All animals used for this study received humane care in accordance with the &#39;&#39;Principles of Laboratory Animal Care&#39;&#39; formulated by the National Society for Medical Research and the &#39;&#39;Guide for the Care of Laboratory Animals&#39;&#39; published by the National Institutes of Health, Ontario, Canada. All studies were approved by the Animal Care Committee of the Toronto General Research Institute.
NOTE: This study protocol is based on a porcine model. The graft is stored in the cold for 2 h and then undergoes normothermic machine perfusion for 3 h prior to transplantation (Figure 1).
1. Animals
<ol>
	<li>Use male Yorkshire pigs (40-50 kg).</li>
</ol>
2. Organ procurement
NOTE: The preoperative procedure and part of the surgical procedure are the same as previous papers published by our group15 and is as follows:
<ol>
	<li>House the pigs in the research facility for a minimum of 7 days to allow for acclimation and to reduce their stress level.</li>
	<li>Fast the pigs for a minimum of 6 h before induction of the anesthesia.</li>
	<li>Sedate the pig with an intramuscular (IM) injection of midazolam (0.15 mg/kg), ketamine (25 mg/kg), and atropine (0.04 mg/kg). 
	NOTE: This is done in the housing facility.</li>
	<li>Transfer the animal from the housing facility to the operating room (OR), where recovery of the organ will be performed.</li>
	<li>Position the pig in a supine position on the OR table and place a face mask with 2 L of oxygen and 5% of isoflurane until the jaw is relaxed.</li>
	<li>Visualize the vocal cords using laryngoscope and spray them with 2% lidocaine to prevent spasm during intubation. Replace the mask with oxygen and isoflurane for at least 30 s before attempting intubation.</li>
	<li>Introduce a 7 mm endotracheal tube and block the cuff with 5 mL of air. Use capnometry to ensure that the tube is in the correct position.</li>
	<li>Decrease the isoflurane gas to 2.5%. Turn on the ventilator and set it to 15-20 breaths/min and the tidal volume to 10-15 mL/kg bodyweight. Monitor heart rate and oxygen saturation constantly.</li>
	<li>Using the Seldinger technique16, introduce an 8.5 Fr. x 10 cm catheter into the jugular vein (either right or left).</li>
	<li>Use the jugular vein catheter to start an infusion of fentanyl (2.5 mL in 500 mL of Ringer) at 250 mL/ hr.</li>
	<li>Check muscular reflexes to determine the depth of anesthesia. Jaw tone is the most reliable muscular reflex 17. 
	NOTE: If the rigidity of mandibular muscles is noted, increase isoflurane and/or fentanyl infusion.</li>
</ol>
3. Surgical procedure
<ol>
	<li>Disinfect and cover the surgical field. Perform a midline incision from the xyphoid to the pubic symphysis. Extend the surgical field with a left lateral incision for better exposure.</li>
	<li>Dissect the inferior vena cava (IVC) from the abdominal aorta. Further free the aorta from the surrounding tissue and ligate the small lumbar aortic branches. Identify and place ligatures around both renal arteries. 
	NOTE: The ligatures must not be tied at this timepoint.</li>
	<li>Once the back of the aorta is free, pass two ligatures around it. The lower ligature will eventually be tied just above the iliac artery bifurcation and upper ligature will be tied 5 cm above the previous tie.</li>
	<li>Dissect the hepatic hilum. Tie all arteries as close to the liver as possible. Identify the common bile duct, place two ligatures close to the liver, and divide the structure.</li>
	<li>Dissect around aorta but do not cut at this timepoint. Identify and dissect around the suprahepatic portion of aorta and place a tie around it. 
	NOTE: The ligatures must not be tied at this timepoint.</li>
	<li>Open the lesser sac to allow ice to cool pancreas. Mobilize the pancreas as little as possible before flush.</li>
	<li>Administer 500 IU of heparin per kg of donor weight through the central line. Wait 5 min and start blood collection in citrate, phosphate, dextrose, saline, adenine, glucose, and mannitol (CPD/SAG-M) bags using the jugular catheter.</li>
	<li>Tie the inferior aortic ligature, cannulate the aorta with a flush line above the iliac bifurcation tie, and secure the cannula with an upper tie. Ligate both renal arteries.</li>
	<li>Tie the suprahepatic aorta (crossclamp) once enough blood has been collected (600 mL). Administer 10 mL of potassium chloride for sacrifice.</li>
	<li>Initiate a flush with University of Wisconsin (UW) preservation solution. Cut an opening in the portal vein (as high as possible) and cava for venting. Place ice in the abdominal cavity.</li>
	<li>Assess the pancreas tail and duodenum C-loop after flushing 1 L of UW solution. If adequate flush, begin dissection, identify, and clamp mesenteric vessels. Slow down flushing for the second liter of UW.</li>
