Porcine Normothermic Isolated Liver Perfusion

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 p....

Protocol

All experiments were conducted after KU Leuven animal care committee approval and in line with European guidelines.

1. Animal information

NOTE: Male TOPIGS TN70 pigs, aged 3 months, with a body weight of approximately 30 kg and liver weight of 600-700 g are used for this study protocol.

1. Keep the animals under a 12 h day/night rhythm in single pens with free access to food and tap water and visual, olfactory, and auditory contact between .......

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.......

Materials

Name	Company	Catalog Number	Comments
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