This research work was financially supported by grants provided by Innovatief Actieprogramma Groningen (IAG-3), Jan Kornelis de Cock Stichting and Tekke Huizingafonds, all in the Netherlands. We are appreciative to all the Dutch transplantation coordinators for identifying the potential discarded livers and obtaining informed consent.

This protocol has been approved by the Medical Ethical Committee (Medisch Ethische Toetsingscommissie) of the University Medical Center Groningen, the Netherlands.
1. Preparation of the Perfusion Fluid
Note: The total volume of the perfusion fluid prepared for normothermic machine perfusion according to this protocol is 2,233 ml and the targeted osmolarity of the perfusion fluid is 302 mOsmol/L.
<ol>
	<li>From the components of the perfusion fluid described in Table 1, keep the human packed red blood cells, fresh frozen plasma and human albumin separated. Mix the rest of the components in a sterile manner and store the solution in a sterile bag for transportation to the operating room (OR). Do this in a sterile environment (ideally a Good Manufacturing Practice facility) or in a laminar flow cabinet in a culture room.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Components</td>
			<td fo:width="2in" style="width:80px">Quantity</td>
		</tr>
		<tr>
			<td>Packed red blood cell (Hematocrit 60%)</td>
			<td style="text-align:right">840 ml</td>
		</tr>
		<tr>
			<td>Fresh frozen plasma</td>
			<td style="text-align:right">930 ml</td>
		</tr>
		<tr>
			<td>Human albumin 200 g/L (Albuman, Sanquin)</td>
			<td style="text-align:right">100 ml</td>
		</tr>
		<tr>
			<td>Modified parenteral nutrition (Clinimix N17G35E, Baxter International Inc.)</td>
			<td style="text-align:right">7.35 ml</td>
		</tr>
		<tr>
			<td>Multivitamins for infusion (Cernevit, Baxter international Inc.)</td>
			<td style="text-align:right">7 &#956;l</td>
		</tr>
		<tr>
			<td>Concentrated trace elements for infusion (Nutritrace , B. Braun Melsungen AG)</td>
			<td style="text-align:right">7.35 ml</td>
		</tr>
		<tr>
			<td>Metronidazol for i.v. administration (5 mg/ml) (Flagyl, Sanofi-Aventis)</td>
			<td style="text-align:right">40 ml</td>
		</tr>
		<tr>
			<td>Cefazolin 1,000 mg flask 5 ml powder for i.v. administration (Servazolin, Sandoz)</td>
			<td style="text-align:right">2 ml</td>
		</tr>
		<tr>
			<td>Fast-acting insulin (100 IU/ml) (Actrapid&#174;, Novo Nordisk)</td>
			<td style="text-align:right">20 ml</td>
		</tr>
		<tr>
			<td>Calcium glubionate, intravenous solution 10%, 137.5 mg/ml (Sandoz)</td>
			<td style="text-align:right">40 ml</td>
		</tr>
		<tr>
			<td>Sterile H2O</td>
			<td style="text-align:right">51.3 ml</td>
		</tr>
		<tr>
			<td>NaCl 0.9% solution</td>
			<td style="text-align:right">160 ml</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate 8.4% solution</td>
			<td style="text-align:right">31 ml</td>
		</tr>
		<tr>
			<td>Heparin 5,000 IE/ml for i.v. administration</td>
			<td style="text-align:right">4 ml</td>
		</tr>
		<tr>
			<td>Total</td>
			<td style="text-align:right">2,233 ml</td>
		</tr>
	</tbody>
</table>


Table 1: Components of the perfusion fluid16.
<ol start="2">
	<li>Transfer human packed red blood cells (840 ml), fresh frozen plasma (930 ml), human albumin 200 g/L (100 ml) and the solution prepared in step 1.1 to the OR to be administered to the perfusion device.</li>
</ol>