	<li>Retrieve the pancreatic graft and a segment of cava or iliac vein for extension of portal vein. 
	NOTE: The pancreatic graft is removed with the spleen.</li>
	<li>Place the organ inside an organ bag that is placed inside a basin filled with ice.</li>
</ol>
4. Back table preparation of the pancreatic graft (Figure 2A)
<ol>
	<li>Remove the flush line from distal part of the aorta and close with a tie. Fill the organ bag with the remaining UW solution. Free the pancreas from adherent tissue, including the spleen.</li>
	<li>Perform portal vein extension using previously recovered cava or iliac vein with 6-0 Prolene. Cannulate the portal vein and proximal aorta with a &#188; in x 3/8 in reducer.</li>
	<li>Cannulate the distal part of duodenum with Malecot catheter and tie. Clamp the end of catheter to avoid spilling of duodenal content. Oversaw mesenteric vessels with 4-0 Prolene.</li>
	<li>Register the weight of the graft. Keep the graft in static cold storage (SCS) until the start of the NEVPP.</li>
</ol>
5. Normothermic ex vivo pancreas perfusion (NEVPP)
NOTE: The perfusion circuit is made of neonatal cardiopulmonary bypass equipment (Figure 3).
<ol>
	<li>Attach the corresponding tubing to the oxygenator and to the venous reservoir, as well as the arterial line to the outflow of the oxygenator and place the bubble filter in its holder. Connect the purge line that goes from bubble filter to the venous reservoir. Open the bubble filter cap to let all the air out.</li>
	<li>Connect the venous line to the inlet of the venous reservoir. Connect the dialysis filter and tubing where dialysate will be infused. Connect the flow meter sensor, pressure lines, and the temperature probe. Connect the arterial and venous sample lines to the sample ports.</li>
	<li>Place the pancreas chamber (Figure 3) on a Mayo table and introduce the arterial and venous tubing through the holes intended for this purpose. Connect and turn on external heater unit.</li>
	<li>Place the suction tubing inside the roller pump and connect one end into the tubing that comes out from the chamber to collect the fluids, and the other end to the venous reservoir to collect all organ loses of perfusate.</li>
	<li>Connect the oxygen tubing (green) to the gas tank containing the carbogen mixture (95% O2/5% CO2) and the oxygenator. Connect the heater pump unit tubing to the oxygenator.</li>
	<li>Clamp arterial and venous outflow lines, as well as the outflow of the venous reservoir.</li>
</ol>
6. Preparation of the perfusate and priming of the circuit
<ol>
	<li>Fill the venous reservoir with the perfusate (Table 1).</li>
	<li>Use one syringe pump for continuous administration of the vasodilator (epoprostenol) at 8 mL/h into the arterial line. Use a second syringe pump for continuous administration of the enzyme inhibitor directly into the venous reservoir (15 mg, 10 mL/h).</li>
	<li>Turn on the heart lung machine (HLM) and start up the pressure, temperature, and timer panels. Turn on heater pump to warm the perfusion solution to 38 &#176;C. Open the O2/CO2 supply.</li>
	<li>Remove the tubing clamp placed on the outflow of the venous reservoir, start the centrifugal pump, and take it up to 1,500 rpm. Clamp the tubing, bypassing the arterial filter and release air from the arterial filter. Zero the arterial and venous pressure lines.</li>
</ol>
7. Pancreas graft perfusion (Figure 2B)
<ol>
	<li>Open the organ bag where the pancreas is stored. Flush with 200 mL of albumin through the arterial cannula. Remove the pancreas from the ice and position inside the organ chamber. Confirm that the arterial and the venous tubing are air-free.</li>
	<li>Release clamp from the arterial side and clamp the shortcut between the arterial and venous tubing. Once blood starts coming out of arterial tubing, connect line to arterial cannula. Set arterial pressure to 20-25 mmHg, by regulating the speed of the centrifugal pump. Connect venous tubing once blood starts coming out from the venous cannula.</li>
	<li>Administer one vial of verapamil (2.5 mg/mL) directly on the arterial side, when the pancreas is completely connected and no major bleeds are observed.</li>
	<li>Record pressures, arterial flow, temperature, and duodenum secretion continuously. Collect blood, record duodenal output every hour, and assess hourly macroscopically for edema. Record the perfusion parameters and take samples for analysis (venous and arterial blood gas samples, as well as samples for amylase, lipase, and LDH).</li>
	<li>Disconnect arterial and venous tubing when perfusion is over, remove the graft from organ chamber, and flush with cold UW and weigh. Store graft on ice in a sterile organ bag until the moment of transplantation.</li>
</ol>

