Development of new strategies for liver ex

vivo preservation during transplantation

Développement de nouvelles méthodes pour la préservation ex vivo du
foie au cours de la transplantation

Thèse de doctorat de l'université Paris-Saclay

École doctorale n° 569, Innovation Thérapeutique du fondamental à l’appliqué (ITFA)

Spécialité de doctorat : Physiologie, Physiopathologie
Graduate School : Santé et médicaments. Référent : Faculté de Pharmacie.

Thèse préparée dans l’unité de recherche Chronothérapie, cancers et

transplantation (Université Paris-Saclay), sous la direction de René ADAM, PU-PH,
la co-supervision de Alexandre LOPEZ, Directeur R&D

Thèse soutenue à Villejuif, le 9 décembre 2022, par

Njikem ASONG FONTEM

THESE DE DOCTORAT

Composition du Jury
Membres du jury avec voix délibérative

Mickaël LESURTEL
Président
PU-PH, Université Paris Cité
NNT : 2022UPASQ054

Pasquale INNOMINATO
Rapporteur & Examinateur
PU-PH, Université de Warwick
Jacques PIRENNE
PU-PH, Université Catholique de Rapporteur & Examinateur
Leuven
Daniel CHERQUI
Examinateur
PU-PH, Université Paris-Saclay
Joan ROSELLO-CATAFAU
Examinateur
PI, Université de Barcelone
Eric SAVIER
Examinateur
PH, Hôpital Pitié Salpêtrière
Titre : Développement de nouvelles méthodes pour la préservation ex vivo du foie au cours de la
transplantation

Mots clés : transplantation, ischémie-reperfusion, solution de préservation, machine de perfusion

Résumé : 43% des dysfonctionnements précoces
dans la transplantation hépatique sont liés au
syndrome d’ischémie-reperfusion, à l’origine d’une
accumulation des lésions entre le prélèvement du
foie chez un donneur et sa transplantation chez un
receveur. Les foies marginaux (présentant des
maladies guérissables), utilisés pour répondre à la
pénurie d’organes, sont très sensibles à ce syndrome.
Tout l’enjeu aujourd’hui est de réussir à diminuer les
complications postopératoires en améliorant la
qualité de leur préservation.
Par conséquent, l’essentiel de nos recherches a
consisté à développer de nouvelles stratégies de
préservation ex vivo du foie. Les méthodes les plus
utilisées sont la conservation du greffon dans un
liquide de préservation froid dite « préservation
statique » et sa conservation sur une machine de
perfusion oxygénée dite « préservation dynamique
». Utilisant un protocole de transplantation
hépatique chez le rat, nous avons développé une
nouvelle solution de préservation mais aussi un
nouveau protocole de machine de perfusion.

Title: Development of new strategies for liver ex vivo preservation during transplantation

Keywords: transplantation, ischemia-reperfusion, preservation solution, perfusion devices

Abstract: 43% of early dysfunctions in liver
transplantation are linked to the ischemia-
reperfusion syndrome, describing the accumulation
of lesions between liver removal from a donor and its
transplantation into a recipient. Marginal livers (with
curable diseases) are very susceptible to ischemia-
reperfusion. Still, they are used to address organ
shortages. The challenge today is to reduce
postoperative complications by improving the
quality of their preservation.
Therefore, our research aims to develop new ex vivo
liver preservation strategies. The most commonly
used methods are the conservation of the graft in
a cold preservation liquid called "static
preservation" and its preservation on an
oxygenated perfusion machine called "dynamic
preservation." Using a rat liver transplantation
model, we investigated the role of a new
preservation solution and a new perfusion machine
protocol.
 ©2022

Njikem Asong Fontem

ALL RIGHTS RESERVED

ii
ACKNOWLEDGMENTS

First, I would like to thank the Most High for infusing in me the passion
for biology and giving me the strength and the right amount of focus
and perseverance throughout my studies.

I also wish to express my appreciation to my thesis director, Pr. René

Adam for allowing me to join his laboratory to conduct my PhD and
for sharing his expertise on liver transplantation during these 3-years.

Many thanks to my thesis supervisor, Alexandre Lopez, for the

opportunity to collaborate with Institut Georges Lopez, but also for his
scientific expertise and support at every level of my thesis.

I am also thankful to my thesis jury: Mickael Lesurtel, Jacques Pirenne,

Pasquale Innominatio, Daniel Cherqui, Eric Savier, Joan Rosello-
Catafau for agreeing to revise my thesis.

Special thanks to my team “Chronotherapy, Cancer & Transplantation,”

namely Xiao Mei, Amal, Sandrine, Francis, Nassiba, Marc-Antoine, and
Mathilde, for your warm welcome since day one and enlighting
sciences-related and non-sciences-related talks.

I also want to thank the Experimental Hepatic Ischemia-Reperfusion

Unit of Barcelona, namely Joan Rosello-Catafau and Arnau Panisello-
Rosello, for the great scientific talks and revisions of my work.

This endeavor would not have been possible without my Mom,

Elizabeth Abonjo Akwalabeng, who should also receive a PhD in
“Coaching for PhD students”. Your daily attentions and motivations
were crucial in the completion of this work. I also want to thank you
for the microscope I asked for and got for my 8th birthday. Until that
day, I knew I wanted to work in a field where I’ll be able to use
innovative tools to EXPERIMENT, and here I am.

iii
I am grateful for my ancestors who kept an eye on me from where they
rest and gave me the mental support and power necessary to complete
this work.

Big thanks to my brother and sister Akwa, Atem, and Nkia, who all
pushed me to pursue what was passionating me and somehow
inspired me by their professional and personal accomplishments.

I want to extend my sincere thanks to my friends in France: my high

school babies, my favorite Youth Ambassadors, Kongossa Familly, my
day-one Evry friends, and all my close friends I met during my
bachelor's and master’s program. I’m thankful you did not choose to
follow biological studies (for the most part) like me, making you the
best person to relax, laugh, free my mind, and chill.

Thanks should also go to all my close friends living abroad for the 24/7
mental support and for facilitating the holiday process.

I would be remiss in not mentioning my different networks and

support groups for helping me lay aside my PhD life for a minute (when
I needed it the most) and keep all my other passions alive: The more
factory, ASHSR, 45.

Lastly, I want to thank my favorite artists (big shoutouts to my favorite

RnB singers) for accompanying me during sleepless nights and always
setting the mood for productivity.

iv
“Don’t tell me the sky is the limit when they are
footprints on the moon.”
Paul Brandt

“Remember one thing through every dark night;

there’s a bright day after that. So no matter how
hard it get, stick your chest out. Keep your head
up and handle it.”
Tupac Shakur

23:59:59

Romans 8:28

v
 SUMMARY

Acknowledgments ....................................................................................................................iii
Summary ............................................................................................................................... vi
Table of Contents.....................................................................................................................vii
List of Figures ............................................................................................................................ix
List of Tables............................................................................................................................... x
List of Abbreviations ................................................................................................................xi
1 Introduction ..................................................................................................................1
1.1 The liver: An essential organ for life ............................................................................................... 1
1.2 Orthotopic Liver Transplantation (OLT) ......................................................................................... 9
1.3 Ischemia Reperfusion Injury (IRI): A detrimental yet unavoidable syndrome during
OLT ................................................................................................................................................ 23
1.4 Liver ex-vivo Preservation Strategies to decrease IRI ........................................................... 29
1.5 Research Objectives ........................................................................................................................... 49
2 Results ......................................................................................................................... 53
2.1 Article 1 ................................................................................................................................................ 53
2.2 Article 2 ................................................................................................................................................ 69
2.3 Article 3 ................................................................................................................................................ 87
3 Discussion ................................................................................................................. 108
3.1 Introduction ......................................................................................................................................... 108
3.2 Oxygen: a crucial element at the crossroad of multiple preservation strategies..... 109
3.3 Glycocalyx integrity, a relevant biomarker for machine perfusion strategies ........... 111
3.4 HOPE-PRE, D-HOPE, NMP, NRP, COR: towards ischemia-free liver transplantation? ..
.............................................................................................................................................. 113
3.5 Robustness of experimental models to study extended criteria donors' ex vivo
preservation ......................................................................................................................................... 115
3.6 From a general preservation protocol to a personalized approach ............................. 117
4 Conclusion ................................................................................................................ 120
Résumé en Français .............................................................................................................. 121
Summary in English .............................................................................................................. 123
5 Bibliography ............................................................................................................. 125

vi
 TABLE OF CONTENTS

Acknowledgments ....................................................................................................................iii
Summary ............................................................................................................................... vi
Table of Contents.....................................................................................................................vii
List of Figures ............................................................................................................................ix
List of Tables............................................................................................................................... x
List of Abbreviations ................................................................................................................xi
1 Introduction ..................................................................................................................1
1.1 The liver: An essential organ for life ............................................................................................... 1
1.1.1 Liver Anatomy ......................................................................................................................................... 1
1.1.2 Liver Physiology ...................................................................................................................................... 4
1.1.3 Histology ......................................................................................................................................... 6
1.1.4 Chronic Liver Failure and End-stage Liver Diseases ................................................................. 8
1.2 Orthotopic Liver Transplantation (OLT) ......................................................................................... 9
1.2.1 History of Liver Transplantation ....................................................................................................... 9
1.2.2 Liver Transplantation Nowadays ................................................................................................... 12
1.2.3 Extended criteria donors: A solution to organs shortage ................................................... 15
1.2.4 Scoring of liver transplantation ..................................................................................................... 18
1.2.5 Measuring Living Function after Orthotopic Liver Transplantation ................................ 20
1.3 Ischemia Reperfusion Injury (IRI): A detrimental yet unavoidable syndrome during
OLT ................................................................................................................................................ 23
1.3.1 Definitions ...................................................................................................................................... 23
1.3.2 Cellular and Molecular Mechanism During IRI ........................................................................ 24
1.4 Liver ex-vivo Preservation Strategies to decrease IRI ........................................................... 29
1.4.1 Static Cold Storage (SCS) ................................................................................................................. 29
1.4.2 Preservation Solutions ....................................................................................................................... 30
1.4.3 Machine perfusion .............................................................................................................................. 38
1.4.4 Machine Perfusion Devices.............................................................................................................. 43
1.4.5 Other strategies for decreasing IRI............................................................................................... 47
1.5 Research Objectives ........................................................................................................................... 49
1.5.1 Evaluation of a novel preservation solution additive for fatty liver graft: Hemarina®
(M101) ...................................................................................................................................... 49
1.5.2 Evaluation of a novel timing for Hypothermic Oxygenated Perfusion (HOPE): HOPE-
PRE ...................................................................................................................................... 50
1.5.3 Evaluation of a novel preservation solution for static and dynamic preservation
protocols: IGL-2.................................................................................................................................... 51
2 Results ......................................................................................................................... 53
2.1 Article 1 ................................................................................................................................................ 53
2.1.1 Objective and hypotheses of this work ...................................................................................... 53
2.1.2 Summary ...................................................................................................................................... 54
2.1.3 Publication ...................................................................................................................................... 54

vii
2.2 Article 2 ................................................................................................................................................ 69
2.2.1 Objective and hypotheses of this work ...................................................................................... 69
2.2.2 Summary ...................................................................................................................................... 69
2.2.3 Publication ...................................................................................................................................... 70
2.3 Article 3 ................................................................................................................................................ 87
2.3.1 Objective and hypotheses of this work ...................................................................................... 87
2.3.2 Summary ...................................................................................................................................... 87
2.3.3 Publication ...................................................................................................................................... 88
3 Discussion ................................................................................................................. 108
3.1 Introduction ......................................................................................................................................... 108
3.2 Oxygen: a crucial element at the crossroad of multiple preservation strategies..... 109
3.3 Glycocalyx integrity, a relevant biomarker for machine perfusion strategies ........... 111
3.4 HOPE-PRE, D-HOPE, NMP, NRP, COR: towards ischemia-free liver transplantation? ..
.............................................................................................................................................. 113
3.5 Robustness of experimental models to study extended criteria donors' ex vivo
preservation ......................................................................................................................................... 115
3.6 From a general preservation protocol to a personalized approach ............................. 117
4 Conclusion ................................................................................................................ 120
Résumé en Français .............................................................................................................. 121
Summary in English .............................................................................................................. 123
5 Bibliography ............................................................................................................. 125

viii
LIST OF FIGURES

Figure 1. Liver Morphological Anatomy

Figure 2. Scheme of Couinaud’s classification: Liver lobes and

segments

Figure 3. Rat Liver Anatomy

Figure 4. Hepatocytes hexagonal structures

Figure 5. The structure of liver lobules

Figure 6. Role of Disse Space in normal conditions and injured liver

Figure 7. Stages of Liver Failure

Figure 8. Evolution of Liver Transplantation per year in Europe

Figure 9. Evolution of the Living Donor Liver Transplantation in France

Figure 10. Histology of steatotic livers

Figure 11. Glycocalyx modifications induced by ischemia-reperfusion

injuries

Figure 12. Overview of ischemia-reperfusion injuries

Figure 13. Key components of organ preservation solutions and their

functions in protecting against edema and ischemia perfusion injuries

Figure 14. Experimental ex vivo machine perfusion system

ix
LIST OF TABLES

Table 1. MELD score evolution based on three months’ mortality rate

Table 2. Overview of the composition of preservation solutions

Table 3. Risks & Benefits of Machine Perfusion

Table 4. Overview of the different machine perfusion systems available

for liver transplantation

Table 5. Proposition of personalized preservation approaches

x
LIST OF ABBREVIATIONS

ALT Alanine Amino Tranferase

AST Aspartate Amino Transferase
ATP Adénosine Triphosphate
cAMP cyclic Adenosine Mono Phosphate
CE Celsior Preservation Solution
cHOPE Continuous Hypothermic Oxygenated Perfusion
CIT Cold Ischemia Time
COR Controlled Oxygenated Rewarming
DAMP Danger Associated Molecular Pattern
DBD Death Brain Donor
DCD Death Cardiac Donor
D-MELD Donor - Model of End-Stage Liver Disease
DNA Desoxyribonucleic Acid
EAD Early Allograft Dysfunction
EC Eurocollins
ECD Extend Criteria Donor
eNOS endothelial Nitric Oxide Synthase
ESC Endothelial Sinusoidal Cells
GCX Glycocalyx
HBV Hepatite B Virus
HCC Hepatocellular Carcinoma
HCV Hepatite C Virus
HES Hydroxy Ethyl Starch
HMP Hypothermic Machine Perfusion
HO Heme Oxygenase
HOPE Hypothermic Oxygenated Perfusion
HSC Hepatic Stellar Cells
HSPG Heparan Sulfate Proteoglycan
HTK Histidine Tryptophan Ketoglutarate
ICU Intensive Care Unit
IGL Institut Georges Lopez
IL Interleukine
INR International Normalized Ration
IPC Ischemic Pre Conditioning
IR Ischemia Reperfusion

xi
IRI Ischemia Reperfusion Injuries
KC Kupffer Cells
LDH Lactates Dehydrogenase
LT Liver Transplantation
MAPK8 Mitogen-Activated Protein Kinase 8
MELD Model of End-Stage Liver Disease
MPP Machine Perfusion Protocols
MPT Membrane/Mitochondrial Permeability Transition
NADH Nicotinamide Adenine Dinucleotide
NFKB Nuclear Factor Kappa B
NKT Natural Killer Cells
NMP Normothermic Machine Perfusion
NO Nitric Oxide
OLT Orthotropic Liver Transplantation
OPTN Organ Procurement and Transplantation Network
PEG Poly Ethylene Glycol
PFC Perfluorocarbons
pH Potential Hydrogen
PNF Primary Non-Function
PNN Poly Nuclear Neutrophil
PO2 Partial Pressure of Oxygen
PPAR Peroxisome Proliferator-Activated Receptor
PS Preservation Solution
ROS Reactive Oxygen Species
SCS Static Cold Storage
SDC1 Syndecan-1
SNMP Sub Normothermic Machine Perfusion
SOD Super Oxide Dismutase
TMZ Trimetazidine
TNF Tumor Necrosis Factor
TRAIL Tumor necrosis factor Related Apoptosis Inducing Ligand
UNOS United Network for Organ Sharing
UW University of Wisconsin
WBC White Blood Cells
WIT Warm Ischemia Time

xii
1 INTRODUCTION

1.1 THE LIVER: AN ESSENTIAL ORGAN FOR LIFE

Orthotopic liver transplantation (OLT) is a life-saving medical therapy

performed by transplant surgeons to treat end-stage liver diseases.
The liver is a vital organ controlling the homeostasis of the entire
organism. The liver involves crucial functions such as protein synthesis,
metabolism, degradation, and excretion.