2. Priming of the Perfusion Device
<ol>
	<li>Add the components of the perfusion fluid, including the human packed red blood cells, fresh frozen plasma, human albumin and the solution prepared in step 1.1 to the machine via the connector on top of the oxygenators and remove all the air bubbles from the tubing.</li>
	<li>Switch on the venous pump and follow the manufacturer&#8217;s instructions on the screen. Then turn on the arterial pump and follow the manufacturer&#8217;s instructions on the screen.</li>
	<li>Null the pressure meters against atmospheric pressure by following the instructions on the screen. This ensures that the pressure measured during the perfusion is the real pressure at the level of the portal vein and the hepatic artery.</li>
	<li>Start the oxygenation using carbogen (95% O2&#160;+ 5% CO2) at a flow rate of 4 L/min. The air flow will be divided among the two oxygenators (2 L/min per oxygenator) and this should result in a pO2 of around 60 kPa (or 450 mmHg) in the perfusion fluid. For longer perfusions, it is advisable to use separate sources of oxygen and carbon dioxide. This allows for small adjustments in the O2/CO2 ratio, which can be used to adjust the pH and pCO2 of the perfusion fluid.</li>
	<li>Take a perfusion sample for blood gas measurement 15-20 min after the device has been primed and monitor the pH and electrolytes accordingly. 
	NOTE: Be sure to discard about 3 ml of perfusion fluid before taking the samples, as this fluid is in the peripheral tubing and does not represent the perfusion fluid in the system. Add an 8.4% sodium bicarbonate solution for buffering capacity, aiming for a physiological pH (7.35-7.45). For example, add 25-35 ml of an 8.4% sodium bicarbonate solution and check the pH and bicarbonate levels in the perfusion fluid by taking samples for blood gas measurement at regular intervals.</li>
</ol>
3. Procurement and Preparation of Donor Livers
Note: Procure the organ using the standard technique of in situ cooling and flush out with cold preservation fluid (0-4 &#176;C)19. To facilitate cannulation of the artery, leave a segment of the supratruncal aorta attached to the hepatic artery (Figure 2A).
<ol>
	<li>Flush out the bile ducts with the preservation fluid (i.e., University of Wisconsin solution). Ligate the cystic duct with a surgical suture.</li>
	<li>Pack and store the organ in a standard sterile donor organ bag and box with crushed ice for subsequent transportation to the MP center.</li>
	<li>Start the back table procedure immediately upon arrival of the donor liver in the operating room.
	<ol>
		<li>Take a sample of at least 10 ml of the preservation fluid for microbiological testing.</li>
		<li>Remove the diaphragmatic attachments to the bare area of the liver as well as any remaining cardiac muscle from the upper cuff of the vena cava with surgical scissors.</li>
		<li>Dissect the artery and portal vein using dissecting scissors and ligate side branches using surgical sutures or hemoclips.</li>
		<li>Close the distal end of the supratruncal aorta segment using a non-absorbable monofilament suture (e.g., 3-0 Prolene). Insert the arterial cannula into the proximal end of the supratruncal aorta and secure with sutures (Figure 2A). Use the cannula provided in the disposable package as supplied by the manufacturer of the perfusion device.</li>
		<li>Insert the venous cannula in the portal vein and secure with sutures. Use the cannula provided in the disposable package. The hepatic vein remains uncannulated.</li>
		<li>Flush out the bile duct with the preservation solution. Insert a silicon catheter into the bile duct and secure with sutures. 
		NOTE: Do not insert the catheter too deeply into the bile duct as this may cause injury to the biliary epithelium.</li>
		<li>Flush out the liver with 0.9% NaCl solution via the portal vein cannula as follows:
		<ol>
			<li>If the graft has been preserved in University of Wisconsin solution as the preservation solution, flush out the liver with 2,000 ml of cold (0-4 &#176;C) 0.9% NaCl solution followed by 500 ml of warm (37 &#176;C) 0.9% NaCl solution.</li>
			<li>If the graft has been preserved in Histidine-Tryptophan-Ketoglutarate solution as the preservation solution, flush out the liver with 1,000 ml of cold (0-4 &#176;C) 0.9% NaCl solution followed by 500 ml of warm (37 &#176;C) 0.9% NaCl solution. The purpose of the warm flush is to prevent a significant drop in the temperature of the perfusion fluid.</li>