<ol>
	<li>. National Diabetes Statistics Report | Diabetes | CDC Available from: <a target="_blank" href="https://www.cdc.gov/diabetes/data/statistics-report/index.html">https://www.cdc.gov/diabetes/data/statistics-report/index.html</a> (2022)</li><li>Shyr, Y. M., Wang, S. E., Chen, S. C., Shyr, B. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reappraisal+of+pancreas+transplantation.">Reappraisal of pancreas transplantation.</a> Journal of the Chinese Medical Association JCMA. 82 (7), 531-534 (2019).</li><li>Dholakia, 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=Pancreas+transplantation:+past,+present,+future.">Pancreas transplantation: past, present, future.</a> American Journal of Medicine. 129 (7), 667-673 (2016).</li><li>Johnson, P., Sharples, E., Sinha, S., Friend, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+and+islet+transplantation:+pancreas+and+islet+transplantation+in+diabetes+mellitus.">Pancreas and islet transplantation: pancreas and islet transplantation in diabetes mellitus.</a> Transplantation Surgery. , 205-217 (2021).</li><li>Kopp, W. 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=Pancreas+transplantation+with+grafts+from+donors+deceased+after+circulatory+death:+5+years+single-center+experience.">Pancreas transplantation with grafts from donors deceased after circulatory death: 5 years single-center experience.</a> Transplantation. 102 (2), 333-339 (2018).</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>Nasralla, 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=A+randomized+trial+of+normothermic+preservation+in+liver+transplantation.">A randomized trial of normothermic preservation in liver transplantation.</a> Nature. 557 (7703), 50-56 (2018).</li><li>Selzner, 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+liver+perfusion+using+steen+solution+as+perfusate+for+human+liver+transplantation:+First+North+American+results.">Normothermic ex vivo liver perfusion using steen solution as perfusate for human liver transplantation: First North American results.</a> Liver Transplantation: Official Publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. 22 (11), 1501-1508 (2016).</li><li>Hosgood, S. A., Thompson, E., Moore, T., Wilson, C. H., 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=Normothermic+machine+perfusion+for+the+assessment+and+transplantation+of+declined+human+kidneys+from+donation+after+circulatory+death+donors.">Normothermic machine perfusion for the assessment and transplantation of declined human kidneys from donation after circulatory death donors.</a> The British Journal of Surgery. 105 (4), 388-394 (2018).</li><li>Urbanellis, P., 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+kidney+perfusion+improves+early+dcd+graft+function+compared+with+hypothermic+machine+perfusion+and+static+cold+storage.">Normothermic ex vivo kidney perfusion improves early dcd graft function compared with hypothermic machine perfusion and static cold storage.</a> Transplantation. 104 (5), 947-955 (2020).</li><li>Barlow, A. 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=Use+of+ex+vivo+normothermic+perfusion+for+quality+assessment+of+discarded+human+donor+pancreases.">Use of ex vivo normothermic perfusion for quality assessment of discarded human donor pancreases.</a> American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 15 (9), 2475-2482 (2015).</li><li>Kumar, R., 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+porcine+pancreas:+A+physiological+model+for+preservation+and+transplant+study.">Ex vivo normothermic porcine pancreas: A physiological model for preservation and transplant study.</a> International journal of surgery. 54, 206-215 (2018).</li><li>Hamaoui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pancreatic+machine+perfusion:+translational+steps+from+porcine+to+human+models.">Development of pancreatic machine perfusion: translational steps from porcine to human models.</a> The Journal of Surgical Research. 223, 263-274 (2018).</li><li>Prudhomme, 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=Successful+pancreas+allotransplantations+after+hypothermic+machine+perfusion+in+a+novel+diabetic+porcine+model:+a+controlled+study.">Successful pancreas allotransplantations after hypothermic machine perfusion in a novel diabetic porcine model: a controlled study.</a> Transplant International: Official Journal of the European Society for Organ Transplantation. 34 (2), 353-364 (2021).</li><li>Kaths, 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=Normothermic+ex+vivo+kidney+perfusion+for+the+preservation+of+kidney+grafts+prior+to+transplantation.">Normothermic ex vivo kidney perfusion for the preservation of kidney grafts prior to transplantation.</a> Journal of Visualized Experiments: JoVE. (101), e353 (2015).</li><li>Graham, A. S., Ozment, C., Tegtmeyer, K., Lai, S., Braner, D. A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+venous+catheterization.">Central venous catheterization.</a> The New England Journal of Medicine. 356 (21), 21 (2009).</li><li>Swindle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swine in the Laboratory Surgery, Anesthesia, Imaging, and Experimental Techniques. , (2015).</li><li>Mazilescu, L. 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=Normothermic+ex+situ+pancreas+perfusion+for+the+preservation+of+porcine+pancreas+grafts.">Normothermic ex situ pancreas perfusion for the preservation of porcine pancreas grafts.</a> American Journal of Transplantation. , (2022).</li><li>Fridell, J. 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=Preparation+of+the+pancreas+allograft+for+transplantation.">Preparation of the pancreas allograft for transplantation.</a> Clinical transplantation. 25 (2), (2011).</li><li>Nassar, A., Liu, Q., Walsh, M., Quintini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normothermic+ex+vivo+perfusion+of+discarded+human+pancreas.">Normothermic ex vivo perfusion of discarded human pancreas.</a> Artificial Organs. 42 (3), 334-335 (2018).</li></ol>

