Name: normothermic ex vivo pancreas perfusion for the preservation of pancreas allografts before transplantation
Uploaded: 2022-07-27T13:20:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation at JoVE.com

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><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&amp;ouml;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&#039;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&amp;nbsp;</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 ...

normothermic-ex-vivo-pancreas-perfusion-for-preservation-pancreas

Research

JoVE Journal

Medicine

1.9K 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

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 ...

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing modified cannulas and techniques adapted from conventional commercial ex vivo perfusion equipment. The system was utilized to evaluate the preservation outcomes following 12 h of perfusion. C57BL/6J mice served as liver donors, and the livers were explanted by cannulating the portal vein (PV) and bile duct (BD), and subsequently flushing the organ with warm (37 &#176;C) heparinized saline. Then, the explanted livers were transferred to the perfusion chamber and subjected to normothermic oxygenated machine perfusion (NEVLP). Inlet and outlet perfusate samples were collected at 3 h intervals for perfusate analysis. Upon completion of the perfusion, liver samples were obtained for histological analysis, with morphological integrity assessed using modified Suzuki-Score through Hematoxylin-Eosin (HE) staining. The optimization experiments yielded the following findings: (1) mice weighing over 30 g were deemed more suitable for the experiment due to the larger size of their bile duct (BD). (2) a 2 Fr (outer diameter = 0.66 mm) polyurethane cannula was better suited for cannulating the portal vein (PV) when compared to a polypropylene cannula. This was attributed to the polyurethane material&#39;s enhanced grip, resulting in reduced catheter slippage during the transfer from the body to the organ chamber. (3) for cannulation of the bile duct (BD), a 1 Fr (outer diameter = 0.33 mm) polyurethane cannula was found to be more effective compared to the polypropylene UT - 03 (outer diameter = 0.30 mm) cannula. With this optimized protocol, mouse livers were successfully preserved for a duration of 12 h without significant impact on the histological structure. Hematoxylin-Eosin (HE) staining revealed a well-preserved morphological architecture of the liver, characterized by predominantly viable hepatocytes with clearly visible nuclei and mild dilation of hepatic sinusoids.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

This protocol presents an optimized erythrocytes-free NEVLP system using mouse livers. Ex vivo preservation of mouse livers was achieved by employing ...

Engineering

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 ...

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

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

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

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

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

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

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

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

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

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Bioengineering

Orthotopic Pig Liver Transplant Preservation Using Machine Perfusion

Preservation of Porcine Donation after Circulatory Death (DCD) Liver by Perfusion and Orthotopic Liver Transplantation

Conventional static cold storage (SCS) exacerbates ischemic injury in the DCD liver, leading to severe complications for transplant recipients. To address this issue, clinical application of MP technology for donor liver preservation is underway. Simultaneously, efforts are focused on the development of various MP instruments, validated through relevant animal model experiments. Effective large animal trials play a pivotal role in clinical applications. However, challenges persist in the ex vivo preservation of DCD livers and the transplantation procedure in pigs. These hurdles encompass addressing the prolonged preservation of donor livers, conducting viability tests, alleviating ischemic injuries, and shortening the anhepatic phase. The use of a variable temperature-controlled MP device facilitates the prolonged preservation of DCD livers through sequential Dual Hypothermic Oxygenated Machine Perfusion (DHOPE) and Normothermic Machine Perfusion (NMP) modes. This protocol enhances the porcine OLTx model by improving the quality of DCD livers, optimizing the anastomosis technique, and reducing the duration of the anhepatic phase.

Author Spotlight: Advancements and Challenges in Machine Perfusion for Liver Transplantation

This protocol outlines the methodology of establishing a porcine model utilizing variable temperature-controlled Machine Perfusion (MP) for the preservation of the donor liver, followed by orthotopic liver transplantation (OLTx). It aims to promote the success rate of OLTx using donor donation after circulatory death (DCD) liver and establish a stable model.

Conventional static cold storage (SCS) exacerbates ischemic injury in the DCD liver, leading to severe complications for transplant recipients. To address ...

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 ...

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.

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

Summary

Explore More Videos

Chapters in this video

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

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

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

Ex Situ Normothermic Machine Perfusion of Donor Livers

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

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

Isolation and Decellularization of a Whole Porcine Pancreas

Preservation of Porcine Donation after Circulatory Death (DCD) Liver by Perfusion and Orthotopic Liver Transplantation

Avoiding Ischemia Reperfusion Injury in Liver Transplantation

Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation