The authors thank Britta Trautewig, Corinna L&#246;bbert, Astrid Dinkel, and Ingrid Meder for their diligence and commitment. Furthermore, the authors thank Tom Figiel for producing the picture material.

This study was performed at the Laboratory for Animal Science of Hannover Medical School after approval by the Lower Saxony regional authority for consumer protection and food safety (Nieders&#228;chsisches Landesamt f&#252;r Verbraucherschutz und Lebensmittelsicherheit [LAVES]; 19/3146)
1. Donor liver procurement
NOTE: The liver donors were female domestic pigs (Sus scrofa domesticus), aged 4-5 months old and with an average body weight of approximately 50 kg, which had already been in quarantine at the animal research facility for a minimum of 10 days prior to surgery.
<ol>
	<li>Perform premedication by intramuscular injection of atropine (0.04-0.08 mg/kg body weight), zolazepam (5 mg/kg body weight), and tiletamine (5 mg/kg body weight). After establishing an intravenous access (e.g., ear vein) induce anesthesia with an injection of propofol (1.5 - 2.5 mg/kg body weight).</li>
	<li>Perform intubation with an 8.0-8.5 mm endotracheal tube, depending on the animal size and anatomy. Establish monitoring of electrocardiography, measurement of respiratory gases and peripheral oxygen saturation, and non-invasive blood pressure measurement.</li>
	<li>Maintain anesthesia in pigs during donor liver procurement via inhalation of isoflurane (0.8-1.5 vol%) and intravenous application of fentanyl (0.003-0.007 mg/kg body weight). Perform volume-controlled ventilation throughout the procedure.</li>
	<li>After placement of the donor pig in a supine position and fixation of the limbs at the base of the operation table with elastic bands, scrub the skin with antiseptic agent, e.g., povidone-iodine or isopropyl alcohol, and cover the animal with sterile drapes.</li>
	<li>Confirm an adequate depth of anesthesia by loss of the withdrawal response to toe pinch. Perform a midline laparotomy beginning at the xiphoid process by using monopolar cautery. Place an abdominal retractor and mobilize the intestine to the right of the donor.</li>
	<li>Perform a splenectomy by dissection of the splenocolic ligament, the gastrosplenic ligament, and the phrenicosplenic ligament. Clamp the splenic vein and splenic artery near the splenic hilum with an Overholt clamp and place ligatures (3-0 polyfilament suture) after severing the vessels. Sever additional (smaller) vessels either by bipolar forceps or by ligation. 
	NOTE: A splenectomy during donor liver procurement is not obligatory but reduces the efflux of blood during and after perfusion.</li>
	<li>Mobilize the intestine to the left side of the donor and sever the falciform ligament and the triangular ligaments using scissors and bipolar cautery.</li>
	<li>After sufficient dissection of the liver, incise the left portion of the diaphragm over a distance of 5-10 cm using scissors to locate the thoracic segment of the descending aorta. Encircle and place a ligature (3-0 polyfilament suture) without tightening.</li>
	<li>Incise the right portion of the diaphragm over a distance of 5-10 cm using scissors and identify the suprahepatic vena cava inferior.</li>
	<li>Relocate the intestine to the upper left of the donor and enter the retroperitoneal space by transverse incision of the peritoneum over a distance of 5-10 cm using scissors.</li>
	<li>Locate the abdominal aorta and inferior vena cava just above the iliac bifurcation and separate both vessels over a length of approximately 6 cm. Place two 3-0 polyfilament ligatures&#160;around the abdominal aorta: one cranial of the iliac bifurcation and one approximately 3 cm cranially, without tightening. Place another&#160;ligature around the intrahepatic vena cava inferior without tightening.</li>
	<li>Intravenously inject heparin (25,000 I.E.). Choose an appropriate cannula and de-air the drip line with cooled preservation solution.</li>
	<li>Tighten the caudally located first ligature around the abdominal aorta. After occluding the abdominal aorta cranially of the second ligature (either manually or by placing an atraumatic vascular clamp), make a transverse incision between both the ligatures using scissors.</li>
	<li>Insert the cannula into the incision and secure it with the remaining ligature. Sever the suprahepatic inferior vena cava far cranially (close to the right atrium) using scissors.</li>
	<li>After blood loss of approximately 1,500-2,000 mL, cross-clamp the thoracic segment of the descending aorta by tying the ligature and start antegrade perfusion. 
	NOTE: For the possible need for blood (transfusions) during engraftment or for normothermic machine perfusion, whole blood (approximately 1,500 mL) can be collected using a container containing citrate-based anticoagulant.</li>
	<li>Tighten the ligature placed around the infrahepatic vena cava inferior, incise the vessel cranially of the ligature, and insert a surgical aspirator. Inject a lethal dose of pentobarbital sodium (5,000 mg). Place crushed sterile ice into the thoracic and abdominal cavity without compromising the liver tissue.</li>
	<li>After perfusion with 3,500 mL of preservation solution over a course of approximately 10-15 min, sever the incised suprahepatic vena cava inferior. Sever the infrahepatic vena cava inferior at the level of the left renal vein.</li>
	<li>Sever the bile duct cranial of the pancreatic tissue between two ligatures (3-0 polyfilament) to avoid bile spillage. Sever the portal vein cranial of the pancreas.</li>
	<li>Locate the celiac artery after blunt preparation and follow dorsally to the abdominal aorta. Excise the respective aortic segment in order to create a patch for later engraftment.</li>
	<li>Excise the diaphragm around the suprahepatic vena cava inferior and sever remaining adhesions using scissors. Extract the liver.</li>