1.1.1 Liver Anatomy

1.1.1.1 Morphological Anatomy

The liver is an essential organ for life. Indeed, this digestive gland is
formed at the end of 3rd week during development and originates
from the stomach (duodenum)1. Indeed, as the most prominent
compound glands, the liver has exocrine and endocrine functions both
performed by its highly specialized cells: hepatocytes. The liver is
located in the right upper quadrant of the abdomen, below the
diaphragm to the right of the stomach on top of the gallbladder
(Figure 1.). The liver is a homogeneous quadri-lobular organ (Anterior:
right/left lobes; Posterior: quadrate/caudate lobe) of reddish-brown
color. Its smooth surface is covered by the peritoneum and a fibrous
capsule, the Glisson’s capsule. Its consistency is firm, and the liver
weights 2% of the entire body mass (~1,5 kg), from which ~800g
corresponds to blood volume. The liver ensures the metabolism of
most substances and nutrients absorbed by the body, namely
carbohydrates, proteins, and lipids.
 Figure 1. Liver morphological anatomy (National Cancer Institute,
2011; Teach me Anatomy, 2022)

1.1.1.2 Functional Anatomy: Couinaud Classification

While morphological anatomy is needed to understand global liver

structure and interactions with other organs, functional anatomy is
critical to comprehend liver intrahepatic anatomy. Claude Couinaud
proposed a classification based on the anatomy of the portal venous
system in 1954. Indeed, Couinaud’s subdivided into eight segments (I-
VIII) the portal vein distribution within the liver (Figure 2.). Nowadays,
this classification is widely accepted, and it is the basis of numerous
liver resection surgical protocols.

2
 Figure 2. Scheme of Couinaud’s classification: Liver lobes and
segments (Stammers, M. (2018) Imaging the Liver)

1.1.1.3 Human/Rat Comparative Liver Anatomy

The experimental model used throughout the experiment conducted

in this thesis solely uses rats to investigate different technic of liver
preservation. This section highlights fundamental differences between
human and rat liver anatomy. In rats, the liver is located below the
diaphragm. It spans the entire upper part of the abdomen (Figure 3A).
In humans, the liver is located in the upper right abdominal quadrant
and is smaller (as a percentage of total body mass) than in rats. In
humans, abdominal ligaments delineate the different lobes. The
ligaments are less prominent in rats. Both humans and rats have four
lobes (left, right, caudate, median/quadrate) (Figure 3B). However, rats’
lobes have multiple segments. Compared to humans, rats do not have
a gallbladder.

3
 A

Figure 3. (A) Rat liver spans the entire abdomen (B) Posterior view of
a rat liver2

1.1.2 Liver Physiology

1.1.2.1 Endocrine Function

The liver ensures the storage of carbohydrates through glycogen,

which is then released upon body needs. It synthesizes hormones from
lipids stored in intracytoplasmic lipids vesicles of the smooth
endoplasmic reticulum in hepatocytes. The liver also degrades
peptides and amino acids from the intestine, which are transformed

4
into urea. Moreover, the liver synthesizes many structural proteins such
as albumin, fibrinogen, globulin, transferrin, and coagulation factors).

1.1.2.1.1 Lipid Metabolism

The liver metabolizes lipids coming from daily food intake. Lipids are
absorbed by intestine cells and enterocytes and transformed into
chylomicrons. Chylomicrons are lipoproteins produced during
digestion carrying exogenous lipids from the intestine to adipose
tissue. Chylomicrons reach the lymphatic system, go to the arterial
circulation, and end up in the liver. Lipids are stored in the
perisinusoidal space, also known as the space of Disse (See section).
Lipids are then metabolized for energy production through the Kreb
cycle and β-oxydation. Still, lipids are also utilized for glycoproteins
and lipoproteins synthesis.

1.1.2.2 Exocrine Function

The liver is responsible for bile secretion in the biliary canaliculus. Bile
contains cholesterol, phospholipids, pigments, bilirubin, and biliary
acids. It ensures the organism's detoxification through the enzymatic
process and resident macrophage cells (Küpffer cells). During fetal life,
the liver is also involved in hematopoiesis.

1.1.2.2.1 Bile Secretion

Bile contains water, biliary acids, and bilirubin. 10% of biliary acids are
synthetized in hepatocytes’ smooth endoplasmic reticulum, while 90%
of biliary acids are absorbed by enterocytes and transported by
hepatocytes from blood to the biliary canaliculus. The latest is called
the enterohepatic cycle.

1.1.2.2.2 Bilirubin Metabolism

Bilirubin comes from senescent red blood cells, more precisely from
the catabolism of heme by Küpffer cells (KCs). Heme is transformed by
Küpffer cells (liver resident macrophages) in biliverdin, then
unconjugated bilirubin. Bilirubin goes through a glycuroconjugaison
inside the hepatocytes. Conjugated bilirubin will then be excreted in
the bile and flow into the digestive tract, where bacteria will be

5
transformed into feces or urines.

1.1.3 Histology

1.1.3.1 Hepatocytes

Hepatocytes are epithelial cells, representing 85% of the total hepatic

cells. They are organized into bundles and have a central nucleus with
highly dense cytoplasm (Figure 4.). Organelles present in the
cytoplasm are the nucleus, nucleolus, endoplasmic-reticulum (rough
and smooth), Golgi apparatus, and cytoplasm rich in glycogen vesicles.
Hepatocytes have highly developed capabilities of synthesis and
degradation. They are polar cells with two main poles—a vascular pole
to sinusoids and a biliary basolateral pole to hepatocytes leading to
biliary canaliculus.

Figure 4. Hepatocytes hexagonal structures (Alamy Images)

1.1.3.2 Hepatic Lobules

The hepatic parenchyma is organized in functional lobules. The hepatic

lobule comprises the portal triad: portal vein, hepatic artery, and bile
duct surrounding hepatocytes (Figure 5.). A hepatic lobule has six units
of the portal triad. These vessels allow nutrient-rich blood (coming
from the portal vein) to enter the hepatic lobules. Therefore, thanks to
sinusoids, nutrients and oxygen (through the hepatic artery) can enter
hepatocytes. The central vein, located in the middle of the hepatic

6
lobule, will take the nutrient and oxygen-poor blood and meet the
hepatic vein.

Figure 5. The structure of liver lobules (Southern Illinois University)

1.1.3.3 Sinusoids

Endothelial sinusoidal cells (ESCs) form the hepatic capillary system,

also called sinusoids. These capillaries are fenestrated, meaning that
they are perforated with tiny holes to ensure improved communication
between the blood and the liver. KCs are liver-resident macrophages
in which phagocyte microbes penetrate the liver or other metabolic
waste to detoxify the blood of unwanted and old red blood cells.

1.1.3.4 Peri Sinusoidal Space: Disse Space

Hepatic stellar cells (HSC), also called perisinusoidal cells or stellate

cells, are located between the sinusoid capillaries and the hepatocytes
(Figure 6.). They are highly involved in the evolution of hepatic
pathologies like cirrhosis because, when activated, they differentiate
into myofibroblast, collagen-producing cells under the influence of
growth factor (fibrogenesis). The excessive presence of collage fibers
in the extra cellular matrix favors the progression of cirrhosis and
hepatocellular carcinoma (HCC).

7
 Figure 6. Role of Disse Space in normal conditions and injured liver.
KC Kupffer cells; qHSCs: quiescent hepatic stellate cells, NK: natural
killers, LSEC: liver sinusoidal endothelial cells, aKC: activation of KCs,
aHSC: activated hepatic stellate cells, NKT: natural killer T-cells 3

1.1.4 Chronic Liver Failure and End-stage Liver Diseases

Fibrosis is a pathological mechanism that kills cells over time. If not

rapidly managed by pharmacological treatment, this process will cause
liver failure and, ultimately, cirrhosis, where the whole liver shrinks and
gets hard 4. The most common causes of cirrhosis are extreme alcohol
consumption, viruses (e.g., Hepatitis C), a fat diet causing excessive
lipid storage by the liver (steatosis), or drugs.

Figure 7. Stages of Liver Failure (Shutterstock)

8
 In healthy patients, there is no collagen fiber in the liver. Chronic
hepatitis is characterized by the abnormal presence of collagen fiber
(fibrosis) in the liver. Hepatic stellate cells play a crucial role in fibrosis.
In the presence of pro-inflammatory cytokines, they can’t stock vitamin
A and differentiate into myofibroblast. Therefore, myofibroblasts start
to synthesize extra cellular matrix and collagen. Fibrosis is
characterized by an extreme scarification process invading the entire
parenchyma and replacing hepatocytes. Fibrosis is not an irreversible
phenomenon if the cause is well-identified and neutralized.

Cirrhosis is the final common end point in patients with progressive

liver disease of various causes. It is the second indication of liver
transplantation in France. Cirrhosis is advanced fibrosis completely
disrupting the architecture of the liver functional unit: the lobule.
Indeed, cirrhosis leads to hepatic failure and portal hypertension by
forming collagen bundles. Chronic liver infection can cause cirrhosis,
usually occurring multiple years after the first contact. Chronic liver
infections can be of different origins: Viral (Hepatitis B, C), autoimmune
diseases, genetic diseases, vascular diseases, metabolic diseases (iron
overload, cooper overload/Wilson diseases), or steatosis.

Chronic inflammation and cirrhosis can lead to carcinogenesis. Indeed,

cirrhosis is responsible for 80-90% of hepatocellular carcinomas5. In
France, hepatocellular carcinomas have been the first indication for
liver transplantation since 2014.6

1.2 ORTHOTOPIC LIVER TRANSPLANTATION (OLT)

1.2.1 History of Liver Transplantation

The first described transplantation in a human was performed by John

Hunter (1728-1793), a Scottish surgeon and scientist who performed
animal transplants and human tooth transplants7. Research on vascular
surgeries was the prelude to organ transplantation. In 1890, Mathieu
Jaboulay performed a vascular anastomosis. In 1901, Alexis Carrel
performed animal organ transplantation using the triangulating vessel

9
technique for anastomosis 8.

In 1933, Vornoy, a Soviet surgeon, performed the first human renal

transplantation from a 60-year-old donor to a 26-year-old recipient.
The recipient did produce some urines, but the graft failed. In 1954,
Moore, Murray, Merrill, and Harrison from United Stated performed
the first successful renal transplantation between twins with lifelong
survival. In 1950-60, Sir Peter Medawar got a Nobel Prize for his work
on transplantation immunology. He became the leading figure in
setting the basics for understanding basic transplantation
immunology.

In 1963, Thomas Starzl performed the first human liver transplant in

the United States. The success was only technical; indeed, no patient
benefit was observed. Around the same time, Roy Calne and Roger
Williams performed human liver transplantation in Europe. From then,
the number of transplantation started to increase. In 1970, they were
35 liver transplantation teams globally. Starzl transplanted 57 patients,
while Calne transplanted 28 patients 9.

Transplant immunosuppression arose in 1960 with Azathioprine, in

1962 with steroids, and in 1976 with cyclosporine. A Sandoz (Novartis)
scientist, named Jean-François Borel, discovered the latter in 1969 and
isolated it from the fungus Tolypocladium.

From 1970 to 1980, Starzl got 30% success in a hundred cases.

However, the poor status of the recipient was observed as well as
operative difficulties. Moreover, no satisfactory protocols for
immunosuppression were obtained.

In 1983, the American National Institute of Health consensus

conference on liver transplantation discussed the benefits of liver
transplantation as an excellent therapeutic option based on previous
results. The results were conclusive. From now on, insurance
companies will start to cover liver transplantation fees.

Improvements in immunosuppression arose in 1990 and 2000 with an

alternative to cyclosporine, respectively Tacrolimus, and Sirolimus
(which have different side effect profiles, of interest for some patient
groups).

10
Organ preservation solution (UW in 1987) transformed
transplantation. Indeed, the first preservation solution allowed
surgeons to optimize the time window between procurement and
implantation. It increased the understanding of how to get the best
outcomes in transplantation by improving patient selection, surgical
technique, and immunosuppressive protocol.

From the mid-1980 significant technical development was observed.

Indeed, in 1987, Bismuth performed the first split liver transplant,
involving one donor and two recipients. Tanaka, a Japanese surgeon,
performed the first living donor experiments in 1997. In this case, a
piece of liver from a living donor was collected and implanted in a
child. This technic is now extended to adults as well (See section).

8000
Number of liver transplantation

7000
6000
5000
4000
3000
2000
1000
0

Year

Figure 8. Evolution of Liver Transplantation per year in Europe (1980-

2020)10

Although phenomenal progress has been observed during the past 30

years, with some excellent results, significant challenges remain.
Increased indication for transplantation increases the number of
people needing liver transplantation, leading to organ shortage. The
lack of available organs opens the potential use of extended criteria
donors, which are high risks. Indeed, the most controversial aspect of

11
extended criteria donors relies on non-heart-beating liver donors (See
section). For these susceptible grafts, novel organ preservation
strategies must be investigated.

1.2.2 Liver Transplantation Nowadays

1.2.2.1 Innovations in Surgical Procedures

Split liver transplantation (SLT) and Living Donor Transplantation

(LDLT) are surgical techniques that significantly increase the pool of
organs available, particularly for the pediatric population.

1.2.2.1.1 Split Liver Transplantation

SLT is a surgical procedure where a single dead brain donor liver is

divided into right and left portions and implanted into two recipients
simultaneously. Rudolf Pichlmayr performed the first SLT in 1988,
where one donor liver was transplanted into a pediatric (left lobe) and
an adult (right lobe) patient. Two main types of SLT exist, differing on
the age of the recipients. The “classical split” technic is performed to
achieve a right extended graft (Couinaud’s segments I, IV–VIII) and a
left lateral graft (Couinaud’s segments II and III) for one adult and one
small pediatric recipient. The second technic describes the split along
the line of Cantilie, resulting in one right (Couinaud’s segments V–VIII)
and one left (Couinaud’s segments I–IV) to supply two adult recipients
can be performed.

Even though the pediatrics mortality waitlist went from 40% in the
1980s to 10% in 201811, SLT remains a highly demanding surgical
procedure in adult patients and is associated with increased
perioperative complications. Because of these high complication rates
associated with SLT, ethical concerns were raised: Does splitting a liver
would turn an excellent organ into two high-risk grafts? Even though
some transplant centers have published favorable SLT outcomes
similar to those obtained in full organ LT, demonstrating that under
controlled conditions, SLT should continue to be performed.

12
1.2.2.1.2 Living Donors Transplantation

LDLT is a procedure in which a healthy living person donates a portion

of his liver to another person. LDLT is one of the most remarkable steps
in the field of LT. It was first introduced for pediatric patients in 1989.
Its implementation in adults was successfully performed ten years later.
LDLT provides life-saving therapy for many patients who would
otherwise die awaiting a cadaveric organ. In recent years, LDLT helped
reduce the pediatric patient mortality waiting list from 40% in 1989 to
5% in 2018. LDLT is a clinically safe addition to deceased donor liver
transplantation and has been able to extend the scarce donor pool
significantly.

45
40
35
30
25
20
15
10
5
0
2020
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019

Right Left

Figure 9. Evolution of the Living Donor Liver Transplantation in France

(1998-2020)6

1.2.2.1.3 Organ Tissue Engineering and Stem Cells

The tissue engineering approach for organ replacement consists of the

decellularization of deceased donor organs, which will be used as
acellular 3D biological scaffolds to grow a whole new organ using stem
cells, hepatic progenitor cells, or induced pluripotent stem cells.

13
It is currently under investigation for the liver, mainly for in vitro
testing. Indeed, bioengineered liver represents an opportunity to test
preservation solutions and machine perfusion methods, saving the use
of precious livers that could be “resuscitated” and potentially used in
clinical settings. The first method for liver, pancreas, and kidney
decellularization and recellularization was published in 2009 by
Bastista et al.12. This year's exponential growth of publications was
observed in this field. Though bioengineered organs are not available
to the transplant surgeon as alternative grafts, they can already use for
a wide range of applications like drug metabolism 12, tissue physiology
13
, and stem cell biology 14.

