We want to thank Sorin Group (Milano, Italy) for providing us with custom made kidney perfusion circuits. Furthermore we thank XVIVO Perfusion Inc. (Goteborg, Sweden) for providing us with Steen solution, BBraun AG (Melsungen, Germany) for the supply with syringe pumps, and Rieber GmbH &#38; Co KG (Reutlingen, Germany).

Note: A schematic overview of the study protocol is presented in Figure 1.
<img alt="Figure 1" src="/files/ftp_upload/52909/52909fig1.jpg" />
Figure 1. Study Protocol.&#160;This study protocol of normothermicex vivokidney perfusion is based on a porcine model. After surgical dissection of the vessels of the kidney graft and flushing with 500 ml of histidine-tryptophan-ketoglutarate (HTK), the graft can be retrieved. After cold storage (SCS) for 3 hrs, the kidney graft is perfused normothermic&#160;ex vivo&#160;(NEVKP) for multiple hours until the designated transplantation.
All animals received humane care in compliance with the &#8216;&#8216;Principles of Laboratory Animal Care&#8217;&#8217; formulated by the National Society for Medical Research and the &#8216;&#8216;Guide for the Care of Laboratory Animals&#8217;&#8217; published by the National Institutes of Health, Ontario, Canada. The Animal Care Committee of the Toronto General Research Institute approved all studies.
1. Animals
<ol>
	<li>Use male Yorkshire pigs (27 - 33 kg) in this protocol.</li>
</ol>
2. Organ Retrieval
<ol>
	<li>Preoperative Procedure
	<ol>
		<li>House the male Yorkshire pigs in a research facility for at least one week to reduce their stress level. Fast the pigs for a minimum of 6 hrs before induction of the anesthesia.</li>
		<li>Initiate anesthetization of the pig by an intramuscular injection of ketamine (25 mg/kg), atropine (0.04 mg/kg), and midazolam (0.15 mg/kg). Subsequently, transport the animal from the housing facility to the operating room (OR).</li>
		<li>Place the pig in a supine position on the OR table. Let it breathe 2 L of oxygen with 5% of isoflurane spontaneously. After relaxation, expose the vocal cords with a laryngoscope and spray them with 2% lidocaine to prevent a spasm caused by intubation. After intubation with a 6.5 mm tube, block the cuff with 3 - 5 ml of air. 
		Note: Capnometry reveals the correct position of the tracheal tube.</li>
		<li>Lower the isoflurane gas to 2.5%. Set the ventilator to 14 - 16 breaths/min and the tidal volume to 10 - 15 ml/kg bodyweight. Monitor the pig closely. Heart rate and oxygen saturation are recorded by pulse oxymetry.</li>
		<li>Under sterile conditions, introduce an 8.5 Fr. x 10 cm catheter into the jugular vein in Seldinger technique13. Therefore, use a needle to puncture the venous vessel. After introducing a wire, replace the needle with a catheter. Eliminate the wire and fix the catheter to the skin. Administer 200 ml of Ringer&#8217;s lactate solution per hour throughout the surgery.</li>
	</ol>
	</li>
	<li>Surgical Procedure
	<ol>
		<li>Following disinfection and coverage of the surgical field, perform a midline incision from xyphoid to pubic symphysis. To ameliorate the exposure, extend the surgical approach with a left lateral incision. Cover the large and small bowels with a towel and position them to the left side for optimal access to the right kidney.</li>
		<li>Separate the inferior vena cava (IVC) from the abdominal aorta. Ligate aortic branches from the back of the aorta.</li>
		<li>After complete aortic dissection to the back, pass a ligature around the aorta cranial to the renal branches. Additionally, place two ligatures cranial of the iliac bifurcation. Place a tie around the left renal artery.</li>
		<li>Free the right kidney from its adherent tissue. Dissect the renal vein, artery, and ureter.</li>
		<li>Open the diaphragm and administer 1,000 IU heparin per kg donor weight into the heart. For a DCD model, inject 40 mval of KCl intracardially 3 min after systemic heparinization to induce cardiac arrest. The cardiac arrest is valued as the starting point of warm ischemia.</li>
		<li>Meanwhile, for collection of blood, connect the lines of CPDA bags (citrate, phosphate, dextrose, adenosine) to the catheter introduced to the left upper jugular vein. Perform a soft spin (1,500 x g without brake). Remove plasma and buffy coat under sterile condition (biosafety cabinet class II) and store the erythrocytes for transfusion.</li>
		<li>Cannulate the aorta with an organ flush line above the iliac bifurcation. Tie the ligatures at the aorta and the left renal artery.</li>
		<li>Flush the kidney with histidine-tryptophan-ketoglutarate (HTK) solution with a pressure of 100 cm H2O. Clamp the thoracic cava and collect the blood via the jugular catheter. Cut the abdominal cava below the renal vein to secure an optimal flush of the kidney.</li>
		<li>After a complete flush of the right kidney, retrieve the graft with a segment of the aorta. Cut the renal vein and leave the ureter long.</li>
	</ol>
	</li>
	<li>Back Table Preparation of the Kidney Graft for the Perfusion
	<ol>
		<li>Free the kidney from adherent tissue. Close the cranial part of the aorta with a tie and cannulate the lower part with a 1/4&#8221; x 3/8&#8221; reducer. Tie off smaller arterial branches coming from the aorta.</li>
		<li>Cannulate the renal vein with a 1/4&#8221; x 3/8&#8221; reducer directly. Intubate the ureter with an 8 Fr. feeding tube.</li>
		<li>Place the kidney on ice until the start of the NEVKP.</li>
	</ol>
	</li>
</ol>
3. Normothermic Ex vivo Kidney Perfusion (NEVKP)
<ol>