	<li>Perform a cholecystectomy or tighten a ligature around the cystic duct and flush the common bile duct with at least 20 mL of preservation solution. Place the perfusion cannula into the portal vein and flush the graft with a further 500 mL of preservation solution. Place the graft in a sterile bowl placed on ice. 
	&#8203;NOTE: Depending on the scientific objective, the organ can be immediately prepared for engraftment or kept on ice for an indefinite amount of time (20 h in this protocol) before beginning back-table preparation and engraftment.</li>
</ol>
2. Back-table preparation of the liver
<ol>
	<li>Remove the lymphatic tissue beginning at the aortic segment and thereby identify and occlude the arterial side branches and lymphatic vessels with either clips, ligatures (4-0 polyfilament), or sutures (5-0 monofilament; Figure 1A). Likewise, remove the lymphatic tissue around the portal vein and occlude the side branches with sutures (5-0 monofilament).</li>
	<li>Identify the suprahepatic vena cava inferior and place sutures around both diaphragmatic veins (5-0 monofilament) after removing surrounding diaphragmatic tissue. Flush all the vessels with cold saline or preservation solution to identify any remaining leakages. Perform shortening of the vessels and preparation of the aortic patch only upon engraftment to take into account the individual anatomic circumstances.</li>
</ol>
3. Recipient hepatectomy, donor liver engraftment, and perioperative management
NOTE: As liver recipients, female domestic pigs (Sus scrofa domesticus) aged 4-5 months old and with an average body weight of approximately 50 kg, were used. Analogously to the liver donors, the recipients had been in quarantine at the animal research facility for a minimum of 10 days prior to transplant.
<ol>
	<li>Anesthesia and perioperative management
	<ol>
		<li>Perform premedication by intramuscular injection of atropine (0.04-0.08 mg/kg body weight), zolazepam (5 mg/kg body weight), and tiletamine (5 mg/kg body weight). After establishing an intravenous access (e.g., ear vein), induce anesthesia with an injection of propofol (1.5-2.5 mg/kg body weight).</li>
		<li>Perform intubation with an 8.0-8.5 mm endotracheal tube, depending on the animal size and anatomy. Establish monitoring of electrocardiography, measurement of respiratory gases and peripheral oxygen saturation, and non-invasive blood pressure measurement. In the case of a chronic model, apply eye ointment to avoid dryness after the surgical intervention.</li>
		<li>Place the recipient animal on a heating base in a supine position and fix the limbs on the base of the operation table with elastic bands.</li>
		<li>For extended monitoring, under ultrasound guidance, place a three-lumen central venous catheter and a large-bore venous catheter (7 Fr.)&#160;into the internal jugular vein and a large-bore venous catheter (7 Fr.) for volume therapy. In addition, insert an arterial catheter into the internal carotid/cervical artery under ultrasound control for invasive blood pressure measurement (Figure 1B).</li>
		<li>Maintain anesthesia during organ retrieval via inhalation of isoflurane (0.8-1.5 vol%) and intravenous application of fentanyl (0.003-0.007 mg/kg body weight). Perform volume-controlled ventilation throughout the procedure. Apply 2,000 mg of sultamicillin for perioperative antibiosis and 250 mg of methylprednisolone intravenously.&#160;</li>
		<li>Administer a vasopressor such as norepinephrine intravenously to achieve a target mean arterial pressure of 60 mmHg. In addition, apply crystalloid solutions such as Ringer&#39;s lactate solution or colloid solutions such as fluid gelatins if necessary.</li>
		<li>Apply calcium gluconate (10%) and sodium bicarbonate (8.4%), glucose (40%), or potassium chloride (7.45%) intravenously with respect to blood gas analyses obtained every 30 min.</li>
	</ol>
	</li>
	<li>Recipient hepatectomy
	<ol>
		<li>Scrub the skin with antiseptic agent, e.g., povidone-iodine or isopropyl alcohol, and cover the animal with sterile drapes.</li>
		<li>Confirm an adequate depth of anesthesia by loss of the withdrawal response to toe pinch. Perform a midline laparotomy beginning at the xiphoid process by using monopolar cautery. Place an abdominal retractor and mobilize the intestine to the left of the donor. Cover the intestine with a moistened cloth.</li>
		<li>Place a suprapubic urinary catheter for the optimization of intraoperative volume management.</li>
		<li>Sever the falciform ligament and the triangular ligaments using scissors and bipolar cautery. After sufficient dissection of the liver, encircle both the suprahepatic and infrahepatic vena cavae inferior close to the liver parenchyma.</li>
		<li>Dissect and sever the common bile duct below the junction of the cystic duct between two ligatures (3-0 polyfilament).</li>
		<li>Incise the superficial peritoneal layer covering the hepatoduodenal ligament and identify the hepatic arteries shortly before entering the liver parenchyma. Dissect using bipolar cautery or the placement of clips, ligatures, or sutures.</li>
		<li>Dissect the abdominal aorta by incision in the midline (avascular layer) of the right and left diaphragmatic muscles. Prepare the aorta for the aortic anastomosis by removal of the surrounding tissue. 
		NOTE: This step only is required if an aortic anastomosis is performed. Otherwise, further dissect the hepatic artery/the hilar region to prepare for a conventional end-to-end anastomosis between the donor and recipient hepatic arteries.</li>
		<li>Perform recipient hepatectomy by placing an atraumatic vascular clamp on the portal vein, followed by atraumatic vascular clamps on the suprahepatic vena cava inferior (including the surrounding diaphragm while caudally retracting the liver) and the infrahepatic vena cava inferior.</li>
		<li>Sever all three vessels close to the liver parenchyma. Remove the recipient liver from the abdominal cavity. 
