The study was supported by the Key Scientific Research Program for the development of ex vivo Liver Perfusion System of Foshan City, China[(2020)A007]; Guang Dong Basic and Applied Basic Research Foundation (2020B1515120031); Guang Zhou Scientific Research Foundation (202002030201).

All animal experiments were conducted in accordance with the Experimental Animal Management Ordinance (Ministry of Science and Technology of the People&#39;s Republic of China, 2017). Bama female miniature pigs (40-45 kg) were used. The study protocol was approved by the Institutional Animal Care and Use Committee of the General Hospital of Southern Theater Command of PLA, China. The pigs were housed in the research facility for 1 week before transplantation and then fasted but with free access to water for 12 h before the experiment. The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Donor acquisition
<ol>
	<li>Induction of anesthesia and analgesia: Administer atropine intramuscularly at a dosage of 0.02 mg/kg. Subsequently, administer the Zoletil 50 intramuscularly in the range of 2-3.5 mg/kg to induce sedation. For analgesia, administer Tramadol hydrochloride intravenously at a dose of 2 mg/kg.</li>
	<li>Induce general anesthesia by an intravenous infusion of propofol at a rate of 2-3 mg/kg/h, using a 24 G butterfly cannula inserted into an external marginal ear vein.</li>
	<li>Position the pig supine on the surgical table. Perform endotracheal intubation and mechanical ventilation. Continuous monitoring of the heart rate and oxygen saturation is carried out via pulse oximetry placed on the tail. Set the concentration of isoflurane in the vaporizer at 2%.</li>
	<li>Apply 3% iodine solution to disinfect the skin in the area. After allowing the iodine solution to air dry naturally, wipe it off with 70% alcohol. Repeat the disinfection process three times.</li>
	<li>Performed a midline laparotomy, extended laterally to the right. Release the liver from its ligamentous attachments, isolate the bile duct, and sever near the duodenum post-ligation.</li>
	<li>Carefully dissect the hepatic artery (HA) and portal vein (PV) from the surrounding tissue. Mobilize the celiac axis and trace to the abdominal aorta.</li>
	<li>Dissect the abdominal aorta (AA) and the inferior vena cava (IVC) to facilitate blood collection. Administer an intravenous bolus of heparin sodium (25,000 U) for anticoagulation.</li>
	<li>Cannulate the AA and IVC sequentially and collect the blood into acid-citrate dextrose bags for subsequent use for NMP (approximately 1800-2000 mL).&#160;Fit the PV with a specific catheter. Store the collected blood at a temperature of 4 &#176;C.</li>
	<li>Induce the cardiac arrest through the intracardiac infusion of potassium chloride (20 mEq). 
	NOTE: The time interval commencing with cardiac arrest is recorded as Warm Ischemia Time (WIT). The liver undergoes 30 min of WIT without any manipulation, followed by in situ flushing through the abdominal aorta (AA) and portal vein (PV) with 2 L of cold hypertonic citrate adenine solution.</li>
	<li>Excise the liver, ensuring all remaining vessels are long. Preserve a section of abdominal aorta tissue for arterial cannulation. Fit the PV with a specific catheter.</li>
	<li>Place the liver in a sterile organ bag on ice. Ligate all distal arterial branches of the liver and cannulate the common bile duct.</li>
</ol>
2. Initiation with DHOPE mode
<ol>
	<li>Connect the catheter of the portal vein and the abdominal aorta to the MP device. Perfuse the liver with 1.5 L of the University of Wisconsin Machine Perfusion Solution (UW-MPS, see Table of Materials), enriched with heparin sodium (6250 U) and cefoxitin sodium (1 g).</li>
