- Email: [email protected]
Application of Perfusate With Human-Derived Oxygen Carrier Solution Under Subnormothermic Machine Perfusion for Donation After Cardiac Death Liver Grafts in Pigs
Accepted Manuscript The application of perfusate with human-derived oxygen carrier solution under subnormothermic machine perfusion for donation after cardiac death (DCD) liver grafts in pigs Tatsuya Shonaka, Naoto Matsuno, Hiromichi Obara, Ryo Yoshikawa, Yuji Nishikawa, Mikako Gouchi, Masahide Otani, Hiroyuki Takahashi, Hiroshi Azuma, Hiromi Sakai, Hiroyuki Furukawa PII:
S0041-1345(18)30447-0
DOI:
10.1016/j.transproceed.2018.02.184
Reference:
TPS 28428
To appear in:
Transplantation Proceedings
Received Date: 4 January 2018 Accepted Date: 19 February 2018
Please cite this article as: Shonaka T, Matsuno N, Obara H, Yoshikawa R, Nishikawa Y, Gouchi M, Otani M, Takahashi H, Azuma H, Sakai H, Furukawa H, The application of perfusate with human-derived oxygen carrier solution under subnormothermic machine perfusion for donation after cardiac death (DCD) liver grafts in pigs, Transplantation Proceedings (2018), doi: 10.1016/j.transproceed.2018.02.184. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
The application of perfusate with human-derived oxygen carrier solution under
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subnormothermic machine perfusion for donation after cardiac death (DCD) liver grafts
in pigs
My manuscript is submitted for the following meeting: CAST 2017 (15th Congress of the Asian
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Society of Transplantation)
Tatsuya Shonakaa, Naoto Matsunoa, Hiromichi Obarab, Ryo Yoshikawab, Yuji Nishikawac,
Mikako Gouchia, Masahide Otania, Hiroyuki Takahashia, Hiroshi Azumad, Hiromi Sakaie, Hiroyuki
Department of Surgery, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
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a
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Furukawaa
b
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Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2503 FAX: +81-166-68-2193
Department of Mechanical Engineering, Tokyo Metropolitan University. 1-1 Minami-Osawa,
Hachioji-shi, Tokyo, 192-0397 Japan. Tel/Fax : +81-42-677-2711
c
Department of Pathology, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2371 FAX: +81-166-68-2379 1
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d
Department of Pediatrics, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
e
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Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2481 FAX: +81-166-68-2489
Department of Chemistry, Nara Medical University. 840 Shijo-cho, Kashihara-shi, Nara,
E-mail addresses of authors:
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Tatsuya Shonaka: [email protected]
Naoto Matsuno: [email protected]
Hiromichi Obara: [email protected]
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634-8521 Japan. TEL +81-744-29-8810 FAX +81-744-29-8810
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Ryo Yoshikawa: [email protected]
Yuji Nishikawa: [email protected]
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Mikako Gouchi: [email protected]
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Masahide Otani: [email protected]
Hiroyuki Takahashi: [email protected]
Hiroshi Azuma: [email protected]
Hiromi Sakai: [email protected]
Hiroyuki Furukawa: [email protected] 2
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Corresponding author: Naoto Matsuno. Email: [email protected]
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Department of Surgery, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2503 FAX: +81-166-68-2193 Grant Information: This work was supported by the Grants-in-Aid for Scientific Research (C) (KAKENHI) No.17K10503.
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Key words: liver transplantation, subnormothermic machine perfusion, hemoglobin-based
Tables: 0
Figures: 4 (color - No)
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Declarations of interest: None
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oxygen carriers, pig
Abbreviations:
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adenosine triphosphate; ATP
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aspartate transaminase; AST
carbonyl hemoglobin; HbCO
donation after cardiac death; DCD
hematoxylin-eosin; HE
hemoglobin; Hb 3
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hemoglobin-based oxygen carriers; HBOCs
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hemoglobin vesicle; HbV
ischemia-reperfusion injury; IRI
nucleic-acid amplification testing; NAT
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red blood cells; RBC
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University of Wisconsin; UW
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EP
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warm ischemic time; WIT
4
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Abstract
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Oxygenation is necessary for aerobic metabolism, which maintains adenosine triphosphate
(ATP) within the graft organ. In recent years, some studies have demonstrated that
has the potential to improve oxygen metabolism.