Scientists are also pursuing human hepatocyte production. Animals

are being genetically engineered to “grow” human hepatocytes.
Hepatocyte “farms” in chimeric animal models may soon be a reality
from which we can recover and grow new livers. However, the number
of cells required to generate a gram of human tissue is approximately
1 billion. Deriving 1 to 100 billion human hepatocytes to generate liver
lobes is currently prohibitively expensive and challenging, explaining
the focus on small animals 15. Although no successful large animal in
vivo studies have been reported, progress is being made that suggests
the future of organ tissue engineering is promising.

Mesenchymal stem cell administration has been proposed as a novel

way to attenuate IRI16. Mesenchymal stem cell administration during
machine perfusion could downregulate the alloimmune response and
promote engraftment after transplantation. Even though this has been
shown in rat kidneys, it could also interest other solid organs17.

1.2.2.2 Limitations of the system during a pandemic (COVID-19)

At the beginning of 2020, the sanitary crisis caused by the Covid-19

pandemic considerably impacted organ harvesting and transplant
activity in France and worldwide. Even though most non-urgent
surgeries were postponed, the French “Agence de la Biomédecine”
recommended the maintenance of essential organ transplantation,
including heart, liver, and lung transplants. Moreover, France did not
exclude Covid-19 positive patients from receiving liver transplantation
compared to the US.

14
Despite these recommendations, the number of liver transplantation
decreased by 17% (n=235 transplantations) compared to the same
period in 2017-2019. This reduction in activity is comparable to other
European countries.

To this day, the main impact of the Sars-Cov-2 pandemic is the

decrease in liver procurement in deceased patients. This is mainly
explained due to fewer road traffic accidents because of national
lockdown periods and ICU’s congestion. In France, this led to an
increased number of patients that were excluded from the waiting list
due to aggravation (39%) or died on the waiting list (26,6%)6. The
previous population described patients with Hepatocellular carcinoma
or Cirrhosis with a MELD score of 20-29 (See section).

1.2.3 Extended criteria donors: A solution to organs shortage

In 2020 in France, 1089 liver transplant procedures from death brain

donors (DBD) were performed on 1052 livers resected (37 livers were
rejected after procurement). DBB represents the most significant and
safest source of organs available for organ transplantation. 840
patients remained on the waiting list, 257 patients died while on the
waiting list, and 352 were removed because they were too sick for a
transplant. Mortality rates on the liver transplant waiting list are
increasing6.

The shortage of organs has resulted in higher utilization of extended

criteria donors (ECDs) to increase the pool of donors. Grafts that would
have been discarded 25 years ago are now reaching the ECD level of
acceptancy as we understand better their conditions and pathologies.
Indeed, ECD grafts were considered lower quality and associated with
poor transplant outcomes. However, the rationale behind their use
relies on carefully assessing donor and recipient risks.

Though no consensus exists regarding the definition of ECD, the most

frequent items used are donor age, steatosis, donation after cardiac
death (DCD), and donors with an increased risk of disease transmission.
The use of ECD was concomitant to the development of machine
perfusion technologies which offered new possibilities (See section).
For example, machine perfusion could be involved in the “defatting” of

15
steatosis grafts or the repair of injuries in DCD grafts. Moreover,
machine perfusion, thanks to continuous circulation, can counteract
the sinusoidal constriction caused by hypothermia.

1.2.3.1 Donor Age

Donor age is one of the most influential donor risk factors. Over the
past two decades, the use of older donors has increased significantly,
and centers are stretching the limits of donor age. In France, 35% of
donors were 66 and older in 2020. Graft from an aged donor might
decrease the regenerative capacity of the transplanted liver and make
it more susceptible to ischemia-reperfusion injury, particularly with
increasing cold ischemia time (CIT). The increased prevalence of
steatosis in older livers can further delay graft function. Although there
is heterogeneity in the threshold defining an older donor, several
studies have shown that older donors are associated with increased
mortality and graft loss. In addition to careful donor and recipient
selection, minimizing CIT is a crucial strategy for improving outcomes
with older donors. Optimal liver function is achieved by keeping CIT to
8 hours or less.

1.2.3.2 DCD (Death Cardiac Donors)

Non-heat beating donors or donors after cardiac death are potential

graft sources to increase the pool and decrease the waiting list. Though
the results of the prior experiences were encouraging, a high rate of
PNF and low one-year survival were observed18. Pharmacological pre-
treatment (See section) is of interest in this patient category to increase
the graft tolerance for which the harmful effects of warm ischemia are
enhanced.

In France, non-heart-beating donors are categorized in the Maastricht

Protocol III (M3), which has been authorized since 2015. In 2020, DCD
currently accounted for 7.2% of liver transplants in France. Such donors
have inadequate organ perfusion during the progression to circulatory
arrest. The hypoperfusion period from the arrest to cold flush is called
uncontrolled warm ischemia time (WIT). It represents an additional
injury phase inherent to this form of organ donation.6

16
1.2.3.3 Steatosis

Hepatic steatosis is found in 6-24%19 of grafts and is one of the

significant reasons for declining liver grafts for human
transplantation20. Significant hepatic steatosis is associated with an
elevated rate of primary non-function (See section) and significantly
reduces one-year survival21. The mechanism most likely to explain the
low tolerance of these grafts is the alteration of microcirculation and
mitochondrial dysfunction22. Against steatosis, IGL-1 and Polysol
preservation solutions seem to be the most adapted compared to
current solutions available in the market for the preservation of
steatotic grafts23,24.

Nonalcoholic fatty liver disease (NAFLD) may affect up to 30% of the

population in Western countries and up to 70% to 80% of obese
individuals25. Traditional risk factors for NAFLD include obesity,
diabetes, and metabolic syndromes. Steatosis lowers mitochondrial
membrane potential and causes deterioration in mitochondrial
function. Kupffer cell activity increases, and the sinusoidal lining is
disrupted and narrowed. These changes enhance cell damage during
cold ischemia and potentiate IRI. The amount of steatosis can be
graded based on histology as mild (<30%), moderate (30%-60%), or
severe (>60%). The type of steatosis exerts a differential effect on
transplant outcomes. In macrovesicular steatosis, hepatocytes contain
a single fat vacuole displacing the nucleus to the cell's periphery. In
microvesicular steatosis, hepatocytes contain numerous fatty
inclusions, which do not cause nuclear displacement. The Paris
consensus meeting26 concluded that mild steatosis has minimal impact
on post-transplant outcomes as long as cold ischemia time is kept
short, ideally less than 8 hours. Although livers with severe steatosis
have generally been discarded, there is growing interest in using
increasingly steatotic organs. The European Association for the Study
of the Liver found acceptable outcomes using livers with greater than
30% macrosteatosis 27.

17
Figure 10. Histology of steatotic livers. (A) Nonalcoholic fatty liver
disease (NAFLD) is characterized by macrovesicular fatty changes (B)
Microvesicular steatosis (rarely detected in livers of patients with
NAFLD) (H&E, ×200) (The Koren Journal of Internal Medicine)

1.2.4 Scoring of liver transplantation

The prognosis of patients with chronic hepatic disease is highly

variable. It depends on factors like the severity of the liver dysfunction
and the presence of complications of comorbidities. Therefore, some
prognosis scores were developed to estimate patient survival. In the
context of liver transplantation, their use is essential to sort and
prioritize patients on the waiting list.

1.2.4.1 Model for End-Stage Liver Diseases (MELD)

In 2002 the Model for End-Stage Liver Diseases (MELD) was developed
to be a more objective assessment of clinical liver grading. MELD uses
three objective variables: serum bilirubin measuring the biliary vesicle
function; serum creatinine measuring the kidney function; and
international normalized ratio (INR), which uses a prothrombin time
test to measure blood coagulation rate. Initially developed to predict
survival after intrahepatic surgery, it was later shown to be efficient in
predicting chronic hepatic disease mortality, becoming a reference in
the field of LT.

In 1998 the Institute of Medicine mandated that patients be allocated

organs based on their disease severity and risk of death rather than on
waiting time. February 27, 2002, was a historic day when the United

18
Network for Organ Sharing (UNOS) adopted and approved the MELD
score as a score to allocate organs for patients awaiting liver
transplantation in the United States. Liver allocation is currently based
on the MELD score. The most at risk for mortality would be at the
highest priority for organ allocation. The introduction of the MELD
score in the US in the first year resulted in a 12% reduction in waitlist
mortality.

MELD score is calculated using serum bilirubin, serum creatinine, and

INR (coagulation) and is given by the formula: R = 9.57 x ln(creatinine
mg/dL) + 3.78 x ln(total bilirubin mg/dL) + 11.2 x ln (INR) + 6.43.
Sodium was recently added to the MELD score, as it was shown that
liver disease patient tends to have low Na+ in their blood. If the MELD
score is between 15-20, the patient will be considered for liver
transplantation.

Table 1. MELD score evolution based on three months’ mortality rate

1.2.4.2 Donor Age - Model for End-Stage Liver Diseases (D-MELD)

D-MELD score assesses the long-term benefit of transplantation.

Though patients and surgeons understand that accepting an older
donor may result in added risk after transplantation, no tool exists to
quantify it in survival terms. Therefore, D-MELD calculates the impact
of donor age on recipient survival. This tool uses a given donor age
and calculates three potential recipients (expressed by values of

19
bilirubin, creatinine, INR, and by recipient age, HCV, HBV, portal
thrombosis, retransplant status) the patient survival at ninety days, one
year, three years. This is relevant to improve the matching
donor/recipient and to identify possible unsustainable matches (e.g.,
donor‐recipient matches with predicted patient survival of less than
50% at five years). This innovative approach allows the selection of the
best recipient for each referred donor, avoiding the allocation of a
high‐risk graft to a high‐risk recipient.

1.2.4.3 French Organ Allocation System

The Agence de la biomédecine (ABM) is responsible for the allocation

of liver grafts in France. ABM manages the national organ waiting list
and offers organs to transplant teams for specific patients in the order
established by the liver allocation score29.

The allocation of liver grafts distinguishes three categories: “high

urgency” (for patients in critical conditions), liver score (since 2007),
and “graft that nobody wanted” (after some extended-criteria livers
have been refused five times, they are offered to a team for a patient
of their choice to optimize the donor-recipient matching).

The liver score is calculated for each patient registered on the waiting
list. The score represents each patient's priority rating and therefore
allows the attribution of an organ to a specific patient, not to a team.
The liver score considers the diagnosis type, the gravity of the patient’s
condition (MELD score for cirrhosis, the alpha-fetoprotein (AFP) score
for hepatocellular carcinomas), the time spent waiting list and the
distance between the procurement and transplant centers.

The liver score considers the MELD score only for candidates with
isolated cirrhosis without cancer (49.6% of the candidates on the
waiting list in 2014) or TNM1 stage hepatocellular carcinomas (HCCs).

1.2.5 Measuring Living Function after Orthotopic Liver

Transplantation

Dynamic biochemical quantitative liver function tests measure the

elimination of a substance in time. Because the substances used for

20
these tests are cleared and metabolized almost exclusively by the liver,
the dynamic quantitative liver function tests constitute an accurate
measure of the specific aspect of liver function.

1.2.5.1 Biochemical Test

1.2.5.1.1 Transaminases (AST/ALT)

Aspartate Amino Transferase (AST) and Alanine Amino Transferase

(ALT) are enzymes that catalyze the transfer of α-amino groups from
aspartate and alanine to the α-keto group of ketoglutarate to generate
oxaloacetate and pyruvate respectively, which are essential
contributors to the citric acid cycle (Kreb’s cycle). Both enzymes require
pyridoxal- 5’-phosphate (vitamin B6) to carry out this reaction,
although the effect of pyridoxal-5’-phosphate deficiency is more
significant on ALT activity than on that AST. AST is a ubiquitous enzyme
in the heart, skeletal muscle, kidneys, brain, and red blood cells. ALT
concentrations in skeletal muscle and kidney are deficient. Therefore,
an increase in ALT serum levels is more specific for liver damage. ALT
is localized primarily in the cellular cytoplasm in the liver, whereas AST
is cytosolic (20%) and mitochondrial (80%). Patients with a significant
increase in aminotransferase levels (>10 times the upper reference
limit) most likely have an acute hepatic injury. In ischemic or toxic liver
injury, AST levels usually peak before those of ALT because of the
enzyme’s asymmetric distribution. A decrease in aminotransferase
levels alone after a marked increase does not have predictive meaning
since both resolutions, and massive hepatic necrosis may draw a
similar biochemical picture. In this setting, bilirubin and prothrombin
time should be investigated to assess the risk of hepatic failure (See
section).

1.2.5.1.2 Lactates

Lactate is a critical intermediary product in numerous metabolic

processes. In healthy livers, lactate levels increase caused by
gluconeogenesis, and the liver exhibits a high lactate clearance. In
contrast, it has previously been shown that patients with acute liver
failure have an abdominal release of lactate. The hepatic lactate
production in acute liver failure is thought to be due to accelerated
abdominal organ glycolysis, leading to the release of lactate in

21
abdominal organs. Defects in hepatic pyruvate metabolism with a
reduction in hepatic glycolysis due to severe hepatic necrosis may also
contribute to the lactate release. 80% of patients with ischemic injury
may reach very high concentrations of lactate dehydrogenase (LDH), a
marker of ischemic damage.

1.2.5.1.3 Plasma Bilirubin

Bile secretion is an important end point of liver function, and bile

production immediately ceases when the liver's perfusion is arrested.
Plasma bilirubin concentration provides indirect information on liver
bile production capacities' uptake, conjugation, and excretion
function. Elevated plasma concentrations of bilirubin are specific
markers for severe liver injury and liver function loss. The plasma
bilirubin concentration is often used with other laboratory markers of
liver pathology, such as albumin levels or liver aminotransaminase
levels.

1.2.5.1.4 Albumin and Coagulation Factor Synthesis

A change in serum albumin level or prothrombin time (blood

coagulation time) may be associated with a decrease in liver
functioning mass, although neither is specific to liver disease. Albumin
and proteins involved in secondary coagulation, including vitamin K-
dependent coagulation proteins (factors II, VII, IX, X, protein C, protein
S, and protein Z) and factor V, XIII, fibrinogen, antithrombin, α2-
plasmin inhibitor, and plasminogen, are exclusively synthesized by the
liver. Their plasma concentrations are, therefore, used as indirect
indicators of liver synthesis function. Albumin, clotting factors, and
coagulation parameters such as the international normalized ratio,
which uses prothrombin time to test and measure blood coagulation
rate) are calculated by routine clinical chemistry. In liver disease, there
is a decrease in the synthesis of albumin and coagulation factors,
increasing prothrombin time and international normalized ratio.

1.2.5.2 Post-transplant syndrome: Acute dysfunctions

1.2.5.2.1 Primary Non-Function

Primary Non-Function (PNF), it’s an acute dysfunction following

22
 revascularization, without any defined causes, leading to
retransplantation in an emergency or patient death. Its incidence is
between 2-8% at the beginning of 1990; now, it has decreased
significantly, reaching 1-4%30,31. The precise mechanism at the origin
of this complication remains undetermined and is probably from many
sources. The length of ICU stay, the donor age, the duration of cold
ischemia, the type of preservation solution, and the mismatch between
the donor and the recipient sex are the factor that is the most linked
to PNF32,33. IRI causes 10% of PNF, which is more verified in the case of
ECDs 33.

1.2.5.2.2 Early Allograft Dysfunction

Early Allograft Dysfunction (EAD) is diagnosed between day two and

day seven after reperfusion, following which the analysis of at least one
of the following parameters bilirubin > 170 μmol/l; prothrombin time
< 50 %; hepatic encephalopathy. Its incidence is located between 15-
20%.31,34

1.3 ISCHEMIA REPERFUSION INJURY (IRI): A DETRIMENTAL YET

UNAVOIDABLE SYNDROME DURING OLT

1.3.1 Definitions

Ischemia-reperfusion injury (IRI) is a syndrome describing the complex

molecular changes arising between the blood flow interruption during
liver procurement until reperfusion, where blood vessels are
reconnected to the recipient. Liver IRI involves many key players, such
as hepatocytes, immune cells, endothelial cells, and mitochondria, but
it also triggers a global response affecting the intestine, pancreas, and
kidneys. IRI can be segmented into three distinct phases:

Cold Ischemia starts when the liver is perfused with a cold preservation
solution following procurement. It is intentionally performed to reduce
graft metabolic activity before its implantation and its reperfusion in
the recipient. During this phase, ischemia causes functional changes
facilitating cellular injury. Indeed, though damage to hepatocytes is
limited, endothelial cells and KCs are susceptible to this phase, during

23
which ATP concentrations are low and anaerobic glycolysis is
increased.

