This work was supported by grants from the Canadian National Transplant Research Program. SH is the recipient of a Faculty of Medicine and Dentistry Motyl Graduate Studentship in Cardiac Sciences. DHF is a recipient of a Collaborative Research Projects (CHRP) grant in aid from the National Sciences and Engineering Research Council and Canadian Institutes of Health Research.

All the procedures in this manuscript were performed in compliance with the guidelines of the Canadian Council on Animal Care and the guide for the care and use of laboratory animals. The protocols were approved by the institutional animal care committee of the University of Alberta. This protocol has been applied in female juvenile Yorkshire pigs between 35&#8211;50 kg. All individuals involved in ESHP procedures had received proper biosafety training.
1. Pre-surgical Preparations
<ol>
	<li...

<ol>
	<li>Ardehali, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex-vivo+perfusion+of+donor+hearts+for+human+heart+transplantation+(PROCEED+II):+a+prospective,+open-label,+multicentre,+randomised+non-inferiority+trial.">Ex-vivo perfusion of donor hearts for human heart transplantation (PROCEED II): a prospective, open-label, multicentre, randomised non-inferiority trial.</a> Lancet. 385 (9987), 2577-2584 (2015).</li><li>Collins, M. J., Moainie, S. L., Griffith, B. P., Poston, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserving+and+evaluating+hearts+with+ex+vivo+machine+perfusion:+An+avenue+to+improve+early+graft+performance+and+expand+the+donor+pool.">Preserving and evaluating hearts with ex vivo machine perfusion: An avenue to improve early graft performance and expand the donor pool.</a> European Journal of Cardiothoracic Surgery. 34 (2), 318-325 (2008).</li><li>Freed, D. H., White, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+heart+preservation:+Straight+up,+or+on+the+rocks?.">Donor heart preservation: Straight up, or on the rocks?.</a> Lancet. 385 (9987), 2552-2554 (2015).</li><li>Guibert, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+preservation:+Current+concepts+and+new+strategies+for+the+next+decade.">Organ preservation: Current concepts and new strategies for the next decade.</a> Transfusion Medicine and Hemotherapy. 38 (2), 125-142 (2011).</li><li>Collins, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+diffusion+tensor+imaging+to+predict+myocardial+viability+after+warm+global+ischemia:+Possible+avenue+for+use+of+non-beating+donor+hearts.">Use of diffusion tensor imaging to predict myocardial viability after warm global ischemia: Possible avenue for use of non-beating donor hearts.</a> Journal of Heart and Lung Transplantation. 26 (4), 376-383 (2007).</li><li>White, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cardioprotective+preservation+strategy+employing+ex+vivo+heart+perfusion+facilitates+successful+transplant+of+donor+hearts+after+cardiocirculatory+death.">A cardioprotective preservation strategy employing ex vivo heart perfusion facilitates successful transplant of donor hearts after cardiocirculatory death.</a> Journal of Heart and Lung Transplantation. 32 (7), 734-743 (2013).</li><li>Iyer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+ex+vivo+perfusion+provides+superior+organ+preservation+and+enables+viability+assessment+of+hearts+from+DCD+donors.">Normothermic ex vivo perfusion provides superior organ preservation and enables viability assessment of hearts from DCD donors.</a> American Journal of Transplantation. 15 (2), 371-380 (2015).</li><li>Peltz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+preservation+maintains+myocardial+ATP+levels+and+reduces+apoptosis+in+an+ex+vivo+rat+heart+transplantation+model.">Perfusion preservation maintains myocardial ATP levels and reduces apoptosis in an ex vivo rat heart transplantation model.</a> Surgery. 138 (4), 795-805 (2005).</li><li>Liao, R., Podesser, B. K., Lim, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+continuing+evolution+of+the+Langendorff+and+ejecting+murine+heart:+New+advances+in+cardiac+phenotyping.">The continuing evolution of the Langendorff and ejecting murine heart: New advances in cardiac phenotyping.