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subnormothermic machine perfusion (SNMP) with hemoglobin-based oxygen carriers (HBOCs)
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【Aim】The aim of this study was to evaluate the effectiveness of perfusate with human-derived
hemoglobin vesicles (HbV) under SNMP in a pig model of donation after cardiac death (DCD).
【Materials and methods】In this study, pig livers were procured with a warm ischemic time of 60
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minutes and were preserved in 3 groups for 240 minutes. The preservation conditions were as
follows: 4℃ cold storage (Group 1); SNMP with University of Wisconsin (UW) perfusate alone
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(Group 2); SNMP (21℃) with UW and HbV (Hb; 0.6mg/dl) perfusate (Group 3). All livers were
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perfused for 120 minutes using pig autologous blood machine perfusion (reperfusion phase).
We investigated the aspartate transaminase (AST) level, hemodynamics (portal vein resistance
[PVR] and oxygen consumption) in the preservation and reperfusion phases. A histological
study (Hematoxylin-Eosin [HE] staining) was performed after 240 minutes of preservation.
【Results】The PVR of Group 3 was not increased in comparison to Group 2. During 1
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preservation, the oxygen consumption of Group 3 was higher than that of Group 2. However, the
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level of AST did not differ between Groups 2 and 3.
【Conclusion】The present study revealed that perfusate with HbV increased the oxygen
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consumption of the donor liver during SNMP.
2
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Introduction
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Despite strategies to expand the organ donor pool, the number of liver transplantations
performed over the past decade has not increased [1,2]. The utilization of grafts from donors
after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool.
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ranging from 20% to 40% overall, and >50% for DCD [3].
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However, liver utilization remains suboptimal, with organ discard rates in the United States
Ischemic reperfusion injury after liver transplantation is a major problem in liver transplantation.
Several studies have demonstrated that subnormothermic machine perfusion (SNMP) is an
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effective method for reducing hepatocellular injury during organ preservation [4–6]. However,
SNMP relies on physically dissolved oxygen [7, 8]. In recent years, some studies have shown
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that the use of perfusate with a hemoglobin-based oxygen carrier during the preservation of the
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liver graft improves the graft function during machine perfusion [9, 10].
The aim of this study was to evaluate and the effectiveness of perfusate with human-derived
hemoglobin vesicles (HbV) under SNMP in a pig model of donation after cardiac death.
Materials and Methods 1
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Domestic female cross-bred Large-Yorkshire, Landrace, and Duroc pigs (approximately 30 kg, 2
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or 3 months of age) were purchased from Sankyo Labo Service Corporation, Inc (Sapporo,
Japan). Ten pigs were used in the present study. The animal care was in accordance with the
Declaration of Helsinki. The study was approved by the Asahikawa Medical University Animal
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Care and Use Committee.
Perfusion Preservation Machine (Fig. 1)
We developed an organ preservation system with temperature-controlled machine perfusion.
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The system has 2 circulating perfusion circuits for the portal vein and hepatic artery. Each circuit
included a roller pump, flow meter, and pressure sensor. The circuits for the hepatic artery
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provided pulsatile flow and installed an oxygenator; the circuits for the portal vein provided no
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pulsatile flow. Both circuits were connected via plastic connectors to the hepatic vessels. This
system has waterproof thermocouples, which measure the solution and the organ temperatures.
A computer included in this system records data and controls the flow conditions and
temperatures of the preservation solution (Fig.1). The temperature in the organ chamber is
controlled by a heat exchanger and ice-cold water. The portal vein and hepatic artery rate are 2
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controlled. The oxygen supply was 300 mmHg during preservation and 380 mmHg during
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reperfusion.
