Normothermic ex vivo machine perfusion (NEVP) has scarcely been explored for the preservation of pancreas allografts. We present an innovative preservation technique for pancreas allografts before transplantation.
Pancreas transplantation (PTx) is a curative treatment for people who live with the burden of a diagnosis of diabetes mellitus (DM). However, due to organ shortages and increasing numbers of patients being listed for PTx, new strategies are needed to increase the number of available grafts for transplantation.
Static cold storage (SCS) is considered the gold standard for standard criteria organs. However, standard criteria donors (SCD) are becoming scarce and new strategies that can increase the rate of organ acceptance from extended criteria donors (ECD) are urgently needed.
Normothermic ex vivo perfusion (NEVP) is one of the strategies that has become increasingly popular over the past couple of decades. This preservation method has already been used successfully in other organs (liver, kidneys, and lungs) but has been minimally explored in pancreas transplantation. The few papers that describe the method for pancreas show little success, edema being one of the major issues. The following manuscript describes the successful NEVP method and setup developed by our group to perfuse swine pancreas.
According to the National Diabetes Statistics Report, a total of 28.7 million people in the United States were living with a diagnosis of diabetes in 2019. Approximately 1.8 million of these had a diagnosis of type 1 diabetes1. PTx is currently the most efficacious and only curative treatment for complicated type 1 diabetes mellitus2, and is a procedure that both increases life expectancy and quality of life of these patients3.
The pancreas is the most often discarded organ after retrieval from deceased donors4. With ongoing organ shortages and the increasing waiting list times, transplant centers are using more pancreas grafts from ECDs, including donation after circulatory death (DCD)5. Strategies to safely preserve, perfuse, assess, and repair allografts coming from extended criteria donors are needed.
NEVP has proven to be successful in the preservation of lung6, liver7, 8, and kidney grafts9,10. However, the number of groups working on machine perfusion for the pancreas, both hypothermic or normothermic, and the number of publications, are few and limited due to graft edema and injury11,12,13,14.
The objective of this study is to present a protocol for normothermic ex vivo pancreas perfusion (NEVPP), using a porcine model with the goal of eventually providing a platform for prolonged preservation, organ assessment, and repair before transplantation. This will allow other research groups to establish a perfusion model for the study of pancreas allografts.
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
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:
3. Surgical procedure
4. Back table preparation of the pancreatic graft (Figure 2A)
5. Normothermic ex vivo pancreas perfusion (NEVPP)
NOTE: The perfusion circuit is made of neonatal cardiopulmonary bypass equipment (Figure 3).
6. Preparation of the perfusate and priming of the circuit
7. Pancreas graft perfusion (Figure 2B)
The upcoming data shows the representative results of seven experiments using a model of heart-beating donor pancreas retrieval. After cannulation of the aorta, flush with UW solution, and retrieval of the pancreas, the grafts were kept on SCS for 2 h while the red blood cells were prepared. NEVPP was performed in this model for 3 h, what we considered the least amount of time necessary for perfusion if graft assessment and repair are intended in the future. Samples and measurements were recorded at hourly timepoints. (0 = baseline, right after organ is connected to the circuit, 1 = 1 h, 2 = 2 h, 3 = 3 h).
Pancreas grafts were placed on an organ chamber that was custom designed for this purpose and includes a heater (Supplemental File). The purpose of NEVPP is to provide a near physiological environment for the organ. For this purpose, arterial pressure was set to remain between 20-25 mmHg in all perfusions. Pressure and flow were measured throughout the whole perfusion and remained stable (Figure 4). Metabolic activity was estimated by calculating the oxygen consumption of the graft using the following formula: [(pO2art-pO2ven) * flow / weight] (Figure 5). Measurements of pH, sodium, calcium, and HCO3 were within physiological values during the whole perfusion (Figure 6). Lactate and potassium levels decreased during the perfusion, and achieved close to normal values at 3 h (Figure 7). Since the circuit is a closed system, amylase and lipase levels are expected to increase during the perfusion (Figure 8). However, the increase in levels does not seem to correlate with the damage to the graft (Figure 9). A semiquantitative scale was used to score fat and parenchyma necrosis as well as islet cell integrity. (0 – no changes, 1 – mild changes, 2 – moderate changes, 3 – severe changes). This was done by a pathologist blinded to the experimental groups, and no signs of pancreatitis were observed.
Pancreas allografts were weighed before and after perfusion to assess edema (Table 2).
Figure 1. Study protocol. Please click here to view a larger version of this figure.
Figure 2. Pancreas before and after perfusion. (A) Before perfusion. (B) After perfusion. Please click here to view a larger version of this figure.
Figure 3. Schematic drawing of the perfusion circuit. With the use of Neonatal cardiopulmonary bypass technology; the perfusate is poured into the venous reservoir and then propelled with help of a centrifugal pump into the oxygenator. After leaving the oxygenator, the circuit divides into tubing that sends perfusate to the dialysis cassette and back to the reservoir and tubing that goes to the arterial filter. After passing the arterial bubble filter, the perfusate is driven with a pressure of 20-25 mmHg through the aorta into the pancreas. The venous outflow leads the perfusate back into the venous reservoir. (Adapted from 18). Please click here to view a larger version of this figure.
Figure 4. Mean arterial flow with standard deviation (mL/min). Please click here to view a larger version of this figure.
Figure 5. Mean oxygen consumption with standard deviation (mL/min/g). Please click here to view a larger version of this figure.
Figure 6. (A) Mean pH, (B) HCO3, (C) sodium, and (D) calcium measurements with standard deviations. Please click here to view a larger version of this figure.
Figure 7. (A) Mean lactate and (B) potassium measurements with standard deviations. Please click here to view a larger version of this figure.