	<li>Set the perfusion parameters that HA is maintained under pressure control at 25 mmHg, while PV is under flow control at 200 mL/min. Ensure the oxygen saturation by continuously pumping 100% oxygen into the perfusate at a rate of 1 L/min. 
	NOTE: The system autonomously monitors and logs key parameters such as the perfusate temperature, HA and PV pressures, and flow rates throughout the perfusion process.</li>
	<li>Set the perfusion mode at a temperature of 4&#176; C. Perfuse the liver graft for 8 h (Figure 1).</li>
</ol>
3. Perfusion with NMP mode
<ol>
	<li>Transit the machine perfusion device to the NMP mode. Elevate the system&#39;s temperature to 37 &#176;C. Prime the machine with 2 L of a mixture consisting of whole blood and perfusate of Machine Perfusion.</li>
	<li>Flush the donor with 1.5 L of normal saline solution at 4 &#176;C and place in a bowl with ice. Change the perfusion fluid and prime the same machine for NMP mode. Transfer the liver to the device once the warming phase is complete for 10 min (Figure 1).</li>
	<li>Establish a constant oxygen flow to both the portal vein and hepatic artery prior to liver placement. Maintain a Fraction of Inspired Oxygen (FiO2) at 60%.</li>
	<li>Set the arterial perfusion pressures at 80/60 mmHg (systolic pressure/diastolic pressure). Set the portal vein perfusion to a constant flow of 0.5 mL/min/g (liver weight), subsequently increasing it to 0.75 mL/min/g (liver weight) after the first hour.</li>
	<li>Throughout the 6 h of NMP, consistently monitor parameters such as pressure, flow rates, and temperature. Perform a viability test via a biochemical assessment of the perfusate at 1 h intervals, including Blood gas analysis, lactic acid, blood glucose, and liver function.</li>
</ol>
4. Recipient hepatectomy
<ol>
	<li>Inject a Zoletil 50 (2-3.5 mg/kg) and atropine (0.02 mg/kg) intramuscularly to the recipient pig. Administer an intravenous dose of Tramadol hydrochloride (2 mg/kg) for pain management.</li>
	<li>Induce general anesthesia via an intravenous propofol infusion (2-3 mg/kg/h). The pig is then oriented in a supine position on a surgical table equipped with a heating mat. The isoflurane vaporizer is set to 2%.</li>
	<li>Perform a midline laparotomy, extended laterally to the right. Cover the large and small intestines with a sterile towel. Facilitate the placement of an abdominal retractor for full visibility.</li>
	<li>Free the liver from its ligamentous attachments. Isolated the bile duct, ligate, and sever. Dissect the hepatic artery retrogradely up to the division of the gastroduodenal artery. Clamp the common hepatic artery proximally to the gastroduodenal artery using a bulldog clamp. Free the portal vein (PV) from the adherent tissue and clamp it on the distal side.</li>
	<li>Clamp the upper side of the vena cava, followed by the dissection of the upper part of the vena cava on the diaphragm side, reserving some intrahepatic vena cava tissue for subsequent suturing. The inferior part of the vena cava is similarly treated, conserving some liver tissue on the vena cava. Remove the liver.</li>
	<li>Perform immunosuppression by intravenously injecting a 500 mg dose of methylprednisolone.</li>
</ol>
5. Orthotopic graft placement and vascular anastomosis
<ol>
	<li>Remove the donor liver from the machine perfusion device. Fit the PV with a specific catheter and subsequently perfuse with cold saline at 4 &#176;C from the PV catheter. Perfuse with 5% Albumin combined with cold saline at 4 &#176;C from the PV catheter. This process is crucial for the elimination of blood and the cooling down of the liver.</li>
	<li>Insert a specialized catheter into the portal vein and secure it by ligation prior to removal of the recipient&#39;s original liver, enabling connectivity to the matching catheter on the donor PV. Concurrently, clamp the inferior vena cava of the donor graft (Figure 2B).</li>