		NOTE: The clamping of the vessels marks the start of the anhepatic phase. During the anhepatic phase, the pigs are hemodynamically instable and require relevant amounts of vasopressors/catecholamines. The anesthesiologist should be prepared to apply norepinephrine and epinephrine. Keep the phase until reperfusion of the liver as short as possible. Communicate well with the anesthesiologist.</li>
	</ol>
	</li>
	<li>Donor liver engraftment
	<ol>
		<li>Place the donor liver into the abdominal cavity. Shorten the donor and/or recipient suprahepatic vena cava inferior to an adequate length while avoiding kinking or too much tension on the anastomosis.</li>
		<li>Place a single suture as a supporting thread (5-0 monofilament), adapting the right corner of the donor and recipient suprahepatic vena cavae inferior. Begin the dorsal side of the anastomosis from the left corner of the vessel(s) with a running suture (5-0 monofilament, double-armed).</li>
		<li>When reaching the right corner, remove the supporting thread, secure the running suture with a clamp, and continue with the ventral side of the anastomosis, again beginning from the left corner of the vessel(s). Tighten the suture with multiple knots without constricting the vessel diameter in order to avoid stenosis.</li>
		<li>Shorten the donor and/or recipient portal vein to an adequate length while avoiding kinking or too much tension on the anastomosis.</li>
		<li>Perform a vascular anastomosis of the donor and recipient portal vein analogous to steps 3.3.2-3.3.3 using a 6-0 monofilament, double-armed suture.</li>
		<li>Perform the porto-venous reperfusion by removing the vascular clamp, occluding the recipient portal vein, and occlude the donor infrahepatic vena cava inferior with a vascular clamp after draining approximately 200-400 mL of blood. Slowly remove the vascular clamp occluding the recipient suprahepatic vena cava inferior and search for active bleeding. 
		&#8203;NOTE: The removal of both clamps marks the end of the anhepatic phase. The amount of catecholamines required should significantly decrease shortly thereafter.</li>
		<li>Shorten the donor and/or recipient infrahepatic vena cava inferior. Perform a vascular anastomosis of the donor and recipient infrahepatic vena cavae inferior analogous to steps 3.3.2-3.3.3 using a 5-0 monofilament, double-armed suture. Remove the clamps occluding the donor and recipient infrahepatic vena cavae inferior.</li>
		<li>Prepare an elliptic aortal patch (Carrel patch) with a diameter of approximately 1-1.5, cm depending on anatomical circumstances, using scissors. Clamp the abdominal aorta with an atraumatic Cooley vascular clamp and make an incision by using a scalpel. Enlarge the incision using scissors to fit the patch.</li>
		<li>Begin the aortic anastomosis with a running suture (6-0 monofilament, double-armed) at the cranial corner of the incision/patch. When reaching the caudal corner, secure the running suture with a clamp and complete the anastomosis again beginning at the cranial corner. Tighten the suture with multiple knots and slowly remove the vascular clamp. 
		NOTE: Clamping of the abdominal aorta will significantly affect the blood pressure of the pig. Communicate well with the anesthesiologist.</li>
		<li>Place a hemostatic gauze around the arterial anastomosis. Place a catheter in the common bile duct and secure it with a single ligature. Make sure not to occlude the diameter of the catheter.</li>
		<li>Close the abdomen temporarily by adapting the muscular fascia and the skin with a running suture and cover the abdomen with cling film and/or drapes to avoid thermal loss. 
		NOTE: If the scientific objectives require a chronic model, perform an end-to-end anastomosis between the donor and recipient bile duct, close the abdomen with separate running sutures for the peritoneum and the muscular fascia, and close the skin with single sutures.</li>
		<li>At the end of follow-up, inject a lethal dose of 5,000 mg of pentobarbital sodium for intraoperative euthanasia.</li>
	</ol>
	</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+alterations+during+orthotopic+liver+experimental+transplantation+in+pigs.">Hemodynamic alterations during orthotopic liver experimental transplantation in pigs.</a> Acta Cirurgica Brasileria. 23 (2), 135-139 (2008).</li><li>Canedo, B. F., 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=Liver+autotransplantation+in+pigs+without+venovenous+bypass:+A+simplified+model+using+a+supraceliac+aorta+cross-clamping+maneuver.">Liver autotransplantation in pigs without venovenous bypass: A simplified model using a supraceliac aorta cross-clamping maneuver.</a> Annals of Transplantation. 20, 320-326 (2015).</li><li>Battersby, C., Hickman, R., Saunders, S. J., Terblanche, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+function+in+the+pig.+1.+The+effects+of+30+minutes'+normothermic+ischaemia.">Liver function in the pig. 1. The effects of 30 minutes' normothermic ischaemia.</a> The British Journal of Surgery. 61 (1), 27-32 (1974).</li><li>Kaiser, G. M., Heuer, M. M., Fr&#252;hauf, N. R., K&#252;hne, C. A., Broelsch, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+handling+and+anesthesia+for+experimental+surgery+in+pigs.">General handling and anesthesia for experimental surgery in pigs.</a> Journal of Surgical Research. 130 (1), 73-79 (2006).</li><li>Oike, F., 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=Simplified+technique+of+orthotopic+liver+transplantation+in+pigs.">Simplified technique of orthotopic liver transplantation in pigs.</a> Transplantation. 71 (2), 328-331 (2001).</li><li>Heuer, 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=Liver+transplantation+in+swine+without+venovenous+bypass.">Liver transplantation in swine without venovenous bypass.</a> European Surgical Research. 45 (1), 20-25 (2010).</li><li>Fondevila, C., 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=Step-by-step+guide+for+a+simplified+model+of+porcine+orthotopic+liver+transplant.">Step-by-step guide for a simplified model of porcine orthotopic liver transplant.</a> The Journal of Surgical Research. 167 (1), 39-45 (2011).</li></ol>