	<li>Preparation of the Perfusion Circuit 
	The perfusion circuit consists of neonatal cardiopulmonary bypass equipment (Figure 2). 
	<img alt="Figure 2" src="/files/ftp_upload/52909/52909fig2.jpg" /> 
	Figure 2. Schematic Drawing of the Perfusion Circuit. The circuit consists of neonatal cardiopulmonary bypass technology. The perfusion solution is collected in the venous reservoir. A centrifugal pump propels the solution into the oxygenator, where it is enriched with oxygen and warmed to 37 &#176;C. After passing the arterial bubble filter, the perfusate is driven with a pressure of 60 &#8211; 80 mmHg through the renal artery into the kidney. The venous outflow (0 - 3 mmHg) leads the perfusate back into the venous reservoir. Syringe and infusion pumps secure the supply with additional compounds. The urine is collected throughout the perfusion. Perfusion circuit characteristics are recorded continuously. Hourly venous and arterial blood gas samples, and kidney injury markers are analyzed.
	<ol> 
		<li>Connect the custom made perfusion circuit to the kidney perfusion device.</li>
		<li>Connect the tubing to the venous reservoir and oxygenator. Connect the arterial line tubing to the arterial outflow of the oxygenator and position the bubble filter in its holder. Connect the purge line. Connect the venous line tubing to the inlet of the venous reservoir.</li>
		<li>For assessment during the perfusion, plug the temperature probe into the arterial outlet, connect the flow meter and the bubble sensor to the arterial line tubing, and connect the pressure lines. Connect the level sensor.</li>
		<li>Connect the venous and arterial sample lines to the venous and arterial sample ports.</li>
		<li>Position the organ chamber on a stand and introduce the venous and arterial tubing through the prepared holes. Fix the tubing to the table and chamber firmly.</li>
		<li>Insert the suction tubing into the roller pump and position one end into the chamber to collect the fluids.</li>
		<li>Connect the oxygen tubing to the gas tank containing 95% O2 / 5% CO2 and the oxygenator. Connect the heating unit tubing to the oxygenator and the organ chamber.</li>
		<li>Use tubing clamps to close the venous and arterial outflow lines. Apply another tubing clamp to the outflow of the venous reservoir.</li>
	</ol>
	</li>
	<li>Preparation of Perfusion Solution, Additional Supplements, and Priming the Circuit
	<ol>
		<li>Use one infusion pump to replace the produced urine with Ringer&#8217;s lactate.</li>
		<li>Use one syringe pump to administer nutrition (glucose 0.5 ml/hr, amino acids 0.5 ml/hr) and insulin (5 IU/hr) into the venous reservoir. Utilize a second syringe pump to infuse a vasodilator (verapamil, 0.25 mg/hr) directly into the arterial line.</li>
		<li>Fill the venous reservoir with the perfusion solution. Therefore, pour Ringer&#8217;s Lactate (175 ml), STEEN solution (200 ml), dRO (27 ml), heparin (1,000 IU), sodium bicarbonate to adjust the pH, and calcium gluconate into the venous reservoir. Finally, add washed erythrocytes (125 ml).</li>
		<li>Switch on the heart lung machine (HLM). Activate the pressure, temperature, level, and bubble sensor panels. Activate the Data Management System (DMS) to record the data throughout the perfusion. Activate the heating unit to warm the perfusion solution and the organ chamber to 37 &#176;C. Open the O2 supply.</li>
		<li>Open the tubing clamp behind the venous reservoir and free the centrifugal pump head from air completely. Start the centrifugal pump at 1,000&#160;rpm and allow the solution to be propelled throughout the circuit. Clamp the tubing bypassing the arterial filter and release air from the arterial filter.</li>
		<li>Zero the pressure lines. Activate the syringe and infusion pumps.</li>
	</ol>
	</li>
	<li>Kidney Graft Perfusion
	<ol>
		<li>Remove the kidney from the ice, and position the kidney on bedding in the organ chamber. Place the urine catheter into the urine collector. After ensuring that the venous and the arterial tubing are free of air, plug the connectors to the tubing.</li>
		<li>Close the shortcut between the arterial and venous tubing lines. Set the arterial pressure to 75 mmHg by regulating the speed of the centrifugal pump.</li>
		<li>Record pressures, arterial flow, temperature, and presence of bubbles continuously with the DMS. Observe the values carefully throughout the perfusion. During the perfusion, blood leaking into the chamber is collected via the suction tubing back into the venous reservoir.</li>
		<li>Record the quantity of urine produced. Collect venous blood and urine samples hourly. Monitor the perfusion by taking venous and arterial blood gas samples and aspartate aminotransferase (AST), and lactate analysis.</li>