The authors do not have anything to disclose.

This study demonstrates that stable NEVPP can be achieved for pancreas allografts with minimal histological damage after 3 h of perfusion with the setup previously presented. Perfusion parameters like arterial flow, pressure, pH, HCO3, and Na remain stable during perfusion, and we observed a decrease and stabilization of K and lactate.
It is of critical importance to manipulate the graft as little as possible during procurement, back table preparation, and perfusion. It is also very important to keep tight control of the arterial pressure. Since the pancreas is a low-pressure organ, an increase in the pressure may cause irreversible damage to the organ.
Back-table preparation for this study is different from human grafts preparation (Figure 2A). Since the pancreas was the only organ procured from the pigs, we were able to take the portion of the aorta that includes the celiac trunk and superior mesenteric artery. As for the portal vein, an extension using iliac vein was performed. In case of human grafts, back-table preparation will have to be done in the same manner as it is done for transplantation, using iliac grafts for arterial reconstruction and portal lengthening19.
This method might be limited by the complexity of the setup. We decided to add a dialysis cassette after noticing severe edema of the graft when done without it. A custom-made organ chamber was also constructed for these experiments which contained an external heating source that proved to be instrumental for the optimal perfusion of the grafts.
There are few studies describing normothermic ex vivo pancreas perfusion. In most of these studies, edema appears to be the major limiting factor. To our knowledge, this method is the only report of using a dialysis cassette to control edema.
Normothermic ex vivo perfusion for the pancreas is still in its infancy compared to other organs. Current protocols are focusing on extended criteria donors (DCD), perfusate improvement, longer perfusion times, and biomarkers to assess graft damage during perfusion. Amylase and lipase levels don&#39;t seem to be reliable markers, since we are using a closed system, and don&#39;t seem to correlate with the histopathology20. So far, our group has also managed to transplant pancreas allografts after perfusion with good results18.
With continued improvements in this technology, we hope this technology will be applicable to clinical transplantation and allow for assessment and repair of pancreas allografts. This will hopefully ultimately result in more graft utilization, decreased waiting time for patients, and better patient outcomes