The authors have nothing to disclose.

Recent technical developments such as the introduction of machine perfusion have the potential to revolutionize the field of liver transplantation. In order to translate graft reconditioning or modification concepts into clinical settings, reproducible transplant models in large animals are inevitable.
After the initial introduction of porcine orthotopic liver transplantation, several authors have worked on the improvement of these techniques over the past five&#160;decades. Differences within the reported surgical approaches are often minor and concern vascular and biliary anastomoses, anesthesia, and perioperative management. Nonetheless, in contrast to the current situation in clinical liver transplantation in which the usage of veno-venous bypass is still common but optional 28, an active or passive porto-caval bypass during the anhepatic phase in pigs is regarded as a necessity to reduce intestinal congestion and, thus, subsequent intestinal ischemia with hemodynamic instability and perioperative mortality, as described in a well-elaborated work by Esmaeilzadeh et al.25.
Apart from the implicated additional costs and technical challenges of a veno-venous bypass, e.g., catheters, a pump device, the need for additional anticoagulation, and potential complications such as air embolism or hemorrhage, and depending on the chosen approach, the need for a simultaneous splenectomy has prompted groups to describe modified techniques without veno-venous bypasses25,29,30.
Torres et al.31 observed severe hemodynamic instability in animals undergoing engraftment without the use of a veno-venous bypass in comparison to recipients with a passive porto-caval shunt and, thus, performed temporary clamping of the supraceliac aorta in these animals, which was also described by others in models of porcine liver auto-/allo-transplantation23,31,32. However, the induction of warm ischemia by cross-clamping of the recipient aorta bears the risk of relevant release of pro-inflammatory molecules and tissue damage upon reperfusion and should, therefore, be avoided at all costs in order to produce reliable scientific results, especially when evaluating ischemia-reperfusion injury. Furthermore, this approach does not resemble clinical practice in humans, which, hence, limits the translation of results obtained in these models.
To avoid such detrimental supporting measures, we believe that two points are crucial. (1) The anhepatic phase should be kept at an absolute minimum, i.e., below 30 min, as has already been shown in the early phases of porcine liver transplantation by Battersby et al.33. We believe that running sutures (double-armed) and a maximum of one supporting thread are sufficient to create a simple and safe anastomosis for both the suprahepatic vena cava inferior and the portal vein. Obviously, the portal-venous reperfusion should commence before anastomosing the infrahepatic vena cava inferior. (2) Anesthetic management should be performed by an experienced anesthesiologist, ideally familiar with liver surgery or transplantation in human patients34. Sophisticated volume management and therapy with vasopressors, i.e., norepinephrine and epinephrine, in combination with a simplified surgical technique are the basis for the successful implementation of this model.
Interestingly, only a small number of surgical groups have provided data on successful porcine orthotopic liver transplantation without veno-venous bypass and concomitant supraceliac aortic clamping. To our knowledge Oike et al., Heuer et al., and, most recently, Fondevila et al. were the only groups to report their (promising) results, with survival rates of 87%, 80%, and 100%, respectively35,36,37. The median anhepatic time in our cohort was 25 min and was, thus, identical to the data presented by Heuer et al.36. During the anhepatic phase, Oike et al.35 reported a 50%-60% reduction in the arterial blood pressure, similar to the observations made in this cohort, leading to increased doses of vasopressors to avoid a decrease in the MAD below 60 mmHg. Heuer et al.36 did not mention the use of catecholamine therapy in their publication but non-specifically mentioned transfusion of whole blood to improve hemodynamic stability. The latter was not required in this model. Fondevila et al., who reported a mean anhepatic time of less than 20 min, solely relied on the administration of crystalloid solutions and did not apply vasoactive substances during engraftment37.
Of note, as opposed to recent publications applying end-to-end anastomosis from the donor to the recipient hepatic artery19,22, this model includes an end-to-side anastomosis with a Carrel patch of the donor aorta being anastomosed to the supraceliac aorta of the recipient. Especially for experimental settings, with the use of organs fulfilling extended donor criteria, e.g., prolonged cold ischemic time, it might be favorable to rule out any problems with the arterial perfusion of the graft. The application of a Carrel patch will help to avoid stenosis of the arterial anastomosis that might become functionally relevant in the case of concomitant peripheral vasospasms frequently observed following reperfusion. Nevertheless, this approach will be more time-consuming than the conventional end-to-end anastomosis due to the more elaborated access of the aorta.
As our representative experiments focused on the immediate postoperative phase and ischemia-reperfusion injury, recipients were kept under anesthesia and were euthanized 6 h after reperfusion. Although beneficial with respect to animal welfare, this constitutes a significant limitation to the validation of our technique regarding graft and recipient survival. We believe, however, that on the basis of the vital parameters, hemodynamics, lactate clearance, bile production, and especially the real-time liver-specific CYP1A2 metabolism (LiMAx test) observed throughout the follow-up, the long-term application of our model should be feasible, especially as the grafts used within the experiments underwent static cold storage for 20 h prior engraftment, in contrast to the successful application of comparable transplant models by others35,36,37. Furthermore, the mentioned previous reports demonstrated that perioperative mortality was exclusively observed during and up to 6 h after surgery, except for one recipient dying from a pulmonary embolism on the first postoperative day in the study by Oike et al.35.
In this work, we demonstrate that a simplified approach to porcine orthotopic liver transplantation without the use of a veno-venous bypass during engraftment can be performed safely and is more cost-effective, without significant hemodynamic alterations or intraoperative mortality even after prolonged static cold storage of the donor organ. Such a model should be of special interest for (surgical) workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, the reconditioning of extended criteria donor organs, and associated immunological mechanisms.

Liver transplantation remains to be the only chance for survival in a variety of different diseases leading to acute or chronic hepatic failure. Since its first successful application in mankind in 1963 by Thomas E. Starzl, the concept of liver transplantation has evolved into a reliable treatment option applied worldwide, mainly as a result of advancements in the understanding of the immune system, the development of modern immunosuppression, and the optimization of perioperative care and surgical techniques1,2. However, aging populations and a higher demand for organs have resulted in donor shortages, with increased use of marginal grafts from extended criteria donors and the emergence of new challenges in the past decades. The introduction and widespread implementation of organ machine perfusion is believed to open up an array of possibilities with regard to graft reconditioning and modulation and to help mitigate organ shortages and reduce waiting list mortality3,4,5,6.
In order to evaluate these concepts and their effects in vivo, translational transplant models are necessary7. In 1983, Kamada et al. introduced an efficient orthotopic liver transplant model in rats that has since been extensively modified and applied by workgroups around the globe8,9,10,11. The orthotopic liver transplant model in mice is technically more demanding, but also more valuable in terms of immunological transferability, and was first reported in 1991 by Qian et al.12. Despite advantages regarding availability, animal welfare, and costs, rodent models are limited in their applicability in clinical settings7. Hence, large animal models are required.
In recent years, pigs have become the main animal species used for translational research due to their anatomical and physiological similarities with humans. Furthermore, current progress in the field of xenotransplantation might further increase the importance of pigs as research objects13,14.
Garnier et al. described a liver transplant model in pigs as early as 196515. Several authors, including Calne et al. in 1967 and Chalstrey et al. in 1971, subsequently reported modifications, ultimately leading to a safe and feasible concept of experimental porcine liver transplantation in the decades to follow16,17,18,19,20,21.
More recently, different work groups have provided data with regard to current issues in liver transplantation using a technique of porcine orthotopic liver transplantation, almost invariably including an active or passive veno-venous, i.e., porto-caval, bypass19,22. The reason for this is a species-specific intolerance to the clamping of the vena cava inferior and the portal vein during the anhepatic phase due to a comparatively larger intestine and fewer porto-caval or cavo-caval shunts (e.g., lack of a vena azygos), resulting in increased perioperative morbidity and mortality23. Vena cava inferior-sparing transplant techniques applied in human recipients as an alternative are not feasible as the porcine vena cava inferior is encased by hepatic tissue23.
However, the usage of a veno-venous bypass further increases technical and logistical complexity in an already demanding surgical procedure, therefore possibly preventing workgroups from attempting implementation of the model altogether. Apart from the direct physiological and immunological effects of a bypass, some authors have pointed out the significant morbidity such as blood loss or air embolism during shunt placement and the need for a simultaneous splenectomy, potentially affecting short- and long-term results after engraftment24,25.
The following protocol describes a simple technique of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h, representing extended criteria donor conditions without the usage of a veno-venous bypass during engraftment, including donor liver procurement, back-table preparation, recipient hepatectomy, and anesthesiological pre- and intraoperative management.
This model should be of special interest for surgical workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, the reconditioning of extended criteria donor organs, and associated immunological mechanisms.

