This method can help answer key questions in the field of transplantation and organ preservation. Applications of this method include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and performing experiments designed to repair organs. The main advantage of this technique is that the normothermic ex vivo liver perfusion model presented here is simple, easily replicable, low cost, and has a broad range of applications.
Start by preparing the 16 gauge portal cuff. Cut a five millimeter section of tubing, and determine the midpoint of the section by measuring 2.5 millimeters. Incise at the midpoint and remove the anterior half of the tubing.
Use a hemostat to crush this now flat portion. Use a lighter to melt the other end of the angiocatheter to create a lip. Next, prepare the bile duct canula by cutting the injection port from a 27 gauge angiocatheter, leaving only the catheter.
Connect this to a 10 centimeter section of 27 gauge cannula tubing. Position an anesthetized rat with its nose in the anesthesia nose cone and its four extremities immobilized. Monitor vital signs by attaching the monitor to the left hind extremity.
Perform a toe pinch to confirm appropriate depth of anesthesia. Spray the animal's abdomen with 70%isopropyl alcohol and allow it to dry. Place a sterile drape over the animal.
Then, after making a midline incision from the xiphoid to the pubis using sharp scissors, gently enter the peritoneum and incise the muscle. Extend the incision laterally to the left and right to form a cross at the level of the inferior border of the liver. At this point, turn the isoflurane anesthesia down to 2%Retract the xiphoid process using a curved mosquito clamp and the ribs using rib retractors.
Then use sharp scissors to cut the falciform, phrenic, and gastrohepatic ligaments. Locate and tie off the phrenic vein with a 7-0 suture as close to its origin as possible to prevent leaking. Next, use a sterile moistened cotton-tip applicator to eviscerate the rat.
Wrap the bowel in gauze moistened with 0.9%normal saline, taking care not to stretch the vasculature. Then dissect over the inferior vena cava to remove excess tissue. Dissect behind the IVC just superior to the bifurcation and pass a loop of a 4-O silk suture for later use.
Next, retract the right kidney to provide exposure to the right adrenal vein and retract the right lobe of the liver superiorly with gauze. Tie off the right adrenal vein with a 7-0 silk suture as close to the IVC as possible and cauterize across it distal to the tie. Then carefully dissect out the splenic vein.
Tie if off using two 7-0 silk sutures and cut across it between the two sutures. After dissecting around the gastroduodenal artery, tie off the gastroduodenal artery with a 7-0 silk suture and ligate. Then dissect around the hepatic artery and loosely place a 7-0 silk suture tie around it.
Next, dissect out the bile duct. Then check the length of the bile duct, tie it off at the distal end, and place a loop of suture around the bile duct and proximally as possible. Use small scissors to cut a hole that is half the diameter of the duct, and place a 27 gauge catheter into the bile duct proximally.
Tie the catheter in place using a roman sandal tie suture. Next, use a 27 gauge needle to inject 0.5 milliliters of heparin into the penial vein, or IVC. Then clamp and tie off the IVC using the previously placed 4-0 silk suture and tie off the hepatic artery using the previously placed 7-0 silk suture.
Now, use a microsurgical clip to clamp off the portal vein. Cannulate the portal vein using a 22 gauge angiocatheter and flush with 60 milliliters of cold 0.9%normal saline containing 100 units of heparin until the liver blanches. After flushing the liver, expose the suprahepatic IVC and cut across it as high in the chest as possible.
Perform the hepatectomy by cutting around the diaphragm and then cutting the hepatic artery, the IVC, the portal vein, and any additional ligaments. Lift out the liver and place an ice-cold 0.9%normal saline. Lastly, place the 16 gauge vascular cuff in the portal vein and connect the liver to the ex vivo normothermic liver perfusion circuit.
Before beginning each perfusion, visual inspection of the circuit should be performed to identify and damage or build-up on circuit components or tubing. If there is build-up of bacteria or other substances on the circuit, parts should be replaced or cleaned. Start by flowing the perfusate at two milliliters per minute.
Watch the monitor for any spikes in portal vein pressure, as this may indicate the vessel has become occluded and necessitate repositioning of the cannula. Insert the protal vein cannula into the cuffed portal vein for the return flow of the perfusate and suture in using a 7-0 silk suture. Once both cannulas are in place, begin turning up the flow of the perfusate by one milliliter per minute until the physiological pressure in the range of 10 to 16 centimeters of water is reached.
Remove one milliliter samples from the pre-port and post-port during the perfusion. Divide each one milliliter sample into two 0.5 milliliter samples. Snap freeze one of the 0.5 milliliter aliquots in cryogenic tubes in liquid nitrogen and run an arterial blood gas analysis using the remaining 0.5 milliliters of perfusate.
Then, measure the pH and buffer the perfusate as needed to return to pH 7.4. After four hours of perfusion, disconnect the liver from the perfusion circuit. Divide the liver into 0.5 gram segments, transfer to cryogenic tubes, and snap freeze in liquid nitrogen.
ALT was measured at zero, 30, 60, 90, 120, 150, 180, 210, and 240 minutes of perfusion. There is significantly less ALT in the base perfusate plus PEG-CAT group compared to base perfusate, which is shown in black at 150, 180, 210, and 240 minutes. Tissue ATP was maintained in the base perfusate plus PEG-CAT group in comparison to the base perfusate alone group.
Tissue MDA production was significantly higher in the base perfusate group than in the base perfusate plus PEG-CAT group. Total GSH was maintained in the base perfusate plus PEG-CAT in comparison to the base perfusate alone group. Each proposed application of normothermic ex vivo liver perfusion will need to be methodically tested in animal models prior to testing in discarded human organs and then in human livers.
The model presented here is ideal as it is easily replicable, eliminates extraneous tests, and is low-cost. After watching this video, you should have a good understanding of how to execute an inexpensive, easily replicable, normothermic ex vivo liver perfusion model using rats.
