Normothermic Ex Vivo Pancreas Perfusion for the Preservation of Pancreas Allografts before Transplantation

All animals used for this study received humane care in accordance with the ''Principles of Laboratory Animal Care'' formulated by the National Society for Medical Research and the ''Guide for the Care of Laboratory Animals'' published by the National Institutes of Health, Ontario, Canada. All studies were approved by the Animal Care Committee of the Toronto General Research Institute.

NOTE: This study protocol is based on a porcine model. The graft is stored in the cold for 2 h and then undergoes normothermic machine perfusion for 3 h prior to transplantation (Figure 1).

1. Animals

1. Use male Yorkshire pigs (40-50 kg).

2. Organ procurement

NOTE: The preoperative procedure and part of the surgical procedure are the same as previous papers published by our group¹⁵ and is as follows:

1. House the pigs in the research facility for a minimum of 7 days to allow for acclimation and to reduce their stress level.
2. Fast the pigs for a minimum of 6 h before induction of the anesthesia.
3. Sedate the pig with an intramuscular (IM) injection of midazolam (0.15 mg/kg), ketamine (25 mg/kg), and atropine (0.04 mg/kg).
   NOTE: This is done in the housing facility.
4. Transfer the animal from the housing facility to the operating room (OR), where recovery of the organ will be performed.
5. Position the pig in a supine position on the OR table and place a face mask with 2 L of oxygen and 5% of isoflurane until the jaw is relaxed.
6. Visualize the vocal cords using laryngoscope and spray them with 2% lidocaine to prevent spasm during intubation. Replace the mask with oxygen and isoflurane for at least 30 s before attempting intubation.
7. Introduce a 7 mm endotracheal tube and block the cuff with 5 mL of air. Use capnometry to ensure that the tube is in the correct position.
8. Decrease the isoflurane gas to 2.5%. Turn on the ventilator and set it to 15-20 breaths/min and the tidal volume to 10-15 mL/kg bodyweight. Monitor heart rate and oxygen saturation constantly.
9. Using the Seldinger technique¹⁶, introduce an 8.5 Fr. x 10 cm catheter into the jugular vein (either right or left).
10. Use the jugular vein catheter to start an infusion of fentanyl (2.5 mL in 500 mL of Ringer) at 250 mL/ hr.
11. Check muscular reflexes to determine the depth of anesthesia. Jaw tone is the most reliable muscular reflex ¹⁷.
    NOTE: If the rigidity of mandibular muscles is noted, increase isoflurane and/or fentanyl infusion.

3. Surgical procedure

1. Disinfect and cover the surgical field. Perform a midline incision from the xyphoid to the pubic symphysis. Extend the surgical field with a left lateral incision for better exposure.
2. Dissect the inferior vena cava (IVC) from the abdominal aorta. Further free the aorta from the surrounding tissue and ligate the small lumbar aortic branches. Identify and place ligatures around both renal arteries.
   NOTE: The ligatures must not be tied at this timepoint.
3. Once the back of the aorta is free, pass two ligatures around it. The lower ligature will eventually be tied just above the iliac artery bifurcation and upper ligature will be tied 5 cm above the previous tie.
4. Dissect the hepatic hilum. Tie all arteries as close to the liver as possible. Identify the common bile duct, place two ligatures close to the liver, and divide the structure.
5. Dissect around aorta but do not cut at this timepoint. Identify and dissect around the suprahepatic portion of aorta and place a tie around it.
   NOTE: The ligatures must not be tied at this timepoint.
6. Open the lesser sac to allow ice to cool pancreas. Mobilize the pancreas as little as possible before flush.
7. Administer 500 IU of heparin per kg of donor weight through the central line. Wait 5 min and start blood collection in citrate, phosphate, dextrose, saline, adenine, glucose, and mannitol (CPD/SAG-M) bags using the jugular catheter.
8. Tie the inferior aortic ligature, cannulate the aorta with a flush line above the iliac bifurcation tie, and secure the cannula with an upper tie. Ligate both renal arteries.
9. Tie the suprahepatic aorta (crossclamp) once enough blood has been collected (600 mL). Administer 10 mL of potassium chloride for sacrifice.
10. Initiate a flush with University of Wisconsin (UW) preservation solution. Cut an opening in the portal vein (as high as possible) and cava for venting. Place ice in the abdominal cavity.
11. Assess the pancreas tail and duodenum C-loop after flushing 1 L of UW solution. If adequate flush, begin dissection, identify, and clamp mesenteric vessels. Slow down flushing for the second liter of UW.
12. Retrieve the pancreatic graft and a segment of cava or iliac vein for extension of portal vein.
    NOTE: The pancreatic graft is removed with the spleen.
13. Place the organ inside an organ bag that is placed inside a basin filled with ice.