</a> American Journal of Physiology-Heart and Circulatory Physiology. 303 (2), H156-H167 (2012).</li><li>Rivard, L., Gallegos, R., Ogden, I., Bianco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+Preservation+of+the+Donor+Heart:+Basic+Science+to+Pre-Clinical.">Perfusion Preservation of the Donor Heart: Basic Science to Pre-Clinical.</a> Journal of Extra Corporeal Technology. 41 (3), 140-148 (2009).</li><li>Dhital, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+heart+transplantation+with+distant+procurement+and+ex+vivo+preservation+of+donor+hearts+after+circulatory+death:+A+case+series.">Adult heart transplantation with distant procurement and ex vivo preservation of donor hearts after circulatory death: A case series.</a> Lancet. 385 (9987), 2585-2591 (2015).</li><li>Messer, S., Ardehali, A., Tsui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+donor+heart+perfusion:+Current+clinical+experience+and+the+future.">Normothermic donor heart perfusion: Current clinical experience and the future.</a> Transplant International. 28 (6), 634-642 (2015).</li><li>White, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+donor+heart+viability+during+ex+vivo+heart+perfusion.">Assessment of donor heart viability during ex vivo heart perfusion.</a> Canadian Journal of Physiology and Pharmacology. 93 (10), 893-901 (2015).</li><li>Messer, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+and+transplantation+of+the+donor+heart+after+circulatory+death.">Functional assessment and transplantation of the donor heart after circulatory death.</a> Journal of Heart and Lung Transplantation. 35 (12), 1443-1452 (2016).</li><li>Hatami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum+stress+in+ex+vivo+heart+prfusion:+A+comparison+between+working+vs+non-working+modes.">Endoplasmic reticulum stress in ex vivo heart prfusion: A comparison between working vs non-working modes.</a> Canadian Journal of cardiology. 33 (10), (2017).</li><li>White, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+perfusion+in+a+loaded+state+improves+the+preservation+of+donor+heart+function.">Ex vivo perfusion in a loaded state improves the preservation of donor heart function.</a> Canadian Journal of cardiology. 31 (10), s202 (2015).</li><li>White, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wholeblood-based+perfusate+provides+superior+preservation+of+myocardial+function+during+ex+vivo+heart+perfusion.">A wholeblood-based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion.</a> Journal of Heart and Lung Transplantation. (14), (2014).</li><li>Lips, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+pressure-volume+measurements+in+mice:+comparison+of+closed-chest+versus+open-chest+approach.">Left ventricular pressure-volume measurements in mice: comparison of closed-chest versus open-chest approach.</a> Basic Research in Cardiology. 99 (5), 351-359 (2004).</li><li>Morita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+there+a+crystal+ball+for+predicting+the+outcome+of+cardiomyopathy+surgery?+Preload+recruitable+stroke+work,+may+be+a+possible+candidate.">Is there a crystal ball for predicting the outcome of cardiomyopathy surgery? Preload recruitable stroke work, may be a possible candidate.</a> Journal of Cardiology. 71 (4), 325-326 (2018).</li><li>Hatami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Canadian Society for Transplantation. , (2017).</li><li>Anthony, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+coronary+angiographic+evaluation+of+a+beating+donor+heart.">Ex vivo coronary angiographic evaluation of a beating donor heart.</a> Circulation. 130 (25), e341-e343 (2014).</li><li>Sandha, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroids+Limit+Myocardial+Edema+During+Ex+vivo+Perfusion+Of+Hearts+Donated+After+Circulatory+Death.">Steroids Limit Myocardial Edema During Ex vivo Perfusion Of Hearts Donated After Circulatory Death.</a> Annals of Thoracic Surgery. , (2018).</li></ol>

