Summary
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.
Abstract
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 °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.
Introduction
Liver transplantation is the sole definitive treatment for end-stage liver failure; however, its success is limited by a persistent imbalance between patients on the waitlist and the availability of potential donor organs1. To increase the donor pool, donor criteria have been gradually extended in the last decade, including older donor age, liver steatosis, and donation after circulatory death (DCD)2,3. During a DCD procedure, the liver invariably suffers a period of warm ischemia between the withdrawal of life-sustaining therapy, declaration of death, and in situ cooling and preservation, aggravating ischemia-reperfusion injury (IRI)4. As a result, DCD livers are associated with an increased incidence of early allograft dysfunction and biliary complications5,6.
For these high-risk donor livers, conventional preservation with static cold storage does not offer sufficient protection against IRI. Hereto, alternative preservation strategies such as normothermic machine perfusion (NMP) have gained considerable traction. During normothermic machine perfusion, the liver is connected ex situ to an isolated circuit and perfused with an oxygenated and nutrient-enriched perfusate at body temperature. Clinical trials suggest that NMP reduces hepatocellular injury, as reflected by reduced peak transaminase release and early allograft dysfunction7. However, little is known about liver cell biology during NMP8.
Animal models have been pivotal in the evolution of liver transplantation. In contrast to rodent models, the pig is considered to be of higher translational value as the porcine liver is anatomically and physiologically close to humans, with similar organ size and bile composition. Nevertheless, porcine liver transplant models are labor-intensive, difficult to standardize, and carry a significantly higher financial cost.
Porcine liver NMP can be used to serve different purposes. It can be applied to mimic transplantation ex situ when using a whole blood-based perfusate, to preserve a donor liver in a protective environment with a leukocyte-depleted red blood cell-based perfusate, to assess potential biomarkers predicting liver function ex situ prior to transplantation, or as a platform to investigate regenerative therapy9,10,11.
The adoption of porcine liver NMP models is challenging, while surgical and perfusion-related technical aspects are scarcely described. In our research laboratory, we adopted the NMP setup originally described by Butler et al.12 to develop and validate a 24 h porcine ex situ isolated liver perfusion model that could be used to both preserve a liver graft for transplantation and to mimic a transplant. Here, we describe a step-by-step protocol; a methodological framework and potential pitfalls are published elsewhere9.
Protocol
Representative Results
Discussion
Here, we have detailed our experience with porcine liver NMP. The advantages of this technique include high translational value and versatility. Porcine liver NMP can be applied either to investigate and increase one's understanding of this enhanced preservation technique, or alternatively, to mimic transplantation. This setup allows manual control over every aspect of the perfusion, enabling adjusting both portal and arterial pressure and flow in various ways.
To simulate clinical practic…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank all research students from the faculty of medicine of KU Leuven involved in these experiments.
Materials
| Alaris GH Plus syringe pump | BD Care Fusion | 80023 UN 01-G | |
| Anesthesia device | Dräger | Titus | |
| Arterial catheter Cavafix Certo | Braun, Melsungen, Germany | BRAU4152557 | |
| Blood gas analyzer | Radiometer | ABL815 | |
| Calcium gluconate 10% | Braun, Melsungen, Germany | 570/13596667/1214 | |
| Capnograph | Dräger | Scio | |
| Cell saver | Medtronic | AutoLog | |
| Centrifugal pump Biomedicus | Medtronic | 85315 REV 3.0 | |
| Centrifuge Rotina 420R Hettich | VWR | 521-1156 | |
| Custom made perfusion circuit | Medtronic | M323901C | |
| Disposable set cell saver | Medtronic | ATLS24 | |
| DLP Single stage venous cannula, straight 20F | Medtronic | 66120 | |
| Epoprostenol | GlaxoSmithKline Belgium, Wavre, Belgium | Flolan | |
| Fentanyl-Janssen 0.05 mg/mL | Janssen | HK-08700 | |
| Flow sensor BioPro TT | Em-Tec | 12271 | |
| Formaldehyde 4% | VWR | VWRK4078.9005 | |
| Freezer -80 °C | New Brunswick Scientific | U570-86 | |
| Fridge | Liebherr | CUP 3513 | |
| Geloplasma | Fresenius-Kabi, Bad Homburg, Germany | freeflex | |
| Heater cooler | Stöckert-Shiley, Sorin group | 16-02-1950 | |
| Heparin 5000 IE/mL | Leo Pharma, Ballerup, Denmark | HeparinLeo | |
| Hepatic artery canula | Medtronic | BIO-MEDICUS 12F | |
| IGL-1 organ preservation solution | Institut Georges Lopez | IGL-1/1000/D | |
| In-line blood gas analyzer | TERUMO | Calibrator 3MCDI 540/CDI 500 | |
| Insulin 200 IU Actrapid | Novo Nordisk, Dagsvaerd, Denmark | MEDI-00018 | |
| Isoflurane 1000 mg/g Inhalation vapour | Chanelle Pharma | Iso-Vet | |
| IV catheter BD Insyte-W 20 G | BD | 381334 | |
| Liquid nitrogen tank | KGW Isotherm | S22 | |
| Mersilene 250CM M3 USP2/0 non needled ligapak | JNJ medical | F4503 | |
| Mersilene 250CM M3.5 USP0 non needled ligapak | JNJ medical | F4504 | |
| Mersilene 5X70CM M3.5 USP0 non needled | JNJ medical | EH6935H | |
| Mersilene 6X45CM M3 USP2/0 non needled | JNJ medical | EH6734H | |
| Micro pipettes 1000 µL | Socorex | 82,51,000 | |
| Monitoring | Siemens | SC 8000 | |
| Plasmalyte Viaflo | Baxter | Plasmalyte Viaflo | |
| Portal vein canula | CALMED LABS | 18F RV-40018 | |
| Pressure sensor | Stöckert-Shiley, Sorin group | 22-06-2000 | |
| Pressure servo regulator | Medtronic | BM 9505-2 | |
| Prolene 4-0 | JNJ medical | EH7151H | |
| Roller pump | Cobe Century USA | 468048-000 REV C | |
| Sodium bicarbonate 8.4% | Braun, Melsungen, Germany | 362 2339 | |
| Sodium taurocholate | Sigma Aldrich, Burlington, USA | 86339 | |
| Surgical scalpel nr 24 | Swann Morton | 0211 | |
| Venous catheter, 3-lumen; 12FR | ARROW | AK-12123-F | |
| Vicryl Vio 250CM M2 USP3/0 non needled gigapak | JNJ medical | V1205G | |
| Xylazine 2% | VMD Livestock pharma | XYL-M 2% | |
| Zinacef Cefuroxime 750 mg | GlaxoSmithKline Belgium, Wavre, Belgium | NDC 0173-0353-32 | |
| Zoletil 100 | Virbac | Zoletil 100 |
References
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