Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

The aim of this procedure is to preserve and assess a liver graft under nearly physiological conditions. This is accomplished by first retrieving the liver graft from a donor pig. In the second step, the liver is cannulated, and then the graft is connected to the ex vivo perfusion circuit.

In the final step, the perfusion properties and liver graft characteristics are monitored for the desired perfusion time. Ultimately, aspartate amino transferase or a ST levels and histological analyses of parenchymal biopsies are used to show the preservation of the injury and viability of the liver graft. The main advantage of this technique over existing preservation methods, like called static storage, is that preservation under physiological condition results in less preservation injury and allows the functional assessment and treatment of the graft before the actual organ transplantation.

Immediately after inducing cardiac death by potassium chloride administration collect 1.6 liters of blood from a 30 to 35 kilogram male Yorkshire pig into CPDA bags close the abdomen, leave the liver intra abdominally for 45 minutes to induce warm ischemic injury immediately before the end of warm ischemia. Time cannulate the aorta and portal vein with organ flush lines at this time as well. Use dual perfusion via aorta pressure and portal gravity driven bags to flush the liver with University of Wisconsin solution.

Then cut the liver outta the pig, leaving all the remaining vessels long and place it in an organ bag. Cannulate the supra and infra hepatic parts of the inferior vena caver with half inch by three eighths of an inch reducers with a lure lock. Cannulate the portal vein and liver artery with three eighths of an inch by quarter inch and quarter inch by three eighths of an inch.

Reduces with a lure lock at this time as well. Then close the bag and store the liver on ice until the perfusion. To set up the ex vivo perfusion circuit, connect the outflow of a main reservoir to a centrifugal pump and a commercial oxygenator behind the oxygenator.

Split the tubing into two lines. Connect one line to a dialyzer that drains back into the main reservoir. Connect the other line to a leukocyte reduction filter.

Split the line after the leukocyte reduction Filter into an arterial line for supplying perfuse eight to the hepatic artery and into a portal venous line for delivering perfuse eight to a second reservoir that drains into the portal vein by gravity outflow. Clamp the portal inflow and then connect the arterial line to a vena caver loop draining into the main reservoir. For fluid recollection, set the starting temperature of the circuit and the water bath to 20 degrees Celsius.

Then cover the bath with an impermeable membrane to collect ascites or leakage of the perfuse eight. Connect a roller pump driven suction line to the main reservoir. Then release the outflow clamp from the main reservoir and fill the circuit with the perfusion solution.

Start the centrifugal pump at 1, 500 rounds per minute, making sure all the air is driven out of the circuit. Then turn on the gas supply to the oxygenator. Connecting the liver to the circuit is the trickiest part of the procedure.

Take care to avoid kinking of the tubing and to remove any bubbles to prevent impediment of the liver outflow. The level of the portal reservoir also has to be followed attentively. It must never run empty.

Remove the liver from the ice and flush out the University of Wisconsin solution with saline. Then place the liver onto the membrane over the water bath. Reduce the speed of the centrifugal pump to 1000 rounds per minute, and place two clamps at the arterial side of the vena caver loop.

Then cut the tubing in between the clamps and connect both vena caver outflows to the vena caver line. Release the clamp from the arterial line. Pour perfuse eight into the arterial cannula to get rid of the bubbles and connect the line to the cannula.

Increase the centrifugal pump to 1, 500 rounds per minute and release the second clamp from the venner cave line and release the clamp from the portal venous reservoir to fill it up. Let the perfusion solution pour into the portal cannula and then connect it. Taking care to keep the fluid levels in the portal reservoir stable.

After connecting the liver to the circuit, raise the temperature of the water bath to 33 degrees Celsius over the next 60 minutes. Aim for a starting arterial flow of about 250 milliliters per minute at 40 millimeters of mercury. This may reach 700 milliliters per minute during perfusion.

Once the pressure is increased up to 70 millimeters of mercury, aim for a port vein flow of 500 to 600 milliliters per minute at three to five millimeters of mercury. After raising the temperature, the port venous flow will increase up to 1, 100 milliliters per minute. Lower the main reservoir to set the outflow to minus two milligrams of mercury to prevent liver congestion by functional outflow obstruction, making sure no air bubbles are caught in the caval tubing.

Set the dialyze eight flow to 500 milliliters per hour. Taking care to adjust dialysis outflow so that the PERFUSE eight is neither diluted nor concentrated. Maintain a low pH during the start of the perfusion to protect the organ.

Adjusting the partial pressure of the carbon dioxide continuously down to 25 to 30 millimeters of mercury so that the pH will reach a physiologic level within an hour. To mimic physiological conditions, continuously inject glucose and insulin into the portal vein. Carefully inject sodium or potassium bicarbonate into the circuit under repetitive blood, gas and electrolyte control, and monitor the perfusion by periodical venous and arterial blood gas and a ST analyses.

Monitor the vascular flow and the pressure. A stable perfusion will maintain constant vascular resistance at the end of the ex vivo perfusion period. Call the perfusion system down, and then after disconnecting the tubing from the liver, flush the PERFUSE eight dually out the liver with ice cold University of Wisconsin solution.

Finally store the liver on ice in a sterile organ bag before transplantation. Achieving constant vascular flows with stable pressures is a good indicator of adequate oxygenation. Once the target temperature of 33 degrees Celsius is reached the flow values level at a constant, nearly physiological range for the rest of the perfusion period, at the same time the organ becomes metabolically active.

This graph shows the venous partial oxygen pressure, a marker of oxygen consumption. Within the initial two hours, the venous partial oxygen pressure declines to a then constant plateau. At this metabolically active state, the liver starts producing bile and the dialyzer provides a balanced electrolyte homeostasis.

The online A ST measurement serves as a monitor of hepatic cellular damage. For example, in this experiment, only a shallow A ST increase over the entire perfusion period is observed. H and e staining after six hours of perfusion reveals a hepatocyte necrosis of less than 5%with an intact lobular and sinusoidal structure.

BAS staining of an ex vivo perfused liver at the same time point demonstrates a replenished cellular glycogen storage compared to the exhausted storage observed in cold preserved donor cardiac death grafts. Following this procedure, an organ reperfusion model like a liver transplantation can be performed to study the effect of the ex vivo liver perfusion on reperfusion injury and organ function.