		<li>At the end of perfusion, disconnect the tubing from the renal artery and vein, flush the graft with cold HTK, and store it on ice in a sterile organ bag until transplantation.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Wolfe, R. 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=Comparison+of+mortality+in+all+patients+on+dialysis,+patients+on+dialysis+awaiting+transplantation,+and+recipients+of+a+first+cadaveric+transplant.">Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant.</a> The New England journal of medicine. 341 (23), 1725-1730 (1999).</li><li>Rabbat, C. G., Thorpe, K. E., Russell, J. D., Churchill, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mortality+risk+for+dialysis+patients+and+cadaveric+first+renal+transplant+recipients+in+Ontario,+Canada.">Comparison of mortality risk for dialysis patients and cadaveric first renal transplant recipients in Ontario, Canada.</a> Journal of the American Society of Nephrology : JASN. 11 (5), 917-922 (2000).</li><li>Fuquay, R., Teitelbaum, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplant+outcomes+and+dialysis+modality.">Transplant outcomes and dialysis modality.</a> Contributions to nephrology. 178, 251-257 (2012).</li><li>Ingsathit, A., Kamanamool, N., Thakkinstian, A., Sumethkul, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+advantage+of+kidney+transplantation+over+dialysis+in+patients+with+hepatitis+C:+a+systematic+review+and+meta-analysis.">Survival advantage of kidney transplantation over dialysis in patients with hepatitis C: a systematic review and meta-analysis.</a> Transplantation. 95 (7), 943-948 (2013).</li><li>Davis, A. 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=The+extent+and+predictors+of+waiting+time+geographic+disparity+in+kidney+transplantation+in+the+United+States.">The extent and predictors of waiting time geographic disparity in kidney transplantation in the United States.</a> Transplantation. 97 (10), 1049-1057 (2014).</li><li>Perico, N., Cattaneo, D., Sayegh, M. H., Remuzzi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+graft+function+in+kidney+transplantation.">Delayed graft function in kidney transplantation.</a> Lancet. 364 (9447), 1814-1827 (2004).</li><li>Maggiore, U., Cravedi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginal+kidney+donor.">The marginal kidney donor.</a> Current opinion in organ transplantation. 19 (4), 372-380 (2014).</li><li>Dittrich, 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=Influence+of+cold+storage+on+renal+ischemia+reperfusion+injury+after+non-heart-beating+donor+explantation.">Influence of cold storage on renal ischemia reperfusion injury after non-heart-beating donor explantation.</a> Nephron. Experimental nephrology. 96 (3), e97-e102 (2004).</li><li>Summers, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+donor+age+and+cold+storage+time+on+outcome+in+recipients+of+kidneys+donated+after+circulatory+death+in+the+UK:+a+cohort+study.">Effect of donor age and cold storage time on outcome in recipients of kidneys donated after circulatory death in the UK: a cohort study.</a> Lancet. 381 (9868), 727-734 (2013).</li><li>Hosgood, S. 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=et+al.+pilot+study+assessing+the+feasibility+of+a+short+period+of+normothermic+preservation+in+an+experimental+model+of+non+heart+beating+donor+kidneys.">et al. pilot study assessing the feasibility of a short period of normothermic preservation in an experimental model of non heart beating donor kidneys.</a> The Journal of surgical research. 171 (1), 283-290 (2011).</li><li>Hosgood, S. A., Patel, M., Nicholson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conditioning+effect+of+ex+normothermic+perfusion+in+an+experimental+kidney+model.">The conditioning effect of ex normothermic perfusion in an experimental kidney model.</a> The Journal of surgical research. 182 (1), 153-160 (2013).</li><li>Nicholson, M. L., Hosgood, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+transplantation+after+ex+vivo+normothermic+perfusion:+the+first+clinical+study.+American+journal+of+transplantation.">Renal transplantation after ex vivo normothermic perfusion: the first clinical study. American journal of transplantation.</a> official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 13 (5), 1246-1252 (2013).</li><li>Seldinger, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheter+replacement+of+the+needle+in+percutaneous+arteriography;+a+new+technique.">Catheter replacement of the needle in percutaneous arteriography; a new technique.</a> Acta radiologica. 39 (5), 368-376 (1953).</li><li>Stubenitsky, B. 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O., 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=Effects+of+warm+ischaemia+combined+with+cold+preservation+on+the+hypoxia-inducible+factor+1&#945;+pathway+in+an+experimental+renal+autotransplantation+model.">Effects of warm ischaemia combined with cold preservation on the hypoxia-inducible factor 1&#945; pathway in an experimental renal autotransplantation model.</a> The British journal of surgery. , (1002).</li><li>Cypel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+ex+vivo+lung+perfusion+in+clinical+lung+transplantation.">Normothermic ex vivo lung perfusion in clinical lung transplantation.</a> The New England journal of medicine. 364 (15), 1431-1440 (2011).</li><li>Knaak, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnormothermic+Ex+vivo+liver+perfusion+reduces+endothelial+cell+and+bile+duct+injury+after+DCD+pig+liver+transplantation.">Subnormothermic Ex vivo liver perfusion reduces endothelial cell and bile duct injury after DCD pig liver transplantation.</a> Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. , (2014).</li><li>Hosgood, S. A., Nicholson, M. L. <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+perfusion+of+declined+human+kidneys+after+inadequate+in+situ+perfusion.">Ex vivo normothermic perfusion of declined human kidneys after inadequate in situ perfusion.</a> American journal of transplantation official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 14 (2), 490-491 (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> American journal of transplantation official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 13 (5), 1327-1335 (2013).</li></ol>