According to the National Diabetes Statistics Report, a total of 28.7 million people in the United States were living with a diagnosis of diabetes in 2019. Approximately 1.8 million of these had a diagnosis of type 1 diabetes1. PTx is currently the most efficacious and only curative treatment for complicated type 1 diabetes mellitus2, and is a procedure that both increases life expectancy and quality of life of these patients3.
The pancreas is the most often discarded organ after retrieval from deceased donors4. With ongoing organ shortages and the increasing waiting list times, transplant centers are using more pancreas grafts from ECDs, including donation after circulatory death (DCD)5. Strategies to safely preserve, perfuse, assess, and repair allografts coming from extended criteria donors are needed.
NEVP has proven to be successful in the preservation of lung6, liver7, 8, and kidney grafts9,10. However, the number of groups working on machine perfusion for the pancreas, both hypothermic or normothermic, and the number of publications, are few and limited due to graft edema and injury11,12,13,14.
The objective of this study is to present a protocol for normothermic ex vivo pancreas perfusion (NEVPP), using a porcine model with the goal of eventually providing a platform for prolonged preservation, organ assessment, and repair before transplantation. This will allow other research groups to establish a perfusion model for the study of pancreas allografts.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alburex 5</td>
			<td>CSL Behring AG</td>
			<td>187337</td>
			<td>25 g of Albumin (human) in 500 mL of buffered diluent</td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung</td>
			<td>Sigma-Aldrich</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr>
			<td>Belzer UW Cold storage solution</td>
			<td>Bridge to life Ltd</td>
			<td>4055</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium gluconate (10%)</td>
			<td>Fresenius Kabi Canada Ltd (Toronto, ON)</td>
			<td>C360019</td>
			<td></td>
		</tr>
		<tr>
			<td>Composelect (blood collection bags)</td>
			<td>Fresenius Kabi Canada Ltd</td>
			<td>PQ31555</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoprostenol</td>
			<td>GlaxoSmithKline Inc.</td>
			<td>218761</td>
			<td></td>
		</tr>
		<tr>
			<td>Heart lung machine, St&#246;ckert S3</td>
			<td>Sorin Group Canada Inc.</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>Hemoflow, Fresenius Polysulfone</td>
			<td>Fresenius Medical Care North America</td>
			<td>0520165A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin (10000 IU/10 mL)</td>
			<td>Fresenius Kabi Canada Ltd</td>
			<td>C504710</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated Ringer&#39;s solution</td>
			<td>Baxter</td>
			<td>JB2324</td>
			<td></td>
		</tr>
		<tr>
			<td>Neonatal cardiopulmonary bypass techonolgy</td>
			<td>Sorin Group Canada Inc</td>
			<td>Custom made</td>
			<td>Dideco perfusion tubing systems, centrifugal blood pump (Revolution), arterial blood filter, microporous hollow fibre memebrane oxygenator), cannulas</td>
		</tr>
		<tr>
			<td>Pancreas chamber</td>
			<td></td>
			<td>Custom made</td>
			<td>With external heater</td>
		</tr>
		<tr>
			<td>Percutaneous Sheath Introducer Set with Integral Hemostasis Valve/side Port for use with 7-7.5 Fr Catheters&#160;</td>
			<td>Arrow International LLC</td>
			<td>SI-09880</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (8.4%)</td>
			<td>Fresenius Kabi Canada Ltd</td>
			<td>C908950</td>