			<li>Perform the warm flush immediately before connecting the liver to the perfusion device. 
			NOTE: Always keep the duration between warm flush and start of NMP less than 1-2 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Donor characteristics (N = 12)</td>
			<td>Number (%) or Median (IQR)</td>
		</tr>
		<tr>
			<td>Age (years)</td>
			<td>61 (50-64)</td>
		</tr>
		<tr>
			<td>Gender (male)</td>
			<td>8 (67%)</td>
		</tr>
		<tr>
			<td>Type of donor 
			DCD, Maastricht type III 
			DBD</td>
			<td>&#160; 
			10 (83%) 
			2 (17%)</td>
		</tr>
		<tr>
			<td>Body mass index (BMI)</td>
			<td>27 (25-35)</td>
		</tr>
		<tr>
			<td>Reason for rejection 
			DCD+ age &#62;60 years 
			DCD+ high BMI 
			DCD+ various reasons* 
			Severe steatosis</td>
			<td>&#160; 
			5 (41%) 
			3 (25%) 
			2 (17%) 
			2 (17%)</td>
		</tr>
		<tr>
			<td>Preservation solution 
			&#160;&#160;UW solution 
			&#160;&#160;HTK solution</td>
			<td>&#160; 
			6 (50%) 
			6 (50%)</td>
		</tr>
		<tr>
			<td>Donor warm ischemia time in DCD (min)</td>
			<td>14 (17 - 20)</td>
		</tr>
		<tr>
			<td>Cold ischemia time (min)</td>
			<td>389 (458-585)</td>
		</tr>
		<tr>
			<td>Donor risk index (DRI)</td>
			<td>2.35 (2.01-2.54)</td>
		</tr>
	</tbody>
</table>
Table 2: Donor characteristics. * donor history of intravenous drug abuse for one graft and prolonged donor sO2 &#60;30% after withdrawal of life support for another graft. Abbreviations: DCD, donation after circulatory death; DBD, donation after brain death; UW, University of Wisconsin; HTK, Histidine-tryptophan-ketoglutarate
<img alt="Figure 2" src="/files/ftp_upload/52688/52688fig2.jpg" /> 
Figure 2: (A) Pictures of a human donor graft that has been prepared on the back table and (B-D) was subsequently perfused normothermically. (A) The arterial cannula is inserted into the surpratruncal aorta and the venous cannula is inserted into the portal vein. The bile duct is cannulated with a silicon biliary catheter. (B) The liver is positioned in the organ chamber with its anterior surface facing downwards and cannulas are connected to the tubings of the perfusion device. (C) 30 min after the start of normothermic machine perfusion. (D) 6 hr after the start of normothermic machine perfusion. During operation the organ chamber is covered by a transparent cover to maintain a sterile moist environment for the liver (not shown in these pictures). <a href="https://www.jove.com/files/ftp_upload/52688/52688fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Normothermic Machine Perfusion
<ol>
	<li>Position the liver in the organ chamber with the anterior surface facing downward. Immediately connect the liver to the primed perfusion device by connecting the portal vein cannula to the portal inflow tube of the perfusion device and the arterial cannula to the arterial inflow tube of the device.</li>
	<li>Start perfusion on both portal and arterial side by following the manufacturer&#8217;s instructions on the screen. Set the mean arterial pressure at 70 mmHg and the mean portal venous pressure at 11 mmHg.</li>
	<li>Take perfusion fluid samples every 30 min for immediate analysis of blood gas parameters (pO2, pCO2, sO2, HCO2- and pH) and biochemical parameters (glucose, calcium, lactate, potassium and sodium) using a conventional blood gas analyzer. Be sure to discard about 3 ml of perfusion fluid before taking the samples, as this fluid is in the peripheral tubing and does not represent the perfusion fluid in the system.
	<ol>
		<li>To take these samples aspirate the perfusion fluid using a 1 ml syringe from the sampling connectors that are part of the disposable tubing set of the perfusion device. For each sample use a new syringe and immediately remove any air bubbles from the syringe upon aspiration of perfusion fluid. Then insert the syringe in the blood gas analyzer and follow the manufacturer&#8217;s instructions provided in the manual of the analyzer.</li>
	</ol>
	</li>
	<li>Collect plasma from the perfusion fluid, freeze and store at -80 &#176;C for determination of alkaline phosphatase (AlkP), gamma-glutamyl transferase (gamma-GT), alanine aminotransferase (ALT), urea and total bilirubin. Collect plasma after 5 min of centrifugation of the perfusion fluid at 1,500 x g and 4 &#176;C.</li>
</ol>

