintain adequate LA pressurization. Moreover, through this pressurization approach, the requirement for a secondary pump is eliminated, facilitating the incorporation of loading capabilities into current Langerdoff setups, as only an extra reservoir is needed. The integration of loading capabilities into clinically utilized machine perfusion systems would amplify the application of cardiac NMP devices by facilitating detailed assessment of cardiac grafts during the preservation period. In effect, maximizing the utility of a system that poses significant financial commitment for patient care due to transportation and device utilization29.
This protocol demonstrates the feasibility of employing both passive afterload and passive LA pressurization during left ventricular loading. Through the validation of passive afterload/LA pressurization as a loading method, this protocol also provides an easy and effective manner of incorporating loading capabilities into established Langendorff perfusion systems. Importantly, it highlights the capability of functional assessment to uncover differences in viable versus failing hearts after extended periods of preservation (&#707;6 h).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Smiths Medical&#160;</td>
			<td>MX9341L</td>
			<td></td>
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			<td>4-0 Prolene sutures&#160;</td>
			<td>Ethicon&#160;</td>
			<td>8711</td>
			<td></td>
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			<td>5-0 Suture</td>
			<td>Fine Scientific Tools</td>
			<td>18020-50</td>
			<td></td>
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			<td>Aortic Connector&#160;</td>
			<td>VentriFLO Inc&#160;</td>
			<td></td>
			<td>Custom Made</td>
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			<td>Aortic root cannula</td>
			<td>Medtronic Inc&#160;</td>
			<td>10012</td>
			<td></td>
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			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
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			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
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			<td>Cell Saver&#160;</td>
			<td>Medtronic Inc&#160;</td>
			<td>ATLG</td>
			<td></td>
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			<td>Cell Saver cartridges&#160;</td>
			<td>Medtronic Inc&#160;</td>
			<td>ATLS00</td>
			<td></td>
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			<td>Dextran</td>
			<td>Sigma</td>
			<td>31389</td>
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			<td>EKG epicardial leads&#160;</td>
			<td>VentriFLO Inc&#160;</td>
			<td></td>
			<td>Custom Made</td>
		</tr>
		<tr>
			<td>Equipment stand and brackets&#160;</td>
			<td>VentriFLO Inc&#160;</td>
			<td></td>
			<td>Custom Made</td>
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			<td>External Pace maker&#160;</td>
			<td>Medtronic Inc&#160;</td>
			<td>5392</td>
			<td></td>
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			<td>Falcon High Clarity 50mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
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			<td>Flow Probes&#160;</td>
			<td>TranSonic Sytems inc&#160;</td>
			<td>1828</td>
			<td></td>
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			<td>Medplus</td>
			<td>G-0409-2720-0409-2721</td>
			<td></td>
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			<td>Hollow fiber oxygenator and Venous&#160; Resevior</td>
			<td>Medtronic Inc&#160;</td>
			<td>BBP241</td>
			<td>Affinity Pixie, 1L</td>
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			<td>HTP 1500&#160; Heat Therapy Pump</td>
			<td>HTP</td>
			<td>6826619</td>
			<td></td>
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			<td>Insulin</td>
			<td>Humulin R</td>
			<td></td>
			<td>MGH Pharmacy</td>
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			<td>Iworx Data Acquisition System</td>
			<td>Iworx</td>
			<td>IX-RA-834</td>
			<td></td>
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			<td>Krebs-Henseleit Buffer</td>
			<td>Sigma</td>
			<td>K3753</td>
			<td></td>
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			<td>Leukocyte Filter</td>
			<td>Haemonetics</td>
			<td>SB1E</td>
			<td></td>
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			<td>Organ Chamber</td>
			<td>VentriFLO Inc&#160;</td>
			<td></td>
			<td>Custom Made</td>
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			<td>Pacing Wires Biopolar</td>
			<td>Medtronic Inc&#160;</td>
			<td>6495</td>
			<td></td>
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			<td>Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
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			<td>Pressure Trasnducers&#160;</td>
			<td>Iworx</td>
			<td>BP100</td>
			<td></td>
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			<td>Pulsatile Pump</td>
			<td>VentriFLO Inc&#160;</td>
			<td>2100-0270</td>
			<td></td>
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			<td>PVC Tubing&#160;</td>
			<td>Medtronic Inc&#160;</td>
			<td>HY10Z49R9</td>
			<td></td>
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			<td>Right Angle Metal Tip Cannula 20F</td>
			<td>Medtronic Inc&#160;</td>
			<td>67318</td>
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			<td>Sigma</td>
			<td>5761</td>
			<td></td>
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			<td>Standard PHD ULTRA CP Syringe Pump</td>
			<td>Harvard Aparatus</td>
			<td>88-3015</td>
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			<td>Medtronic Inc&#160;</td>
			<td>79006</td>
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			<td>TranSonic Sytems Inc&#160;</td>
			<td>T402</td>
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			<td>Medtronic Inc&#160;</td>
			<td>CB841</td>
			<td>Affinity Fusion, 4L</td>
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		<tr>
			<td>WIndKessel Bag</td>
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cardiac loading using passive left atrial pressurization and passive afterload for graft assessment