			<td></td>
		</tr>
		<tr>
			<td>Solu-Medrol</td>
			<td>Pfizer Canada Inc.</td>
			<td>52246-14-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Steen</td>
			<td>XVIVO</td>
			<td>19004</td>
			<td></td>
		</tr>
		<tr>
			<td>Urethral catheter</td>
			<td>Bard Inc</td>
			<td>86020</td>
			<td>20 Fr, malecot model drain</td>
		</tr>
		<tr>
			<td>Verapamil</td>
			<td>Sandoz Canada Inc.</td>
			<td>8960</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The upcoming data shows the representative results of seven experiments using a model of heart-beating donor pancreas retrieval. After cannulation of the aorta, flush with UW solution, and retrieval of the pancreas, the grafts were kept on SCS for 2 h while the red blood cells were prepared. NEVPP was performed in this model for 3 h, what we considered the least amount of time necessary for perfusion if graft assessment and repair are intended in the future. Samples and measurements were recorded at hourly timepoints. (0 = baseline, right after organ is connected to the circuit, 1 = 1 h, 2 = 2 h, 3 = 3 h).
Pancreas grafts were placed on an organ chamber that was custom designed for this purpose and includes a heater (Supplemental File). The purpose of NEVPP is to provide a near physiological environment for the organ. For this purpose, arterial pressure was set to remain between 20-25 mmHg in all perfusions. Pressure and flow were measured throughout the whole perfusion and remained stable (Figure 4). Metabolic activity was estimated by calculating the oxygen consumption of the graft using the following formula: [(pO2art-pO2ven) * flow / weight] (Figure 5). Measurements of pH, sodium, calcium, and HCO3 were within physiological values during the whole perfusion (Figure 6). Lactate and potassium levels decreased during the perfusion, and achieved close to normal values at 3 h (Figure 7). Since the circuit is a closed system, amylase and lipase levels are expected to increase during the perfusion (Figure 8). However, the increase in levels does not seem to correlate with the damage to the graft (Figure 9). A semiquantitative scale was used to score fat and parenchyma necrosis as well as islet cell integrity. (0 - no changes, 1 - mild changes, 2 - moderate changes, 3 - severe changes). This was done by a pathologist blinded to the experimental groups, and no signs of pancreatitis were observed.
Pancreas allografts were weighed before and after perfusion to assess edema (Table 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63905/63905fig01.jpg" /> 
Figure 1. Study protocol. <a href="https://www.jove.com/files/ftp_upload/63905/63905fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63905/63905fig02.jpg" /> 
Figure 2. Pancreas before and after perfusion. (A) Before perfusion. (B) After perfusion. <a href="https://www.jove.com/files/ftp_upload/63905/63905fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63905/63905fig03.jpg" /> 
Figure 3. Schematic drawing of the perfusion circuit. With the use of Neonatal cardiopulmonary bypass technology; the perfusate is poured into the venous reservoir and then propelled with help of a centrifugal pump into the oxygenator. After leaving the oxygenator, the circuit divides into tubing that sends perfusate to the dialysis cassette and back to the reservoir and tubing that goes to the arterial filter. After passing the arterial bubble filter, the perfusate is driven with a pressure of 20-25 mmHg through the aorta into the pancreas. The venous outflow leads the perfusate back into the venous reservoir. (Adapted from 18). <a href="https://www.jove.com/files/ftp_upload/63905/63905fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63905/63905fig04.jpg" /> 
