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

Abstract

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.

Introduction

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 model^6,7.

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.

Protocol

All animal experiments were conducted in accordance with the Experimental Animal Management Ordinance (Ministry of Science and Technology of the People'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

1. 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.
2. 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.
3. 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%.
4. 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.
5. 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.
6. Carefully dissect the hepatic artery (HA) and portal vein (PV) from the surrounding tissue. Mobilize the celiac axis and trace to the abdominal aorta.
7. 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.
8. Cannulate the AA and IVC sequentially and collect the blood into acid-citrate dextrose bags for subsequent use for NMP (approximately 1800-2000 mL). Fit the PV with a specific catheter. Store the collected blood at a temperature of 4 °C.
9. 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.
10. 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.
11. Place the liver in a sterile organ bag on ice. Ligate all distal arterial branches of the liver and cannulate the common bile duct.

2. Initiation with DHOPE mode

1. 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).
2. 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.
3. Set the perfusion mode at a temperature of 4° C. Perfuse the liver graft for 8 h (Figure 1).

3. Perfusion with NMP mode

1. Transit the machine perfusion device to the NMP mode. Elevate the system's temperature to 37 °C. Prime the machine with 2 L of a mixture consisting of whole blood and perfusate of Machine Perfusion.
2. Flush the donor with 1.5 L of normal saline solution at 4 °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).
3. 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%.
4. 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.
5. 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.

4. Recipient hepatectomy

1. 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.
2. 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%.
3. 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.
4. Free the liver from its ligamentous attachments. Isolated the bile duct, ligate, and severe. 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.
5. 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.
6. Perform immunosuppression by intravenously injecting a 500 mg dose of methylprednisolone.

5. Orthotopic graft placement and vascular anastomosis

1. Remove the donor liver from the machine perfusion device. Fit the PV with a specific catheter and subsequently perfuse with cold saline at 4 °C from the PV catheter. Perfuse with 5% Albumin combined with cold saline at 4 °C from the PV catheter. This process is crucial for the elimination of blood and the cooling down of the liver.
2. Insert a specialized catheter into the portal vein and secure it by ligation prior to removal of the recipient'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).
3. Perform an end-to-end anastomosis of the suprahepatic cava using double-armed 4-0 monofilament polypropylene sutures.
4. 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.
5. 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.
6. 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.
7. Insert a catheter into the biliary tract at both donor and recipient ends, with catheters securely sutured to ensure position stability.
8. Remove the portal vein catheter. Perform an end-to-end anastomosis of the portal vein using a 4-0 monofilament polypropylene suture.

6. Post-transplantation care and monitoring

1. Maintain the ventilation of the recipient pig for another 2 h.
2. 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.
3. Collect the blood samples regularly to test blood gas and liver and kidney function at 24 h interval.
4. 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.
5. Terminate the ventilation once the pig is able to breathe fully.
6. 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's body, and the temperature is maintained by a 24 h heater in the pigsty.
7. Inject the methylprednisolone at an initial dose of 250 mg from POD 1, followed by a gradual dose reduction for Immunosuppression.
8. Administer pain medication intravenously on POD 1 (buprenorphine 0.01-0.05 mg/kg). Provide oral dose cephalosporin (2 mg/kg) twice a day from POD 2, and add fluids of nutrition. Water intake is not limited.
9. Remove the venous channel about 5 days after surgery.
10. Observe the urine volume and color. Observe the color change of stool after the operation.
11. Sacrifice the pigs if they are suffered from persistent acidosis, hypoglycemia, signs of hemorrhage or liver failure.
12. Perform the euthanasia at 5 days after OLTx by exsanguination under deep isoflurane anesthesia (5%, >2.5 MAC).

7. Technique of SCS control porcine model

1. 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).
2. Obtain the donor liver promptly following 30 min of WIT, preserving adequate tissue from the proximal hepatic inferior vena cava.
3. Place the liver graft in a preservation bag and immerse it in cold University of Wisconsin (UW) solution for preservation at 4 °C for 8 h.
4. Preserve the liver in NMP at 37 °C for 6 h, while perform OLTx directly with the other froup (Figure 1).

