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 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.
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.
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
2. Initiation with DHOPE mode
3. Perfusion with NMP mode
4. Recipient hepatectomy
5. Orthotopic graft placement and vascular anastomosis
6. Post-transplantation care and monitoring
7. Technique of SCS control porcine model
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.
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 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 nutrients5,12,13,14. 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 viability16,17. 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 rate1,19. 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.
The authors have nothing to disclose.
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).
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. |
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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 |
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