<table><tbody><tr><td>Abdominal retractor</td><td>No Company Name available</td><td>No Catalog Number available</td><td></td></tr><tr><td>Aortic clamp, straight</td><td>Firma Martin</td><td>No Catalog Number available</td><td></td></tr><tr><td>Arterial Blood Sampler Aspirator (safePICOAspirator) 1.5 mL</td><td>Radiometer Medical ApS</td><td>956-622</td><td></td></tr><tr><td>Atropine (Atropinsulfat 0.5 mg/1 mL)</td><td>B.Braun</td><td>648037</td><td></td></tr><tr><td>Backhaus clamp</td><td>Bernshausen</td><td>BF432</td><td></td></tr><tr><td>Bipolar forceps, 23 cm&amp;nbsp;</td><td>SUTTER</td><td>780222 SG</td><td></td></tr><tr><td>Bowl 5 L, 6 L, 9 L</td><td>Chiru-Instrumente</td><td>35-114327</td><td></td></tr><tr><td>Braunol Braunoderm</td><td>B.Braun</td><td>3881059</td><td></td></tr><tr><td>Bulldog clamp</td><td>Aesculap</td><td>No Catalog Number available</td><td></td></tr><tr><td>Button canula</td><td>Krauth + Timmermann GmbH</td><td>1464LL1B</td><td></td></tr><tr><td>Calcium gluconate (2.25 mmol/10 mL (10%))</td><td>B.Braun</td><td>2353745</td><td></td></tr><tr><td>Cell Saver (Autotransfusion Reservoir)</td><td>Fresenius Kabi AG</td><td>9108471</td><td></td></tr><tr><td>Central venous catheter 7Fr., 3 Lumina, 30 cm 0.81 mm</td><td>Arrow</td><td>AD-24703</td><td></td></tr><tr><td>Clamp</td><td>INOX</td><td>B-17845&amp;nbsp; /&amp;nbsp; BH110&amp;nbsp; / B-481</td><td></td></tr><tr><td>Clamp</td><td>Aesculap</td><td>AN909R</td><td></td></tr><tr><td>Clamp, 260 mm</td><td>Fehling Instruments GMbH &amp;amp;Co.KG</td><td>ZAU-2</td><td></td></tr><tr><td>Clip Forceps, medium</td><td>Ethicon</td><td>LC207</td><td></td></tr><tr><td>Clip forceps, small</td><td>Ethicon&amp;nbsp;</td><td>LC107</td><td></td></tr><tr><td>CPDA-1 solution</td><td>Fresenius Kabi AG</td><td>41SD09AA00</td><td></td></tr><tr><td>Custodiol (Histidin-Tryptophan-Ketogluterat-Solution)</td><td>Dr.Franz K&amp;ouml;hler Chemie GmbH</td><td>2125921</td><td></td></tr><tr><td>Dissecting scissors</td><td>LAWTON&amp;nbsp; 05-0641&amp;nbsp;</td><td>No Catalog Number available</td><td></td></tr><tr><td>Dissecting scissors, 180 mm</td><td>Metzenbaum&amp;nbsp;</td><td>BC606R</td><td></td></tr><tr><td>Endotracheal tube 8.0 mm</td><td>Covetrus</td><td>800764</td><td></td></tr><tr><td>Epinephrine (Adrenalin 1:1000)</td><td>InfectoPharm</td><td>9508734</td><td></td></tr><tr><td>Falcon Tubes 50ml</td><td>Greiner&amp;nbsp;</td><td>227 261 L</td><td></td></tr><tr><td>Femoralis clamp</td><td>Ulrich&amp;nbsp;</td><td>No Catalog Number available</td><td></td></tr><tr><td>Fentanyl 0.1mg</td><td>PanPharma</td><td>00483</td><td></td></tr><tr><td>Forceps, anatomical</td><td>Martin</td><td>12-100-20</td><td></td></tr><tr><td>Forceps, anatomical, 250 mm</td><td>Aesculap</td><td>BD052R</td><td></td></tr><tr><td>Forceps, anatomical, 250 mm</td><td>Aesculap</td><td>BD032R</td><td></td></tr><tr><td>Forceps, anatomical, 250 mm&amp;nbsp;</td><td>Aesculap</td><td>BD240R</td><td></td></tr><tr><td>Forceps, surgical</td><td>Bernshausen</td><td>BD 671</td><td></td></tr><tr><td>Forceps, surgical</td><td>INOX</td><td>B-1357</td><td></td></tr><tr><td>G40 solution</td><td>Serag Wiessner</td><td>10755AAF</td><td></td></tr><tr><td>Gelafundin ISO solution 40 mg/mL</td><td>B. Braun</td><td>210257641</td><td></td></tr><tr><td>Guidewire with marker</td><td>Arrow</td><td>14F21E0236</td><td></td></tr><tr><td>Haemostatic gauze (&quot;Tabotamp&quot;&amp;nbsp; 5 x 7.5 cm)</td><td>Ethicon</td><td>474273</td><td></td></tr><tr><td>Heparin sodium 25,000IE</td><td>Ratiopharm</td><td>W08208A</td><td></td></tr><tr><td>Hico-Aquatherm 60</td><td>Hospitalwerk</td><td>No Catalog Number available</td><td></td></tr><tr><td>Infusion Set Intrafix</td><td>B.Braun</td><td>4062981 L</td><td></td></tr><tr><td>Intrafix SafeSet 180 cm</td><td>B.Braun</td><td>4063000</td><td></td></tr><tr><td>Introcan Safety, 18 G&amp;nbsp;</td><td>B.Braun</td><td>4251679-01</td><td></td></tr><tr><td>Isofluran CP</td><td>CP-Pharma</td><td>No Catalog Number available</td><td></td></tr><tr><td>Large-bore venous catheter, 7Fr.