DHF holds patents on ex situ organ perfusion technology and methods. DHF and JN are founders and major shareholders of Tevosol, Inc.

Successful perfusion is defined according to the aims of the study; however, this should include uninterrupted ESHP for the desired amount of time and complete collection of the data on cardiac function during the perfusion. For this purpose, a few critical steps in the protocol must be followed.
The heart is an organ with high oxygen and energy demands, and minimizing the ischemic time before cannulation and perfusion is an important principle that must be followed. The process of procurement...

Clinical relevance
While most aspects of cardiac transplantation have evolved significantly since the first heart transplant in 1967, cold storage (CS) remains the standard for donor heart preservation1. CS exposes the organ to a period of cold ischemia that limits the safe preservation interval (4&#8211;6 hours) and increases the risk of primary graft dysfunction2,3,4. Due to the static nature of CS, assessments of function or therapeutic interventions are not possible in the time between the organ procurement and transplantation. This is a particular limitation in extended criteria donors including hearts donated after circulatory death (DCD), creating an obstacle for overcoming the considerable gap between demand and the current donor pool5,6. To address this limitation, ex situ heart perfusion has been proposed as a novel, semi-physiologic method of preserving donated hearts, minimizing exposure to cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart during preservation time1,7,8.
Ex situ heart perfusion
One of the most frequently used methods for ex situ examination of the isolated heart is Langendorff perfusion. In this method, introduced by Oskar Langendorff in 1895, the blood flows into the coronary arteries and out the coronary sinus of the isolated heart, with the heart in an empty and beating state9,10. Clinical ESHP in a Langendorff mode with the Transmedics Organ Care System apparatus (OCS) has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts1, and has facilitated the clinical transplantation of DCD hearts11. However, there are concerns about the ability of the device to evaluate organ viability, as a number of donor hearts initially thought to be transplantable were discarded after perfusion on the OCS3. The OCS supports the heart in the Langendorff (non-working) mode, and thus possesses a limited capacity for evaluation of the pumping function of the heart3,12. A growing body of evidence suggests that functional parameters offer a better way to assess organ viability, suggesting that assessments of cardiac function may become a reliable tool for the evaluation and selection of hearts for transplantation during ESHP3,12,13,14, Furthermore, our studies on ex situ perfused porcine hearts suggest that ESHP in working mode provides enhanced functional preservation of the heart during the perfusion interval15,16.
An ESHP apparatus capable of preserving the heart in a working mode must possess a level of automation to safely and precisely maintain preload, afterload and flow rates. Also, such a system should possess the flexibility to facilitate comprehensive assessments of cardiac function to be undertaken. The ESHP apparatus used here is equipped with custom software that 1) provides and maintains desired aortic (Ao) and left atrial (LA) pressure/flow and 2) provides real-time analysis of functional parameters and visual evaluation of pressure waveforms with minimal need for supervision. Pressure data is acquired with standard fluid-filled pressure transducers, and flow data is acquired with transit-time doppler flow probes. These signals are digitized with a bridge and analog input, respectively. The heart is positioned horizontally with a slight elevation to the great vessels on a soft silicone membrane. The cannulation attachments pass through the membrane, incorporating a compliance chamber for dampening ventricular ejection. The goal of this work is to provide researchers in the field of cardiac transplantation with a protocol for ex situ perfusion and evaluation of the heart, under normothermic, semi-physiologic conditions in working mode, in a large mammal (Yorkshire pig) model.