<ol>
	<li>Plaats, 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=Hypothermic+machine+preservation+in+liver+transplantation+revisited:+Concepts+and+criteria+in+the+new+millennium.">Hypothermic machine preservation in liver transplantation revisited: Concepts and criteria in the new millennium.</a> Ann. Biomed. Eng. 32 (4), 623-631 (2004).</li><li>op den Dries, S., Sutton, M. E., Lisman, Y., Porte, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+bile+ducts+in+liver+transplantation:+Looking+beyond+ischemia.">Protection of bile ducts in liver transplantation: Looking beyond ischemia.</a> Transplantation. 92 (4), 373-379 (2011).</li><li>Seehofer, D., Eurich, D., Veltzke-Schlieker, W., Neuhaus, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+complications+after+liver+transplantation:+Old+problems+and+new+challenges.">Biliary complications after liver transplantation: Old problems and new challenges.</a> Am. J. Transplant. 13 (2), 253-265 (2013).</li><li>Buis, C. I., 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=Nonanastomotic+biliary+strictures+after+liver+transplantation,+part+1:+Radiological+features+and+risk+factors+for+early+vs.+late+presentation.">Nonanastomotic biliary strictures after liver transplantation, part 1: Radiological features and risk factors for early vs. late presentation.</a> Liver. Transpl. 13 (5), 708-718 (2007).</li><li>Karimian, N., op den Dries, S., Porte, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+biliary+strictures+after+liver+transplantation:+Is+it+the+amount+of+epithelial+injury+or+insufficient+regeneration+that+counts.">The origin of biliary strictures after liver transplantation: Is it the amount of epithelial injury or insufficient regeneration that counts.</a> J. Hepatol. 58 (6), 1065-1067 (2013).</li><li>Gastaca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+complications+after+orthotopic+liver+transplantation:+A+review+of+incidence+and+risk+factors.">Biliary complications after orthotopic liver transplantation: A review of incidence and risk factors.</a> Transplant. Proc. 44 (6), 1545-1549 (2012).</li><li>Sanchez-Urdazpal, L., 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=Ischemic-type+biliary+complications+after+orthotopic+liver+transplantation.">Ischemic-type biliary complications after orthotopic liver transplantation.</a> Hepatology. 16 (1), 49-53 (1992).</li><li>Dubbeld, 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=Similar+liver+transplantation+survival+with+selected+cardiac+death+donors+and+brain+death+donors.">Similar liver transplantation survival with selected cardiac death donors and brain death donors.</a> Br. J. Surg. 97 (5), 744-753 (2010).</li><li>Guarrera, J. V., 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=Hypothermic+machine+preservation+in+human+liver+transplantation:+The+first+clinical+series.">Hypothermic machine preservation in human liver transplantation: The first clinical series.</a> Am. J. Transplant. 10 (2), 372-381 (2010).</li><li>Henry, S. D., 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=Hypothermic+machine+preservation+reduces+molecular+markers+of+ischemia/reperfusion+injury+in+human+liver+transplantation.">Hypothermic machine preservation reduces molecular markers of ischemia/reperfusion injury in human liver transplantation.</a> Am. J. Transplant. 12 (9), 2477-2486 (2012).</li><li>Schlegel, A., Rougemont, O., Graf, R., Clavien, P. A., Dutkowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+mechanisms+of+end-ischemic+cold+machine+perfusion+in+DCD+liver+grafts.">Protective mechanisms of end-ischemic cold machine perfusion in DCD liver grafts.</a> J. Hepatol. 58 (2), 278-286 (2013).</li><li>op den Dries, 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=Hypothermic+oxygenated+machine+perfusion+prevents+arteriolonecrosis+of+the+peribiliary+plexus+in+pig+livers+donated+after+circulatory+death.">Hypothermic oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated after circulatory death.