</td><td>Edwards Lifesciences</td><td>I301F7</td><td></td></tr><tr><td>Ligaclip, medium</td><td>Ethicon</td><td>LT200</td><td></td></tr><tr><td>Ligaclip, small</td><td>Ethicon&amp;nbsp;</td><td>LT100</td><td></td></tr><tr><td>Material scissors</td><td>Martin&amp;nbsp;</td><td>11-285-23</td><td></td></tr><tr><td>Methylprednisolone (Urbason solubile forte 250 mg)</td><td>Sanofi</td><td>7823704</td><td></td></tr><tr><td>Monopolar ERBE ICC 300</td><td>Fa. Erbe</td><td>No Catalog Number available</td><td></td></tr><tr><td>NaCl solution (0.9%)</td><td>Baxter</td><td>1533</td><td></td></tr><tr><td>Needle holder</td><td>Aesculap</td><td>BM36</td><td></td></tr><tr><td>Needle holder</td><td>Aesculap</td><td>BM035R</td><td></td></tr><tr><td>Needle holder</td><td>Aesculap</td><td>BM 67</td><td></td></tr><tr><td>Neutral electrode</td><td>Erbe Elektromedizin GmbH T&amp;uuml;bingen</td><td>21191 - 060</td><td></td></tr><tr><td>Norepinephrine (Sinora)</td><td>Sintetica GmbH</td><td>04150124745717</td><td></td></tr><tr><td>Omniflush Sterile Filed 10 mL</td><td>B.Braun</td><td>3133335</td><td></td></tr><tr><td>Original Perfusorline 300 cm</td><td>B.Braun</td><td>21E26E8SM3</td><td></td></tr><tr><td>Overhold clamp</td><td>INOX</td><td>BH 959</td><td></td></tr><tr><td>Overhold clamp</td><td>Ulrich</td><td>CL 2911</td><td></td></tr><tr><td>Pentobarbital sodium(Release 500 mg/mL)</td><td>WDT, Garbsen</td><td>21217</td><td></td></tr><tr><td>Perfusers</td><td>B.Braun</td><td>49-020-031</td><td></td></tr><tr><td>Perfusor Syringe 50 mL</td><td>B.Braun</td><td>8728810F</td><td></td></tr><tr><td>Petri dishes&amp;nbsp; 92 x 17 mm</td><td>Nunc</td><td>150350</td><td></td></tr><tr><td>Poole Suction Instrument Argyle flexibel</td><td>Covidien, Mansfield USA</td><td>20C150FHX</td><td></td></tr><tr><td>Potassium chloride (7.45%)</td><td>B.Braun</td><td>4030539078276</td><td></td></tr><tr><td>Pressure measurement set</td><td>Codan pvb Medical GmbH</td><td>957179</td><td></td></tr><tr><td>Propofol (1%)</td><td>CP-Pharma</td><td>No Catalog Number available</td><td></td></tr><tr><td>S-Monovette 2.6 mL K3E</td><td>Sarstedt</td><td>04.1901</td><td></td></tr><tr><td>S-Monovette 2.9 mL 9NC</td><td>Sarstedt</td><td>04.1902</td><td></td></tr><tr><td>S-Monovette 7.5 mL Z-Gel</td><td>Sarstedt</td><td>11602</td><td></td></tr><tr><td>Sartinski clamp</td><td>Aesculap</td><td>No Catalog Number available</td><td></td></tr><tr><td>Scalpel&amp;nbsp; No.11</td><td>Feather Safety Razor Co.LTD</td><td>02.001.40.011</td><td></td></tr><tr><td>Scissors</td><td>INOX&amp;nbsp;</td><td>BC 746</td><td></td></tr><tr><td>Seldinger Arterial catheter</td><td>Arrow</td><td>SAC-00520</td><td></td></tr><tr><td>Sodium bicarbonate (8.4%)</td><td>B.Braun</td><td>212768082</td><td></td></tr><tr><td>Sterilization Set (&quot;ProSet Preparation Kit CVC&quot;)</td><td>B.Braun</td><td>4899719</td><td></td></tr><tr><td>Sterofundin ISO solution</td><td>B.Braun</td><td>No Catalog Number available</td><td></td></tr><tr><td>Suction</td><td>Dahlhausen</td><td>07.068.25.301</td><td></td></tr><tr><td>Suction Aesculap Securat 80</td><td>Aesculap</td><td>No Catalog Number available</td><td></td></tr><tr><td>Suction catheter</td><td>ConvaTec</td><td>5365049</td><td></td></tr><tr><td>Sultamicillin (Unacid: 2000 mg Ampicillin/1000 mg Sulbactam)</td><td>Pfizer</td><td>DL253102</td><td></td></tr><tr><td>Suprapubic urinary catheter, &quot;bronchialis&quot;, 50 cm</td><td>ConvaTec</td><td>UK&amp;nbsp; 1F02772</td><td></td></tr><tr><td>Suprasorb (&quot;Toptex lite RK&quot;)</td><td>Lohmann &amp;amp; Rauscher</td><td>31654</td><td></td></tr><tr><td>Suture Vicryl 3-0</td><td>Ethicon</td><td>VCP 1218 H</td><td></td></tr><tr><td>Suture Vicryl 4-0</td><td>Ethicon</td><td>V392H</td><td></td></tr><tr><td>Suture, Prolene 4-0</td><td>Ethicon</td><td>7588 H</td><td></td></tr><tr><td>Suture, Prolene 5-0, double armed</td><td>Ethicon&amp;nbsp;</td><td>8890 H</td><td></td></tr><tr><td>Suture, Prolene 5-0, single armed</td><td>Ethicon&amp;nbsp;</td><td>8720 H</td><td></td></tr><tr><td>Suture, Prolene 6-0, double armed</td><td>Ethicon&amp;nbsp;</td><td>7230 H</td><td></td></tr><tr><td>Suture, Prolene 6-0, single armed</td><td>Ethicon</td><td>EH 7406 H</td><td></td></tr><tr><td>Suture, Prolene: blau 3-0&amp;nbsp;</td><td>Ethicon</td><td>EH 7499H</td><td></td></tr><tr><td>Suture, Safil 2/0</td><td>Aesculap</td><td>C 1038446</td><td></td></tr><tr><td>Suture, Terylene 0</td><td>Serag Wiessner</td><td>353784</td><td></td></tr><tr><td>Syringe 2 mL, 5 mL, 10 mL, 20 mL</td><td>B.Braun</td><td>4606027V</td><td></td></tr><tr><td>TransferSet &quot;1D/X-double&quot; steril 330 cm</td><td>Fresenius Kabi AG</td><td>2877101</td><td></td></tr><tr><td>Ultrasound Butterfly IQ+</td><td>Butterfly Network Inc.</td><td>850-20014</td><td></td></tr><tr><td>Ventilator &quot;Oxylog Dr&amp;auml;ger Fl&quot;</td><td>Dr&amp;auml;ger Medical AG</td><td>No Catalog Number available</td><td></td></tr><tr><td>Yankauer Suction</td><td>Medline</td><td>RA19GMD</td><td></td></tr><tr><td>Zoletil 100 mg/mL&amp;nbsp; (50 mg Zolazepam, 50 mg tiletamin)</td><td>Virbac</td><td>794-861794861</td><td></td></tr></tbody></table>

