Normothermic Ex Vivo Liver Machine Perfusion in Mouse

Normothermic Ex-Vivo Liver Machine Perfusion in Mouse. Normothermic oxygenated liver perfusion is a promising strategy to rescue marginal organs and has recently been introduced in clinical practice. However, the underlying molecular mechanisms are not well-understood.

A mouse model is needed to further study the molecular pathways, taking advantage of genetically modified animals, such as transgenic mice, therefore waiting for establishing a normothermic oxygenated machine perfusion system for mouse livers. Here is a scheme of our entire system. First, the entire system is controlled at 37 degrees centigrade by a thermostatic controller through water circulation.

The perfusate is pumped from the reservoir by a peristaltic pump into an oxygenator. There is an uninterrupted gas flow of 95%oxygen and 5%carbon dioxide in the oxygenator. The perfusate then passes through a bubble trap.

The perfusate with air bubbles is pumped back to the reservoir under pressure. The remaining perfusate flows to the organ chamber where the tube is connected to the portal vein. The perfusate is drained from the liver through the suprahepatic cava into the organ chamber.

The perfusate outflow from the organ chamber is directed back to the reservoir by the peristaltic pump. A bile drainage tube is connected to the organ chamber to collect bile. This is our operating table.

All surgeries are done under a microscope. We maintain semi-sterile operating conditions throughout the surgical procedure. Hold the French tube cannula with forceps.

Penetrate the cannula wall with a 30G needle in one-centimeter distance from the end of the cannula. Push the needle through the cannula until the tip of the needle becomes visible. Here is a comparison picture of 26G catheter and self-made cannula;both are suitable for mouse portal cannulation.

Prepare 25 milliliter of heparinized saline with a final concentration of 2, 500 international units per milliliter, and place syringe in incubator set at 40 degrees centigrade. Here are the main components of the ex-vivo normothermic machine perfusion system. The components mainly include the organ chamber, thermostatic machine, roller pump, oxygenator, and reservoir.

Here, you can see a detailed setup of our organ chamber. Basically, it is composed of an organ chamber body, a pressure sensor, a heat exchanger, and a bubble trap. We set up a perfusate inlet, a perfusate outlet, and a bile drainage port.

This is the ADI monitoring system we use to monitor the portal vein pressure. The ADI system consists of a pressure sensor, a central controller, and displays. Turn on LabChart program for pressure monitoring.

Calibration and zeroing are required before each use of the pressure sensor. Use zero-millimeter mercury and 20-millimeter mercury as marks to calibrate the pressure sensor for the two-step calibration. Based on the constant monitoring of the portal pressure, we can adjust the portal flurane according to pressure changes.

At equal volumes of perfusate to the reservoir and the organ chamber to prime the system, special attention must be paid to maintain sterility during the filling process. Connect induction chamber with a wall socket. Turn oxygen to 0.5 liter per minute.

Turn isoflurane to 3%Place animal in chamber until deep anesthesia is reached. Use micro syringe to apply body weight-adapted dose of analgesia. Here, seven microliter of buprenorphine.

Now, we start the surgery operation. Make a transverse incision in the abdominal area of the mouse. The skin incision extends to the bilateral meat axillary line on both sides.

Carefully make a longitudinal incision along the linea alba. Cut through the abdominal muscle layer with electrocoagulation and scissors. Carefully place a piece of wet gauze to protect the liver from electrocoagulation.

Fully expose the abdominal cavity of the mouse. Carefully move the small intestine out of the abdominal cavity with a wet cotton swab to fully expose the hilum. Shown here is a schematic diagram of portal vein cannulation and bile duct cannulation.

Basically, we choose the appropriate cannula according to the size of the mouse, and we use 6-0 silk suture for the fixation of the cannula. Carefully free the common bile duct using fine curved forceps without teeth. The common bile duct is very easily damaged and break.

Once it breaks, it cannot be cannulated. Due to the direction of the anatomical position, curved forceps are easier to operate. Place two 6-0 silk suture loop over the common bile duct in preparation for the next step.

Carefully puncture the bile duct with a 30G needle. Use pointed curved forceps to enlarge the small holes, so that it can feed bile duct cannulation. Use vessel cannulation forceps across the bile duct cannula and push it into the bile duct.Note.

At the moment of cannulation, you will feel the resistance brought by bile. If the force is not well-controlled, the cannula will be pushed out of the biliary tract by the pressure of bile outflow. Carefully adjust the depth of the cannula.

If it is too deep, it may damage the bile duct, and if it is not deep enough, it may slip out. Bile flow can be observed in the cannula after successful cannulation. Clamp the portal vein with flat forceps and carefully free the connective tissue with curved forceps.

Do not pull hard here, so as not to cause tearing of the portal vein. Once the portal vein is damaged, it is difficult to rec cannulate the portal vein. Place two 6-0 silk suture Loop over the free portal vein.

Use an arterial clip to close the distal portal vein. Very carefully puncture the portal vein with one of the above portal vein cannula. Blood flow can be clearly observed within the cannula after a successful puncture.

Take prewarmed heparin saline solution from incubator. Remove all air bubbles. Fix syringe with prewarmed heparinized saline into the syringe pump.

Connect the extension tube of the syringe pump to the cannula of the portal vein. Adjust the speed to two milliliter per minute and start the liver flushing. Increase isoflurane to 5%and euthanize the mouse with an overdose isoflurane inhalation.

Observe the color of the liver at the end of the flushing procedure. Excise the liver once the color turned to a homogeneous yellow. Carefully transfer the liver into the organ chamber using a Petri dish.

Keep a small amount of saline in the Petri dish to prevent a liver from being crushed.Note. Portal vein and bile duct can easily be twisted during this procedure, which may affect liver perfusion and bile collection. Slowly infuse normal saline into the portal vein cannula with a syringe to evacuate the air bubbles in the cannula.

Connect the portal vein cannula into the perfusate outflow tube in the organ chamber. The mouse bile duct cannula is guided through a valve of the organ chamber, which is closed with a rubber cap. Then, the bile tube is inserted into a pre-prepared 0.5 milliliter micro tube with a small hole in the lead.

After everything is connected, turn on the peristaltic pump. Check the portal vein pressure reading to adjust the flow rate. Maintain the portal vein pressure between seven to 10-millimeter mercury by adjusting the flow rate.Note.

Nominal flow rate can slightly vary depending on age and positioning of tubes. This is the selection of perfusate medium, perfusate volume, and perfusion pressure based on a literature workup of normothermic oxygenated rat liver machine perfusion studies. This is a step-by-step trial to establish the mouse perfusion model.

It took about 14 experiments to select the most suitable catheters. In our hands, using two French rubber catheters for the portal vein, together with one French rubber catheter for the bile duct, is the best combination to achieve homogeneous perfusion with all catheters in place. pH level and potassium level were stable throughout 12-hour perfusion.

pH level ranged from 7.26 to 7.71, and potassium level ranged from 5.9 to 6.8 millimole per liter. HE staining shows that after 12 hours perfusion, the histology integrity of mouse liver was relatively well-preserved. Taken together, we demonstrated here the successful establishment of a normothermic oxygenated machine perfusion system for the mouse liver.

Perfusion of a mouse liver for 12 hours resulted in a good preservation of the liver morphology, although not throughout the whole organ. Further effort is needed to achieve complete homogeneous perfusion over a longer period of time. Once this is achieved, the use of genetically modified animals can be considered, such as the use of IL-2-knockout mice to investigate the role of inflammation.