The authors have nothing to disclose.

This study demonstrates that NEVKP with an erythrocyte-based solution can be performed with excellent results for a prolonged period of time in a porcine model. During the 10 hrs&#160;ex vivo perfusion the kidneys demonstrated stable perfusion parameters, active renal metabolism, homeostasis, and minimal renal injury.
Urine production and kidney injury depend on the composition of the perfusion solution. It is important to keep oncotic pressure and osmolarity of the perfusate within a physiologic range. In particular, a low oncotic pressure will result in an unphysiologically high urine production with significant kidney edema and increasing markers of kidney injury. STEEN solution containing albumin is chosen in this model to regulate the oncotic pressure and to simulate physiologic conditions for the kidney. Sodium bicarbonate and calcium gluconate are added to the system to achieve physiological values of pH, HCO3, sodium, potassium, calcium, and chloride. The selection and dosage of the vasodilator is important to secure sufficient blood flow and oxygen supply.
The technique of normothermic ex vivo kidney perfusion has several limitations. Ex vivo perfusion is not associated with hormonal support of the kidney, which could negatively impact longer perfusion periods. In addition, the new technology, at this point in time, is associated with increased costs. Future improvements might simplify the technology and reduce the costs. The development of a portable kidney perfusion device might allow to completely avoid cold kidney storage in the future.
The severe and persisting organ shortage leads to an increased use of marginal organs (ECD or DCD kidney grafts) 7. Currently, organ preservation is based on static cold storage or hypothermic machine perfusion. As a prolonged cold ischemic time has a significant impact on the outcome of kidney function of standard criteria15 and marginal grafts8,9, new preservation techniques minimizing cold storage are of particular interest16-19.
A major obstacle to use marginal grafts more extensively is the inability to assess the quality and viability of organs prior to transplantation. Currently, only clinical parameters such as donor age, donor related diseases, and warm ischemia time of the grafts are used for the decision of whether an organ is accepted or declined for transplantation. By preserving the graft under normothermic conditions, graft assessment based on perfusion characteristics and data is possible. Real-time parameters such as renal vascular flow, pressure, intrarenal resistance, urine production, oxygen consumption, and kidney injury parameters (such as AST and lactate) are supposed to be useful parameters to assess the viability of the graft.
In addition, the active metabolism during NEVKP allows the application of repair strategies to improve marginal kidney grafts prior to transplantation. For example, inhibition of pro-inflammatory pathways, immunomodulation, gene transfer, as well as stem cell administration could be future techniques to modify kidney grafts during the preservation time and improve recipient outcome.