	<li>Perform an end-to-end anastomosis of the suprahepatic cava using double-armed 4-0 monofilament polypropylene sutures.</li>
	<li>Connect both ends of the portal vein with matching cannulas to restore blood flow. Heparin saline is flushed into the cannula prior to insertion. Intermittent opening of the portal vein and temporary unclamping of the inferior vena cava facilitate the flushing of residual perfusion fluid.</li>
	<li>Remove the clamp from the suprahepatic cava, and perform an end-to-end anastomosis of the inferior vena cava with 4-0 monofilament polypropylene sutures.</li>
	<li>Flush the donor hepatic artery with 10 mL of heparinized saline and posit an additional bulldog clamp distally to prevent back bleeding. Perform an end-to-end anastomosis using a 7-0 monofilament polypropylene suture.</li>
	<li>Insert a catheter into the biliary tract at both donor and recipient ends, with catheters securely sutured to ensure position stability.</li>
	<li>Remove the portal vein catheter. Perform an end-to-end anastomosis of the portal vein using a 4-0 monofilament polypropylene suture.</li>
</ol>
6. Post-transplantation care and monitoring
<ol>
	<li>Maintain the ventilation of the recipient pig for another 2 h.</li>
	<li>Turn on the air-conditioning of the intensive care unit to raise the indoor temperature. A heating pad is used to keep the pig warm.</li>
	<li>Collect the blood samples regularly to test blood gas and liver and kidney function at 24 h interval.</li>
	<li>Seal the indwelling needle with heparin saline. The indwelling needle of the pig auricular vein must be properly managed to avoid falling off after pig movement.</li>
	<li>Terminate the ventilation once the pig is able to breathe fully.</li>
	<li>Return the recipient pig to the piggery and place it in a wooden bed about 10 cm high until awake from anesthesia. The bottom of the wooden bed is padded with a towel to prevent urine from accumulating around the animal&#39;s body, and the temperature is maintained by a 24 h heater in the pigsty.</li>
	<li>Inject the methylprednisolone at an initial dose of 250 mg from POD 1, followed by a gradual dose reduction for Immunosuppression.</li>
	<li>Administer pain medication intravenously on POD 1 (buprenorphine 0.01-0.05 mg/kg). Intramuscularly administer tramadol on POD2-POD5 (100 mg/kg every 12 h). Provide oral dose cephalosporin (2 mg/kg) twice a day from POD 2, and add fluids of nutrition. Water intake is not limited.</li>
	<li>Remove the venous channel about 5 days after surgery.</li>
	<li>Observe the urine volume and color. Observe the color change of stool after the operation.</li>
	<li>Sacrifice the pigs if they are suffered from persistent acidosis, hypoglycemia, signs of hemorrhage or liver failure.</li>
	<li>Perform the euthanasia at 5 days after OLTx by exsanguination under deep isoflurane anesthesia (5%, &#62;2.5 MAC).</li>
</ol>
7. Technique of SCS control porcine model
<ol>
	<li>Employ identical procedures for anesthesia as above. Dissect the abdominal aorta (AA) and inferior vena cava (IVC) to aid blood drainage, utilizing the same heparinization method. Sequentially cannulate the AA and IVC. Rapidly induce cardiac arrest through intracardiac infusion of potassium chloride (20 mEq).</li>
	<li>Obtain the donor liver promptly following 30 min of WIT, preserving adequate tissue from the proximal hepatic inferior vena cava.</li>
	<li>Place the liver graft in a preservation bag and immerse it in cold University of Wisconsin (UW) solution for preservation at 4 &#176;C for 8 h.</li>
	<li>Preserve the liver in NMP at 37 &#176;C for 6 h, while perform OLTx directly with the other froup (Figure 1).</li>
</ol>