</a> PLoS. One. 9 (2), e88521 (2014).</li><li>Tolboom, H., 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=Subnormothermic+machine+perfusion+at+both+20+degrees+C+and+30+degrees+C+recovers+ischemic+rat+livers+for+successful+transplantation.">Subnormothermic machine perfusion at both 20 degrees C and 30 degrees C recovers ischemic rat livers for successful transplantation.</a> J. Surg. Res. 175 (1), 149-156 (2012).</li><li>Gringeri, 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=Subnormothermic+machine+perfusion+for+non-heart-beating+donor+liver+grafts+preservation+in+a+swine+model:+A+new+strategy+to+increase+the+donor+pool?+Transplant.">Subnormothermic machine perfusion for non-heart-beating donor liver grafts preservation in a swine model: A new strategy to increase the donor pool? Transplant.</a> Proc. 44 (7), 2026-2028 (2012).</li><li>Bruinsma, B. G., 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=Subnormothermic+machine+perfusion+for+ex+vivo+preservation+and+recovery+of+the+human+liver+for+transplantation.">Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation.</a> Am. J. Transplant. 14 (6), 1400-1409 (2014).</li><li>op den Dries, 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=Ex+vivo+normothermic+machine+perfusion+and+viability+testing+of+discarded+human+donor+livers.">Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.</a> Am. J. Transplant. 13 (5), 1327-1335 (2013).</li><li>Sutton, M. 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=Criteria+for+viability+assessment+of+discarded+human+donor+livers+during+ex+vivo+normothermic+machine+perfusion.">Criteria for viability assessment of discarded human donor livers during ex vivo normothermic machine perfusion.</a> PLoS. One. 9 (11), (2014).</li><li>Minor, T., Efferz, P., Fox, M., Wohlschlaeger, J., Luer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+oxygenated+rewarming+of+cold+stored+liver+grafts+by+thermally+graduated+machine+perfusion+prior+to+reperfusion.">Controlled oxygenated rewarming of cold stored liver grafts by thermally graduated machine perfusion prior to reperfusion.</a> Am. J. Transplant. 13 (6), 1450-1460 (2013).</li><li>Makowka, L., 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=Surgical+technique+of+orthotopic+liver+transplantation.">Surgical technique of orthotopic liver transplantation.</a> Gastroenterol. Clin. North. Am. 17 (1), 33-51 (1988).</li><li>Hansen, T., 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=Histological+examination+and+evaluation+of+donor+bile+ducts+received+during+orthotopic+liver+transplantation--a+morphological+clue+to+ischemic-type+biliary+lesion?.">Histological examination and evaluation of donor bile ducts received during orthotopic liver transplantation--a morphological clue to ischemic-type biliary lesion?.</a> Virchows Arch. 461 (1), 41-48 (2012).</li><li>Monbaliu, D., Brassil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Machine+perfusion+of+the+liver:+Past,+present+and+future.">Machine perfusion of the liver: Past, present and future.</a> Curr. Opin. Organ. Transplant. 15 (2), 160-166 (2010).</li><li>Oldani, G., 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=Pre-retrieval+reperfusion+decreases+cancer+recurrence+after+rat+ischemic+liver+graft+transplantation.">Pre-retrieval reperfusion decreases cancer recurrence after rat ischemic liver graft transplantation.</a> J. Hepatol. 61 (2), 278-285 (2014).</li><li>Tromans, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+pressure+dependent+solubility+of+oxygen+in+water:+A+thermodynamic+analysis.">Temperature and pressure dependent solubility of oxygen in water: A thermodynamic analysis.</a> Hydrometallurgy. 48 (3), 327-342 (1998).</li></ol>