Kidneys are the most frequently transplanted solid organs. For patients suffering from end-stage renal disease, kidney transplantation offers better life expectancy, and improved quality of life when compared to dialysis1-4. The persisting organ shortage represents a severe problem in the field of transplant medicine (Table 1)&#160;5.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>United States*</td>
			<td>Eurotransplant region**</td>
		</tr>
		<tr>
			<td>Patients on kidney transplant waiting list</td>
			<td>101,563 (February, 2015)</td>
			<td>10,689 (December, 2014)</td>
		</tr>
		<tr>
			<td>Deceased donor kidneys transplanted in 2014</td>
			<td>10,650</td>
			<td>3,119</td>
		</tr>
		<tr>
			<td>Median waiting time to deceased donor kidney transplant (in years)</td>
			<td>Up to 5 years*</td>
			<td>Up to 4 years**</td>
		</tr>
	</tbody>
</table>
Table 1. Kidney Graft Shortage in US and Eurotransplant Region.
The outcome of kidney transplantation is negatively affected by the waiting time, with poorer outcome for patients subjected to prolonged dialysis6. This has triggered interest in marginal kidney grafts as an additional donor source, such as kidneys from older donors, donors with multiple comorbidities (extended criteria donors (ECD), and kidneys donated after cardiac death (DCD). Marginal donor kidneys that would have been declined in the past are now considered for transplantation7.
A major obstacle for the use of marginal kidney grafts is the preservation technique of cold anoxic storage. Currently, kidney grafts are stored statically on ice or perfused at 4 &#176;C without oxygen. The cold anoxic preservation technique is associated with ongoing graft injury during kidney preservation and does not allow graft assessment because of the lack of metabolism and urine production. In particular, marginal kidney grafts tolerate cold storage poorly, resulting in significant kidney injury, and high rates of delayed graft function (DGF)&#160;8,9. DGF is a prognostic factor for poor long-term graft function.
Extracorporeal kidney perfusion represents an alternative method for the preservation, assessment and repair of organs. In a porcine model, beneficial results were presented for kidneys perfused ex vivo under normothermic conditions10,11. The first clinical trial performed in 2013 demonstrated a lower rate of delayed graft function when kidneys retrieved from extended criteria donors were perfused for 1 hr immediately before transplant12.
This paper presents a model of normothermic ex vivo kidney perfusion (NEVKP). The goal of this study is to reduce the applied cold ischemia time to a minimum and extend the period of NEVKP. NEVKP is an alternative preservation method that aims to reduce the damage that can be caused by cold storage techniques.