Human-derived HbV as hemoglobin-based oxygen carriers (HBOCs)
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Sakai et al. demonstrated HBOCs as artificial oxygen carriers, which encapsulate a purified and
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concentrated hemoglobin solution in phospholipid vesicles, mimicking the cellular structure of
red blood cells (RBC) [11]. HbV were prepared from out-of-date nucleic-acid amplification
testing (NAT)-inspected RBC provided by the Japanese Red Cross. The carbonyl hemoglobin
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(HbCO) purification procedure includes pasteurization and nanofiltration for the utmost safety
from infection. Liposome encapsulation protects against the toxic effects of molecular
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hemoglobin. It is encapsulated with liposomes composed of four lipids. These lipids were
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selected based on their stability, encapsulation efficiency, and biocompatibility [12]. Organ
preservation would likely be improved if the oxygen metabolism can be supported by perfusion
with a fluid such as HbV [13, 14]. Human-derived HbV were used as hemoglobin (Hb)-based
HBOCs. We also used HbV that had been created 18-20 months previously to confirm the
molecular stability. 3
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The Preparation and Preservation of the DCD Liver
Under inhalation anesthesia with isoflurane (Forane, Abbott, Japan), cardiac arrest was induced
by the intravenous injection of potassium chloride and the removal of ventilation. The donor
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livers were exposed to a warm ischemic time (WIT) of 60 minutes. After donor liver extirpation,
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we took 240 minutes for preservation. During preservation, all livers were assigned to the
following three groups: Group 1 (N=3), cold storage (4 ) with University of Wisconsin (UW)
solution; Group 2 (N=4), SNMP (20-22 ) with UW perfusate only; Group 3 (N=3), SNMP with
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HbV and UW perfusate. The perfusate of Group 2 consisted of 1500 ml of UW solution. The
perfusate of Group 3 consisted of 1400 ml of UW solution and 100 ml of HbV. The concentration
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of hemoglobin was 0.6-0.7mg/dl in the perfusate. To assess organ viability and
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ischemia–reperfusion injury (IRI) after preservation, all livers were reperfused with autologous
diluted blood for 120 minutes. We assessed the aspartate aminotransferase (AST) level (a
marker of hepatocellular injury). Portal vein (PV) resistance was calculated as the portal vein
pressure and flow. The PV flow divided by the PV pressure was called the PV resistance (PVR,
per 100g liver, wet liver weight). It measures the oxygen pressure between the portal vein and 4
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perfusate. After correcting the temperature, the difference in oxygen pressure was determined
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to calculate the oxygen consumption. We evaluated the PVR and oxygen consumption as
hemodynamic parameters (per 100g liver, wet liver weight). A histological analysis
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(Hematoxylin-Eosin [HE] staining) was performed after 240 minutes of preservation.
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Statistical analysis
All measures were expressed as the mean± SD within each group. All analyses were performed
using the R software program (version 3.1.2) and the EZR software program [15]. Two group
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comparisons of continuous measures between each group were conducted using a two-sample
t-test at a given time point. Repeated measures were used for data with more than three time
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points. When the comparison between groups were performed a univariate Type
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repeated-measures ANOVA was performed with the assumption of sphericity. P values of <0.05
were considered to indicate statistical significance.
Results
Fig. 2 shows the portal vein resistance (PVR) during preservation. During preservation, the PVR 5
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gradually increased in Group 2. However, there was no significant difference between Groups 2
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and 3. The PVR values were 0.010±0.009 (Group 1), 0.008±0.007 (Group 2), 0.008±0.005
(Group 3) mmHg/ml/min/100g liver after 120 minutes of reperfusion. The PVR value of Group 1
was slightly higher than the values of Groups 2 and 3. However, the differences among the
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groups were not statistically significant.
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Fig.3 shows the oxygen consumption during preservation. The oxygen consumption of Group 3
was significantly higher during preservation (P=0.03). The oxygen consumption in Groups 1, 2,
and 3 was 0.65±0.09, 0.68±0.05, and 0.63±0.14 mg/min/100g liver, respectively after 120
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minutes of reperfusion. The differences were not statistically significant.
The AST enzyme levels of Groups 2 and Group 3 did not differ to a statistically significant extent
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during preservation. Fig.4 shows the AST level after 120 minutes of reperfusion. The level of
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AST in the perfusate of Groups 1, 2, and 3 was 4016.7±1598.9, 1388±198.7, and 1598.9±288.0
IU/L, respectively. The level of AST in Group 1 was higher than that in Groups 2 and 3; however,
the difference was not statistically significant. Hepatocellular reperfusion injury was not
prevented in this model. A histological examination revealed was no evidence of HbV or venous
thrombosis at the end of the preservation phase. 6
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Discussion
The present study revealed that perfusate with human-derived HbV increased the oxygen
dose of HbV did not improve hepatocellular injury.
