Porcine Normothermic Isolated Liver Perfusion

The scope of our research is to improve preservation of high-risk liver grafts by increasing our understanding of normothermic machine perfusion, or NMP, which allows reconditioning of the graft, assessment before transplantation, and provides a platform for regenerative therapy. Recent clinical trials suggest that normothermic machine perfusion reduces hepatocellular injury, whereas graft assessment can increase organ utilization rates. However, little is known about the liver cells'biology during NMP.

Animal models have been pivotal in the evolution of liver transplantation. And in our lab, we established a validating model of porcine isolated liver perfusion, which can be applied to study NMP as a preservation strategy, as well as mimic transplantation. The main advantage of our model is the high translational value compared to rodent models.

Additionally, our setup allows researchers to adjust all individual settings to have full control over the perfusion. Our own research has focused on the effect of perfusate composition and extent of injury on the innate immune response of liver grafts during normothermic machine perfusion. In the future, we want to explore NMP as a platform for regenerative therapy, for example, by means of administration of paracrine factors of stem cells.

Begin by inserting a 22-gauge catheter into the ear vein of a sedated pig. Connect the catheter to a three-way valve, and induce anesthesia by starting an intravenous drip of Plasma-Lyte at 40 milliliters per hour. Make a seven-centimeter-long incision from the lateral left side of the top of the sternum to the sternocleidomastoid muscle.

After placing an orthostatic retractor, locate the extrajugular vein and dissect it free by ligating any side branches. Place two 2-0 ligatures around the external jugular vein, and tie off the cranial ligature. Cut open the vein caudal to the tied ligature, and insert a 12 French venous catheter.

After exposing and repeating the ligature on the carotid artery, caudally insert the arterial line in the carotid artery, and fixate with 2-0 ligature. Make an incision along the midline from the xiphoid to the pubic bone. Transect the umbilical ligament and place an abdominal retractor.

Pull the intestines to the left laterally and cranially to visualize the aorta and vena cava. Now dissect the free three centimeters of the aorta cranial to the iliac bifurcation, and place two ligatures around the aorta. Dissect the vena cava free at the same level as the aorta, and place two ligatures around it.

Starting from the lateral side of the gastroduodenal ligament, dissect the common bile duct free and encircle it with a vessel loop. Remove a large lymph node on the lateral side of the portal vein, and transect a branch from the stomach to the pancreas to free the vein. Encircle the portal vein with a vessel loop on the liver's side and a ligature on the pancreatic side.

Retract the portal vein laterally, ensuring that it is not closed off. Locate the common hepatic artery and encircle it with a vessel loop. All hilar structures are now identified.

Retract the esophagus to the right side, exposing the thoracic aorta. After administering 500 units per kilogram of heparin to the pig, tie a caudal ligature around the aorta, and then insert a 20 French cannula into the aorta and fixate. Mimic DCD by clamping the thoracic aorta for about 60 minutes to induce warm ischemia.

After flushing the aortic cannula with ice cold preservation fluid, topically place slush ice on the abdomen to cool it. Once flushing is complete, remove the aortic cannula, tie the ligature around the portal vein, cannulate and fixate the cannula in the portal vein with a vessel loop. Flush the liver with two liters of ice cold preservation fluid via the portal vein.

Next, expose the infrahepatic vena cava by retracting the intestines to the left. Dissect the vena cava free from the retroperitoneum, and divide it cranially from the renal veins. Now dissect the common hepatic artery up to the celiac artery and the aorta.

Next, divide the gastroduodenal artery and cut out the celiac artery with a patch of the aorta. After cutting out the suprahepatic vena cava and any other remaining attachments, remove the freed liver and weigh it. Cannulate the portal vein of the weighed liver with a 25 French cannula, and secure it with ligatures.

Next, cannulate the hepatic artery with a 14 French reinforced cannula and fixate. Cannulate and fixate the infrahepatic vena cava. Cannulate the bile duct and fixate with a purse string.

De-air the portal cannula and test for leaks by back flushing the portal vein with 250 milliliters of cold plasma expander. Place the excised porcine liver in the receptacle. Next, connect the cannulas to the respective inflow and outflow tubes using a T-connector.

Install three-way taps on the T-connector and connect them to the pressure lines. Place the bile duct cannula in a container under mineral oil and start the perfusion. The portal vein contributes to approximately 75%of total hepatic blood flow, while the hepatic artery contributes to 25%of total hepatic flow.

Stable intrahepatic vascular resistance was seen, with the portal vein showing lower resistance relative to the hepatic artery. Stable and consistent perfusion led to steady levels of aspartate transaminase release in six to 12 hours. Perfusate pH was self-regulated and maintained within normal ranges by the liver.

Perfusate lactate was observed to be cleared one hour from the perfusion start, and bile secretion was maintained for 24 hours. Improper placement of the cannula at the left vena cava led to impaired outflow, causing a doubled portal vein resistance, leading to decreased portal vein blood flow and a compensatory increase in the hepatic artery flow. Outflow obstruction was also associated with intrahepatic hemorrhage and hepatocellular necrosis, as shown by increased concentration of aspartate transaminase in the perfusate.