4. Back table preparation of the pancreatic graft (Figure 2A)

1. Remove the flush line from distal part of the aorta and close with a tie. Fill the organ bag with the remaining UW solution. Free the pancreas from adherent tissue, including the spleen.
2. Perform portal vein extension using previously recovered cava or iliac vein with 6-0 Prolene. Cannulate the portal vein and proximal aorta with a ¼ in x 3/8 in reducer.
3. Cannulate the distal part of duodenum with Malecot catheter and tie. Clamp the end of catheter to avoid spilling of duodenal content. Oversaw mesenteric vessels with 4-0 Prolene.
4. Register the weight of the graft. Keep the graft in static cold storage (SCS) until the start of the NEVPP.

5. Normothermic ex vivo pancreas perfusion (NEVPP)

NOTE: The perfusion circuit is made of neonatal cardiopulmonary bypass equipment (Figure 3).

1. Attach the corresponding tubing to the oxygenator and to the venous reservoir, as well as the arterial line to the outflow of the oxygenator and place the bubble filter in its holder. Connect the purge line that goes from bubble filter to the venous reservoir. Open the bubble filter cap to let all the air out.
2. Connect the venous line to the inlet of the venous reservoir. Connect the dialysis filter and tubing where dialysate will be infused. Connect the flow meter sensor, pressure lines, and the temperature probe. Connect the arterial and venous sample lines to the sample ports.
3. Place the pancreas chamber (Figure 3) on a Mayo table and introduce the arterial and venous tubing through the holes intended for this purpose. Connect and turn on external heater unit.
4. Place the suction tubing inside the roller pump and connect one end into the tubing that comes out from the chamber to collect the fluids, and the other end to the venous reservoir to collect all organ loses of perfusate.
5. Connect the oxygen tubing (green) to the gas tank containing the carbogen mixture (95% O₂/5% CO₂) and the oxygenator. Connect the heater pump unit tubing to the oxygenator.
6. Clamp arterial and venous outflow lines, as well as the outflow of the venous reservoir.

6. Preparation of the perfusate and priming of the circuit

1. Fill the venous reservoir with the perfusate (Table 1).
2. Use one syringe pump for continuous administration of the vasodilator (epoprostenol) at 8 mL/h into the arterial line. Use a second syringe pump for continuous administration of the enzyme inhibitor directly into the venous reservoir (15 mg, 10 mL/h).
3. Turn on the heart lung machine (HLM) and start up the pressure, temperature, and timer panels. Turn on heater pump to warm the perfusion solution to 38 °C. Open the O₂/CO₂ supply.
4. Remove the tubing clamp placed on the outflow of the venous reservoir, start the centrifugal pump, and take it up to 1,500 rpm. Clamp the tubing, bypassing the arterial filter and release air from the arterial filter. Zero the arterial and venous pressure lines.

7. Pancreas graft perfusion (Figure 2B)

1. Open the organ bag where the pancreas is stored. Flush with 200 mL of albumin through the arterial cannula. Remove the pancreas from the ice and position inside the organ chamber. Confirm that the arterial and the venous tubing are air-free.
2. Release clamp from the arterial side and clamp the shortcut between the arterial and venous tubing. Once blood starts coming out of arterial tubing, connect line to arterial cannula. Set arterial pressure to 20-25 mmHg, by regulating the speed of the centrifugal pump. Connect venous tubing once blood starts coming out from the venous cannula.
3. Administer one vial of verapamil (2.5 mg/mL) directly on the arterial side, when the pancreas is completely connected and no major bleeds are observed.
4. Record pressures, arterial flow, temperature, and duodenum secretion continuously. Collect blood, record duodenal output every hour, and assess hourly macroscopically for edema. Record the perfusion parameters and take samples for analysis (venous and arterial blood gas samples, as well as samples for amylase, lipase, and LDH).
5. Disconnect arterial and venous tubing when perfusion is over, remove the graft from organ chamber, and flush with cold UW and weigh. Store graft on ice in a sterile organ bag until the moment of transplantation.