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. Despite the immense opportunity for assessment due to the beating state of the heart, current clinical practice depends on limited metabolic trends for graft evaluation. Hemodynamic measurements obtained from left ventricular loading have garnered significant attention within the field due to their potential as objective assessment parameters. In effect, this protocol provides an easy and effective manner of incorporating loading capabilities to established Langendorff perfusion systems through the simple addition of an extra reservoir. Furthermore, it demonstrates the feasibility of employing passive left atrial pressurization for loading, an approach that, to our knowledge, has not been previously demonstrated. This approach is complemented by a passive Windkessel base afterload, which acts as a compliance chamber to maximize myocardial perfusion during diastole. Lastly, it highlights the capability of capturing functional metrics during cardiac loading, including left ventricular pulse pressure, contractility, and relaxation, to uncover deficiencies in cardiac graft function after extended periods of preservation times (&#707;6 h).

Author Spotlight: Enhancing Graft Viability Assessment Through Quantitative Metrics and Innovative Reservoir Systems

Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment

The scope of this research covers two important areas of cardiac ex vivo machine perfusion. First, it highlights the shortcomings of current clinically-used assessment metrics in determining graft viability and transplantability. Second, it provides an alternative assessment technique capable of discerning between viable and unviable grafts, even when conventional metrics fail to do so.Through this work, we developed a gravity-dependent loading reservoir that significantly simplifies and standardize the incorporation of loading capabilities into existing ex vivo machine perfusion systems. This, in combination with passive afterload, seems to significantly increase the predictive capabilities of the technique. The field has been in dire need of quantitative assessment metrics that eliminate the subjectivity of current practices.This work offers a method to obtain quantitative objective metrics for assessing graft variability. This assessment method is expected to significantly improve the ability to draw conclusions from both experimental and clinical setups.

Hearts from 4 Yorkshire pigs (30 - 35 kg) were harvested and preserved via Langendorff NMP for 6 h prior to 4 h of continuous loading. This experimental condition was chosen since 6 h is the average clinical preservation duration (5.1 &#177; 0.7 h)34. Through the addition of 4 extra hours of continuous loading (total of 10 h ex vivo time), some degree of heart failure was expected as a clear correlation between perfusion time and myocardial function decline has been previously reported<su...

The protocol describes a porcine ex vivo heart perfusion system in which direct loading of the left ventricle may serve as an assessment technique for graft health while simultaneously providing a holistic evaluation of graft function. A discussion of the system design and possible assessment metrics is also provided.

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. ...

cardiac-loading-using-passive-left-atrial-pressurization-passive

Research

JoVE Journal

Medicine

Designing a Porcine Ex Vivo Heart Perfusion System for Graft Health Assessment

To begin, start mounting the organ chamber on its stand, followed by the windkessel bag and the smaller reservoir. Verify that all components are securely connected. Then attach one end of a quarter inch silicone tubing to the outflow port of the oxygenator, and a Y connection at the distal end.To one side of the Y connector, attach another quarter inch silicone tubing with a lure connection and connect the other end to the bottom port on the windkessel bag. After that, fix a three-way lure valve to the expander's lure connection for adenosine drip delivery. Place a Hoffman clamp on an additional piece of quarter inch silicone tubing.For loaded profusion, attach one end of this line to the second end of the Y connector, and the other end to the top of the smaller reservoir tubing to control reservoir filling. Ensure that the clamp is fully engaged at this time. Then connect an overflow line from the top of the loading reservoir to the larger reservoir.Fit a quarter inch tube to the bottom of the loading reservoir and connect it to a port at the base of the organ chamber. Ensure that the line has a one-way valve. Next, insert two three-way lure valves midway through the tubing for adenosine delivery and a one-way valve before reaching the organ chamber.Fit the second overflow port at the bottom of the windkessel bag with three by eight inch tubing, connecting it to any inflow port of the venous reservoir. Afterward, close the Hoffman clamp completely during Langendorff mode and adjust it during loaded mode to modulate aortic pressures. Then connect a three-way lure valve to the overflow port at the top of the windkessel bag.Use a quarter inch tubing to connect the overflow port at the top windkessel bag to any inflow port in the venous reservoir. Connect the third outflow port on the windkessel bag to the aortic port on the organ chamber, inserting a temperature probe in the lower three-fourths of the tubing length.