Warm Ischemia or Rewarming begins when the liver returns to

physiological temperature during transplantation right before the
vascular anastomoses have been completed. It is the most harmful
phase. Indeed, this phase targeting primarily hepatocytes, is marked
by oxidative stress and mitochondrial dysfunction.

Early reperfusion is when the liver is connected to the recipient’s blood

supply. This period marks the activation of Kupffer Cells and
endothelial cells resulting in ROS generation. Late reperfusion
describes the period between 6-48 hours after reperfusion. This period
is caused by the infiltration of polynuclear neutrophils (PNN) and T-
CD4+ which release protease and cytotoxic enzymes promoting
hepatocyte degradation. During this phase, all lesions accumulated
during the previous steps are exacerbated. i

IRI describes the accumulation of molecular, cellular, and histological

modifications during these three phases. Before implantation, there
are limited tools to assess the extent of IRI, as it is only highlighted
during reperfusion.

1.3.2 Cellular and Molecular Mechanism During IRI

1.3.2.1 Anaerobic glycolysis and acidosis

During liver procurement, occlusion of the hepatic artery and portal

vein is performed, causing hypoxia (oxygen deficit). Therefore, the
metabolism of liver cells (hepatocytes, Kuppfer cells, endothelial cells)
transitions from aerobic to anaerobic. The oxidative phosphorylation
can no longer assure ATP production in the mitochondria. Anaerobic
glycolysis in the cytosol takes over, even though its ATP production
yield is much lower than oxidative phosphorylation. As a consequence,
lactic acid is produced. Even though this alternative ATP production
pathway maintains cell survival by sustaining ATP production and
preventing mitochondrial membrane permeability transition (MPT), it
increases intracellular hydrogen ions and, therefore, the pH. During the
acute ischemic period, an acidic environment is highly protective for
liver cells as it inhibits proteases and phospholipases, usually active at

24
neutral pH35. However, the reactivation of all enzymes causes major
tissue injuries at reperfusion. This is called the pH paradox. Increased
H+ concentration also increases the activity of the ATP-independent
Na+/H+ exchanger to alkalize the cytosol (which is key to DNA
synthesis).

Moreover, decreased activity of the ATP-dependent Na+/K+ pump is

observed. Na+ intracellular concentration increases drastically, leading
to cellular swelling (water entrance in the cell) and cell death. As liver
cell volume increases, the endothelial vessels' width narrows, reducing
the microcirculatory blood flow.

1.3.2.2 Calcium overload

Calcium overload is a direct consequence of Na+/K+ ATPase pump

dysfunction. Indeed, as the Na+ level increases, the Na+/Ca2+ ATP-
independent exchanger causes a flow of Ca2+ into the cell. High
concentrations of Ca2+ lead to the activation of Ca2+-dependent
enzymes such as calpains, protein kinase C and phospholipases and
ultimately leads to liver cells apoptosis as they attack cells' structural
component. Ca2+ homeostasis disruption also leads to mitochondrial
permeability transition leading to pore formation, promoting
apoptosis or necrosis.

1.3.2.3 Mitochondrial dysfunction

Mitochondria are the powerhouse of the cell. Indeed, these organelles

are present in high numbers in all liver cells. Within its double
membrane, the oxidative phosphorylation process composed of a five-
electron pump can convert oxygen into ATP molecules. Ionic
unbalances (Ca2+, H+, Na+) during ischemia-reperfusion contributes to
mitochondrial dysfunction as they promote mitochondrial
permeability transition. Indeed, hypoxia promotes the uncoupling of
the electron transport chain, which is responsible for mitochondrial
membrane depolarisation. Mitochondria, therefore, form a mega
channel, allowing soluble molecules to pass through the membrane
freely. During the early stage of ischemia, a small number of
hepatocyte mitochondria are degraded though mitophagy. This
process clears abnormal mitochondria. Impaired mitophagy

25
accumulates dysfunctional mitochondria leading to uncontrolled
increases in ROS and increased ATP consumption. When many
mitochondria go through MPT, cytochrome c release increases,
activating caspases and, ultimately, the apoptotic pathway.
Mitochondria are the largest source of tissue ROS generation upon
reperfusion in hepatic IRI36.

1.3.2.4 Oxidative Stress

Reactive oxygen species (ROS) are highly reactive molecules produced

during physiological reactions such as oxidative phosphorylation, lipid
degradation, and inflammation. Cells have antioxidant systems against
intracellular sources of ROS. Antioxidants in the liver are catalase,
glutathione, glutathione reductase, and superoxide dismutase. These
antioxidants neutralize ROS in physiological conditions. Four primary
sources of ROS exist. The first one is directly linked to electron
transport chain uncoupling, leading to superoxide anion accumulation.
Secondly, AMP, a by-product of ATP degradation, produces
hypoxanthine when delaminated. In hypoxic conditions, hypoxanthine
transformation in xanthine oxidase produces hydroxyl radicals.
Hypoxia also activates the NADPH oxidase system at the surface of
polynuclear neutrophils (PNN), transforming H2O molecules into
hydrochloric acid (H2O2). Finally, the cyclo-oxygenase pathway,
activating phospholipase A2 (a consequence of calcium overload), will
also generate free oxygen radicals promoting PNN aggregation. At
reperfusion, ROS generation increases drastically, surpassing liver
antioxidant capabilities. ROS excessive production causes oxidative
stress. ROS can be harmful in many ways. Indeed, ROS can cause
protein oxidation, lipid peroxidation, and DNA damage. Their role in
PNN can damage endothelial cells damaging the microvasculature.

1.3.2.5 Role of NO and glycocalyx

Nitrite oxide (NO) plays a vital role in microvasculature. NO is

synthetized from L-arginine by the enzyme NO synthase (NOS). Two
isoforms exist the inducible isoform (iNOS) and the endothelial isoform
(eNOS). It has been demonstrated that eNOS was protective against
IRI as it neutralized ROS, prevented inflammation and apoptosis, and
promoted vasodilation and PNN adhesion, allowing hepatic stellate

26
cells (HSCs) to stretch, and facilitating blood flow in liver sinusoids. On
the other hand, iNOS activation has been shown to mitigate the effects
of hepatic IRI.

NO production was also linked to glycocalyx (GCX), a thin-barrier

covering blood vessels' lumen. It is mainly composed of proteoglycan
(e.g., heparan sulfate) and phospholipid (e.g., syndecan-1, glypican). It
was demonstrated that the internalization of heparan sulfate from the
endothelial glycocalyx enhanced the production of eNOS during IRI in
an animal model37. Therefore, glycocalyx integrity is not only key to
transducing key signals such as NO production but also regulates the
inflammatory pathway. Indeed, as the first contact with inflammatory
cells, its integrity could enhance or prevent inflammation. That is why
glycocalyx disruption can lead to chronic inflammation, vascular
permeability, and edema. It has been shown that GCX during IRI can
be damaged through enzymatic cleavage of proteoglycans or by
oxidative stress by ROS38. Glycocalyx thickness could also be used as
a biomarker to measure the extent of IRI. Therefore, maintaining
glycocalyx integrity is an attractive therapeutic approach to increase
NO production and mitigate IRI.

Figure 11. Glycocalyx modifications induced by ischemia-reperfusion

injuries. DAMPs, damage-associated molecular patterns; GCX,
glycocalyx; IRI, ischemia/reperfusion injury; NO, nitric oxide; ROS,
reactive oxygen species.39

27
1.3.2.6 Kupffer Cells and Neutrophils

Kupffer cells (KCs) are liver-resident macrophage cells. They are in the
liver sinusoids, endothelial cells, and hepatic stellate cells. Their
interplay with white blood cells (WBC), the body's most abundant
innate immune cells that act as the first defense against infections, is
instrumental in developing hepatic IRI. KCs are activated during early
reperfusion and produce ROS. Activated KC also has pro-inflammatory
cytokines, which stimulate adhesion molecule expression at the surface
of endothelial cells. WBC bind to this adhesion molecule, allowing
them to penetrate from the endothelial lumen to the hepatic
parenchyma. Once in the parenchyma, they secrete proteases and ROS
at the origin of hepatocytes' apoptosis and necrosis. Rats' treatment
with an anti-ICAM-1 antibody against an adhesion molecule reduced
hepatic IRI 40. This is in line with the vital role of WBC during IRI.

1.3.2.7 Role of cytokines and chemokines

Activated KC and hepatic stellate cells mainly produce cytokines and

chemokines. They sustain liver inflammation which is a significant
cause of IRI. The inflammatory cascade begins with the production of
IL-1b, IL-12, and IL-23. TNF-a and NF-Kb are also two crucial mediators
of the inflammatory response.

1.3.2.8 Apoptosis and Necrosis

Oncotic necrosis is the result of ATP depletion. Indeed, mitochondrial

and cellular edema are observed as it causes ionic imbalances and
water entry into the cells. Before cellular death, liver cell instability is
initiated through mitochondrial permeability, lysosomal dislocation,
ionic imbalance, and cellular edema. Plasmic membrane permeability
releases the intracellular component into the liver sinusoids triggering
and inflammatory response through macrophage (KCs) activation. On
the other side, apoptosis can be triggered in two ways. The intrinseque
pathway goes through the mitochondria. Indeed, during IRI,
mitochondrial permeability increases, releasing cytochrome c and
activating the caspase cascade leading to DNA fragmentation. The
extrinseque pathway involves the extracellular death receptors FAS,

28
 TRAIL (Tumor necrosis factor Related Apoptosis Inducing Ligand), and
the cytokine TNF-alpha. This will also activate the caspase cascade.
Hepatocyte death occurs more frequently through oncotic necrosis
compared to apoptosis.

Figure 12. Overview of ischemia-reperfusion injuries 41

1.4 LIVER EX-VIVO PRESERVATION STRATEGIES TO DECREASE IRI

The ultimate goal of liver preservation is to maintain the graft function

throughout storage steps to improve graft viability at reperfusion. For
liver grafts, two methods exist Static Cold Storage (SCS) and Machine
Perfusion (MP).

1.4.1 Static Cold Storage (SCS)

Static cold storage (SCS) is the gold-standard of graft preservation

during liver transplantation. Indeed, in clinical practice, the graft is
washed in situ with a cold (5°C ± 3°C) preservation solution (PS)
through the portal vein or the portal vein and the hepatic artery. This
step allows the preservation solution to be homogenously distributed

29
across the organ through the vessels replacing the blood content. The
graft is then immerged into the same PS at 5°C ± 3°C and transported
to the transplant centers. The relevance of this method relies on the
diminution of metabolism and energy consumption by the liver cells
under the effect of hypothermia. Indeed, at 4°C, cell activity amounts
to around 10% of its physiological activity. Based on the Arrhenius
equation, the cellular metabolic rate is reduced by 50% for every 10°C
drop in temperature. Although hypothermia is a crucial element for
tissue preservation, it has harmful repercussions due to the induction
of cell swelling42 and cytoskeletal alteration43. This observation
triggered the development of preservation solutions to counteract
many pathophysiological pathways induced by cold ischemia.

1.4.2 Preservation Solutions

Preservation solutions development was initially investigated by Belzer

and Southard44, who suggested the late 1980s fundamental
composition of a flush-out solution to ensure adequate organ
protection. The ideal solution should include components that prevent
hypothermic induced cell swelling (e.g., impermeant/colloids such as
HES or PEG-35), minimize intracellular acidosis (e.g., p.H buffers), stop
the expansion of the interstitial space (e.g., impermeant/colloids such
as HES or PEG-35), prevent injury from oxygen free radicals (e.g.,
antioxidants such as glutathione or allopurinol) and provides
substrates for regenerating high-energy phosphate compounds (e.g.,
amino acids, ATP precursors, adenosine, phosphate) during
reperfusion.

Currently, more than ten perfusion solutions for the liver are
commercialized. These can be separated into two main groups
mimicking the intracellular or extracellular environment based on their
ionic content. The following section will detail the most used
preservation solution.

30
 Figure 13. Critical components of organ preservation solutions and
their functions in protecting against edema and ischemia perfusion
injuries. 45

1.4.2.1 EuroCollins

Euro-Collins (EC) is an intracellular solution (high K+-low Na+)

developed in the 1970s46. Oncotic agents are absent from its
formulation except for glucose. Glucose, though being an active
metabolic component, is impermeable to renal cells. Therefore, EC was
appropriate for kidney preservation. However, liver cells are highly
permeable to glucose. Thus, the osmotic effect was lost, and glucose
was metabolized in anaerobic conditions leading to intracellular
acidosis activation, which is highly detrimental to long-term
preservation. For this reason, glucose was replaced by other larger
sugar molecules, such as lactobionate and raffinose, in the UW
solution.

1.4.2.2 University of Wisconsin (UW)/Belgen

University of Wisconsin (UW), also known as Belgen, is currently one of

the most used preservation solutions for the liver. Indeed, the 3-year
survival of the whole liver preserved with UW is 75%47. Developed in

31
the late 1980s for renal transplantation, it impacted the logistics of liver
transplantation, increasing the preservation time from 6h to 16h48. The
essential advantage of this low Na+/high K+ solution, characteristic of
an intracellular PS, relies on the adequate concentration of
impermeant (raffinose, lactobionate), counteracting hypothermic
induced cell swelling. However, three critical limitations exist. The first
one is the absence of components balancing the deleterious calcium
overload. Even though lactobionate can chelate calcium by reducing
calcium-dependent enzymes, its action within UW is not explicit. Yet,
lactobionate seems to be a key component as its only replacement
with a similar substance decreases the efficacy of the solution49. The
second drawback concerns hydroxyethyl starch (HES), an oncotic agent
associated with hyper aggregation of red blood cells, causing tissue
saturation with the PS50,51. This might reduce the efficiency of the
blood flush out. Finally, an elevated concentration of K+ is associated
with the activation of voltage-dependent channels that could highly
disturb the ionic equilibrium and induce cardiac arrest52. Novel
preservation solutions were developed to overcome these limitations.

1.4.2.3 UW-MPS (Belzer-MPS)/Perfgen

Belzer-MPS, also known as Perfgen, is a modified version designed

specifically for perfusions53,54. In this solution, lactobionate is replaced
by gluconate, and the viscosity is divided into two. Indeed the viscosity
of UW is 5.70 Cp while UW-MPS is 2.40 Cp (Table 2).

1.4.2.4 Histidine Tryptophan Ketoglutarate (HTK)

Histidine Tryptophan Ketoglutarate (HTK) is based on using a powerful

buffer system formed by histidine and its two substrates, tryptophan
and ketoglutarate55, using the principal of equilibration of the
extracellular space without any impermeant. HTK solutions have a low
viscosity, providing a faster and more homogeneous temperature
decrease and improved blood washout during procurement. For short
preservation length, its efficacy is similar to UW 56. Its low concentration
of K+ minimizes cardiac risk during revascularisation. However, HTK
solution was associated with reduced graft survival in case of
additional risk factors such as DCD, cold ischemia time over 8 h, and
donors over 70 years compared to UW solution 57.

32
1.4.2.5 Celsior

Celsior, or CE, was developed in the 1990s as a cardiac preservation

solution with a low K+/high Na+ (extracellular composition). Celsior is
a combination of the best advantages of UW and HTK. Indeed, it uses
the same impermeant agents as UW regulating osmotic pressure
(lactobionate and mannitol) coupled with histidine58. Unlike HTK, an
antioxidant (glutathione) is also added in its reduced form. Cellular
edema prevention is assured by lactobionate and mannitol. Without
any impermeant, the viscosity of the Celsior solution is low, enhancing
its perfusability, which is protective against endothelial lesions. Celsior
solution expresses similar performances for liver preservation (primary
non-function frequency and one-year survival) as UW 59.

1.4.2.6 Polysol

Polysol is a PS developed in Amsterdam in 2005 60 sets explicitly for

machine perfusion protocols. The Polysol mechanism maintains a
metabolism at 4°C61. Indeed, the solution is enriched in amino-acids,
vitamins, and antioxidants and supplemented with nutrients required
for liver cells (histidine, tryptophan, glutamine, arginine), robust
buffers, and antioxidants (glutathione and vitamin such as ascorbic
acid). Polysol is also enriched in a tissue culture medium, favoring
recovery. In Bessems’ 2005 study 60, it was demonstrated that liver
preserved in machine perfusion using Polysol resulted in better-quality
liver preservation than static cold storage with UW and machine
perfusion using UW-G (Belzer-MPS).