Figure 4. Mean arterial flow with standard deviation (mL/min). <a href="https://www.jove.com/files/ftp_upload/63905/63905fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63905/63905fig05.jpg" /> 
Figure 5. Mean oxygen consumption with standard deviation (mL/min/g). <a href="https://www.jove.com/files/ftp_upload/63905/63905fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63905/63905fig06.jpg" /> 
Figure 6. (A) Mean pH, (B) HCO3, (C) sodium, and (D) calcium measurements with standard deviations. <a href="https://www.jove.com/files/ftp_upload/63905/63905fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63905/63905fig07.jpg" /> 
Figure 7. (A) Mean lactate and (B) potassium measurements with standard deviations. <a href="https://www.jove.com/files/ftp_upload/63905/63905fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63905/63905fig08.jpg" /> 
Figure 8. (A) Mean amylase and (B) lipase measurements with standard deviations. <a href="https://www.jove.com/files/ftp_upload/63905/63905fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63905/63905fig09.jpg" /> 
Figure 9. Core biopsies before and after perfusion. (A) Normal pancreatic parenchyma before machine perfusion18. (B) Post perfusion biopsy with good preservation of pancreatic acini and islet cells. <a href="https://www.jove.com/files/ftp_upload/63905/63905fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Ringer&#8217;s lactate</td>
			<td>260 mL</td>
		</tr>
		<tr>
			<td>Steen Solution</td>
			<td>195 mL</td>
		</tr>
		<tr>
			<td>Washed erythrocytes</td>
			<td>162.5 mL</td>
		</tr>
		<tr>
			<td>Double Reverse Osmosis Water (DRO)</td>
			<td>35 mL</td>
		</tr>
		<tr>
			<td>Heparin (10000 IU / 10 mL)</td>
			<td>1.3 mL</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (8.4%)</td>
			<td>10.4 mL</td>
		</tr>
		<tr>
			<td>Calcium gluconate (10%)</td>
			<td>1.3 mL</td>
		</tr>
		<tr>
			<td>Metylprednisolone (Solu-Medrol)</td>
			<td>325 mg</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>15 mg</td>
		</tr>
	</tbody>
</table>
Table 1. Perfusate composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Weight before</td>
			<td>Weight after</td>
			<td>Gain</td>
			<td>% difference</td>
		</tr>
		<tr>
			<td>Case 1</td>
			<td>244 g</td>
			<td>240 g</td>
			<td>-4 g</td>
			<td>-1.63</td>
		</tr>
		<tr>
			<td>Case 2</td>
			<td>154 g</td>
			<td>164 g</td>
			<td>10 g</td>
			<td>6.49</td>
		</tr>
		<tr>
			<td>Case 3</td>
			<td>184 g</td>
			<td>245 g</td>
			<td>61 g</td>
			<td>33.15</td>
		</tr>
		<tr>
			<td>Case 4</td>
			<td>190 g</td>
			<td>226 g</td>
			<td>36 g</td>
			<td>18.94</td>
		</tr>
		<tr>
			<td>Case 5</td>
			<td>198 g</td>
			<td>307 g</td>
			<td>109 g</td>
			<td>55.05</td>
		</tr>
		<tr>
			<td>Case 6</td>
			<td>205 g</td>
			<td>315 g</td>
			<td>107 g</td>
			<td>51.44</td>
		</tr>
		<tr>
			<td>Case 7</td>
			<td>193 g</td>
			<td>256 g</td>
			<td>63 g</td>
			<td>32.64</td>
		</tr>
	</tbody>
</table>
Table 2. Weight before and after perfusion. 
Supplemental File: Custom made pancreas chamber for perfusion. Designed in collaboration with the Machine Shop of the Medical Physics - Radiation Medicine Program at Princess Margaret Cancer Centre. <a href="https://www.jove.com/files/ftp_upload/63905/RightsLink Printable License.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation at JoVE.com