Graft Preparation for Loading in Ex Vivo Normothermic Machine Perfusion Systems

To begin, take the isolated pig's heart and cut the branches of the aortic arch to form a single outflow tract. Insert the aortic cannula through this tract and secure it using a zip tie and 4-0 silk suture. Then place a bipolar pacing wire on the posterior wall of the right ventricle.Create two per string 4-0 prolene sutures around the perimeter of the left atrium tract. Secure the sutures with tourniquet snares, but do not tie them until loading. Lastly, close the left atrium appendage with a simple continuous 4-0 prolene suture.Then record the initial heart weight.

Cardiac Graft Revival and Left Ventricular Loading in the Langendorff Perfusion System

To begin clamp the 3 by 8 inch tubing right before the aortic port to stop perfusate flow. Position the prepared pig's heart with its posterior wall facing the operator and angle the organ chamber at roughly 20 degrees. Then, place the aortic cannula at a 90 degree angle from the aortic port, and unclamp the aortic line slowly.De-air the aortic cannula by allowing perfusate to flow into it gradually. Now, slowly decrease the angle of the aortic cannula until it aligns with the aortic port, then fully connect it to the aortic line. Once fully connected, gently massage the heart intermittently to prevent distension due to left ventricular filling.Vent the left ventricle through the open left atrium during this period. Start data acquisition and initiate the adenosine drip at 333 microliters per minute. Then, connect the pacing wires to the pacing box and set it to 60 beats per minute for backup pacing.If fibrillation is present, defibrillate the heart using 30 JUULs paddles. Once an organized rhythm is present, discontinue manual venting and place EKG leads directly on the heart using hook needles. Connect the right angle cannula to the atrial port of the organ chamber.Once connected, clamp the cannula and release the Hoffman clamp in the line between the oxygenator and the loading reservoir to allow fluid into the loading reservoir. Then, fill the loading reservoir until the pressure reaches 15 to 20 millimeters of mercury. Increase the pump output to maintain both aortic and atrial pressure.Next, insert half of the right angle metal tip into the left atrium with the tip pointing toward the appendage to promote mitral valve competence. Insert the pressure sensor inside the left ventricle to record left ventricular pressure. After that, release the clamp on the cannula to allow the left atrium to fill.Once de-aired, use the previously placed sutures and tourniquet snares to close the left atrium opening completely. Adjust the cannula and snares as needed to minimize fluid leakage. After securing the cannula in the left atrium, clamp the line from the oxygenator to the windkessel bag to completely stop retrograde perfusion to the aorta.Finally, move the adenosine drip from the line leading to the windkessel bag to the line between the loading reservoir and the atrial port. Vascular resistance, oxygen uptake rate, lactate accumulation, glucose consumption, and potassium accumulation showed no statistical difference throughout the perfusion period. Similarly, no difference was seen in the weight gained by the grafts.However, failing grafts heart rate steadily declined after two hours of loaded time with the area under the curve showing no statistically significant difference until three hours of perfusion. Additionally, loading dependent metrics suggested a loss of cardiac function as early as 30 minutes into loading time with the measure for contractility being the first metric to indicate a loss of function. Like heart rate, left ventricular pulse pressure steadily declined after two hours of loaded perfusion with statistically significant differences between grafts, pulse pressure, and relaxation seen after 1.5 hours of perfusion.

312 Views.  Massachusetts General Hospital, Harvard Medical School.  The protocol describes a porcine ex vivo heart perfusion system in which direct loading of the left ventricle may serve as an assessment technique for graft health while simultaneously providing a holistic evaluation of graft function. A discussion of the system design and possible assessment metrics is also provided.

Enhanced Cardiac Assessment in Ex Vivo Perfusion with Left Ventricular Loading

Summary