1.4.2.7 IGL-1

IGL-1 solution developed in Lyon combines the extracellular

composition (low K+/high Na+) of Celsior with the presence of an
impermeant like UW. Indeed, HES was substituted by polyethylene
glycol with a molecular weight of 35 KDa (PEG35). PEG molecules are
water-soluble polymers with multiple molecular weights, which are
non-immunogenic and non-toxic62. PEGs prevent the generation of
ROS63, enhance cell survival pathways in hypoxia conditions and repair
endothelial cell damage during post-ischemic reperfusion64. PEGs can
restore membrane integrity65, enter cells through the disrupted
membranes, and interact with cellular organelles66, conferring hepato

33
protection. The beneficial effect of IGL-1 against apoptosis,
microcirculation dysfunction, and immune response was
demonstrated in experimental67 and clinical68 settings on liver and
kidney transplantation. IGL-1 is the first solution reported to be
advantageous in SCS of suboptimal livers. Indeed, previous studies of
cold preservation and machine perfusion demonstrated that IGL-1
contributes to more efficient conservation of non-steatotic and
steatotic rat liver grafts than UW 23. The beneficial effects of IGL-1
include the prevention of hepatic damage, oxidative stress, and
mitochondrial injury, which are mediated through nitric oxide (NO)
production. IGL-1 3-year graft survival is similar to UW (75%)47.

1.4.2.8 IGL-2

IGL-2 is a modified version of IGL-1 developed by Institut Georges

Lopez. It differs from IGL-1 as PEG35 and glutathione concentrations
were increased by five-fold and three-fold, respectively. These changes
have been made to improve vasodilation of the vessels and antioxidant
properties. Sodium nitrite (NaNO2) was added to ensure the activation
of the endothelial nitrite oxide synthase (eNOS) involved in NO
production, glycocalyx, and vasodilatation. The viscosity has also been
adapted to better suits machine perfusion systems. Preliminary studies
investigating IGL-2 in static cold storage demonstrated that
mitochondrial protection was enhanced and ROS generation was
decreased69.

34
Table 2. Overview of the composition of preservation solutions45

35
1.4.2.9 Preservation Solution Additives

Preservations solutions have greatly improved the conditions of liver

preservation. As strategies to increase the organ pool are on the rise,
such as using suboptimal grafts, new alternatives must be investigated
to optimize graft quality, particularly during the sensitive cold storage
phase. One of them relies on adding active compounds to current
preservation solutions. Most studies were performed using UW and
IGL-1, solutions in which new additives have been proposed to
improve static liver graft preservation70.

1.4.2.9.1 Oxygen Carriers

The presence of oxygen during the preservation step is critical to

prevent mitochondrial dysfunction and ROS generation. That is why
most machine perfusion protocols, such as HOPE, use oxygen directly
added to the preservation solution. Even though this technic has
proved efficient, the logistic remains complex. Improving oxygen
availability within the graft by adding an oxygen carrier is a more
accessible and cost-effective method to prevent IRI. Two of them have
been investigated for liver preservation purposes.

1.4.2.9.1.1 Hemo2life® (M101)

M101 (HEMO2life®, Morlaix, France) is a powerful oxygen carrier

isolated from extracellular hemoglobin isolated from Arenicola marina,
a marine invertebrate. Unlike human hemoglobin, which can carry four
oxygen molecules, M101 can fix up to 156 molecules. M101 is not only
active over a wide range of temperatures (from 4 °C to 37 °C), but it
also releases O2 through a gradient. This process contributes to
continuous oxygen delivery and cell consumption71,72. M101
supplementation to various preservation solutions reported
encouraging results. Indeed, all groups supplemented with M101
demonstrated improved early function recovery73,74. First-in-human
use on kidneys has shown similar results75.

36
1.4.2.9.1.2 Perfluorocarbons (PFC)

Perfluorocarbons (PFCs) are high-capacity oxygen compounds with a

hundred times higher oxygen solubility than in blood.
Supplementation of PFC in preservation solutions has been studied in
a wide range of organs, including livers. Recent experimental studies
on porcine models showed better preservation of aerobic metabolism
leading to maintenance of mitochondria integrity and decreased
inflammation76. However, not only a high partial O2 pressure is
necessary to maximize O2 content, but it also equilibrates rapidly with
the surrounding environment, quickly losing its oxygenation capacity
77
.

1.4.2.9.2 Others

Trimetazidine (TMZ) is an anti-ischemic drug with anti-oxidant action

in several abdominal organs, including livers 41,78,79. TMZ was tested in
steatotic and non-steatotic rat livers after static and dynamic perfusion
protocols. UW + TMZ reduced hepatic injury by improving
microcirculatory function and decreasing oxidative stress and
mitochondrial damage. TMZ addition was tested in IGL-1 in the same
experimental settings and demonstrated improved graft preservation
along with a significant upregulation of hypoxia-inducible factor-1
alpha and increased NO production80.

Epidermal growth factor (EGF-1) and insulin-like growth factor-1 (IGF-

1) in UW or IGL-1 alleviated the resistance of steatotic livers to
ischemia-reperfusion injuries, partly due to Akt and eNOS signaling
activation, and reduced cytokine release81,82.

Aferetica, an Italian machine perfusion manufacturer (See section),

developed a blood purification cartridge (PerSorb) for their PerfLife ex
vivo machine perfusion. It has been shown that Persorb can reduce
excessive levels of inflammatory mediators within the perfusate,
preventing inflammation pathways' activation during perfusion
protocols.

37
1.4.3 Machine perfusion (MP)

Machine perfusion (MP) techniques consist of continuous controlled

perfusion through the graft with a fluid (e.g., blood, preservation
solutions) using an ex vivo device before transplantation. The perfusion
ensures the constant and homogeneous distribution of the
preservation solution through the organ, washing out the blood
content and providing, in some cases, oxygen to fulfill the organ’s
metabolic demand. MP also regulates the interstitial space ions
balance preventing edema and allowing the delivery of nutrients,
oxygen (optional), and removal of toxic metabolites. This technique
revolutionized liver transplantation protocol as it offers the
opportunity to extend preservation time without increasing IRI and
monitor the functional and biochemical performance in real-time,
adding granularity to the graft quality scores. Though its benefits on
low-risk graft are inconclusive as SCS results are satisfying, its ability to
optimize suboptimal graft preservation is undeniable. Indeed, pilot
studies consistently confirmed their potential to expand the donor
pool, reducing the waiting list83,84.

Different type of MP protocol exists, all characterized by the

temperature of preservation: hypothermic (HMP) at 5°C ± 3°C, sub-
normothermic (SNMP) at 25°C-34°C, and normothermic (NMP) at
37 °C. Also, several flows and pressures (pulsatile or continuous), single
or dual perfusion (hepatic artery and portal vein), oxygenation, or non-
oxygenation are under investigation.

Table 3. Risks & Benefits of Machine Perfusion70

38
1.4.3.1 Hypothermic Machine Perfusion (1-12°C)

Hypothermic Machine Perfusion (HMP) is a dynamic cold preservation

method where the PS oscillates between 1-12°C, which ensures a
homogeneous and continuous supply of metabolic substrates to the
graft during the ex vivo period. HMP hepato-protection relies on ATP
synthesis85 and critical metabolites maintaining the cells' antioxidant
potential, reducing cellular injuries observed during reperfusion86.
HMP represents an excellent way to “rejuvenate” grafts subjected to a
long period of warm ischemia (e.g., a graft from non-heat-beating
donors). This technic offers the possibility of post-condition marginal
graft61,87. These post-conditioning steps performed on grafts that
underwent uncontrolled warm ischemia still show their efficiency after
a long period of cold ischemia 87. Compared to SCS, HMP was shown
to improve graft function and attenuate biomarkers of liver injury
through the decrease of ROS release. Its application to the suboptimal
liver is relevant as it improved hepatocellular and endothelial function
while reducing damage in a rat fatty liver model.

HMP has been used in a small number of patients successfully for years
88
. HMP is a safe technic because, in the event of MP dysfunction, the
graft returns to SCS conditions. However, the lack of consensus on the
optimal perfusion setting remains. Indeed, the challenge is to establish
optimal parameters for HMP to maintain sufficient perfusion without
damaging liver tissue. For example, endothelial cells may be damaged
by the shear stress generated by the fluid flow. This has been
associated with morphological changes occurring in the sinusoidal
endothelial cells compartment, flow heterogeneity, elevated vascular
resistance, and the risk of sinusoidal flow obstruction89. One major
drawback of HMP is the inability to perform real-time liver function
assessment. For example, bile synthesis is only observed under
physiological temperature, preventing hepato-biliary function from
being assessed.

Perfusion usually occurs through the portal vein as this vessel has a
high tolerance to hepatic pressure variation90. Performances through
the hepatic artery were equivalent though they needed to be pulsatile.

39
Belzer-MPS remains the predominant perfusion solution. Other
alternatives are currently under investigation, such as Polysol, which
showed lower enzyme release and bile production than Belzer-MPS60.

The optimal timing and the length of protocol remain active research
fields. Focusing on the timing, three strategies have been investigated:
PRE-SCS, where HMP could be performed at the donor site; END-SCS,
where HMP is performed at the recipient site; and finally, the
continuous perfusion technic, where the graft is connected to the HMP
device throughout the entire preservation phase. END-SCS is the most
investigated and accepted method as it has been associated with a
lower risk of shear stress thanks to its shorter duration91. It is also cost-
effective and convenient because the machine may not be available at
procurement centers. Transferring such machines might not be
feasible until portable devices reach the market. Given these practical
issues, a certain period of SCS would seem unavoidable to move the
liver graft.

1.4.3.2 Hypothermic Oxygenated Perfusion (5°C ± 3°C)

Hypothermic Oxygenated Perfusion (HOPE) is a machine perfusion

technique differentiating from HMP by adding oxygen to the
preservation solution. HOPE was first introduced by Dutkowski et al.
92,93
, who demonstrated the impact of HOPE on isolated rat livers. They
showed that if the isolated liver was transplanted after 3 hours of
HOPE, no ROS burst, and endothelial damage was observed94. By
comparing the impact of 1 hour of HOPE after 5 hours of SCS vs. 5
hours of SCS, they observed that the HOPE group showed decreased
necrosis after reperfusion. In addition, transaminase levels were lower,
and bile production was significantly higher. HOPE showed consistent
results on a larger animal model, the Swiss Landrace pig95. This technic
supported the use of HOPE, especially in non-heart-beating donors
where the deleterious warm ischemia time is unknown. Therefore,
HOPE is highly relevant in the context of donor shortage and the
increasing use of older donors and fatty livers. Such grafts are highly
susceptible to ischemia-reperfusion injury and would be the most
appropriate candidates.

This was reported by Dutkowski et al. in 2015 96, where HOPE was used

40
on donors after cardiac death. Among 25 DCD grafts that underwent
HOPE compared to 50 DCD grafts that underwent SCS, HOPE led to a
significant reduction in graft injury. The 1-year survival rate was 90%.
Guarrera et al. 97 also reported the outcome of 31 recipients whose
graft underwent HMP using marginal grafts rejected by the United
Network for Organ Sharing (UNOS). The Control group was composed
of organs submitted to SCS. HMP led to a lower risk of biliary
complications and decreased kidney injury. The critical differences
between Guarrera and Dutkowski rely on the perfusate's different
oxygenation. Guarrera used a portal vein and hepatic artery, while
Dutkowski used a portal vein only, with an oxygenated perfusion
solution. It has now been shown that both technics lead to comparable
outcomes98,99.

It is known that HOPE technics mitigate oxidative stress despite the

high content of oxygen in the cold, probably due to changes in
mitochondrial electron transfer at temperatures below 15°C (Arrhenius
breakpoint). At the same time, the metabolism of accumulated
electron donors such as NADH and succinate leads to ATP re-synthesis
with surprisingly high efficiency compared to normothermic machine
perfusion conditions. End ischemic HOPE seems to allow a higher
upload of energetic cellular levels in liver grafts procured after
circulatory death compared to NMP. However, NMP has some
potential benefits that could be synergistic with HOPE.

Finally, a recent study demonstrated the benefits of HOPE in

performing split liver transplantation. Indeed, splitting the liver during
HOPE improved the preservation quality of both partial grafts. This
HOPE-SPLIT innovative surgical practice observed no early allograft
dysfunction or graft loss100.

1.4.3.3 Normothermic Machine Perfusion (37°C)

The concept behind normothermic machine perfusion (NMP) is to

maintain a physiological metabolism by providing oxygen, and
essential substrates in an environment kept at a body temperature
(37°C). This allows the preservation of energy reserves such as ATP. Its
significant competitive advantage compared to the hypothermia
method relies on the ability to perform viability assessment before

41
transplantation. Indeed, as the liver metabolism is maintained during
preservation, markers, including bile production and liver enzymes, can
be measured as well as hepatic flow and oxygen ratio. This is important
to improve the accuracy of the donor/recipient matching. As the organ
is fully metabolically active, the perfusate is blood-based.

NMP usually begins when the organ is harvested and continues while
it is in transit. This highlights the need to develop a portable machine
perfusion device to perform NMP directly, bypassing the cold ischemia
intermediary step. The first clinical study was conducted by Ravikumar
et al. in 2016101, where 20 marginal liver grafts (16 DBD and 4 DCD)
were compared to 40 cold-stored livers. They observed a comparable
1-month survival between the two groups. However, the AST peak was
significantly lower during the first week in the NMP group. Following
this, the first multicenter randomized control trial was performed in
Europe by the Consortium for Organ Preservation. Of the study's 272
livers (194 DBD and 78 DCD) involved, 137 (50.3%) underwent NMP.
The AST peak and percentage of early allograft dysfunction were
significantly lower in the NMP-treated livers than in those submitted
to standard SCS102.

Despite the promising results of NMP, device malfunction or user error

can cause substantial damage with more detrimental effects under
NMP than in hypothermic conditions. Indeed, if the device stops
functioning, the graft needs to undergo detrimental warm ischemia for
an unknown period of time, often leading to graft loss103. Moreover,
the use of packed red blood cells as perfusates is more complex to
monitor than cold preservation solution. Finally, NMP devices are more
sophisticated than HMP and require a trained clinical team. Moreover,
disposables needed to run the device are expensive104.
1.4.3.3.1 Defatting Strategies

Researchers also showed a growing interest in NMP, which could be

involved in the “defatting” process, decreasing steatosis in marginal
liver grafts. Diluted blood is replaced by a unique “deffating” perfusion
solution for steatotic grafts tailor-made to stimulate lipid metabolism.
This solution comprises compounds activating receptors such as
PPARs to exert an insulin-mimetic effect to promote intracellular cAMP
and activate lipid catabolism. This composition was added to the cell

42
culture medium and used in the following research studies. The first
one using porcine livers demonstrated a 50% reduction in lipid droplet
size in hepatocytes, reaching the size found in control non-
pathological livers105. The same result was observed in Zucker fatty
liver rats after 3 hours of NMP106. NMP could therefore represent a
step forward in decreasing organ shortage by enabling the use of
discarded livers, especially for those with severe steatosis. Though
defatting results are promising in the animal model, further studies will
need to be conducted on humans.
1.4.3.4 Sub-Normothermic Machine Perfusion (25-34°C)

We presented above that NMP is intended to be performed after SCS.

This subjects the organ to drastic temperature changes at the origin of
oxidative burst. Because of the harmful temperature changes,
researchers developed a novel approach to counteract the detrimental
effects. They hypothesized that maintaining the graft at a sub-thermic
condition (25-34°C) could lower the metabolic demand while
maintaining sufficient metabolism for viability testing. Sub
Normothermic Machine Perfusion (SNMP) requires no complex
temperature control, which is a crucial advantage compared to other
approaches in clinical settings.

SNMP potential was investigated by Okamura et al.107 in a steatotic rat

model. They demonstrated that SNMP improved cell viability. SNMP
(20-25°C) treatment was associated with lower vascular resistance, a
better-preserved microcirculation, and a more substantial
mitochondrial function. This coincided with a higher energy charge
and greater bile production. SNMP also reduced the release of Hmgb1,
which is known to be one of the most dangerous DAMPS released from
a steatotic liver. The first evidence of SNMP efficiency in a human was
brought by Bruinsma et al. in 2017108. This group suggested that SNMP
might be indicated for the human liver with minimal injury.