Representative Results

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 steps involved in the transplantation procedure is depicted in Figure 2A-D. As a comparative measure, liver grafts (n = 8) with 30 min WIT due to cardiac death and preserved in SCS for 8 h were directly transplanted into recipient pigs as a control group. It was observed that three of the control recipient pigs died within 36 h after transplantation (Supplementary Figure 1). The awakening time for these pigs in the control group was prolonged, and during the observation period, they showed a decrease in appetite and a decline in vitality. Five of the control recipient pigs survived until the end of the follow-up period on day 5 after transplantation. However, in the group that underwent DHOPE-NMP, six out of eight recipient pigs survived for 5 days. One pig in the DHOPE-NMP group died due to bile leakage 3 days after transplantation, while another died of cardiac arrest 24 h after OLTx. Blood samples were collected at 24 h intervals after surgery through the indwelling deep vein, and tests for ALT and total bilirubin levels were conducted (Figure 3A,B).

Figure 1: An overview of the protocol. The procedure for preservation of DCD liver grafts with WIT of 30 min in the SCS group (preserved in UW Cold Storage solution), and DHOPE-NMP group before being transplanted into recipient pigs is depicted here. (A) Representative image of a liver graft connected to the DHOPE at 4 °C. (B) Liver graft connected to the NMP at 37 °C. (C) Schematic describing the procedure, settings, and animal groups. Please click here to view a larger version of this figure.

Figure 2: Schematic representation of a porcine OLTx model using MP. (A) The hepatic artery and portal vein of the donor liver are connected to cannulas attached to the MP apparatus, and the bile flow is monitored in real time. (B) The recipient liver is isolated, and a specialized cannula is inserted into the recipient's end of the portal vein. (C) Once the donor liver is correctly positioned, the suprahepatic vena cava is anastomosed. Concurrently, the infrahepatic inferior vena cava is clamped, and the donor's portal vein cannula is connected to the matching recipient-end cannula to restore the portal venous blood flow. (D) The infrahepatic inferior vena cava is anastomosed, and then the hepatic artery and bile duct are anastomosed. Please click here to view a larger version of this figure.

Figure 3: Assessment of total bilirubin and ALT after porcine OLTx in SCS group (n = 5) and DHOPE-NMP group (n = 6). (A,B) Analysis of total bilirubin and ALT after porcine OLTx at the indicated time points. Data represent mean ± SD. Two-way ANOVA was used to analyze the differences between the two groups. DHOPE-NMP group vs. SCS group: *P < 0.05. Please click here to view a larger version of this figure.

Supplementary Figure 1: Kaplan-Meier survival curve of porcine OLTx following SCS group (n = 8) and DHOPE-NMP group (n = 8). Please click here to download this File.

Supplementary Figure 2: Assessment of portal vein pressure (PV pressure), serum lactate, and ALT during NMP after 8 h of DHOPE (n = 6) or SCS (n = 6) treatments. (A-C) Analysis of PV pressure, lactate, and ALT during NMP at the indicated time points. Data represent mean ± SD. Two-way ANOVA was used to analyze the differences between the two groups. DHOPE group vs. SCS group: *P < 0.05, **P < 0.01. Please click here to download this File.

Discussion

Liver MP is currently extensively utilized in clinical trials, but further preclinical research using large animal models remains necessary^5,6. 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 vein^7,8. The implantation phase continues to be a substantial obstacle in porcine OLTx for DCD donors, with complications such as arrhythmia, shock, metabolic disturbances, and hyperkalemia arising from intestinal hyperemia, insufficient venous return, and reperfusion-related injury⁹. Various techniques have been employed to alleviate inferior vena cava blood flow congestion during the anhepatic phase. Passive portal and jugular vein bypasses have demonstrated the ability to maintain hemodynamic stability in pigs during this phase¹⁰. The bypass model, involving active decompression of the inferior vena cava and portal vein, facilitates comfortable anastomosis of the superior hepatic vena cava and portal vein.

In this study, the protocol utilized portal cannulas to connect the portal vein of the recipient directly. It reduces the anhepatic phase duration, thereby maintaining hemodynamic stability. The protocol describes a novel approach that combines DHOPE and NMP to preserve the DCD donor livers, followed by porcine OLTx. DHOPE offers greater stability and does not depend on scarce blood resources, making it a promising technique for the long-term preservation and transportation of donor livers. At present, the longest preservation duration of ex vivo liver preservation through NMP was 7 days observed at the University of Zurich¹¹. This apparatus, grounded in biotechnological principles, integrates additional dialysis and periodic blood product exchange, incurring substantial costs.

The DCD liver with a WIT of 30 min exhibited severe graft dysfunction after 8 h of SCS preservation during NMP (Supplementary Figure 2). This observation aligns with the trend in liver function indicators seen in subsequent OLTx experiments. In the SCS group, the rise in total bilirubin indicated graft dysfunction at the early stage after OLTx. Conversely, in the DHOPE-NMP group, the increase in total bilirubin was effectively attenuated on the fourth-day post-OLTx. This suggests that sequential DHOPE-NMP plays a crucial role in maintaining systemic hemodynamic stability after reperfusion, enabling long-term survival following OLTx.