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consumption of the donor liver during SNMP. However, it also revealed that perfusate with a low
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Perfusate with HbV increased the oxygen consumption of the donor liver during SNMP. Burisma
et al. suggested the performance of SNMP for ex vivo preservation and the recovery of the
human liver for transplantation [16] and investigated the perfusion outcomes with a
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metabolomics analysis [17]. Oxygenated perfusion enables any changes in the tissue energy of
the graft to be restored [18, 19]. Compagnon et al. reported that an oxygenated and
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transportable MP system could fully rescue liver grafts exposed to lethal ischemic damage in a
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pig model of DCD liver transplantation [20]. Thus, we hypothesized that oxygen might be carried
from the perfusate to the donor liver in order to respond to oxygen consumption during perfusion.
P. Fontes et al. [9] reported that a new, cell-free HBOC solution was developed by combining a
stable second-generation bovine-derived hemoglobin compound. They also reported that the
higher hepatic O2 delivery using HBOC solution was associated with an improved graft function. 7
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However, their hemoglobin was derived from bovine, which may have unknown virus infectious
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and will be difficult to use in clinical setting. Liu et al. [10] reported that bile production and
hepatocellular injury were improved by blood donor perfusate normothermic machine perfusion.
In our study, human-derived HbV-containing perfusate was used. It was shown that oxygen
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consumption was significantly higher in the HbV perfusate during perfusion. This indicates that
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oxygen had been transferred from the perfusate to the donor liver by the HbV. We used HbV at
18 to 20 months after its creation. However the HbV was able to transport oxygen. Furthermore,
human-derived hemoglobin and virus removal were performed during the creation of the HbV;
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thus, it would seem easy to use in the clinical setting.
Because of a low dose of HbV, this study could not show to improve the hepatocelular injury.
EP
The perfusate of Fontes et al. [9] was set to Hb 3.5 g/dl, while that of Liu et al. [10] perfusion was
AC C
set to 21-25% hematocrit. In the present study, our HbV content was 0.6-0.7g/dl hemoglobin,
which was lower in comparison to the 2 previous reports. We hypothesized that HbV carried
oxygen but that the amount of oxygen transported was not sufficient to improve the
hepatocellular injury.
8
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The present study is associated with some limitations. The study was performed using an
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animal model. This is an experiment on the transplant model, and the result of the actual
transplant is unknown.
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Conclusion
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The present study demonstrated the use of perfusate with human-derived HbV increased the
oxygen consumption of the donor liver during SNMP. However, this perfusate could not prevent
liver injury because the amount of HbV was insufficient. We plan to perform further studies using
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Figure Captions:
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Fig. 1. We developed an organ preservation system with temperature controlled machine
perfusion. The system had 2 circulating perfusion circuits for the portal vein and hepatic artery.
Oxygen was supplied from the hepatic artery and portal vein through an artificial lung. This
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perfusion device quantified the flow rate and the oxygen supply.
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Fig. 2. The portal vein resistance (PVR) in the preservation phase.
Fig. 3. The oxygen consumption in the preservation phase.
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Fig. 4. The AST level of the perfusate after 120 minutes of reperfusion.
14
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PV
Pressure Gage
Flow meter
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HA
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Optical Oxy. Sensor
Oxygenator and Heat Exchanger
Air Trap
AC C
EP
Liver
Organ holder
Pump
Pump
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mmHg/ml/min/100g liver
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0.025
Group2
0.02
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Group3
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0.015
EP
0.01
AC C
0.005
0 0
30
60
90
120
150
180
210
240
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mg/min/100g liver
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0.7
P=0.03
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0.6
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0.5
0.3
Group2
EP
0.2
TE D
0.4
AC C
Group3
0.1 0 0
30
60
90
120
150
180
210
240
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6000
IU/L
5000
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P=0.09
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P=0.12
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4000
TE D
3000
1000
AC C
EP
2000
0 Group1
Group2
Group3
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1. Hemoglobin vesicles (HbV) is a human-derived oxygen carrier. 2. We evaluated the effectiveness of perfusate with HbV.
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Highlights
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3. Pig liver after cardiac arrest was preserved under subnormothermic machine perfusion.
AC C
EP
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4. The perfusate with HbV increased the oxygen consumption during machine perfusion.