1.4.4 Machine Perfusion Devices

Many perfusion devices are commercialized or included in clinical trials

in Europe and the US. Some specialize in hypothermic protocols,
normothermic protocols, or both. They all monitor vital components
such as pressure flow and temperature. But some of them, such as

43
Organox, differentiate by measuring PO2, PCO2, glucose, and bile
production. As described before, the logistic of machine perfusion is a
significant limitation.

For this reason, even though some devices like Organ Assist are static,
most other devices are transportable and can run on battery for a
limited time. This is key, especially for NMP protocols that might be
performed continuously. Only one machine perfusion device (Airdrive)
has been considered fully portable, but it is still in the preclinical
stages.

44
Table 4. Overview of the different machine perfusion systems
available for liver transplantation

45
1.4.4.1 Experimental Machine Perfusion Device

The entire research work presented in this thesis uses a machine

perfusion model developed by previous Ph.D. students in the
laboratory. This in-house isolated rat liver perfusion system was
developed following Dutkowski's work93. Its main components are an
oscillating pump, an oxygenation membrane, an external circuit to
maintain the perfusates at a chosen temperature, an internal circuit
where the perfusates circulates, and different sensor to probe the flow,
the pressure, and the temperature. This device's particularity is that it
can perfuse two livers simultaneously at two different temperatures,
which is innovative for an experimental model. It is also possible to
perform a wide range of temperatures (0-37°C). Therefore this device
allows us to perform HMP, HOPE, and NMP protocols. This device has
been improved over time by adding a more performant oxygenation
membrane and sensors of improved sensitivity.

Figure 14. Experimental ex vivo machine perfusion system. (A) Bubble

Trap (B) Membrane Oxygenator (C) Peristaltic Pump (D) Liver Perfusion
Chamber (E) Preservation Solution Tank (F) Pressure and Flow Sensors
(G) Bubbling Oxygenation

We also use the isolated rat liver perfusion system to develop a model
of liver transplantation ex vivo. To this end, an NMP-like protocol is

46
performed for two hours at 37°C with a reperfusion perfusate. The
reperfusion perfusate is mainly composed of William E medium
(hepatocytes cell culture medium) and albumin. The solution is
adjusted to physiological pH that is 7.3.

1.4.5 Other strategies for decreasing IRI

1.4.5.1 In situ

1.4.5.1.1 Surgical ischemic pre-conditioning

Surgical ischemic pre-conditioning (IPC) consists of multiple cycles of

vascular occlusion and reperfusion (clamping/declamping) performed
during liver procurement. This ischemic stress should increase graft
tolerability toward long-term ischemia. This paradox was initially
characterized by the heart before being applied to other organs109.

It was demonstrated that surgical IPC performed before a long period

of warm ischemia was protective in rodent livers. Indeed, it reduced
hepatocellular damage and enhanced hepatic function and survival
110,111
. However, though surgical IPC increased the tolerance of warm
ischemia, no effect was observed on cold ischemia. Therefore,
clinicians did not fully adopt the practice112–115. Indeed, it appears that
the number of cycles performed is organ-dependent. Therefore, where
the heart would necessitate multiple cycles of vascular occlusion109, the
liver will only need a unique process of occlusion lasting for 10 min116.
The exact mechanism underlying surgical IPC remains to be elucidated.
Many factors have been directly and indirectly linked to surgical IPC
protection, such as MAPK8, NO, apoptosis, and pro-inflammatory or
oxidative stress markers.

1.4.5.2 Ex situ

1.4.5.2.1 Donor pharmacological pre-conditioning

Donor pharmacological preconditioning aims at improving in situ the

graft physiological capabilities. Indeed, fasting, leading to glycogen
depletion, appeared responsible for significantly decreasing bile and
ATP production at the origin of actual damages during reperfusion.
Therefore, in the clinical setting, glucose was administered

47
intravenously to donors, leading to increased hepatic glycogen
associated with minor hepatocellular lesions 117. Kupffer cells (KC)
could also be inactivated because of fasting, suppressing ROS
generation, and pro-inflammatory molecules. Therefore, KC
inactivation was further investigated as a therapeutic target. In rodents,
they also studied the injection of antioxidant compounds such as
glutathione117 and superoxide dismutase118,119 and demonstrated their
protection potential during cold ischemia. Apoptosis inhibitors were
also investigated as a potential therapeutic targets, such as Caspase 3.
Calpain protein, activated upon calcium accumulation, was also
investigated as a potential target to prevent IRI.

Cold ischemia slows down the metabolism of sinusoidal endothelial

cells120. Indeed, their morphological modification could alter the
hepatic microcirculation at reperfusion. In experimental situations,
many teams reported the protective effect of donor preconditioning
with prostaglandins121,122 for their role in preventing reperfusion
lesions.

However, many questions come with the donor preconditioning:

technical questions (e.g., when is the best time to administer it?);
ethical (e.g., can the pre-treatment begins before the patient's death?);
systemic (e.g., what are the preconditioning effect on other organs?)

1.4.5.2.2 Recipient pre-conditioning

The heme oxygenase-1 (HO-1) system is a crucial cytoprotective

mechanism activated during cellular stress. Indeed, in ischemia-
reperfusion injuries, local overexpression of HO-1 led to the
improvement of antioxidant and anti-apoptotic/inflammatory
processes and maintenance of microcirculation123. For this reason, the
administration of biliverdin (Heme metabolism by-product) in the
recipient right before and a couple of hours after graft implantation
seems to be a promising therapeutic target to prevent complications
after liver transplantation.

1.4.5.2.3 Gene therapy

Gene therapy is also investigated in rodents to transfect anti-apoptotic

and antioxidant genes to reduce IRI. Therefore, the transfection of Bcl2,

48
 an anti-apoptotic gene, could represent an excellent way to increase
the hepatic resistance to SCS by attenuating apoptosis124. SOD
enzymes were transfected to strengthen the immune and antioxidant
defense. Other teams have developed cytoprotective strategies based
on the expression of genes such as HO-1 or IL-13. The alteration of the
inflammatory response through the inhibition of Nfkb was also
investigated. Though preliminary results are promising, the technique
is not fully efficient yet. Indeed, issues regarding the low efficacy of
transfection vectors and the low translation rate of target genes still
need to be overcome.

1.5 RESEARCH OBJECTIVES

1.5.1 Evaluation of a novel preservation solution additive for fatty

liver graft: Hemo2life® (M101)

1.5.1.1 Objective

One of the leading research objectives of this thesis is to assess the

relevance of M101, an oxygen carrier, as a preservation solution
additive to improve steatotic graft preservation.

1.5.1.2 Problem Statement

Using grafts from extended criteria donors represents a huge

opportunity to increase the pool of donors. However, as these grafts
are more sensitive to IRI, they require advanced ex vivo preservation
technics. For this reason, most machine perfusion protocols (MPP) are
prioritized for suboptimal graft preservation. Even though MPPs have
proved their superiority compared to SCS-only in humans, the level of
acceptance by surgeons remains low due to its cost and the complexity
of the logistics. That is why MPP alternatives need to be investigated.

Preservation solution additives seem to be a good alternative. Indeed,

they are less costly and easy to implement during the graft
preservation journey. Still, additives can also be graft-specific based on
the donor characteristic (e.g., steatosis level, inflammation, hepatitis C
virus). For this study, we chose to focus on additives for steatotic grafts
as they are the most observed ECD graft.

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1.5.1.3 Research Question & Hypotheses

As steatotic grafts are characterized by impaired microcirculation and

mitochondrial dysfunction22, oxygen carriers are an excellent
opportunity to potentiate these functions by improving O 2 availability
and ATP production.

M101 is an oxygen carrier extracted from a marine worm that can fix
up to 156 O2 molecules, which is 40 times more than human
hemoglobin. M101 has improved kidney preservation when added to
SCS75.

In this study, we hypothesize that adding M101 directly to the

preservation solution (IGL-1) during SCS might improve the availability
of oxygen molecules which is crucial to ensure basal metabolism and
to optimize the preservation quality, especially for steatotic graft. To
this end, we used rat steatotic liver to investigate the impact of M101
on the quality of their preservation.

1.5.2 Evaluation of a novel timing for Hypothermic Oxygenated

Perfusion (HOPE): HOPE-PRE

1.5.2.1 Objective

The second objective of my thesis is to investigate a novel timing for

the Hypothermic Oxygenated Perfusion protocol.

1.5.2.2 Problem Statement

Machine perfusion, especially HOPE protocols, has been widely studied

in large animal models, including humans. It has been demonstrated
that even though HOPE machine devices are costly and demand strong
logistics, the technics are safe and beneficial for suboptimal grafts.
HOPE is currently performed after static cold storage. Indeed, once the
graft is resected from donors, it is directly placed in the cold
preservation solution for transportation purposes. It is only at the
transplant center that the graft is placed on the machine perfusion
device. Therefore, this sequential order has been commonly accepted
mainly for logistical reasons. Other timing needs to be studied to
investigate if different timing of HOPE could enhance graft protection.

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This research is also relevant because of the increased studies on
portable and transportable machine devices, where HOPE protocols
could be performed anytime, depending on the graft needs.

1.5.2.3 Research Question & Hypotheses

For this second research project, we hypothesized that some grafts

might need HOPE protocol upstream SCS. Indeed, SCS though being
protective for grafts when performed for a limited period, remains in a
deleterious phase as the liver starts to accumulate lesions precisely
because of mitochondrial dysfunction, ATP depletion, and anaerobic
glycolysis caused by hypoxia. Therefore, providing oxygen to the graft
before SCS could boost mitochondrial energy charge and help the
graft cope later with the deleterious SCS phase. To this end, using rat
livers, we analyzed the expression levels of 40 genes involved in
ischemia-reperfusion injuries in two experimental groups: HOPE
performed after SCS (HOPE-END) and HOPE performed before SCS
(HOPE-PRE).

1.5.3 Evaluation of a novel preservation solution for static and

dynamic preservation protocols: IGL-2

1.5.3.1 Objective

Finally, the third objective of my thesis is to evaluate a novel

preservation solution for the static and dynamic preservation of
steatotic grafts.

1.5.3.2 Problem Statement

Novel machine perfusion protocols and devices go along with the

development of innovative perfusates to meet the new technologies.
However, in the field of preservation solutions, little has changed in the
past decades. Indeed, UW-SCS/Belgen developed in the 1980s,
remains one of the most used preservation solutions for static cold
storage. For machine perfusion, Belzer-MPS/Perfgen is the first
solution developed, though being initially made for kidney perfusion.
Therefore, it might not be suitable for liver perfusion. Moreover, the
presence of HES as a colloid has been linked to blood cell aggregation,

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which prevents adequate graft perfusion. This is important specifically
for steatotic grafts where blood microcirculation is already impaired.
Therefore, new preservation solutions to overcome this deleterious
effect are needed. Other preservation solutions, such as Polysol, have
been designed explicitly for perfusion. However, as described before,
HOPE protocols come with SCS. The mixture of two preservation
solutions on a single graft might cause unknown interaction.

1.5.3.3 Research Question & Hypotheses

This third research project focuses on developing a novel preservation

solution that could be used for static and dynamic perfusion protocols,
easing the logistics and avoiding a mixture of preservation solutions.
IGL-2 is a modified IGL-1 solution where PEG35 (colloid agent) and
glutathione (anti-oxidant) concentrations were increased and viscosity
modified. Using the rat model, we studied IGL-2 in SCS and HOPE
settings and compared it to UW-SCS and Belzer-MPS.

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2 RESULTS

2.1 ARTICLE 1

2.1.1 Objective and hypotheses of this work

Although the HOPE protocol is the most promising technique to

rejuvenate marginal grafts during ex vivo preservation, high-cost and
complex logistics prevent its adoption in clinics. Consequently, easy-
to-implement and cost-effective alternatives need to be developed to
improve the salvage of marginal grafts.

Preservations solutions additives represent a way to potentiate the

efficacy of existing preservation solutions during static cold storage.
Besides the perfusion, the key difference between HOPE and SCS
remains the presence of oxygen which is linked to longer and better
preservation of liver grafts. That is why oxygen carriers are currently
strong candidates to be used as additives to improve oxygen
availability and transport within the graft.

Many oxygen carriers are under investigation, as they could enhance

oxygen delivery and potentially create the same effect as HOPE
without the abovementioned drawbacks.

M101, a natural giant extracellular hemoglobin (Hb) extracted from a

marine worm, can fix O2 forty times more than human Hb. M101 is
efficient at physiologic O2 pressure and passively releases O2 offering
an oxygen gradient. This is key to preventing drastic oxygen changes
as it provides the organ with the right amount of oxygen. Moreover,
M101 addition could be of importance for steatotic grafts, where the
microcirculation is altered to promote the homogenous distribution of
oxygen.

The objectives of this research were to investigate the impact of M101

supplementation on liver graft preservation and to appraise the
relevance of this oxygen carrier as an additive to enhance steatotic
graft preservation.

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2.1.2 Summary

We resected 36 livers from obese Zucker rats, which were randomly

divided into three groups: Control, SCS 24H, and SCS 24H + M101 at
1g/L. Normothermic Machine Perfusion (NMP) at 37°C was performed
for 2h to mimic liver transplantation and evaluate the graft quality.
Perfusate and tissues were sampled for functional assessment and
biochemical analysis (transaminases ALT-AST, Glutamate
dehydrogenase -GLDH, lactate, Malondialdehyde -MDA, HMGB1,
Nitrite-Nitrate -NO2-NO3, Bcl-1, Caspase-3).

Transaminases, GLDH, and lactate levels, markers of liver injury, were

significantly lower in the group preserved with M101 (p<0.05) at the
end of NMP. Protection from reactive oxygen species (low MDA and
higher production of NO2-NO3) and less inflammation (HMGB1) were
also observed in this group (p<0.05). Bcl-1 and Caspase-3 were higher
in the 24h SCS group (p<0.05) and presented more histological
damage than those preserved with M101.

These data demonstrate for the first time that adding M101 to the IGL-
1 preservation solution significantly protects steatotic livers from
obese rats during SCS by decreasing reperfusion injury and improving
graft function.

2.1.3 Publication

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2.2 ARTICLE 2

2.2.1 Objective and hypotheses of this work

Static cold storage (SCS), describing the immersion of the liver in a cold
preservation solution (PS), is currently the gold standard for low-risk
liver preservation during transplantation.

Numerous machine perfusion strategies are currently under

investigation for healthy and extended criteria donors' liver
preservation. HOPE, the most promising technique, is performed at
the transplant center after a period of SCS of variable length
depending on the graft transportation time (HOPE-END). Although
HOPE-END preliminary results in the clinic are satisfying, this specific
timing was first adopted as it facilitates the logistics. Indeed, the
transplant team is more likely to be the ones performing HOPE than
the procurement team. Therefore, HOPE must be performed after
transportation (SCS) at transplant centers.

However, we hypothesize that SCS, though slowing down the

metabolism, remains a sensitive step where injuries accumulate and
are exacerbated during reperfusion. Therefore, we postulated that
performing HOPE before SCS (HOPE-PRE) could fuel the graft energy
level by better preserving mitochondria, reducing its sensitivity
through SCS, and protecting it from IRI.

2.2.2 Summary

We resected 27 livers from Sprague Dawley “healthy” rats, which were

randomly divided into three groups: SCS (n= 9), HOPE –END (n=9), or
HOPE-PRE (n=9). NMP at 37°C was performed for 2h to mimic liver
transplantation and evaluate the graft quality.

Then, liver injuries were assessed at three different levels. Histological

analysis demonstrated that the HOPE-PRE group showed significantly
less ischemic necrosis than the HOPE-END and SCS-only groups. From
a biochemical standpoint, transaminases were lower after 2 hours of
reperfusion in the HOPE-PRE group, a marker of decreased liver injury.

qPCR analysis on 37 genes involved in IRI revealed that protection in

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HOPE-PRE and HOPE-END was mediated through similar pathways
(mitochondrial protection, glycocalyx protection, and autophagy
activation). However, the HOPE-PRE group demonstrated increased
transcriptional levels for protective genes.

2.2.3 Publication

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2.3 ARTICLE 3

2.3.1 Objective and hypotheses of this work

The most used protocol in clinical liver transplantation is the graft’s

static cold storage (SCS) in a cold (5°C ± 3°C) using UW/Belgen
preservation solution. During SCS, the graft is deprived of oxygen.
Therefore, the protection of the graft is ensured by the cold (reducing
metabolism activity) and the composition of the preservation solution,
which counteracts ischemia-reperfusion injuries. HES presence in
UW/Belgen has been involved in the hyper aggregation of red blood
cells. This latest characteristic is highly harmful to graft wash-out and
perfusion, especially in steatotic livers where microcirculation is altered
due to lipid infiltration in the hepatocytes.

