The overall goal of this procedure is to perform a subnormal themic machine, perfusion of the human liver for the preservation, recovery, and assessment of the organ. In a preclinical research setting, this is accomplished by first preparing the liver for connection to the device. In the second step, the machine perfusion system is primed, the liver is connected, and the perfusion is started in the final step.
The machine perfused liver will become functionally active at a subnormal thermic state, and produce bile. Ultimately, samples collected from the liver during the profusion can be analyzed to study the metabolic recovery and function of the organ. The main advantage of this technique over existing preservation methods like cold profusion and normal thermic profusion, is that Subnormal themic machine profusion allows functional recovery and assessment of the viability of the liver, while not needing to meet the high metabolic demands of body temperature perfusion.
Many aspects of this technique require experience or a demonstration for successful completion. For example, demonstration of how to perform the back table preparation of the liver, which includes the dissection of the artery vein and valve duct, can be helpful for researchers with no prior surgical training Upon receipt of the liver, place an ice filled organ bowl on a sterile draped surface and remove the liver from the transport box, leaving it mostly submerged in the bag of cold preservation solution. Next, use Metzen balm scissors to reveal the various branches along the arterial supply to the liver from the aorta to the proper hepatic artery.
Then carefully dissect the rest of the artery to prevent severing any vessels that supply the liver without cutting or tying branches that do not have a visible end. When the artery has been isolated, use size zero to four zero silk sutures to tie off all of the arterial branches that do not supply the liver. Tie and cut the splenic and gastric arteries close to their origin on the celiac trunk.
Cut an aortic patch around the celiac trunk. Then use an arterial profusion cannula to cannulate the celiac trunk. Now dissect the portal vein free, tying off any branches or stitching holes in the vein and cannulated with a portal vein perfusion cannula.
Remove sections of the diaphragm from the supra hepatic vena cava without cutting the vein itself. Then cut two two to three millimeter long full circumference tissue samples from the end of the common bile duct. The samples should be handled delicately.
Snap freeze one sample and liquid nitrogen for storage at negative 80 degrees Celsius, and store the other in 10%buffered formin. Then cannulate the common bile duct with a vessel cannula and connect a drain tube made of small diameter tubing. Next, ligate the cystic duct with a zero silk tie.
Connect the flush tubing set to ice cold bags of lactated ringers solution and prime the tubing to remove the air. Set the flow regulator on the flush tubing to a slow trickle and occlude the portal vein at the hilum to remove any air in the vessel. Now fill the cannula in vein with flush solution to remove the air from the portal vein, taking care not to elevate the bag more than 20 centimeters over the height of the liver to avoid excessive pressure on the vein.
Then flush the liver with a total of two liters of ICEC cold lactated ringer solution. During the flush, briefly occlude the portal vein at the lowest point to check for leaks, and then repeat the flush for the hepatic artery with one liter of solution as just demonstrated while the liver is flushing, prime the profusion system with the addition of two liters of room temperature profuse eight to the organ bowl and start the device to prime the tubing. Set the temperature to 21 degrees Celsius.
Then set a pressure of three millimeters, mercury on the portal vein and 30 millimeters mercury on the hepatic artery. Open the carbogen gas tank and set it to a flow of three liters per minute. Next, draw a blood gas sample from the portal vein and hepatic artery inflow sample ports, and run the samples in the blood gas analysis machine according to the manufacturer's instructions to confirm an adequate oxygenation and pH.
Use sodium bicarbonate or hydrogen chloride to correct the pH as necessary. Then before the liver is connected, collect a one milliliter sample of the perfu eight as a times zero measurement and store it at negative 80 degrees Celsius. Use a single edge steel blade to cut two wedge biopsies of about 250 milligrams each from the liver at this time as well.
Snap freezing one sample in liquid nitrogen for negative 80 degrees Celsius storage and fixing the other in 10%buffered formalin. Before starting the profusion, weigh the liver, then transfer the organ to the organ chamber of the device and remove the air from the cannulas. Just demonstrated.
When the cannulas are deed, connect the portal vein and hepatic artery inflows to the appropriate cannulas. Start profusion ensure that the liver is submerged by perfuse eight, covering any dry surfaces, including the inflow vessels with wet sterile gauze. To prevent dehydration, let the bile tubing drain into a collection container.
Taking care that the opening of the bile drain is at the level of the liver or lower to allow the bile to run freely. Once the liver is warmed to 21 degrees Celsius, target the flow rates at 275 to 325 and 50 to 100 milliliters per minute per kilogram for the portal vein and hepatic artery respectively. During the perfusion, take samples from the liver tissue perfused and bile as appropriate.
Continue the perfusion for three hours, monitoring the pressure pH and oxygenation and adjusting the pH with sodium bicarbonate as necessary. At the end of the perfusion, collect the final samples, disconnect the liver and remove the bile duct cannula. Finally, collect two post profusion tissue samples of the bile duct as just demonstrated and discard the human liver.
Following the proper biohazard disposal guidelines. Flow begins low as a result of a higher resistance in the cold liver using a pressure of three millimeters, mercury on the portal vein and 30 millimeters mercury on the hepatic artery. The target flows can generally be achieved once the liver has warmed up to 21 degrees Celsius after 60 minutes of perfusion frequent measurement of the perfuse eight by blood gas analysis is essential for both experimental purposes as well as for maintaining adequate perfusion conditions.
For example, the dissolved oxygen partial pressure should be greater than 700 millimeters mercury on the inflow of both the portal vein and the hepatic artery. Whereas the outflow oxygen pressure measured in the vena cava should generally decrease with the longer perfusion reflecting an increasing oxygen uptake. Hepatic transaminases also can be measured in the PERFU eight, for example.
An increase of alanine, transaminase, or a LT is generally observed in the first 30 minutes of the perfusion with a minimal further increase during the rest of the perfusion. Further evaluation of a TP production in machine perfused livers reveals a 2.8 fold increase in the A TP content of the liver reflecting a recovering energy status. Moreover, h and e histological analysis reveals no additional injuries sustained during the procedure.
Following this procedure allows extensive analysis of the isolated liver providing unique insights into hepatic physiology and pathology. After watching this video, you should have a good understanding of how to maintain a human liver during suberic machine perfusion to facilitate the assessment of its function outside the body.