porcine liver transplantation without veno-venous bypass as an extended criteria donor model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

The technique presented in this protocol has provided reliable and reproducible results in terms of hemodynamic stability and animal survival throughout the procedure, as well as graft function in the postoperative course.
Most recently, we applied the model for the study of ischemia-reperfusion injury and therapeutic interventions mitigating detrimental effects in the immediate postoperative course. Upon retrieval and 20 h of static cold storage, liver grafts (with a mean weight of 983.38 g) were implanted in the described manner. The experiments were terminated 6 h after portal-venous reperfusion and sampling of blood and bile as well as liver and bile duct tissue at defined intervals. All recipients survived the engraftment and the subsequent 6 h follow-up under general anesthesia until euthanasia.
Since the focus of this protocol lies in the feasibility of a porcine orthotopic liver transplant model without the use of a veno-venous bypass, the results presented here are limited to the intraoperative vital parameters and the application of vasopressors (Figure 2), as well as graft performance, defined by conventional laboratory parameters, i.e., serum concentrations of lactate, aspartate transaminase (AST), alanine transaminase (ALT), and glutamate dehydrogenase (GLDH), bile production (Table 1), and the liver maximum function capacity (LiMAx) test as described previously in a model of porcine liver resection (Figure 3)26. The LiMAx&#160;test is based on the real-time metabolism of intravenously injected 13C-methacetin by the liver-specific CYP1A2 system. Before and after injection, the ratio of 13CO212CO2 in the exhaled air is determined to quantify the individual hepatic function27.
As expected, recipients required increased concentrations of norepinephrine immediately before and throughout the anhepatic phase in order to stabilize the mean arterial pressure (MAD) at &#8805;60 mmHg. Low concentrations of epinephrine were simultaneously used to additionally increase cardiac output in this vulnerable time period. Upon portal-venous reperfusion, the need for vasopressors quickly declined and even more so during temporary clamping of the abdominal aorta for completion of the aortic anastomosis. After engraftment, the MAD and required doses of vasopressors remained stable.
Mean operation time, defined as the time from skin incision to completion of all vascular anastomosis and reperfusion, was 103.50 min, including a mean anhepatic phase of 27.13 min. Of note, only two recipients underwent an anhepatic phase of more than 30 min. All recipients showed declining lactate serum concentrations 4 h after portal-venous reperfusion, and LiMAx values obtained 6 h after portal-venous reperfusion were comparable to the values measured in the liver donors before organ procurement in all but one recipient (anhepatic phase of 34 min).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64152/64152fig01.jpg" /> 
Figure 1: Graft and recipient preparation. (A) The figure depicts the back-table preparation of the celiac axis and the aortic segment. (B) This figure shows the recipient in a supine position with extended monitoring, including a central venous catheter (blue) in the left internal jugular vein and an arterial catheter (red) in the right internal carotid/cervical artery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64152/64152fig01large.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/64152/64152fig02.jpg" /> 
Figure 2: Mean arterial pressure and concentrations of vasopressors required during engraftment. The figure depicts the mean arterial pressure (MAD in mmHg) measured and the concentrations of norepinephrine and epinephrine (in &#181;g/kg/h) during defined time periods throughout the procedure in all eight recipients. Values are presented as mean &#177; SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64152/64152fig02large.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/64152/64152fig03v3.jpg" /> 
Figure 3: Values of the liver maximum function capacity (LiMAx) test obtained from the donors prior to liver procurement and from the recipients 6 h after engraftment. The figure depicts box plot data (mean and standard error of the mean) from the liver maximum function capacity (LiMAx) test from the donors prior to liver procurement and from the recipients 6 h after engraftment (n = 8). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64152/64152fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td style="min-width: 60px;">Experiment</td>
			<td style="min-width: 60px;">Graft weight</td>
			<td style="min-width: 60px;">Recipient weight</td>
			<td style="min-width: 60px;">GRWR</td>
			<td style="min-width: 60px;">Operation time</td>
			<td style="min-width: 60px;">Anhepatic phase</td>
			<td colspan="3" style="min-width: 180px;">Lactate (mmol/L)</td>
			<td style="min-width: 60px;">Peak AST</td>
			<td style="min-width: 60px;">Peak ALT</td>
			<td style="min-width: 60px;">Peak GLDH</td>
			<td style="min-width: 60px;">Bile volume</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">No.</td>
			<td style="min-width: 60px;">(g)</td>
			<td style="min-width: 60px;">(kg)</td>
			<td style="min-width: 60px;">(%)</td>
			<td style="min-width: 60px;">(min)</td>
			<td style="min-width: 60px;">(min)</td>
			<td style="min-width: 60px;">2 h</td>
			<td style="min-width: 60px;">4 h</td>
			<td style="min-width: 60px;">6 h</td>
			<td style="min-width: 60px;">(U/L)</td>
			<td style="min-width: 60px;">(U/L)</td>
			<td style="min-width: 60px;">(U/L)</td>
			<td style="min-width: 60px;">&#160;(mL)</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">1</td>
			<td style="min-width: 60px;">1082</td>
			<td style="min-width: 60px;">48.8</td>
			<td style="min-width: 60px;">2.22</td>
			<td style="min-width: 60px;">115</td>
			<td style="min-width: 60px;">25</td>
			<td style="min-width: 60px;">5.8</td>
			<td style="min-width: 60px;">4.7</td>
			<td style="min-width: 60px;">3.7</td>
			<td style="min-width: 60px;">677</td>
			<td style="min-width: 60px;">122</td>
			<td style="min-width: 60px;">39</td>
			<td style="min-width: 60px;">48</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">2</td>
			<td style="min-width: 60px;">946</td>
			<td style="min-width: 60px;">51.4</td>
			<td style="min-width: 60px;">1.84</td>
			<td style="min-width: 60px;">125</td>
			<td style="min-width: 60px;">34</td>
			<td style="min-width: 60px;">6.6</td>
			<td style="min-width: 60px;">5.9</td>
			<td style="min-width: 60px;">5.2</td>
			<td style="min-width: 60px;">1207</td>
			<td style="min-width: 60px;">109</td>
			<td style="min-width: 60px;">268</td>
			<td style="min-width: 60px;">15</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">3</td>