Orthotopic Pig Liver Transplant Preservation Using Machine Perfusion

<ol>
	<li>de Goeij, F., Schlegel, A., Muiesan, P., Guarrera, J. V., Dutkowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothermic+oxygenated+machine+perfusion+protects+from+cholangiopathy+in+donation+after+circulatory+death+liver+transplantation.">Hypothermic oxygenated machine perfusion protects from cholangiopathy in donation after circulatory death liver transplantation.</a> Hepatology. 74 (6), 3525-3528 (2021).</li><li>Dutkowski, 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=Evolving+trends+in+machine+perfusion+for+liver+transplantation.">Evolving trends in machine perfusion for liver transplantation.</a> Gastroenterology. 156 (6), 1542-1547 (2019).</li><li>Parente, 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=Machine+perfusion+techniques+for+liver+transplantation:+A+meta-analysis+of+the+first+seven+randomized-controlled+trials.">Machine perfusion techniques for liver transplantation: A meta-analysis of the first seven randomized-controlled trials.</a> J Hepatol. 79 (5), 1201-1213 (2023).</li><li>Schlegel, 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=Outcomes+of+DCD+liver+transplantation+using+organs+treated+by+hypothermic+oxygenated+perfusion+before+implantation.">Outcomes of DCD liver transplantation using organs treated by hypothermic oxygenated perfusion before implantation.</a> J Hepatol. 70 (1), 50-57 (2019).</li><li>Zhang, Z. B., 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+machine+perfusion+protects+against+liver+ischemia-reperfusion+injury+during+reduced-size+liver+transplantation+in+pigs.">Normothermic machine perfusion protects against liver ischemia-reperfusion injury during reduced-size liver transplantation in pigs.</a> Ann Transplant. 24, 9-17 (2019).</li><li>Minor, 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=Hypothermic+reconditioning+by+gaseous+oxygen+improves+survival+after+liver+transplantation+in+the+pig.">Hypothermic reconditioning by gaseous oxygen improves survival after liver transplantation in the pig.</a> Am J Transplant. 11 (12), 2627-2634 (2011).</li><li>Linares-Cervantes, 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=Predictor+parameters+of+liver+viability+during+porcine+normothermic+ex+situ+liver+perfusion+in+a+model+of+liver+transplantation+with+marginal+grafts.">Predictor parameters of liver viability during porcine normothermic ex situ liver perfusion in a model of liver transplantation with marginal grafts.</a> Am J Transplant. 19 (11), 2991-3005 (2019).</li><li>Fu, Y., 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=Porcine+partial+liver+transplantation+without+veno-venous+bypass:+an+effective+model+for+small-for-size+liver+graft+injury.">Porcine partial liver transplantation without veno-venous bypass: an effective model for small-for-size liver graft injury.</a> Transplant Proc. 43 (5), 1953-1961 (2011).</li><li>Brockmann, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+of+reperfusion+influences+ischemia/reperfusion+injury+and+primary+graft+function+following+porcine+liver+transplantation.">Sequence of reperfusion influences ischemia/reperfusion injury and primary graft function following porcine liver transplantation.</a> Liver Transpl. 11 (10), 1214-1222 (2005).</li><li>Spetzler, V. N., 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=Technique+of+porcine+liver+procurement+and+orthotopic+transplantation+using+an+active+porto-caval+shunt.">Technique of porcine liver procurement and orthotopic transplantation using an active porto-caval shunt.