<table>
	<tbody>
		<tr>
			<td>Debakey-Metzenbaum dissecting scissors</td>
			<td>Pilling</td>
			<td>342202</td>
			<td></td>
		</tr>
		<tr>
			<td>MAYO dissecting scissors</td>
			<td>Pilling</td>
			<td>460420</td>
			<td></td>
		</tr>
		<tr>
			<td>THUMB forceps</td>
			<td>Pilling</td>
			<td>465165</td>
			<td></td>
		</tr>
		<tr>
			<td>Debakey straight vascular tissue forceps&#160;</td>
			<td>Pilling</td>
			<td>351808</td>
			<td></td>
		</tr>
		<tr>
			<td>CUSHING Gutschdressing forceps</td>
			<td>Pilling</td>
			<td>466200</td>
			<td></td>
		</tr>
		<tr>
			<td>JOHNSON needle holder</td>
			<td>Pilling</td>
			<td>510312</td>
			<td></td>
		</tr>
		<tr>
			<td>DERF needle holder</td>
			<td>Pilling</td>
			<td>443120</td>
			<td></td>
		</tr>
		<tr>
			<td>Sternal saw</td>
			<td>Stryker</td>
			<td>6207</td>
			<td></td>
		</tr>
		<tr>
			<td>Sternal retractor</td>
			<td>Pilling</td>
			<td>341162</td>
			<td></td>
		</tr>
		<tr>
			<td>Vorse tubing clamp</td>
			<td>Pilling</td>
			<td>351377</td>
			<td></td>
		</tr>
		<tr>
			<td>MORRIS ascending aorta clamp</td>
			<td>Pilling</td>
			<td>353617</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical snare (tourniquet) set</td>
			<td>Medtronic</td>
			<td>CVR79013</td>
			<td></td>
		</tr>
		<tr>
			<td>2-0 SILK black 12 X 18&#34; strands</td>
			<td>ETHICON</td>
			<td>A185H</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 PROLENE blue 18&#34; PS-2 cutting</td>
			<td>ETHICON</td>
			<td>8687H</td>
			<td></td>
		</tr>
		<tr>
			<td>Biomedicus pump drive (modified)</td>
			<td>Medtronic</td>
			<td>540</td>
			<td>Modified to allow remote electronic control of pump speed</td>
		</tr>
		<tr>
			<td>Biomedicus pump</td>
			<td>Maquet</td>
			<td>BPX-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane oxigenator D 905</td>
			<td>SORIN GROUP</td>
			<td>50513</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing flow module&#160;&#160;</td>
			<td>Transonic</td>
			<td>Ts410</td>
			<td></td>
		</tr>
		<tr>
			<td>PXL clamp-on flow sensor</td>
			<td>Transonic</td>
			<td>ME9PXL-BL37SF</td>
			<td></td>
		</tr>
		<tr>
			<td>TruWave pressure transducer</td>
			<td>Edwards</td>
			<td>VSYPX272</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercept tubing 3/8&#34; X 3/32&#34; X 6&#39;</td>
			<td>Medtronic</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercept tubing 1/4&#34; X 1/16&#34; X 8&#39;</td>
			<td>Medtronic</td>
			<td>3108</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated/Refrigerated Bath Circulator&#160;</td>
			<td>Grant</td>
			<td>TX-150</td>
			<td></td>
		</tr>
		<tr>
			<td>ABL 800 FLEX Blood Gas Analyzer</td>
			<td>Radiometer</td>
			<td>989-963</td>
			<td></td>
		</tr>
		<tr>
			<td>5F Ventriculr straight pigtail cathter</td>
			<td>CORDIS</td>
			<td>534550S</td>
			<td></td>
		</tr>
		<tr>
			<td>5F AVANTI+ Sheath Introducer</td>
			<td>CORDIS</td>
			<td>504605A</td>
			<td></td>
		</tr>
		<tr>
			<td>Emerald Amplatz Guidewire</td>
			<td>CORDIS</td>
			<td>502571A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual chamber pace maker</td>
			<td>Medtronic</td>
			<td>5388</td>
			<td></td>
		</tr>
		<tr>
			<td>Defibrilltor</td>
			<td>CodeMaster</td>
			<td>M1722B</td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion pump</td>
			<td>Baxter</td>
			<td>AS50</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical electrocautery device</td>
			<td>Kls Martin</td>
			<td>ME411</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas mixer</td>
			<td>SECHRIST</td>
			<td>3500 CP-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical oxygen tank</td>
			<td>praxair</td>
			<td>2014408</td>
			<td></td>
		</tr>
		<tr>
			<td>Cabon dioxide tank</td>
			<td>praxair</td>
			<td>5823115</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP biomedicals</td>
			<td>218057791</td>
			<td></td>
		</tr>
	</tbody>
</table>