			<td style="min-width: 60px;">957</td>
			<td style="min-width: 60px;">57.6</td>
			<td style="min-width: 60px;">1.66</td>
			<td style="min-width: 60px;">110</td>
			<td style="min-width: 60px;">30</td>
			<td style="min-width: 60px;">8.3</td>
			<td style="min-width: 60px;">5.8</td>
			<td style="min-width: 60px;">8.1</td>
			<td style="min-width: 60px;">742</td>
			<td style="min-width: 60px;">125</td>
			<td style="min-width: 60px;">143</td>
			<td style="min-width: 60px;">73</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">4</td>
			<td style="min-width: 60px;">825</td>
			<td style="min-width: 60px;">49.2</td>
			<td style="min-width: 60px;">1.68</td>
			<td style="min-width: 60px;">87</td>
			<td style="min-width: 60px;">22</td>
			<td style="min-width: 60px;">7.6</td>
			<td style="min-width: 60px;">6.7</td>
			<td style="min-width: 60px;">6.5</td>
			<td style="min-width: 60px;">675</td>
			<td style="min-width: 60px;">99</td>
			<td style="min-width: 60px;">113</td>
			<td style="min-width: 60px;">35</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">5</td>
			<td style="min-width: 60px;">1045</td>
			<td style="min-width: 60px;">53.4</td>
			<td style="min-width: 60px;">1.96</td>
			<td style="min-width: 60px;">101</td>
			<td style="min-width: 60px;">25</td>
			<td style="min-width: 60px;">7.9</td>
			<td style="min-width: 60px;">6.8</td>
			<td style="min-width: 60px;">5.6</td>
			<td style="min-width: 60px;">919</td>
			<td style="min-width: 60px;">86</td>
			<td style="min-width: 60px;">129</td>
			<td style="min-width: 60px;">25</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">6</td>
			<td style="min-width: 60px;">924</td>
			<td style="min-width: 60px;">45.2</td>
			<td style="min-width: 60px;">2.04</td>
			<td style="min-width: 60px;">105</td>
			<td style="min-width: 60px;">32</td>
			<td style="min-width: 60px;">6.7</td>
			<td style="min-width: 60px;">4.6</td>
			<td style="min-width: 60px;">3.7</td>
			<td style="min-width: 60px;">414</td>
			<td style="min-width: 60px;">90</td>
			<td style="min-width: 60px;">114</td>
			<td style="min-width: 60px;">75</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">7</td>
			<td style="min-width: 60px;">785</td>
			<td style="min-width: 60px;">48.2</td>
			<td style="min-width: 60px;">1.63</td>
			<td style="min-width: 60px;">95</td>
			<td style="min-width: 60px;">24</td>
			<td style="min-width: 60px;">6.8</td>
			<td style="min-width: 60px;">4.8</td>
			<td style="min-width: 60px;">4.1</td>
			<td style="min-width: 60px;">557</td>
			<td style="min-width: 60px;">70</td>
			<td style="min-width: 60px;">110</td>
			<td style="min-width: 60px;">1.5</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">8</td>
			<td style="min-width: 60px;">1303</td>
			<td style="min-width: 60px;">54.6</td>
			<td style="min-width: 60px;">2.39</td>
			<td style="min-width: 60px;">90</td>
			<td style="min-width: 60px;">25</td>
			<td style="min-width: 60px;">12.7</td>
			<td style="min-width: 60px;">12.2</td>
			<td style="min-width: 60px;">9.8</td>
			<td style="min-width: 60px;">1011</td>
			<td style="min-width: 60px;">87</td>
			<td style="min-width: 60px;">94</td>
			<td style="min-width: 60px;">10</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">MEAN</td>
			<td style="min-width: 60px;">983.38</td>
			<td style="min-width: 60px;">51.05</td>
			<td style="min-width: 60px;">1.93</td>
			<td style="min-width: 60px;">103.50</td>
			<td style="min-width: 60px;">27.13</td>
			<td style="min-width: 60px;">7.80</td>
			<td style="min-width: 60px;">6.44</td>
			<td style="min-width: 60px;">5.84</td>
			<td style="min-width: 60px;">775.25</td>
			<td style="min-width: 60px;">98.50</td>
			<td style="min-width: 60px;">126.25</td>
			<td style="min-width: 60px;">35.31</td>
		</tr>
		<tr>
			<td style="min-width: 60px;">SEM</td>
			<td style="min-width: 60px;">57.59</td>
			<td style="min-width: 60px;">1.41</td>
			<td style="min-width: 60px;">0.10</td>
			<td style="min-width: 60px;">4.57</td>
			<td style="min-width: 60px;">1.52</td>
			<td style="min-width: 60px;">0.76</td>
			<td style="min-width: 60px;">0.88</td>
			<td style="min-width: 60px;">0.78</td>
			<td style="min-width: 60px;">90.79</td>
			<td style="min-width: 60px;">6.73</td>
			<td style="min-width: 60px;">23.00</td>
			<td style="min-width: 60px;">9.87</td>
		</tr>
	</tbody>
</table>
Table 1: Perioperative graft and recipient variables. The table summarizes graft and recipient weight, as well as the graft-to-recipient weight ratio (GRWR) and the length of the operation (skin incision to completion of all vascular anastomosis and reperfusion) and of the anhepatic phase. Variables indicating graft function, such as conventional laboratory parameters, i.e., serum concentrations of lactate, aspartate transaminase (AST), alanine transaminase (ALT), and glutamate dehydrogenase (GLDH), and bile production are provided for each of the eight transplants performed.

Watch this Scientific Journal Video about Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model at JoVE.com

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

porcine-liver-transplantation-without-veno-venous-bypass-as-an

Nanyang Technological University

Research

JoVE Journal

Medicine

2.3K Views.  Hannover Medical School. Actuele problemen bij levertransplantatie vereisen de implementatie van grote diermodellen. Dit protocol beschrijft de techniek van orthotopische levertransplantatie bij varkens zonder het gebruik van een veno-veneuze bypass. Deze techniek vermindert de logistieke en technische complexiteit van de procedure aanzienlijk.Bovendien, door levers te enten na 20 uur statische koude opslag, simuleert ons model uitgebreide criteria donorcondities. Begin met het plaatsen van het verdoofde donor...

Video: Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

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 ...

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

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 ...

Bioengineering

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Development and Validation of a Porcine Ex Situ Isolated Liver Perfusion Model 

Porcine Normothermic Isolated Liver Perfusion

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost.
In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 &#176;C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.

The porcine model of liver normothermic machine perfusion (NMP), described here, can be successfully used to study NMP as a preservation strategy, a tool for viability assessment, and a platform for organ repair. It holds a high translational value, however it is technically challenging and labor-intensive.

Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

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