</a> J Vis Exp. (99), e52055 (2015).</li><li>Ceresa, C., Nasralla, D., Pollok, J. M., 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=Machine+perfusion+of+the+liver:+applications+in+transplantation+and+beyond.">Machine perfusion of the liver: applications in transplantation and beyond.</a> Nat Rev Gastroenterol Hepatol. 19 (3), 199-209 (2022).</li><li>Martins, P. N., Buchwald, J. E., Mergental, H., Vargas, L., 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=The+role+of+normothermic+machine+perfusion+in+liver+transplantation.">The role of normothermic machine perfusion in liver transplantation.</a> Int J Surg. 82, 52-60 (2020).</li><li>Br&#252;ggenwirth, 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=Prolonged+dual+hypothermic+oxygenated+machine+preservation+(DHOPE-PRO)+in+liver+transplantation:+Study+protocol+for+a+stage+2,+prospective,+dual-arm,+safety+and+feasibility+clinical+trial.">Prolonged dual hypothermic oxygenated machine preservation (DHOPE-PRO) in liver transplantation: Study protocol for a stage 2, prospective, dual-arm, safety and feasibility clinical trial.</a> BMJ Open Gastroenterol. 9 (1), 000842 (2022).</li><li>Mergental, 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=Transplantation+of+discarded+livers+following+viability+testing+with+normothermic+machine+perfusion.">Transplantation of discarded livers following viability testing with normothermic machine perfusion.</a> Nat Commun. 11 (1), 2939 (2020).</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>Melandro, 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=Viability+criteria+during+liver+ex-situ+normothermic+and+hypothermic+perfusion.">Viability criteria during liver ex-situ normothermic and hypothermic perfusion.</a> Medicina (Kaunas). 58 (10), 1434 (2022).</li><li>Warmuzi&#324;ska, N., &#321;uczykowski, K., Bojko, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+current+and+emerging+trends+in+donor+graft-quality+assessment+techniques.">Review of current and emerging trends in donor graft-quality assessment techniques.</a> J Clin Med. 11 (3), 487 (2022).</li><li>OuYang, Q., 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=Evaluation+of+the+ex+vivo+liver+viability+using+a+nuclear+magnetic+resonance+relaxation+time-based+assay+in+a+porcine+machine+perfusion+model.">Evaluation of the ex vivo liver viability using a nuclear magnetic resonance relaxation time-based assay in a porcine machine perfusion model.</a> Sci Rep. 11 (1), 4117 (2021).</li><li>Sampaziotis, 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=Cholangiocyte+organoids+can+repair+bile+ducts+after+transplantation+in+the+human+liver.">Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.</a> Science. 371 (6531), 839-846 (2021).</li><li>van Leeuwen, O. B., 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=Transplantation+of+high-risk+donor+livers+after+ex+situ+resuscitation+and+assessment+using+combined+hypo-+and+normothermic+machine+perfusion:+A+prospective+clinical+trial.">Transplantation of high-risk donor livers after ex situ resuscitation and assessment using combined hypo- and normothermic machine perfusion: A prospective clinical trial.</a> Ann Surg. 270 (5), 906-914 (2019).</li><li>van Leeuwen, O. B., 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=Sequential+hypothermic+and+normothermic+machine+perfusion+enables+safe+transplantation+of+high-risk+donor+livers.">Sequential hypothermic and normothermic machine perfusion enables safe transplantation of high-risk donor livers.</a> Am J Transplant. 22 (6), 1658-1670 (2022).</li></ol>

