Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The overall goal of this procedure is to perform an autologous extra corporeal perfusion of porcine liver and kidney to study the differences from liver only perfusion. This is accomplished by first harvesting the liver and kidney from the sacrificed pig and then preserving the organs on ice. Next, the organs are connected to a custom made perfusion circuit primed with oxygenated normal themic autologous blood.

The pressures and flows through the portal vein, hepatic artery and renal artery are monitored to ensure that they're maintained at physiological levels. Finally, the perfusion is continued for a minimum of six hours with regular measurement of arterial blood gases, liver and renal function, and glucose and cytokine levels. Ultimately, results can be obtained that show improved glycemic control and metabolic acidosis and the ex vivo liver kidney perfusion system.

By comparing data on ex vivo liver only perfusion. The main advantage of this technique over extreme method like liver only ex vivo profusion model is that adding additional kidney to the system adds a dialysis function. Generally, user new to this techniques will struggle because there's a great variation to the hepatic arterial anatomy and it is crucial to ensure good access to the hepatic artery is secured in a timely manner for optimal organ preservation.

Visual demonstration of this technique is critical because in addition to the difficult surgical technique, the orientation of the kidney and the liver priming the circuits and maintaining the circuits are all very difficult. Hopefully, visualizing how we set this up will avoid the pitfalls that we've encountered along the way. After preparing the required solutions and drugs for surgery, assemble a following group of supplies, dissecting scissors, scalpels artery forceps, artery eclipse, 12 to 14 F catheters, Vicryl stitches and ties.

Infusion sets, a large transport container of ice gloves and a large sterile container for the blood collection. Before harvesting the organ, remove the extra corporeal circuit from the packaging and sharing its sterility. It consists of a non-traumatic centrifugal pump that provides the hepatic renal arterial blood flow and pressure readings, an oxygenator, a heat exchanger unit, and a venous reservoir to stimulate the venous flow and pressures, and a central reservoir that supplies pseudo systemic circulation.

After passage through the liver and kidney, the venous blood is collected from the infra and supra hepatic inferior vena cava and renal vein into a container stimulating the systemic venous reservoir and return to the centrifugal pump. Connect the drainage lines from the IVC and renal vein to the systemic reservoir. Then connect the systemic reservoir to the pump and then the oxygenator.

There are three lines exiting the oxygenator connect one to the hepatic artery, one for the renal artery, and one for the reservoirs. Applying the hepatic portal vein After isolating the left kidney and the liver from a euthanized pig for transport to the lab, according to the text protocol, prime the circuit by adding 1.5 to 2.5 liters of heparinized autologous porcine blood to the systemic venous reservoir. During the back bench preparation, start the centrifugal pump and allow the PERFUSE eight to run through the intraluminal and extraluminal compartments and to fill the circuit lines and the hollow fiber filter, add the following to the perfuse eight 20 milliliters of epoprostenol sodium 750 milligrams of rizzi sodium, and 10 milliliters of water, and 10 milliliters of 10 millimolar.

Calcium chloride allow gravity to prime the centrifugal pump and manually tap the circuit towards the venous reservoir. To remove any air bubbles, start the centrifugal pump and set it at a pump speed of 1200 RPM to prime the rest of the circuit with the perfuse eight. Start the flow of oxygen to the oxygenator at a rate of two liters per minute and connect the oxygenator to the water, bath and heat exchanger preset to a temperature of 38 degrees Celsius.

Then zero the pressure monitoring device on the centrifugal pump to air on the kidney and liver. Identify both the ureter and the common bile duct respectively and cannulate and secure them. Infuse one liter of 0.9%sodium chloride solution through the portal vein 500 milliliters through the hepatic artery and 500 milliliters through the renal artery to remove any blood clots from the organs in the cannulas and to flush out the preservation solution.

Once all the lines are primed and the PERFUSE eight is optimized, collect a baseline blood sample for full blood count and biochemical and blood gas analysis before connecting the first organ. Take a tissue biopsy if performing histological examination. Next, transfer the retrieved kidney into the allocated non biogenic perfusion chamber.

Then connects the renal artery catheter to the respective line of the extra corporeal circuit and the catheterized ureter into an hourly urometer for urine output monitoring. Adjust the profusion pressure of the kidney and maintain the renal arterial pressure between 80 to 90 millimeters of mercury. Now transfer the liver into the allocated perfusion chamber and immediately connects the hepatic artery and portal vein to their respective lines of the extra corporeal circuit.

Allow the cannulated bile duct to drain freely into an allocated bile bag for monitoring. Adjust the perfusion pressure and maintain the hepatic artery pressure between 80 to 100 millimeters of mercury and the portal vein pressure at less than 10 millimeters of mercury. Then begin and maintain a continuous infusion of 20 milliliters per hour of epoprostenol sodium and 10 milliliters per hour of 10 units per milliliter of sodium Toro Coate administer boluses of 40 milliliters of 8.4%sodium bicarbonate and 50 units of insulin atropic to maintain physiological parameters of glucose and acid-based balance.

The resistance in the portal venous system is low compared to the arterial channels. Therefore, a clamp is applied on the inlet tubing of the portal reservoir to control the inflow into the portal reservoir, and regulate pressures in the hepatic artery and portal vein to decrease the inflow into the portal reservoir and increase the flow and pressure into the arterial lines. Tighten the clamp to maintain physiological values of the arterial and venous flow and pressure.

Alter the pump speed aiming for a hepatic and renal arterial flow of between 0.2 and 0.3 liters per minute and change the inflow resistance of the portal reservoir to achieve a portal vein flow above one liter per minute. Collect blood samples hourly for hematological biochemical and blood gas analysis and correct any electrolyte glycemic and metabolic changes using the appropriate solution. Although the liver function test remained stable for the first six hours, there were significant changes that occurred beginning at 19 hours during a 24 hour experiment as demonstrated here.

Factor five and 10 progressively diminished during the entire experiment and significant changes were evident from five hours on for factor five and beginning at three hours per factor 10. The overall levels of urea and creatinine concentrations were significantly lower in the liver kidney model and no significant differences were observed over the course of the perfusion. However, in the liver only model urea and creatinine levels significantly increased after the first hour.

This figure reveals that the glucose concentration normalized when the ex vivo porcine liver model was connected with one kidney. When compared to the liver circuit alone, there was no major difference between pre and immediately post profusion blood sugar levels. In the liver kidney model, the amount of insulin required was much lower compared to the liver ex vivo model alone where insulin infusion was required constantly to control high glucose concentration.

Sodium bicarbonate was given as required in the liver kidney model to titrate the acidosis. It was not required as a continuous infusion where it was in the liver model. Blood gas analysis showed significantly lower pH levels in the liver kidney circuits than in the liver circuit alone, where a significant increase in the bicarbonate levels was noted beginning at three hours.

We demonstrated a similar result in base excess levels. These results demonstrate that the difference in the acid-based balance was corrected by the addition of a homeostatic organ in the liver kidney circuit. We also use this model to study the differences between IL six and IL eight in both the liver, kidney, and liver only.

Experiments IL six and IL eight levels increased considerably after the first and second hours respectively and remains high. There were no significant differences between the liver alone and liver kidney model After its development. This technique pa, by the way, for researchers in the field of eye transplantation to explore the initial phase of information due to eye transplanted to the liver.