DHOPE has been proven to enhance the preservation quality of DCD donor livers⁴. Clinical studies consistently show that DHOPE effectively maintains ATP levels in hepatocytes and reduces the mitochondrial oxygen debt during low-temperature preservation. This leads to a decrease in IRI upon reperfusion. Long-term follow-up studies have reported a reduced incidence of biliary tract-related complications in DHOPE-maintained donor liver transplantations⁴. NMP operates under stringent conditions, working at body temperature with oxygenated blood and nutrients^5,12,13,14. It's important to note that while NMP offers benefits such as functional assessment and reduced cold ischemia time, its complexity, cost, and the need for specialized staff trained in NMP can introduce challenges¹⁵. NMP provides a solid platform for assessing ex vivo liver viability; traditional biochemical functional indicators often fall short in reflecting liver viability^16,17. In contrast, less common indicators like bile pH, lactate, and blood glucose have been found more promising in indicating liver vitality. At present, no established indicator accurately assesses ex vivo liver function. In a previous study, gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) was utilized as an MRI contrast agent to monitor real-time liver reserve function. Faster bile excretion of the MRI contrast agent post-reperfusion was observed in DHOPE-preserved livers compared to those preserved using SCS¹⁸.

Biliary duct anastomosis is considered challenging in liver transplantation due to its high complication rate^1,19. The fragility of the biliary tract tissue heightens the risk of inflammatory stenosis and postoperative biliary obstruction²⁰. One of the pigs died due to bile leakage, which may have been caused by excessive tension in the bile duct. For biliary tract anastomosis, we employed an end-to-end biliary stent approach, chosen primarily for its time-saving advantages. The presence of stents effectively prevents early postoperative biliary obstruction. Additionally, postoperative feeding in pigs tended to be low in the SCS group, possibly due to gastric mucosal injury from elevated portal vein pressure. This necessitated acid suppression and gastric mucosal protection therapy.

Other studies have highlighted the potential of sequential hypothermic MP followed by NMP to alleviate the detrimental effects of warm ischemia in DCD livers. One notable investigation assessed the efficacy of combining NMP and DHOPE to rejuvenate and evaluate the viability of high-risk donor livers that were initially deemed unsuitable for transplantation. This research specifically compared two oxygen carriers during the perfusion process: an artificial hemoglobin-based oxygen carrier (HBOC) and red blood cells (RBC)²¹. Remarkably, one-year post-transplant, graft, and patient survival rates were reported to be 94% and 100%, respectively. Only a single patient (3%) exhibited post-transplant cholangiopathy²¹.

The protocol introduces a detailed procedure for preserving porcine DCD liver through sequential DHOPE-NMP mode, followed by the OLTx procedure. This methodology holds promise in extending the duration of ex vivo liver perfusion for DCD grafts and establishing a stable model of OLTx. Collaborative endeavors and comprehensive clinical trials will be pivotal in optimizing protocols, ultimately benefiting patients awaiting liver transplantation.

Materials

Name	Company	Catalog Number	Comments
Anesthesia respirator	Mindray,Shenzhen	WATO EX-20
Automatic biochemical analyzer	MNCHIP, China	Celercare V5
Bama female miniature pigs	Pearl Lab Animal Sci & Tech Co,Ltd (Guangdong, China).		40-45 kg
Blood gas analyzer	Abbott	i-STAT300
ECG monitor	Shenzhen Ericon Medical Equipment Co., LTD China	M-9000S
Fully automatic snowflake ice	Changshu Shenghai Electric Co., Ltd. China
Perfusate in NMP			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.
Potal catheter	Jinxin technology,Shunde,China		Portal vein catheter for custom cannula of varying internal diameter (6-8.5 mm)
Refrigeration centrifuge	hermo Fisher Scientific - CN
The CG8/CG4 blood gas test card	Abbott
The CHEM 8 test card	Abbott
The ex vivo liver machine perfuion device	Devocean Medical Instrument Co., Ltd, Guangdong, China	DEVOCEAN-LIVER 2000	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
UW Cold Storage solution	Bridge to Life, Ltd., USA	Belzer UW	Liver in SCS group were preserved in UW Cold Storage solution
UW Machine Perfusion Solution	Bridge to Life, Ltd., USA	Belzer MPS	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
Vacuum extractor	SMAF	DYX-2A