The authors have nothing to disclose.

Liver MP is currently extensively utilized in clinical trials, but further preclinical research using large animal models remains necessary5,6. Porcine OLTx presents significant challenges that result in low success rates. These challenges encompass warm ischemia of the donor, anatomical variations, and intolerance to prolonged clamping of the vena cava and portal vein7,8. The implantation phase continues...

Liver transplantation remains the sole curative treatment for end-stage liver disease and selected liver cancers. Despite significant advancements in procurement, preservation, operative techniques, and post-transplant immunosuppression, a notable mortality rate persists among patients on the waiting list due to a shortage of suitable donor organs. A primary challenge lies in preserving livers procured from DCD, as these organs necessitate specialized care to mitigate ischemic injuries1. Ex vivo liver machine perfusion offers a unique method to both preserve and evaluate DCD liver grafts before transplantation2. Clinical trials have underscored the feasibility and safety of ex vivo liver machine perfusion for both standard and expanded criteria donors, employing either hypothermic or normothermic conditions3. Importantly, therapeutic interventions during ex vivo liver machine perfusion have shown promise in reducing ischemia-reperfusion injury (IRI)4.
In efforts to extend the preservation duration and enhance the quality of DCD liver grafts, ongoing animal experiments aim to optimize the performance of MP devices and refine the method of ex vivo liver preservation5. Porcine OLTx serves as an optimal model for clinically oriented research, validating the preservative quality of MP. However, ischemic injury to the donor, hemodynamic instability, and intestinal congestion during the anhepatic phase of porcine OLTx collectively impact the survival rate of the porcine model6,7.
A variable temperature-controlled MP device that integrates both NMP and DHOPE modes was utilized to preserve the DCD livers of porcine in the following protocol. This device facilitates extended ex vivo preservation of the DCD livers and alleviates the ischemic injury of the donor liver compared to traditional SCS. The apparatus ensures temperature regulation, supports long-distance transportation, and provides bionic perfusion alongside dynamic and accurate assessment of donor quality. The protocol contains all the information for a stable model of DCD liver preservation utilizing a sequential DHOPE-NMP mode followed by porcine OLTx, including the transition of perfusion settings, optimizing the anastomosis technique, and the procedure of the anhepatic phase.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia respirator</td>
			<td>Mindray,Shenzhen</td>
			<td>WATO EX-20&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic biochemical analyzer</td>
			<td>&#160;MNCHIP, China</td>
			<td>&#160;Celercare V5</td>
			<td></td>
		</tr>
		<tr>
			<td>Bama female miniature pigs&#160;</td>
			<td>Pearl Lab Animal Sci &#38; Tech Co,Ltd (Guangdong, China).&#160;</td>
			<td></td>
			<td>40-45 kg&#160;</td>
		</tr>
		<tr>
			<td>Blood gas analyzer&#160;</td>
			<td>&#160;Abbott</td>
			<td>i-STAT300</td>
			<td></td>
		</tr>
		<tr>
			<td>ECG monitor</td>
			<td>Shenzhen Ericon Medical Equipment Co., LTD China</td>
			<td>M-9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fully automatic snowflake ice</td>
			<td>Changshu Shenghai Electric Co., Ltd. China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusate in NMP</td>
			<td></td>
			<td></td>
			<td>5% Human serum albumin 100-150 mL, 
			Whole blood 1.2-1.5 L, 
			2.5% NaHCO3 21 mL, 
			10% CaCL2 7mL , 
			Heparin 5000 U , 
			Cefoxltin 1 g, 
			Metronidazole 500 mg, 
			Sodium taurocholate 5 g, 
			Short acting insulin 72 U, 
			Total parenteral nutrition solution 250-500 mL.</td>
		</tr>
		<tr>
			<td>Potal catheter</td>
			<td>Jinxin technology,Shunde,China</td>
			<td></td>
			<td>Portal vein catheter for custom cannula of varying internal diameter (6-8.5 mm)</td>
		</tr>
		<tr>
			<td>Refrigeration centrifuge&#160;</td>
			<td>hermo Fisher Scientific - CN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The CG8/CG4 blood gas test card</td>
			<td>&#160;Abbott</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The CHEM 8 test card</td>
			<td>&#160;Abbott</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The ex vivo liver machine perfuion device</td>
			<td>Devocean Medical Instrument Co., Ltd, Guangdong, China</td>
			<td>DEVOCEAN-LIVER 2000</td>
			<td>This is a multi-mode, temperature-controlled, biomimetic ex vivo liver machine perfusion device, capable of preserving the liver outside the body for 24 h</td>
		</tr>
		<tr>
			<td>UW Cold Storage solution&#160;&#160;</td>
			<td>Bridge to Life, Ltd., USA</td>
			<td>Belzer UW</td>
			<td>Liver in SCS group were preserved in UW Cold Storage solution&#160;</td>
		</tr>
		<tr>
			<td>UW Machine Perfusion Solution</td>
			<td>Bridge to Life, Ltd., USA</td>
			<td>Belzer MPS</td>
			<td>&#160;Adenine (free base) 0.68 g, 
			Calcium Chloride (dihydrate) 0.068 g, 
			Dextrose (+) 1.80 g, 
			Glutathione (reduced) 0.92 g, 
			HEPES (free acid) 2.38 g, 
			Hydroxyethyl Starch 50.0 g, 
			Magnesium Gluconate 1.13 g, 
			Mannitol 5.4 g, 
			Potassium Phosphate (monobasic) 3.4 g, 
			Ribose, D(-) 0.75 g, 
			Sodium Gluconate 17.45 g, 
			Sodium Hydroxide 0.70 g, 
			Sterile Water for Injection To 1000 mL Volume</td>
		</tr>
		<tr>
			<td>Vacuum extractor</td>
			<td>SMAF</td>
			<td>DYX-2A</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

DCD livers underwent a DHOPE-NMP procedure as a protective measure before being transplanted into recipient pigs. The procedure of DHOPE-NMP was as follows: DCD livers (n = 8) with 30 min WIT were preserved in DHOPE for 8 h at the first stage, followed by transfer to NMP mode for another 6 h. Subsequently, these grafts were utilized for LT in porcine recipients. The schematic picture describing the groups and the protocol is shown in Figure 1. The schematic representation of the principal st...

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.

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 ...

preservation-porcine-donation-after-circulatory-death-dcd-liver

Research

JoVE Journal

Bioengineering

277 Views.  PLA General Hospital of Southern Theatre Command: People's Liberation Army General Hospital of Southern Theatre Command. 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.

Video: Author Spotlight: Advancements and Challenges in Machine Perfusion for Liver 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 ...

Medicine

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 ...

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 ...

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

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

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

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

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.

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 ...

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.

Author Spotlight: Liver Graft Preservation Through Normothermic Machine Perfusion

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 ...