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.

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

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

Machine perfusion technology has advanced the preservation of donor liver quality, especially for expanded criteria livers, but continuously pumping oxygen and nutrients via a centrifugal pump and oxygenator, it remains organ activity and monitors liver function, improving preservation time and transportation success rates. The main challenges in using machine perfusion for donor livers are replicating liver conditions with sophisticated equipments and skilled operations. Limitations in post-site DCD liver models includes deterioration in liver quality, storage duration, and a hepatic phase, leading to lower success rates in experimental models.To overcome challenges, we have developed a machine profusion device with variable temperature control, and we find such techniques to extend the liver preservation while minimizing ischemic damage. This advancement enhance the success of our post-site liver transplant model and to promote further preclinical use of machine profusion.

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...

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 ...

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

Research

JoVE Journal

Bioengineering

Temperature-Controlled Machine Perfusion for Preservation of Donor Porcine Liver

Begin the procedure on an anesthetized pig placed in a supine position on a surgical table by performing a midline laparotomy, extended laterally to the right on the disinfected skin. Carefully dissect the hepatic artery and portal vein from the surrounding tissue. Then, mobilize the celiac axis and trace it to the abdominal aorta.Dissect the abdominal aorta and the inferior vena cava to facilitate blood collection. Sequentially, cannulate the abdominal aorta and inferior vena cava. Collect the blood into acid citrate dextrose bags for subsequent use.Then, fit the portal vein with a specific catheter. Next, excise the liver from the animal after inducing cardiac arrest. Preserve a section of abdominal aorta tissue for arterial cannulation.Place the liver in a sterile organ bag on ice. Ligate all distal arterial branches of the liver before cannulating the common bile duct. Connect the catheter of the portal vein and abdominal aorta to a machine profusion device.Perfuse the liver with 1.5 liters of perfusion solution. Set the perfusion temperature to 4 degrees celsius and time to 8 hours. Then, set the perfusion parameters so that the hepatic artery is under pressure control of 25 millimeters of mercury, and the portal vein is under flow control of 200 milliliters per minute.While the device is in the normal thermic machine profusion mode, increase the system's temperature to 37 degrees Celsius before priming it. Transfer the porcine donor liver into the device once it has been warmed for 10 minutes. Set the arterial profusion pressures and the portal vein perfusion.Increase the portal vein perfusion pressure to 0.75 milliliters per minute per gram after the first hour. After removing the donor liver from the device, insert a catheter into the portal vein to perfuse it sequentially via the portal vein catheter. Next, insert a specialized catheter into the portal vein of the recipient liver and ligate the catheter to secure it to the matching catheter on the donor portal vein.With double armed 4-0 monofilament polypropylene sutures perform an end-to-end anastomosis of the suprahepatic cava. Connect both ends the portal vein with matching cannulas to restore blood flow. After removing the clamp from the suprahepatic cava, perform an end-to-end anastomosis of the inferior vena cava with 4-0 monofilament polypropylene sutures.Flush the donor hepatic artery with 10 milliliters of heparinized saline and posit an additional bulldog clamp distally to prevent back bleeding. Finally, after removing the portal vein catheter, anastomose the portal vein with a 4-0 monofilament polypropylene suture.

205 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.

Orthotopic Pig Liver Transplant Preservation Using Machine Perfusion

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