normothermic ex situ heart perfusion in working mode: assessment of cardiac function and metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

At the start of the perfusion (in non-working mode), the heart will normally resume a sinus rhythm when the temperature of the system and perfusate approaches normothermia. When entering working mode, as the LA pressures are approaching the desired values, ejection on the Ao pressure tracing should be observed and the LA flow (a reflection of cardiac output) should increase gradually. In a Yorkshire pig model (35&#8211;50 kg) and a starting heart weight of 180&#8211;220 grams, the initial...

Watch this Scientific Journal Video about Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism at JoVE.com

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

This method can help answer key questions about the optimal preservation of donor hearts, as well as their functional and metabolic evaluation for subsequent transplantation suitability. The main advantage of this technique is it allows a functional evaluation of the donor heart that is not based solely on indirect metabolic assessments. The implications of this technique extend towards the therapy of dysfunctional donor hearts, as the heart function can be easily and repeatedly assessed over time.Generally, individuals new to this matter will struggle because some technical expertise is required for connecting the heart in a safe and secure manner. Visual demonstration of this method is critical as the technical aspects of the heart retrieval and connection to the device can be challenging to learn by text instruction alone. After confirming a lack of response to noxious stimulus, perform a median sternotomy on the anesthetized pig and once the heart has been exposed, use Metzenbaum scissors to open the pericardium.Use 1-0 silk sutures to fix the pericardial edges to the sternum and gradually collect 750 milliliters of whole blood from the two-stage venous cannula placed in the right atrium in an autoclaved glass container over a period of 15 minutes while simultaneously replacing the volume with one liter of an isotonic crystalloid solution. Add the blood to a perfusion circuit primed with 750 milliliters of Krebs-Henseleit buffer supplemented with 8%albumin and place a 14 gauge cardioplegia needle and cannula into the ascending aorta. Secure the cardioplegia needle on the aorta with a snare and connect the cardioplegia cannula to the cardioplegia bag.Add 100 milliliters of blood to 400 milliliters of cardioplegia. After the end of exsanguination, cross-clamp the ascending aorta with an aortic clamp. Next, deliver the cardioplegic solution into the aortic root.When all the solution has been administered, remove the clamp and excise the heart, including all of the aortic arch vessels and a segment of the descending aorta. To prevent the deleterious effects of prolonged ischemia, the heart should be procured and mounted on the apparatus for perfusion as quickly as possible. After removing any excess tissue around the left atrium, use a 3-0 polypropylene suture to place a purse string suture around the left atrial orifice.Suture and close the inferior vena cava with a 3-0 polypropylene suture. Place the left atrial cannula into the left atrial orifice and secure the cannula with a snare. Place the left atrial cannula over the silicone membrane embedded magnet of the apparatus, taking care that the magnet and the corresponding metal ring in the left atrial cannula are properly engaged.Attach the aorta to the aortic cannula embedded in the silicone membrane and use a silk tie to secure the aorta around the cannula. Turn the aorta to achieve a proper lie without tension or kinking and increase the aortic pump speed to about 1600 RPM and gently squeeze the ventricles to de-air the heart. The remaining air in the aortic root will be ejected through the innominate and subclavian branches.Connect the aortic purge line to the innominate artery and secure the connection with a silk tie. Snare the left subclavian artery orifice with a silk tie and secure the closure with a snare and a snap. Place a 5 French introducer sheath through the orifice of the subclavian artery, adjusting the length and orientation of the catheter so that the catheter does not interfere with the aortic valve function.Adjust the aortic pump speed to reach a mean pressure of 30 millimeters of mercury to start the perfusion in the non-working Langendorff mode. The appearance of a dark, deoxygenated perfusate in the pulmonary arterial line indicates the re-establishment of coronary flow. Turn the heat exchanger to 38 degrees Celsius to warm the perfusate and use a blood gas analyzer to check the dissolved gas status, adjusting the gas mixture to the appropriate parameters.After an hour in non-working mode, enter a suitable left atrial pressure in the desired left atrial pressure section of the software and initiate the feedback loop. The activated working mode will appear as a green button and the left atrial pump speed will automatically increase and decrease to reach and maintain the selected left atrial pressure. For the assessment and recording of the steady-state data, place a fluid-filled pigtail catheter through the introducer sheath into the left ventricle.For assessment of the preload recruitable stroke work, remove the pigtail catheter from the left ventricle and adjust the desired rate of drop in the left atrial pump speed during the analysis and the desired time period during which the analysis will take place. Click record pressure volume loop. The software will automatically exit the working mode and gradually reduce the left atrial pump RPM, while simultaneously recording the left ventricle stroke work and the left atrial pressure.At the conclusion of data collection, the software will perform a linear regression on the newly acquired data set to yield the preload recruitable stroke work. After the ex situ apparatus heart software has completed the analysis, a message will appear on the main page showing the correlation coefficient of the analysis. Click okay if the coefficient is greater than 0.95.The preload recruitable stroke work analysis results will be recorded. To assess the metabolic state of the heart and the perfusate, use the information obtained from the blood gas analysis of the perfusate samples collected from both the arterial and pulmonary artery lines every one to two hours. In a Yorkshire Pig model with a starting heart weight of 180 to 220 grams, the initial left atrial flow will be approximately 2, 000 milliliters minute, typically approaching about 2, 250 milliliters per minute during the first one to two hours of perfusion in the working mode.Blood gas analysis and metabolic assessments performed on the perfusate samples obtained during ex situ heart perfusion provide extensive information on the metabolic status of the heart over time, indicating the perfusate lactate concentration and the information needed to calculate the myocardial oxygen consumption. In the absence of the organs that naturally metabolize their clear cardiac biomarkers, the accumulation of these factors within the perfusate solution over time is typically observed. In addition to load dependent parameters, the left ventricle preload recruitable stroke work can be evaluated during the computer-controlled linear reduction in the left atrial pressure, as demonstrated.Echocardiography during ex situ heart perfusion can also provide additional information about myocardial function and anatomical parameters. While attempting this procedure, it's important to remember that ex situ heart perfusion is still in its infancy and that more studies are warranted to design better metabolic heart support for better preservation of cardiac viability and function. Following this procedure, other methods like angiographic imaging can be performed to answer additional questions about donor heart's coronary vascular status.After it's development, this technique paved the way for researchers in the field of organ transplantation to explore the possibility of the safe transplantation of extended criteria donated hearts in patients awaiting a heart transplant. Don't forget that working with biomaterials can be extremely hazardous and that precautions, such as wearing gloves and protective glasses, should always be taken while performing this procedure.

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Research

JoVE Journal

Medicine

12.9K vistas.  University of Alberta. Este método puede ayudar a responder preguntas clave sobre la preservación óptima de los corazones de los donantes, así como su evaluación funcional y metabólica para la posterior idoneidad del trasplante. La principal ventaja de esta técnica es que permite una evaluación funcional del corazón del donante que no se basa únicamente en evaluaciones metabólicas indirectas. Las implicaciones de esta técnica se extienden hacia la terapia de corazones de donantes disfuncionales, ya que ...