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

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Normothermic Ex Vivo Pancreas Perfusion for
the Preservation of Pancreas Allografts before
Transplantation
Catherine Parmentier1, Samrat Ray1, Laura Mazilescu1,2, Masataka Kawamura1, Yuki Noguchi1, Emmanuel
Nogueira1, Sujani Ganesh1, Bhranavi Arulratnam1, Sangeetha Kalimuthu3, Markus Selzner1, Trevor Reichman1
1Ajmera Transplant Program, University Health Network, Toronto General Hospital 2Department of General, Visceral, and Transplantation Surgery,
University Hospital Essen 3Department of Pathology, University Health Network, Toronto General Hospital
Corresponding Author
Trevor Reichman
trevor.reichman@uhn.ca
Citation
Parmentier, C., Ray, S., Mazilescu, L.,
Kawamura, M., Noguchi, Y.,
Nogueira, E., Ganesh, S.,
Arulratnam, B., Kalimuthu, S.,
Selzner, M., Reichman, T. Normothermic
Ex Vivo Pancreas Perfusion for the
Preservation of Pancreas Allografts
before Transplantation. J. Vis. Exp. (),
e63905, doi:10.3791/63905 (2022).
Date Published
June 15, 2022
DOI
10.3791/63905
URL
jove.com/t/63905
Abstract
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.
Introduction
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

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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.
Protocol
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

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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).
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

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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

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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.
Representative Results
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).

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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 17 ). Please click here to view a larger version of this figure.

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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.

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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.

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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 perfusion17 . (B)
Post perfusion biopsy with good preservation of pancreatic acini and islet cells. Please click here to view a larger version of
this figure.

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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.
Discussion
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,

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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 lengthening18 .
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 histopathology19 . So far, our group has also managed
to transplant pancreas allografts after perfusion with good
results17 .
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
Disclosures
The authors do not have anything to disclose.
Acknowledgments
None.
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