The authors of this manuscript have no conflict of interest to disclose.

This video provides a step by step protocol for normothermic machine perfusion of human donor livers using a device that enables pressure controlled dual perfusion through the hepatic artery and portal vein. While following this protocol, technical failures of the perfusion machine did not occur and all grafts were well perfused and well oxygenated. The ex situ perfused livers had stable hemodynamics and were metabolically active, as defined by the production of bile16,17.
...

The current method of organ preservation in liver transplantation is flush out with and subsequent storage of donor livers in cold (0-4 &#176;C) preservation fluid (such as University of Wisconsin solution or Histidine-Tryptophan-Ketoglutarate solution). This method is referred to as static cold storage (SCS). Although the metabolic rate of livers at 0-4 &#176;C is very low, there is still demand for 0.27 &#181;mol oxygen/min/g liver tissue, which cannot be provided during SCS1. The conventional method of SCS, therefore, results in some degree of (additional) injury of donor livers. While this amount of preservation injury is not a problem in donor livers of good quality, it can become a critical and limiting factor in suboptimal livers that have already suffered some degree of injury in the donor. For this reason, livers with suboptimal quality or so-called extended criteria donor (ECD) livers are frequently rejected for transplantation as the risk of early graft failure is considered to be too high. High rates of delayed graft function, primary non-function, and non-anastomotic biliary strictures (NAS) have been described in recipients of livers from donation after circulatory death (DCD), older donors or recipients of steatotic grafts2. NAS are a major cause of morbidity and mortality after liver transplantation. NAS may occur in both extra- and intrahepatic donor bile ducts and can be accompanied by intraductal biliary sludge and cast formation3,4. Although the etiology of NAS is thought to be multifactorial, ischemia/reperfusion injury of the bile ducts during graft preservation and transplantation has been identified as a major underlying mechanism2,5. Transplantation of a DCD graft has been identified as one of the strongest risk factors for the development of NAS. The combination of a period of warm ischemia in a DCD donor, cold ischemia during organ preservation, and subsequent reperfusion injury in the recipient is thought to be responsible for irreversible injury of the bile ducts, which, in combination with a poor regenerative capacity of the bile ducts, results in fibrotic scarring and narrowing of the bile ducts after liver transplantation2,5. NAS have been reported in up to 30% of patients receiving a DCD liver6-8 . It has become clear that the current method of SCS of liver grafts for transplantation is insufficient for preinjured ECD livers such as those from DCD donors. Alternative methods are needed to increase and optimize the use of ECD livers for transplantation.
Machine perfusion (MP) is a method of organ preservation that may provide better preservation of donor organs, compared to SCS. MP could be especially relevant for the preservation of ECD grafts. An important advantage of MP is the possibility to provide oxygen to the graft during the preservation period. MP can be performed at various temperatures, which have been classified as hypothermic (0-10 &#176;C), subnormothermic (10-36 &#176;C) and normothermic (36-37 &#176;C) MP (NMP). Depending on the temperature used for MP, the type of perfusion fluid has to be adjusted and with increasing temperature more oxygen should be supplied. The first clinical application of MP in human liver transplantation was based on hypothermic perfusion without active oxygenation of the perfusion fluid9,10. In animal models, hypothermic oxygenated MP (0-10 &#176;C) has been shown to have protective effects against ischemia/reperfusion injury of liver grafts11 and to provide better preservation of the peribiliary vascular plexus of the bile ducts12. Subnormothermic oxygenated MP at 20 &#176;C or 30 &#176;C has also been studied in animal models and was shown to provide earlier recovery of graft function of DCD livers, compared to SCS13,14. The feasibility of subnormothermic oxygenated MP of human livers was recently reported in a series of seven discarded human donor livers15. NMP (37 &#176;C) allows for the assessment of graft viability and functionality prior to transplantation16,17. Additionally, MP allows for gradual rewarming of the liver graft before transplantation, which has been demonstrated to facilitate recovery and resuscitation of the graft18.