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.

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 ...

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1.8K Views.  Toronto General Hospital. 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. 

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

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

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 ...

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 ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

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, ...

Robot-Assisted Kidney Transplantation

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed in the supine Trendelenburg position. Kidney allografts are provided by a living donor. Before vascular anastomosis, the kidney allograft is prepared by inserting a double-J stent in the ureter, and the temperature for the anastomosis is lowered by wrapping it in an ice-packed gauze. A 12 mm or 8 mm port for the robotic camera and three 8 mm ports for robotic arms are placed. A peritoneal pouch is created for the kidney allograft by raising the peritoneal flaps on both sides over the psoas muscle before dissecting the iliac vessels and bladder. A 6 cm Pfannenstiel incision is made to insert the kidney into the peritoneal pouch, lateral to the right iliac vessels. 
After clamping the external iliac vein with Bulldogs clamps, a venotomy is performed, and the graft renal vein is anastomosed to the external iliac vein in an end-to-side continuous manner with a 6/0 polytetrafluoroethylene suture. After clamping the graft renal vein, the iliac vein is declamped. This is followed by clamping of the external iliac artery, arteriotomy, arterial anastomosis with a 6/0 polytetrafluoroethylene suture, clamping of the graft renal artery, and declamping of the external iliac artery. Reperfusion is then carried out, and ureteroneocystostomy is performed using the Lich-Gregoir technique. The peritoneum is closed at a few locations with polymer locking clips, and a closed-suction drain is placed through one of the working ports. After deflating the pneumoperitoneum, all incisions are closed.

This paper provides technical details for robot-assisted kidney transplantation from a living donor.

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed ...

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 ...

Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this state-of-the-art technique remains the paucity of organs deemed fit for transplantation. High intravascular resistance, delicate intraparenchymal capillary framework, and complex lobular anatomy around the mesenteric vasculature are what make this organ more susceptible to injury and less tolerant to trivial trauma compared to organs such as the liver and kidney. Meticulous surgical dissection and judicious tissue handling form the cornerstone of the entire exercise of pancreas transplantation. Owing to morphological similarity between the anatomy of the porcine pancreas to the surrounding mesenteric vessels and the organs when compared to the human anatomy, demonstration of the technique in the porcine model could help to most accurately extrapolate this to a human setting. The present article aims to outline the essential surgical tips and tricks that need to be followed, in order to ensure a higher success rate of transplantation of this highly susceptible organ in a porcine 3-day survival model.

The video article summarizes the technique of pancreatectomy and pancreas allotransplantation in a porcine 3-day survival model with a step-by-step description of the method and emphasis on the surgical tips and tricks to deal with the precarious and delicate porcine visceral anatomy.

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

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

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

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

Purification of Porcine Islets by Surgical Perfusion of Pig Pancreas

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet xenotransplantation offers a promising alternative to allotransplantation, with the potential for large-scale production of on-demand, functional islets. The yield and viability of isolated islets is highly susceptible to the quality of the donor pancreas and the method of procurement, particularly the duration of warm-ischemia time. To improve organ preservation and subsequent islet yield and viability, we have developed a protocol for surgical perfusion and resection of the porcine pancreas. This protocol employs direct infrarenal aortic cannulation and organ perfusion to both minimize warm-ischemia time and simplify the procedure for operators who do not have extensive surgical expertise. Subsequent arterial perfusion of the pancreas via the aorta flushes stagnant blood from the microvasculature, thereby reducing thrombosis and oxidative damage to the tissue. This manuscript provides a detailed protocol for surgical perfusion and resection of the porcine pancreas, followed by islet isolation and purification.

Author Spotlight: Techniques and Challenges in Islet Purification for Xenotransplantation

This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet ...