Name: ex situ normothermic machine perfusion of donor livers
Uploaded: 2015-05-26T15:00:00
Description: Watch this Scientific Journal Video about Ex Situ Normothermic Machine Perfusion of Donor Livers at JoVE.com

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 <st...

<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>

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 ...

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Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

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Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

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 ...

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...

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 ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...