Figure 8. (A) Mean amylase and (B) lipase measurements with standard deviations. Please click here to view a larger version of this figure.
Figure 9. Core biopsies before and after perfusion. (A) Normal pancreatic parenchyma before machine perfusion18. (B) Post perfusion biopsy with good preservation of pancreatic acini and islet cells. Please click here to view a larger version of this figure.
Ingredient | Amount |
Ringer’s lactate | 260 mL |
Steen Solution | 195 mL |
Washed erythrocytes | 162.5 mL |
Double Reverse Osmosis Water (DRO) | 35 mL |
Heparin (10000 IU / 10 mL) | 1.3 mL |
Sodium bicarbonate (8.4%) | 10.4 mL |
Calcium gluconate (10%) | 1.3 mL |
Metylprednisolone (Solu-Medrol) | 325 mg |
Aprotinin | 15 mg |
Table 1. Perfusate composition.
Weight before | Weight after | Gain | % difference | |
Case 1 | 244 g | 240 g | -4 g | -1.63 |
Case 2 | 154 g | 164 g | 10 g | 6.49 |
Case 3 | 184 g | 245 g | 61 g | 33.15 |
Case 4 | 190 g | 226 g | 36 g | 18.94 |
Case 5 | 198 g | 307 g | 109 g | 55.05 |
Case 6 | 205 g | 315 g | 107 g | 51.44 |
Case 7 | 193 g | 256 g | 63 g | 32.64 |
Table 2. Weight before and after perfusion.
Supplemental File: Custom made pancreas chamber for perfusion. Designed in collaboration with the Machine Shop of the Medical Physics – Radiation Medicine Program at Princess Margaret Cancer Centre. Please click here to download this File.
This study demonstrates that stable NEVPP can be achieved for pancreas allografts with minimal histological damage after 3 h of perfusion with the setup previously presented. Perfusion parameters like arterial flow, pressure, pH, HCO3, and Na remain stable during perfusion, and we observed a decrease and stabilization of K and lactate.
It is of critical importance to manipulate the graft as little as possible during procurement, back table preparation, and perfusion. It is also very important to keep tight control of the arterial pressure. Since the pancreas is a low-pressure organ, an increase in the pressure may cause irreversible damage to the organ.
Back-table preparation for this study is different from human grafts preparation (Figure 2A). Since the pancreas was the only organ procured from the pigs, we were able to take the portion of the aorta that includes the celiac trunk and superior mesenteric artery. As for the portal vein, an extension using iliac vein was performed. In case of human grafts, back-table preparation will have to be done in the same manner as it is done for transplantation, using iliac grafts for arterial reconstruction and portal lengthening19.
This method might be limited by the complexity of the setup. We decided to add a dialysis cassette after noticing severe edema of the graft when done without it. A custom-made organ chamber was also constructed for these experiments which contained an external heating source that proved to be instrumental for the optimal perfusion of the grafts.
There are few studies describing normothermic ex vivo pancreas perfusion. In most of these studies, edema appears to be the major limiting factor. To our knowledge, this method is the only report of using a dialysis cassette to control edema.
Normothermic ex vivo perfusion for the pancreas is still in its infancy compared to other organs. Current protocols are focusing on extended criteria donors (DCD), perfusate improvement, longer perfusion times, and biomarkers to assess graft damage during perfusion. Amylase and lipase levels don't seem to be reliable markers, since we are using a closed system, and don't seem to correlate with the histopathology20. So far, our group has also managed to transplant pancreas allografts after perfusion with good results18.
With continued improvements in this technology, we hope this technology will be applicable to clinical transplantation and allow for assessment and repair of pancreas allografts. This will hopefully ultimately result in more graft utilization, decreased waiting time for patients, and better patient outcomes
The authors have nothing to disclose.
None.
Alburex 5 | CSL Behring AG | 187337 | 25 g of Albumin (human) in 500 mL of buffered diluent |
Aprotinin from bovine lung | Sigma-Aldrich | A1153 | |
Belzer UW Cold storage solution | Bridge to life Ltd | 4055 | |
Calcium gluconate (10%) | Fresenius Kabi Canada Ltd (Toronto, ON) | C360019 | |
Composelect (blood collection bags) | Fresenius Kabi Canada Ltd | PQ31555 | |
Epoprostenol | GlaxoSmithKline Inc. | 218761 | |
Heart lung machine, Stöckert S3 | Sorin Group Canada Inc. | Custom made | Centrifugal pump, roller pump, control panel (sensors for pressure, flow, temperature, bubbles, and level), oxygen blender, heater unit |
Hemoflow, Fresenius Polysulfone | Fresenius Medical Care North America | 0520165A | |
Heparin (10000 IU/10 mL) | Fresenius Kabi Canada Ltd | C504710 | |
Lactated Ringer's solution | Baxter | JB2324 | |
Neonatal cardiopulmonary bypass techonolgy | Sorin Group Canada Inc | Custom made | Dideco perfusion tubing systems, centrifugal blood pump (Revolution), arterial blood filter, microporous hollow fibre memebrane oxygenator), cannulas |
Pancreas chamber | Custom made | With external heater | |
Percutaneous Sheath Introducer Set with Integral Hemostasis Valve/side Port for use with 7-7.5 Fr Catheters | Arrow International LLC | SI-09880 | |
Sodium bicarbonate (8.4%) | Fresenius Kabi Canada Ltd | C908950 | |
Solu-Medrol | Pfizer Canada Inc. | 52246-14-2 | |
Steen | XVIVO | 19004 | |
Urethral catheter | Bard Inc | 86020 | 20 Fr, malecot model drain |
Verapamil | Sandoz Canada Inc. | 8960 |