<table><tbody><tr><td>Neonatal cardiopulmonary bypass technology</td><td>SORIN GROUP Canada Inc (Markham, Canada)</td><td>Custom made</td><td>Neonatal venous reservoir D100 (500 ml, 1/16&quot; in- and outflow), neonatal oxygenator D100, centrifugal pump head (Revolution), arterial bubble filter (D130)</td></tr><tr><td>Heart lung machine, St&amp;ouml;ckert S3</td><td>SORIN GROUP Canada Inc (Markham, Canada)</td><td>Custom made</td><td>Centrifugal pump, roller pump, control panel (sensors for pressure, flow, temperature, bubbles, and level), oxygen blender, heater unit</td></tr><tr><td>Tubing</td><td>SORIN GROUP Canada Inc (Markham, Canada)</td><td>01906BPC SG XS</td><td>3/16&quot; x 1/16&quot;</td></tr><tr><td></td><td></td><td>019071PC SG XS</td><td>1/4&quot; x 1/16&quot;</td></tr><tr><td></td><td></td><td>019060PC SG XH</td><td>3/8&quot; x 1/16&quot;</td></tr><tr><td>Tubing connectors</td><td>SORIN GROUP Canada Inc (Markham, Canada)</td><td></td><td>Various sizes</td></tr><tr><td>STEEN solution</td><td>XVIVO (G&amp;ouml;teborg, SWE)</td><td>19004</td><td>200 ml</td></tr><tr><td>Ringer&#039;s lactate</td><td>Baxter (Mississauga, ON, CAN)</td><td>JB2324</td><td>175 ml</td></tr><tr><td>Sodium bicarbonate</td><td>Hospira (Montr&amp;eacute;al, QC, CAN)</td><td>6625050</td><td>pH-dependent</td></tr><tr><td>Calcium gluconate</td><td>Pharmaceutical Partners of Canada (Richmond Hill, ON)</td><td>C31110</td><td>Calcium-dependent</td></tr><tr><td>Heparin</td><td>Sandoz Canada Inc (Toronto, ON, CAN)</td><td>10750</td><td>1,000 IU</td></tr><tr><td>Amino acid and glucose, Travasol 10%</td><td>Baxter (Mississauga, ON, CAN)</td><td>JB6760</td><td>1 ml/hr</td></tr><tr><td>Fast acting insulin, Novorapid</td><td>Novo Nordisk Canada Inc (Mississauga, ON, CAN)</td><td>DS6H748</td><td>5 IE/hr</td></tr><tr><td>Verapamil</td><td>Sandoz Canada Inc (Quebec, QC, CAN)</td><td>52216</td><td>0.25 mg/hr</td></tr></tbody></table>

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.