The perfusion device used in the current protocol for hepatic machine perfusion enables dual perfusion (via the portal vein and the hepatic artery) using two centrifugal pumps, that provide a continuous portal flow and a pulsatile arterial flow. The system is pressure-controlled, allowing auto-regulation of the flow through the liver, depending on the intrahepatic resistance. Two hollow fiber membrane oxygenators allow for the oxygenation of the liver graft, as well as for the removal of CO2. The temperature can be set based on the intended type of MP (minimum temperature of 10 &#176;C). Flow, pressure and temperature are displayed on the device in real-time allowing a continuous control of the perfusion process. A new sterile disposable set of tubing, reservoir and oxygenators is available for the perfusion of each graft (Figure 1).
The aim of this video article is to provide a step by step protocol for ex situ normothermic machine perfusion of human donor livers using this newly developed liver perfusion machine.
<img alt="Figure 1" src="/files/ftp_upload/52688/52688fig1.jpg" /> 
Figure 1: (A) A schematic drawing, (B) a photo of the perfusion machine, (C) a closer view of the oxygenator, and (D) centrifugal pump used for normothermic perfusion of human donor livers. <a href="https://www.jove.com/files/ftp_upload/52688/52688fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Liver Assist</td>
			<td>Organ Assist</td>
			<td>OA.Li.Li.140</td>
			<td>Perfusion device</td>
		</tr>
		<tr>
			<td>Liver Assist disposable package</td>
			<td>Organ Assist</td>
			<td>OA.Li.DP.540</td>
			<td>Disposable set and cannulas</td>
		</tr>
		<tr>
			<td>Meredith No.8</td>
			<td>Vygon Nederlands B.V.</td>
			<td>1362082</td>
			<td>Bile duct cannula</td>
		</tr>
		<tr>
			<td>Human albumin 200g/l / ALBUMAN</td>
			<td>Sanquin</td>
			<td>15522598</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Modified parenteral nutrition</td>
			<td>Baxter Nederland B.V.</td>
			<td>N14G30E</td>
			<td>7.35 ml</td>
		</tr>
		<tr>
			<td>Multivitamins for infusion / CERNEVIT</td>
			<td>Baxter International Inc.</td>
			<td>9800927</td>
			<td>7 ul</td>
		</tr>
		<tr>
			<td>Concentrated trace elements for infusion / NUTRITRACE</td>
			<td>B. Braun Melsungen AG</td>
			<td>14811332</td>
			<td>7.35 ml</td>
		</tr>
		<tr>
			<td>Metronidazole 5mg/ml</td>
			<td>Baxter Nederland B.V.</td>
			<td>98181882</td>
			<td>40 ml</td>
		</tr>
		<tr>
			<td>Cefazoline / SERVAZOLIN</td>
			<td>Sandoz B.V.</td>
			<td>15611337</td>
			<td>2 ml</td>
		</tr>
		<tr>
			<td>Fast acting insulin&#160;</td>
			<td>various vendors</td>
			<td></td>
			<td>20 ml</td>
		</tr>
		<tr>
			<td>Calcium glubionate, intravenous solution 10%, 137.5 mg/mL</td>
			<td>Sandoz</td>
			<td>97038695</td>
			<td>40 ml</td>
		</tr>
		<tr>
			<td>Sterile H2O</td>
			<td>Fresenius Kabi Nederland B.V.</td>
			<td>98084453</td>
			<td>51.3 ml</td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Baxter Nederland B.V.</td>
			<td>15262510</td>
			<td>160 ml</td>
		</tr>
		<tr>
			<td>Heparin 5000 IE/ml for i.v. administration</td>
			<td>LEO Pharma B.V.</td>
			<td>98026178</td>
			<td>4 ml</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate 8,4%</td>
			<td>B. Braun Melsungen AG</td>
			<td>97973874</td>
			<td>The amount depends on the pH</td>
		</tr>
		<tr>
			<td>Packed red blood cell (in SAGM)</td>
			<td>Blood bank (Sanquin)</td>
			<td>N0012000</td>
			<td>750 ml</td>
		</tr>
		<tr>
			<td>Fresh frozen plasma</td>
			<td>Blood bank (Sanquin)</td>
			<td>N04030A0/N04030B0</td>
			<td>900 ml</td>
		</tr>
	</tbody>
</table>

ex situ normothermic machine perfusion of donor livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

12 human livers that were declined for transplantation due to various reasons were used after obtaining informed consent for research from donor families. Donor characteristics are described in Table 2. The human donor livers were perfused normothermically for 6 hr by using the protocol described in this paper. The quality of the liver grafts were evaluated by monitoring the macroscopic homogeneity of liver perfusion (Figure&#160;2A-D). The hemodynamics ...

Watch this Scientific Journal Video about Ex Situ Normothermic Machine Perfusion of Donor Livers at JoVE.com

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

ex-situ-normothermic-machine-perfusion-of-donor-livers

Research

JoVE Journal

Medicine

11.1K Views.  University of Groningen, University Medical Center Groningen. Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

Video: Ex Situ Normothermic Machine Perfusion of Donor Livers

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

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.

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 ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation. 
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed. 
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.

Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.

Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...