Novel strategies need to be developed to meet the requirement of

marginal grafts. The most promising technic is called Hypothermic
Oxygenated Perfusion (HOPE). This perfusion method ensures a
continuous supply of oxygen (dissolved in the preservation solution)
to the graft during the ex vivo period. SCS combined with HOPE was
demonstrated to preserve liver grafts better and longer. The only
preservation solution explicitly designed for perfusion purposes is UW-
MPS/Perfgen, a modified version of UW. Though the viscosity has been
updated, the presence of HES remains a significant issue. In addition,
the mixture of UW and UW-MPS when SCS is combined with HOPE
might cause interactions of unknown effects.

That is why developing a new preservation solution is crucial to satisfy

the HOPE protocol. The present research investigates the efficiency of
the IGL-2 solution, a modified version of IGL-1, where vasodilatation
and antioxidant properties were uplifted by increasing the
concentration of PEG35 (oncotic agent) glutathione. We hypothesized
that these boosted properties are the most suitable to meet static
preservation and dynamic perfusion requirement.

2.3.2 Summary

Using Zucker rat lineage, a rat model of the steatotic liver, we

compared the IGL-2 solution to UW/Belgen and UW-MPS/Perfgen.

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After 24H of static cold storage (for IGL2.SCS vs. BELGEN.SCS
comparison) added to 2h of HOPE (for IGL2.HOPE vs. PERFGEN.HOPE
comparison) and 2h of NMP (for all groups), we measured
transaminases, lactates, and key markers of glycocalyx and apoptosis.

For the first time, we compared IGL-2 to Belzer-MPS solution for fatty
liver using HOPE strategies. In this study, we demonstrated the
suitability of using IGL-2 as an alternative to UW and Belzer-MPS to
improve the quality of steatotic graft preservation subjected to HOPE.
Besides, IGL-2 use permits the combination of hypothermic static and
dynamic preservation strategies, simplifying thus the complicated
logistics involved in clinical liver transplantation to better preserve the
liver graft quality.

2.3.3 Publication

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3 DISCUSSION

3.1 INTRODUCTION

The increasing mortality of patients awaiting a liver transplant urges

the expansion of the donor pool. Even though living donor
transplantation (See section) seems a great option to save pediatric
patients, surgeons and researchers showed increased interest in
marginal grafts from extended criteria donors to target specifically
adult populations. This idea was initiated from clinical observation.
Indeed because of the significant increase in food intake in
westernized countries, medical doctors saw numerous grafts with
macro steatosis that were most of the time rejected for transplantation.
This approach follows the benefit/risk principle. Undoubtedly, a
steatotic graft, though having metabolic disorders, would benefit a
patient with liver cancer. Initially, it was shown that using marginal
grafts increases early mortality in liver transplantation among high-
MELD patients125. This result might be explained by a mistake in a
donor-recipient pairing or inadequate ex vivo preservation protocols.
Indeed, as marginal grafts are more sensitive to IRI, meticulous
preservation procedures are needed. For this reason, developing novel
strategies to optimize liver ex vivo preservation is crucial to optimize
marginal graft preservation, expand the donor pool, and avoid
primary-non-function, early allograft dysfunction, or retransplantation.

Liver ex vivo preservation can be optimized at different levels to

mitigate IRI. The first level that can be modified is the preservation
solution used. Indeed, each composition targets hypothermia-induced
changes but differently. For example, IGL-1, a PEG-based solution, has
been demonstrated to be more suitable for marginal grafts. Instead of
changing the entire composition, researchers have investigated the
addition of active compounds (e.g., anti-inflammatory, oxygen carriers)
to potentiate existing preservation solutions economically. However,
several interrogations remain on this topic: Is historical preservation
solution composition compatible with new strategies? If not, what
aspect should we modify to ensure a good fit?

The second level that can be investigated is the development of

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 innovative machine perfusion protocol. Research on machine
perfusion devices and protocols has increased over the past decade.
NMP and HOPE protocols are the most investigated. Since 2011, all
nine clinical trials completed for both strategies recommend their use
for marginal grafts. On this axis, many questions remain: When is the
best timing to perform machine perfusion? What is the optimal length
of the protocol? What are the most adapted preservation solutions or
perfusate? Are the different protocols/temperatures indication-
specific? While liver physiological biomarkers are well described, little
is known about their behavior in hypothermia. Indeed, liver function is
usually assessed using NMP strategies or reperfusion. However, it
might be too late to detect or correct any abnormalities at that time.
Therefore, across all levels, there is a need to identify novel biomarkers
to assess liver status during preservation, especially in the hypothermic
protocol.

The main objective of this research work is to develop new strategies

for liver ex vivo preservation. For three years, I proposed and studied
several techniques providing preliminary answers to the questions
mentioned above.

3.2 OXYGEN: A CRUCIAL ELEMENT AT THE CROSSROAD OF MULTIPLE

PRESERVATION STRATEGIES

During liver transplantation, warm ischemia initiates progressive but

rapid ATP depletion. Grafts' ATP levels are further decreased during
SCS because of hypothermia. During cold ischemia, even though the
metabolic activity is low (10%), mitochondrial electron transport
dysfunction is observed because of the absence of its final acceptor,
O2. The disruption of the electron transport chain in the mitochondrial
membrane causes succinate accumulation which drives reactive
oxygen species generation126. For this reason, preserving
mitochondrial function and ATP production by managing oxygenation
is crucial to prevent IRI. Whether using oxygen carriers (Article 1) or
hypothermic oxygenated perfusion (Article 2, Article 3), fundamental
questions are still under investigation. What is the best way to provide
oxygen continuously and homogeneously to the graft? How can we

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optimize oxygen bioavailability? To avoid ROS generation, what is the
optimized saturation level of oxygen supply to the graft?

Oxygen carriers are a promising component to improve oxygen

availability, especially when there is no active oxygenation (e.g., during
SCS). In the first study, adding M101, an oxygen carrier from a marine
worm, significantly improves steatosis liver preservation during static
cold storage. Indeed, M101 protected the graft from reactive oxygen
species generation, decreasing inflammation. However, further work is
needed to understand the diffusion of M101 within the graft in static
cold storage conditions. Indeed, as there is no flow within the graft, it
might question the administration of M101. Should it be administered
in the preservation solution during the organ rinsing? In addition, how
can M101 be reuploaded in O2 once in the vessels in static cold storage
conditions? Also, what could be the effect of M101 accumulation in
the vessels? Although many questions remain, these results encourage
investigating the role of M101 in HOPE conditions where M101 will be
homogeneously distributed to the graft. Moreover, the first-in-human
use of M101 in kidneys (OXYOP/NCT02652520) has reported the safety
of the molecule as well as the improvement of graft recovery75. A
randomized clinical trial is currently recruiting patients to assess M101
efficacy (OXYOP2/NCT04181710).

The second and third studies both used HOPE, ensuring continuous
active oxygenation. Whether PRE-HOPE or END-HOPE, all protocols
seemed beneficial for the graft. Indeed, all HOPE-preserved groups
demonstrated lower transaminases and lactates than SCS. Also, most
genes involved in mitochondrial protection and antioxidation were
upregulated in the HOPE groups compared to SCS (Article 2). The
upregulation was substantial for the HOPE-PRE group suggesting a
more appropriate timing for oxygenation to optimize mitochondrial
protection.

Regarding oxygenation, three questions are essential: When to

oxygenate? For how long should the oxygenation last? What is the
optimal quantity of oxygen (pO2) to be used? Previous studies on
kidneys demonstrated that short 2 hours of HOPE lead to similar
results as 22h HOPE127. Moreover, as ATP depletion is drastic after
warm ischemia, providing oxygenation right after could correct the

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 ATP debt128. This previous statement is consistent with our hypothesis
on livers suggesting that providing oxygen early in the preservation
protocol could boost the mitochondrial energy charge helping the
graft better cope with static cold storage and variable cold ischemia
times (Article 2). Recent studies on kidneys from DCD demonstrated
that oxygenation (using a bubbling pre-oxygenation technique of the
perfusate) right after procurement led to improved 1-year survival
compared to non-oxygenated perfusion. Although this study supports
our hypothesis that “the earlier oxygen is provided, the better,” the
amount of oxygen to be used remains unclear 129.

With active oxygenation comes oxygen saturation, which could also

influence graft preservation. Indeed, though intuitively, we might think
that high saturation is protective, it has been linked to ROS production
because of the electron transport chain's intense activity130. Better
characterizing graft oxygenation needs will be critical to providing
adequate active oxygenation without triggering ROS generation.
Although research on O2 consumption of the human pancreas in
hypothermic conditions has been conducted recently, further
investigation is needed for livers.

Other oxygenation methods are also under investigation. A german

randomized control trial on 116 patients (ISRCTN00167887)
demonstrated that 2 hours of liver venous persufflation (gaseous
oxygen perfusion) immediately before transplantation is a safe technic
that significantly decreased the rate of early allograft dysfunction for
marginal grafts (old donors, macrosteatotic donors)131. Another
research team has proposed using an oxygenated – UW solution for
rinsing and static preservation. They demonstrated that their
oxygenation protocol prevented ATP depletion in DCD donors but did
not reduce early reperfusion injuries132.

3.3 GLYCOCALYX INTEGRITY, A RELEVANT BIOMARKER FOR MACHINE

PERFUSION STRATEGIES

NMP permits the real-time assessment of liver function. Indeed, most

biomarkers of hepatic lesions (transaminases, lactates) can be

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measured during normothermia. However, transaminase levels are
highly dynamic. Therefore, if the quantification is performed before the
transaminase peak, surgeons might misevaluate the level of injury.
Consequently, we hypothesize that even if transaminases are sensitive
markers, they may be too variable to assess the level of damage over
time. Moreover, the transaminases value range in physiological and
liver disease conditions highly differs from one center to another as it
depends on the local population133,134. Identifying biomarkers with a
stronger correlation with time is key to predicting graft outcomes.

Glycocalyx (GCX) seems to be a good candidate. Indeed, this layer of

proteoglycans (syndecan-1) and glycoproteins (heparan sulfate)
covers the luminal surface of the endothelium. The glycocalyx is central
in blood vessel integrity, microcirculation, and vasodilatation.
Glycocalyx also acts as a primary barrier to prevent penetration of
immune cells (e.g., leucocytes) into the hepatic parenchyma.
Glycocalyx degradation is observed in many pathological processes,
such as trauma135 and IRI136. ROS massive release during cold ischemia
and reperfusion might damage the glycocalyx. Loss of glycocalyx
integrity is responsible for immune cell recruitment that could cause
sinusoidal vasoconstriction137. Free circulating glycocalyx markers also
act as Damage Associated Molecular Patterns (DAMPs), enhancing
immunological response activation 138. Researchers have measured
glycocalyx markers in human plasma during reperfusion. A rapid
increase of syndecan-1 is observed, suggesting that syndecan-1
release is correlated to reperfusion injury 139.

In the third article, we also measured glycocalyx levels in perfusates.

Indeed, we hypothesized that high glycocalyx markers are linked to a
loss of glycocalyx integrity140. After two hours of reperfusion, low levels
of GCX markers are observed in the IGL-2 groups compared to Belgen
(UW composition) and Perfgen (Belzer-MPS composition). This
suggests improved glycocalyx protection by IGL-2 supported by a
decreased inflammation (low level of Hmgb1). We also assessed
glycocalyx levels in tissue, expecting an inverted correlation with
perfusates results. No GCX level differences were detected in the
tissue, independently of the preservation solution. The ELISA kit
detection method might not suit tissue samples, and concentrations
might have exceeded the sensitivity range.

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 Glycocalyx thickness could also be used to quantify shear stress.
Indeed, shear stress generation is a specificity of perfusion strategies
where sinusoids are constantly exposed to different fluids, unlike static
cold storage strategies. Previous studies have demonstrated that when
the flow is smooth and laminar, GCX is thick and regenerated
efficiently. When the flow was disturbed or obstructed because of
congestion (e.g., immune cell accumulation), loss of GCX integrity was
observed. Hyaluronan, a long polysaccharide anchored to
proteoglycans and glycoproteins, seems crucial for GCX thickness and
regeneration, especially in disturbed flow conditions141. Hyaluronan
supplementation has promoted healing after cardiac ischemia-
reperfusion injuries 142.

3.4 HOPE-PRE, D-HOPE, NMP, NRP, COR: TOWARDS ISCHEMIA-

FREE LIVER TRANSPLANTATION?

All machine perfusion protocols are currently performed after static

cold storage. Though this sequential order was supported by clinical
trials showing IRI protection, it was also chosen for logistical reasons.
Indeed, the transplant team must perform machine perfusion
protocols at transplant centers (after graft transportation). The lack of
a portable machine device with a CE mark prevented other timing from
being investigated in clinical trials.

We hypothesized that performing HOPE after warm ischemia in the

second study could benefit the grafts. This technic combines
hypothermia and oxygen perfusion that favors minimal yet vital
metabolism activity. Indeed, this technic minimized drastic changes
caused by ischemia, such as ROS generation, mitochondrial
dysfunction, and acidosis due to anaerobic glycolysis. The gene
expression analysis on 40 genes involved in ischemia-reperfusion
injuries demonstrated higher mitochondrial, antioxidant, and anti-
apoptotic gene expression in the HOPE-PRE group compared to the
HOPE-END group. Even though both groups had the same cold
storage time (10 hours), the machine perfusion protocols' order seems
critical to enhancing protection. Another compelling result was the
increase of protective and detrimental IRI genes in the HOPE-PRE

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group. This could be explained by reperfusion injury caused by early
perfusion that could trigger inflammation pathways. Therefore, it
seems HOPE-PRE protects ischemic injuries while HOPE-END protects
reperfusion injuries. The perfect balance between these two protocols
is needed to optimize graft preservation.

One approach could be limiting the drastic temperature changes

between hypothermia and reperfusion. Indeed, at 5°C ± 3°C, the
metabolism activity is at around 10%, and the tissue is depleted in
energy. Abrupt blood reperfusion combined with rapid tissue
rewarming could cause irreversible damage to tissues that
accumulated ischemic injury and ATP depletion. This is achieved by
Controlled Oxygenated Rewarming (COR), a machine perfusion
method where the temperature is increased gradually from 4°C to
20°C, leading to a slow increase in metabolism activity has been
proposed to improve graft function, especially for reperfusion-related
injuries. Researchers have investigated optimizing the passage from
SCS to SNMP143. Grafts underwent 18 hours of SCS followed by 90
minutes of SNMP, HMP, or COR before being reperfused ex-situ. COR
was associated with significantly increased bile production and
decreased injury on reperfusion. Including COR in perfusion protocols
could potentially combine the best of cold and warm perfusion
techniques.

Furthermore, combining continuous HOPE with COR might be a good

strategy. In addition, it combines HOPE-END and HOPE-PRE, with the
potential to effectively prevent ischemic and reperfusion injuries
thanks to COR. Unlike normothermia, this technic might allow better
control of metabolism activity during hypothermia and reperfusion.

Normothermic perfusion options have been investigated to reduce or

even eliminate ischemia. Normothermic Regional Perfusion (NRP) is a
method that restores oxygenated blood circulation to all abdominal
organs using an extracorporeal circuit acting like a heart. This technic
performed in situ of interest because it has the potential to transform
DCD organs in DBD. It also allows the surgeons to control the
procurement timing and decrease the cold ischemia time. A
retrospective analysis of DCD donors demonstrated that NRP was
associated with improved outcomes compared to cold storage144. Even

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 though results are encouraging, NRP raises ethical concerns as it
requires pre-mortem cannulation (only in Spain) and heparinization
that could lead to donor resuscitation. Current regulatory guidelines
do not permit NRP in some European countries.

Fully-avoiding ischemia remains a challenge tackled in 2021 by a

Chinese research team145. They reported for the first time ischemia-
free liver transplantation in humans. This technic enables continuous
oxygenated blood supply to the liver from procurement to
implantation. In other words, normothermic machine perfusion (NMP)
was performed ex-situ continuously. Livers from DBD donors treated
with this method were recovering better than livers preserved in UW
under static cold storage conditions. This study presents some
limitations. First, they have excluded all ECD donors (DCD, steatosis).
As machine perfusions indicated for ECD graft, testing this approach
on this subpopulation is crucial.