In the following the results of six experiments using a model of heart-beating kidney retrieval are presented. After in situ flush and kidney retrieval, the grafts were stored on ice for 3 hr (SCS) while the erythrocytes were prepared. For the clinical setting, this simulates the time required for the retrieval and the back table preparation. NEVKP was performed for 10 hr.
To maintain physiological conditions and simulate an in vivo surrounding for the kidney, the organ chamber should be heated and sealed. Perfusion and urine replacement solution should represent physiological values for blood gas analysis, oncotic pressure, and osmolarity. Normal values (baseline values) obtained from Yorkshire pigs in situ, are located in each figure description, respectively (Figures 3 -&#160;13). The aim of NEVKP is to ensure that the graft is supplied with sufficient oxygen and nutrition. As ischemia causes vasoconstriction, thus increasing intrarenal resistance, achieving a constant flow with a stable pressure is a good indicator for adequate oxygenation. After the target graft temperature of 37 &#176;C is reached via rewarming of the organ after SCS, flow values and intrarenal resistance remain stable with a constant physiological pressure of around 60-80 mmHg throughout the whole perfusion (Figures 3 and 4). The quantity of urine production depends mainly on the composition of the perfusion solution (Figure 5).
Hourly measurements of venous and arterial pO2 reveal the metabolic activity of the kidney. The oxygen consumption was calculated using the equation ((pO2art &#8211; pO2ven) x flow / weight) (Figure 6) 14. During the perfusion pH, HCO3, and electrolytes are stable without requiring interventions (Figures 7 - 10). Real-time AST and lactate measurements serve to monitor cellular damage. No increase of parameters of cell injury is detected during the NEVKP period (Figures 11 and 12). The osmolarity of the perfusion solution is stable (Figure 13). Histological assessment reveals minor changes (Figure 14 - 16).
<img alt="Figure 3" src="/files/ftp_upload/52909/52909fig3.jpg" /> 
Figure 3. Mean arterial flow with standard deviation (ml/min). Throughout the perfusion the flow remains in a physiological range. Porcine physiological values, measured in situ: mean art. flow: 170 &#177; 57 ml/min (range 83 &#8211; 325 ml/min).
<img alt="Figure 4" src="/files/ftp_upload/52909/52909fig4.jpg" /> 
Figure 4. Intrarenal resistance (IRR), mean&#160;and standard deviation (mmHg/ml/min). The mean arterial pressure (MAP) remains constant between 60 and 80 mmHg. The intrarenal resistance is below 0.5 mmHg/ml/min constantly.
<img alt="Figure 5" src="/files/ftp_upload/52909/52909fig5.jpg" /> 
Figure 5. Total urine volume, mean&#160;and standard deviation (ml). The total urine volume mainly depends on the composition of the perfusion solution. The higher the oncotic pressure and the osmolarity, the lower the urine production.
<img alt="Figure 6" src="/files/ftp_upload/52909/52909fig6.jpg" /> 
Figure 6. Oxygen consumption, mean and standard deviation (ml/min/g).
<img alt="Figure 7" src="/files/ftp_upload/52909/52909fig7.jpg" /> 
Figure 7. pH venous, mean and standard deviation. The pH remains constant in a physiological range without administration of bicarbonates. Porcine physiological values, measured in situ: pH 7.46 &#177; 0.06 (range 7.34 &#8211; 7.63).
<img alt="Figure 8" src="/files/ftp_upload/52909/52909fig8.jpg" /> 
Figure 8. HCO3 venous, mean and standard deviation (mmol/l). The HCO3 remains in a physiological range without administration of bicarbonates. Porcine physiological values, measured in situ: HCO3 30.3 &#177; 2.4 mmol/L (range 21.6 &#8211; 35.8 mmol/L).
<img alt="Figure 9" src="/files/ftp_upload/52909/52909fig9.jpg" /> 
Figure 9. Venous sodium concentration, mean and standard deviation (mmol/L). The sodium remains in a physiological range. Porcine physiological values, measured in situ: 137.1 &#177; 3.8 mmol/L (range 118.7 &#8211; 140.9 mmol/L).
<img alt="Figure 10" src="/files/ftp_upload/52909/52909fig10.jpg" /> 
Figure 10. Venous potassium concentration, mean and standard deviation (mmol/L). The potassium remains constant in a physiological range. Porcine physiological values, measured in situ: 3.85 &#177; 0.46 mmol/L (range 3.5 &#8211; 5.36 mmol/L).
<img alt="Figure 11" src="/files/ftp_upload/52909/52909fig11.jpg" /> 
Figure 11. Venous aspartate aminotransferase, mean and standard deviation (AST; U/L). In the ex vivo normothermic kidney perfusion, AST demonstrates a cell injury marker. AST values are low throughout perfusion.
<img alt="Figure 12" src="/files/ftp_upload/52909/52909fig12.jpg" /> 
Figure 12: Lactate, mean and standard deviation (mmol/L). In the ex vivo normothermic kidney perfusion, lactate represents a cell injury marker. The values are stable throughout the perfusion.
<img alt="Figure 13" src="/files/ftp_upload/52909/52909fig13.jpg" /> 
Figure 13: Osmolarity of the serum, mean and standard deviation (mosm/L). A constant osmolarity in the perfusion solution secures low but constant urine production. Porcine physiological values, measured in situ: 282 &#177; 1.7 mosm/L (range 279 &#8211; 283 mosm/L).
<img alt="Figure 14" src="/files/ftp_upload/52909/52909fig14.jpg" /> 
Figure 14: Histology (H&#38;E).&#160;50X / 200X magnification of corticomedullary junction showing mild tubular vacuolization. No signs of necrosis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52909/52909fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" src="/files/ftp_upload/52909/52909fig15.jpg" /> 
Figure 15: Histology (PAS). 50X / 200X magnification of corticomedullary junction showing mild tubular vacuolization. No signs of necrosis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52909/52909fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 16" src="/files/ftp_upload/52909/52909fig16.jpg" /> 
Figure 16: Histology (TUNEL staining). 25X / 200X magnification. Very occasionally nuclei are stained demonstrating very low rates of apoptosis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52909/52909fig16large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation at JoVE.com

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.

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 ...

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19.0K Views.  Toronto General Hospital.  Het ernstige tekort aan organen heeft geleid tot een toename van het gebruik van marginale nieren grafts voor transplantatie. Dit heeft de interesse in alternatieve methoden voor de opslag geactiveerd, omdat marginale enten vooral tolereren koude opslag slecht. De techniek van normotherme ex vivo perfusie nier (NEVKP) een nieuwe bewaarmethode voor nieren grafts voorafgaand aan de transplantatie. 

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

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

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.

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 ...

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.

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.

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

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

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 ...

Avoiding Ischemia Reperfusion Injury in Liver Transplantation

Currently, ex situ machine perfusion is a burgeoning technique that provides a better preservation method for donor organs than conventional static cold preservation (0&#8211;4 &#176;C). A continuous blood supply to organs using machine perfusion from procurement and preservation to implantation facilitates complete prevention of ischemia reperfusion injury and permits ex situ functional assessment of donor livers before transplantation. In this manuscript, we provide a step-by-step ischemia-free liver transplantation protocol in which an ex situ normothermic machine perfusion apparatus is used for pulsatile perfusion through the hepatic artery and continuous perfusion of the portal vein from human donor livers to recipients. In the perfusion period, biochemical analysis of the perfusate is conducted to assess the metabolic activity of the liver, and a liver biopsy is also performed to evaluate the degree of injury. Ischemia-free liver transplantation is a promising method to avoid ischemia-reperfusion injury and may potentially increase the donor pool for transplantation.

Presented here is a protocol to provide a step-by-step ischemia-free liver transplantation protocol under ex situ normothermic machine perfusion (37 &#176;C) of human livers from donors to recipients.

Currently, ex situ machine perfusion is a burgeoning technique that provides a better preservation method for donor organs than conventional static cold ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

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 ...

Kidney Procurement in a Preclinical Large Animal Model

Machine perfusion has evolved as a viable strategy for ex vivo organ assessment, monitoring, treatment, optimization, as well as to prolong preservation times. Large animal models have been paramount for the development and optimization of these technologies. However, in order to ensure graft quality and data reproducibility, standardized and clinically translatable surgical techniques for organ and tissue procurement should be followed. Thus, here, we describe an optimized protocol for kidney procurement in a preclinical swine model. Kidney recovery is performed using mixed breed (Yorkshire cross/mix) pigs. Briefly, following sterile disinfection and draping of the surgical field, a complete midline incision is performed to gain optimal access to both kidneys. The ureter, renal vein, and artery are dissected until their origin from the inferior vena cava and the aorta, respectively. After complete renal dissection, the ureter is tied and cut distally. The donor animal is then fully heparinized with 100 IU per kg/body weight. Next, the renal artery is clamped close to the aorta, and the renal vein is clamped close to the vena cava using a Satinsky vascular clamp. The kidney graft is then resected, and the renal artery is immediately cannulated back table. The kidney will then be flushed with an ice-cold preservation solution and stored on ice until either machine perfusion or transplantation. Finally, the renal artery stump is ligated with a 2-0 silk ligature, and the vena cava is closed with a 6-0 polypropylene suture. This technique recovers kidneys and simulates either a living (single kidney) or deceased (dual kidney) donor setting. The single kidney recovery offers the advantage to perform a subsequent autotransplantation. In the deceased donor model, blood can be collected prior to euthanasia by inserting blood bag needles directly into the aorta, thereby exsanguinating the animal and providing blood for ex vivo machine perfusion.

Here, we present a protocol that details a surgical model for kidney procurement in a preclinical swine model for subsequent machine perfusion or transplantation.

Machine perfusion has evolved as a viable strategy for ex vivo organ assessment, monitoring, treatment, optimization, as well as to prolong preservation ...

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

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