Moreover, all livers were procured and implanted in the same centers;
therefore, using static perfusion devices was possible. The
development of portable machine perfusion devices will be critical to
improving the acceptance of this approach. Research on novel
preservation solutions able to combine static cold storage and
perfusion protocol will be crucial to meet the requirement and the
logistics of novel devices.

Finally, when it comes to perfusion protocols, a key question remains:

Should single (portal vein only) or dual (portal vein and hepatic artery)
perfusion be performed? Even though research published earlier this
century demonstrated the superiority of dual perfusion in porcine
model146 as it mimics better physiological conditions, recent studies
did not reveal significant differences between both approaches in
HOPE and SNMP settings 99,147.

3.5 ROBUSTNESS OF EXPERIMENTAL MODELS TO STUDY EXTENDED

CRITERIA DONORS' EX VIVO PRESERVATION

We used an experimental model of the Zucker rat and in-house

machine perfusion throughout all three studies presented in the result
section. This section aims a highlighting the main limitations and

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propose some improvements.

M101 and IGL-2 studies utilized the Zucker rat, a genetically modified
rat model with a mutation in the gene coding for leptin (regulating
satiety)148. Therefore, Zucker rats' food intake is significantly elevated,
and they develop increased obesity. They are described as a model of
choice for studying diabetes. In our case, we used the Zucker rat to
model steatotic liver conditions. While rats in both studies were raised
in an environment with ad libitum food, a heterogeneous level of
steatosis was observed. In humans, grafts are categorized as ECD when
a certain level of steatosis is reached. A heterogeneous level of
steatosis makes it challenging to assess whether the graft is "ECD-like"
or not. Even though the more steatosis seems to be, the better to
characterize a graft "ECD," further research needs to be performed to
investigate rats' "ECD" threshold regarding steatosis. It has also been
suggested that Zucker rats might develop homogenous steatosis
when submitted to a high-fat diet. This could be an option to decrease
heterogeneity.

On the other side, as Zucker rats are costly, we assessed steatosis

induction on wild-type Sprague Dawley rats. We compared steatosis
levels on Sprague Dawley rats fed for ten weeks with a high-fat diet to
Sprague Dawley rats fed with a classic diet. Rats were weighed every
week. Even though no weight difference (rat and liver) was observed
in all groups, the high-fat diet group developed increased steatosis.
This result was consistent with other studies149. Though heterogeneity
in steatosis levels was also observed, it might represent an economical
way to study steatosis and increase the number of rats per group.

Moreover, our sole ECD model was steatosis which, as discussed

above, demonstrates several limitations experimentally. Therefore,
developing a new experimental model of ECD graft is needed.
Controlled DCD rat models seem to be an excellent option for
improving homogeneity among ECD. Less than 40 studies have
investigated the DCD rat model since 2002. Most protocols
recommend heparinizing the animals before provoking cardiac arrest,
a step that is not found in clinical settings. DCD "gravity" is assessed
by the warm ischemia time defined by the time between cardiac arrest
and cold preservation flush. In the rat, warm ischemia generally does

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 not exceed 30 mins. Many studies have demonstrated that HOPE
improved the graft function of rat DCD grafts150–152. Therefore,
approaches indicated for ECD, such as M101 supplementation and
HOPE-PRE, must be validated on the rat ECD model.

Using our experimental perfusion system, we developed a model of

liver transplantation to test liver function recovery. Even though this
NMP model is key to reducing the number of animals involved in the
studies, it does not permit us to test liver function recovery for a long
time. Indeed, after more than two hours of reperfusion, macroscopic
changes in liver damage can be observed in the liver. As utilized in the
NMP protocol, a blood-based solution might be a relevant approach
to expand liver recovery analysis and strengthen our ex vivo model.
However, supplying and conditioning packed red blood cells is costly
and requires additional logistics. For this reason, using artificial
hemoglobin, that is, M101 could represent an opportunity to increase
the availability of oxygen carriers–based perfusate.

3.6 FROM A GENERAL PRESERVATION PROTOCOL TO A PERSONALIZED

APPROACH

In an era where personalized medicines using –omics technology

seems to be a key trend, their use in the field of liver transplantation
remains scarce. Most personalized medicine approach targets
transplant pharmacotherapy and aims at developing specific
immunosuppressive dosing as multiple drugs with varying
pharmacokinetic and iatrogenic interactions can be used. Taking the
example of tacrolimus immunosuppressant agent, the initial dose is
based on the body weight. However, tacrolimus is metabolized by the
liver. Therefore, its efficacy will be closely linked to graft function.
Personalized pharmacotherapy approaches have proved efficient in
determining the adequate dose based on individual graft function
153,154
.

Ex vivo preservation strategies could also benefit from developing a

personalized approach. Indeed, numerous ex vivo strategies presented
above are all relevant and support that there is not a single strategy
above the other but several methods that can be utilized depending
on graft characteristics. These strategies could also be combined to

117
simultaneously prevent different parts of ischemia-reperfusion that
cannot be achieved using unique techniques. The study we conducted
closest to an –omics approach remains the study investigating HOPE-
END and HOPE-PRE. Even though we have analyzed a minimal set of
the entire genome, a "transcriptomic" signature could already be
observed. Indeed, we observed most of the ischemia-reperfusion
pathways that were impaired in each graft. This could lead to specific
supplementation in the preservation solutions or during perfusion,
such as anti-inflammatory agents, to counteract destructive pathways.
Our results also suggest that a donor graft with increased
inflammation should undergo HOPE-END preservation while a donor
with impaired mitochondrial function should undergo HOPE-PRE.

The assessment of the donor graft before procurement presents

several limitations. Indeed, as we do not know when the patient's death
will be confirmed, it is difficult to know when is the optimal timing to
perform invasive methods. For example, liver biopsies can be
performed to assess hepatic energy charge, steatosis level, or fibrosis.
Therefore, the need for a non-invasive biomarker to predict the graft
profile before procurement could add value to the allocation decision.

To perform a personalized approach, detailed proteomics or genomic

profile could be achieved using non-invasive methods and detecting
predictive biomarkers such as miRNAs (miRNAs). Indeed, these single-
stranded noncoding RNA molecules regulate the post-transcriptional
expression of target genes. These stable compounds are organ-
specific and can be quantified in several body fluids, such as serum and
bile. The development of miRNA detection technics has identified
them as potential biomarkers, predictive or diagnostic biomarkers.
Specific hepatic miRNA has been associated with diagnosing different
liver diseases after transplantation155. Other studies demonstrated the
significant correlation between miRNAs expression and graft function
during liver preservation and early graft dysfunction after liver
transplantation156.

The following table summarizes how a personalized approach could

be set based on the learnings of this thesis and literature research. Two
levels of segmentation are proposed: donors' macroscopic graft
characteristics and donors' graft molecular signatures. The first

118
approach determines ex vivo preservation key parameters (e.g.,
preservation solution used, type of protocol available). In contrast, the
molecular signature approach might provide tools to refine
preservation solution composition supplementation (e.g., additives) or
machine perfusion parameters (e.g., flow rate, oxygenation rate). It
offers preliminary insights into what a personalized allocation system
might look like in the future.

Table 5. Proposition of personalized preservation approaches

119
4 CONCLUSION

This thesis investigated novel strategies for liver ex vivo preservation

during transplantation. Indeed, the scarcity of livers for transplantation
urges the development of an innovative method to optimize organ
quality and ultimately increase the pool of organs available.

Our first study demonstrated the efficacy of the oxygen carrier M101
for protecting the steatotic liver during static cold storage against
ischemia-reperfusion injuries. M101 supplementation in the
preservation solution was associated with improved protection against
reactive oxygen species, inflammation, and apoptosis.

Our second study evaluated the benefit of a pre-ischemic hypothermic

oxygenated perfusion (HOPE-PRE) in preserving the liver. Compared
to the gold-standard HOPE-END performed after SCS, we
demonstrated that HOPE-PRE enhanced the transcription of several
genes involved in ischemia-reperfusion protection, such as
antioxidants and glycocalyx.

The final study investigated the role of a novel preservation solution,

IGL-2, during SCS and HOPE. We compared IGL-2 to the most used
preservation solution, UW/BELGEN for SCS and UW-MPS/PERFGEN for
HOPE. We demonstrated that IGL-2 better-preserved glycocalyx
proteins and reduced inflammation, apoptosis, and edema during SCS
and HOPE.

Our findings have shown multiple ex vivo preservation methods

decreasing ischemia-reperfusion injuries. More research is needed on
machine perfusion protocols to optimize their outcomes and
accessibility. In addition, investigating biomarkers adapted to
perfusion strategies will be key to supporting surgeons in decision-
making. Both approaches need to be conducted hand in hand as they
converge towards the same goal: The safe and accessible expansion of
the pool of donors.

120
RÉSUMÉ EN FRANÇAIS

La transplantation hépatique est la seule option pour soigner les

maladies incurables du foie. Cependant, le nombre d'organes
disponibles est limité. Afin d’augmenter le nombre de donneurs, les
chirurgiens ont étudié l'utilisation de greffons provenant de donneurs
à critères élargis (DCE). Ces greffons, qui proviennent de donneurs
atteints de maladies guérissables telles que la stéatose (excès de
gouttelettes lipidiques dans les hépatocytes) sont plus sensibles à
l'ischémie-reperfusion. Ce syndrome inévitable se caractérise par
l'accumulation de lésions entre le prélèvement du foie et sa
transplantation. Pour le limiter et optimiser la qualité des greffons DCE,
le développement de nouvelles stratégies de préservation ex vivo est
crucial.

La préservation statique froide (PSF), immersion d'un organe dans une

solution de préservation (SP) à 5°C ± 3°C, est actuellement le gold-
standard de la préservation du foie. Cette technique adaptée aux
greffons sains, ne l’est pas pour les greffons DCE. La perfusion
hypothermique oxygénée (HOPE) est la technique la plus prometteuse
pour les greffons DCE. En effet, post- PSF, le foie subit 2 heures de
HOPE, où une SP oxygénée froide est recirculée dans le greffon. Les
deux SP les plus utilisées (UW pour PSF et UW-MPS pour HOPE)
utilisent HES comme agent oncotique. Cet agent induit une agrégation
des globules rouges qui peut empêcher le lavage du greffon, en
particulier en cas de stéatose où la microcirculation est altérée.

HOPE, bien qu'efficace, s'accompagne d'une logistique complexe et de

coûts élevés. Les alternatives visant à optimiser PSF pour améliorer
l’intégrité du greffon sont plus abordable. Dans une première étude,
nous avons analysé le potentiel de M101, un transporteur d'oxygène
issu d’un ver marin, comme additif aux SP pendant PSF. Nous avons
démontré que M101 pouvait être bénéfique pour les greffons
stéatosiques, son ajout M101 entraînant moins de lésions hépatiques
chez le rat.

121
Mais HOPE étant toujours associé à PSF, le besoin de deux SP distinctes
pour PSF et HOPE pourrait endommager greffon. Pour cette raison,
nous avons étudié une nouvelle SP, IGL2, avec le potentiel d'être utilisé
à la fois pour PSF et HOPE. IGL2 est une version modifiée d'IGL1 où les
concentrations en PEG (agent oncotique) et glutathion ont été
augmentées, stimulant la vasodilatation et les propriétés
antioxydantes. Nous avons comparé IGL2 avec UW (pendant PSF) et
UW-MPS (pendant HOPE). Durant PSF ou HOPE, la solution IGL2 a
amélioré la protection des foies stéatosiques chez le rat.

Actuellement, HOPE est toujours réalisé après PSF (HOPE-END). Cet

ordre a été adopté principalement pour des raisons logistiques. Au
cours de la PSF, l'hypothermie réduit considérablement le
métabolisme. Pourtant, le manque d'ATP détériore les mitochondries
et favorise la génération d'espèces réactives de l'oxygène (ROS)
pouvant initier l’inflammation. Par conséquent, effectuer HOPE avant
PSF (HOPE-PRE) pourrait stimuler les mitochondries permettant à
l'organe de mieux faire face au PSF. Pour cette raison, nous avons
étudié le niveau d'expression de 40 gènes impliqués dans l'ischémie-
reperfusion en condition HOPE-PRE, HOPE-END et PSF (seule). La
transcription des gènes protecteurs étaient plus élevés dans le groupe
HOPE-PRE, suggérant une protection améliorée.

Au cours des trois études, nous avons amélioré la qualité du greffon à

plusieurs niveaux en utilisant une nouvelle SP, un additif et en revisitant
un protocole de perfusion machine. Ces résultats suggèrent qu’il
n’existe pas un protocole de préservation unique, mais une variété. Par
ailleurs, alors que nous entrons dans l'ère de la médecine
personnalisée, il pourrait être pertinent d'établir un protocole de
conservation personnalisé, particulièrement pour les greffons
marginaux, afin de prédire les complications postopératoires et
d'augmenter le nombre de donneurs.

122
SUMMARY IN ENGLISH

Liver transplantation is the only treatment to cure end-stage liver

diseases. However, the number of organs available is limited, causing
increased patient mortality on waiting lists. To increase the pool of
donors, surgeons have investigated the use of high-risk grafts coming
from extended criteria donors (ECD). ECD grafts come from donors
with curable diseases such as steatosis, characterized by lipid droplets
in the hepatocytes. ECD grafts are more sensitive to ischemia-
reperfusion, this inevitable syndrome characterized by the
accumulation of lesions between liver removal and its transplantation
in a recipient. That is why developing novel ex vivo preservation
strategies is crucial to prevent ischemia-reperfusion and optimizing
marginal graft function.

Static cold storage (SCS), describing organ immersion in a cold

preservation solution, is currently the gold standard of liver
preservation. Even though this technic is suitable for healthy grafts, it
is not optimized for ECD. Hypothermic Oxygenated Perfusion (HOPE)
is the most promising machine perfusion protocol for marginal graft.
Indeed, after SCS, the liver undergoes 2 hours of HOPE, where a cold
oxygenated preservation solution is recirculated within the graft. The
most used preservations solutions are UW for SCS and UW-MPS for
HOPE. Both preservations solutions used HES as a pressure control
(oncotic) agent, which has been linked to the aggregation of red blood
cells. This could prevent graft washouts, especially for steatotic livers
where microcirculation is altered.

The HOPE protocol, though efficient, comes with complex logistics and
high costs. Alternatives aiming at the optimization of SCS represent a
more accessible and cost-effective opportunity to optimize graft
quality. That is why the first study investigated the potential of M101,
a powerful oxygen transporter from marine worms, as a preservation
solution additive during graft SCS. We have demonstrated that M101
could benefit steatotic grafts as M101 addition led to less liver injury.
As the HOPE protocol is always combined with SCS, the need for 2
distinct preservation solutions for SCS and HOPE protocols could be

123
detrimental to the graft. For this reason, we have investigated a novel
preservation solution, IGL2, with the potential to be used for both SCS
and HOPE. IGL2 is a modified version of IGL1 where PEG (oncotic
agent) and glutathione were increased, boosting vasodilation and
antioxidant properties. We compared IGL2 with UW (during SCS) and
UW-MPS (during HOPE). In the SCS or HOPE setting, the IGL2 solution
improved rat steatotic livers' protection.

Currently, the HOPE protocol is always performed after SCS (HOPE-

END). This order was mainly adopted for logistical reasons. During SCS,
hypothermia significantly reduces metabolism activity. Still, the lack of
ATP deteriorates mitochondria and promotes reactive oxygen species
(ROS) generation, which is linked to increased inflammation. Therefore,
performing HOPE before SCS (HOPE-PRE) could boost mitochondria
allowing the organ to better cope with SCS. For this reason, we
performed HOPE-PRE, HOPE-END, and SCS on 27 rat livers. We
investigated the expression level of 40 genes involved in ischemia-
reperfusion. Compared to SCS only, transcriptional levels of protective
genes were higher in the HOPE-PRE group suggesting improved
protection.

In all three studies, we enhanced graft quality on many levels using a

new preservation solution, preservation solution additive, and
challenging well-established machine perfusion protocol. These
research findings proved that they might not be a one-size-fits-all
protocol but a variety of tools that can be leveraged depending on the
initial quality of the graft. Moreover, as we enter the personalized
medicine era, it might also be relevant to establish a customized
preservation protocol, specifically for marginal graft, to predict
postoperative complications better and effectively increase the donor
pool.

124
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