S0041-1345(18)30447-0
DOI:
10.1016/j.transproceed.2018.02.184
Reference:
TPS 28428
To appear in:
Transplantation Proceedings
Received Date: 4 January 2018 Accepted Date: 19 February 2018
Please cite this article as: Shonaka T, Matsuno N, Obara H, Yoshikawa R, Nishikawa Y, Gouchi M, Otani M, Takahashi H, Azuma H, Sakai H, Furukawa H, The application of perfusate with human-derived oxygen carrier solution under subnormothermic machine perfusion for donation after cardiac death (DCD) liver grafts in pigs, Transplantation Proceedings (2018), doi: 10.1016/j.transproceed.2018.02.184. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
The application of perfusate with human-derived oxygen carrier solution under
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subnormothermic machine perfusion for donation after cardiac death (DCD) liver grafts
in pigs
My manuscript is submitted for the following meeting: CAST 2017 (15th Congress of the Asian
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SC
Society of Transplantation)
Tatsuya Shonakaa, Naoto Matsunoa, Hiromichi Obarab, Ryo Yoshikawab, Yuji Nishikawac,
Mikako Gouchia, Masahide Otania, Hiroyuki Takahashia, Hiroshi Azumad, Hiromi Sakaie, Hiroyuki
Department of Surgery, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
EP
a
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Furukawaa
b
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Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2503 FAX: +81-166-68-2193
Department of Mechanical Engineering, Tokyo Metropolitan University. 1-1 Minami-Osawa,
Hachioji-shi, Tokyo, 192-0397 Japan. Tel/Fax : +81-42-677-2711
c
Department of Pathology, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2371 FAX: +81-166-68-2379 1
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d
Department of Pediatrics, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
e
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Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2481 FAX: +81-166-68-2489
Department of Chemistry, Nara Medical University. 840 Shijo-cho, Kashihara-shi, Nara,
E-mail addresses of authors:
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Tatsuya Shonaka: [email protected]
Naoto Matsuno: [email protected]
Hiromichi Obara: [email protected]
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634-8521 Japan. TEL +81-744-29-8810 FAX +81-744-29-8810
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Ryo Yoshikawa: [email protected]
Yuji Nishikawa: [email protected]
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Mikako Gouchi: [email protected]
AC C
Masahide Otani: [email protected]
Hiroyuki Takahashi: [email protected]
Hiroshi Azuma: [email protected]
Hiromi Sakai: [email protected]
Hiroyuki Furukawa: [email protected] 2
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Corresponding author: Naoto Matsuno. Email: [email protected]
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Department of Surgery, Asahikawa Medical University. 2-1-1-1 Midorigaoka-higashi,
Asahikawa-shi, Hokkaido, 078-8510 Japan TEL: +81-166-68-2503 FAX: +81-166-68-2193 Grant Information: This work was supported by the Grants-in-Aid for Scientific Research (C) (KAKENHI) No.17K10503.
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Key words: liver transplantation, subnormothermic machine perfusion, hemoglobin-based
Tables: 0
Figures: 4 (color - No)
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Declarations of interest: None
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oxygen carriers, pig
Abbreviations:
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adenosine triphosphate; ATP
AC C
aspartate transaminase; AST
carbonyl hemoglobin; HbCO
donation after cardiac death; DCD
hematoxylin-eosin; HE
hemoglobin; Hb 3
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hemoglobin-based oxygen carriers; HBOCs
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hemoglobin vesicle; HbV
ischemia-reperfusion injury; IRI
nucleic-acid amplification testing; NAT
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red blood cells; RBC
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University of Wisconsin; UW
AC C
EP
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warm ischemic time; WIT
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Abstract
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Oxygenation is necessary for aerobic metabolism, which maintains adenosine triphosphate
(ATP) within the graft organ. In recent years, some studies have demonstrated that
has the potential to improve oxygen metabolism.
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subnormothermic machine perfusion (SNMP) with hemoglobin-based oxygen carriers (HBOCs)
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【Aim】The aim of this study was to evaluate the effectiveness of perfusate with human-derived
hemoglobin vesicles (HbV) under SNMP in a pig model of donation after cardiac death (DCD).
【Materials and methods】In this study, pig livers were procured with a warm ischemic time of 60
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minutes and were preserved in 3 groups for 240 minutes. The preservation conditions were as
follows: 4℃ cold storage (Group 1); SNMP with University of Wisconsin (UW) perfusate alone
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(Group 2); SNMP (21℃) with UW and HbV (Hb; 0.6mg/dl) perfusate (Group 3). All livers were
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perfused for 120 minutes using pig autologous blood machine perfusion (reperfusion phase).
We investigated the aspartate transaminase (AST) level, hemodynamics (portal vein resistance
[PVR] and oxygen consumption) in the preservation and reperfusion phases. A histological
study (Hematoxylin-Eosin [HE] staining) was performed after 240 minutes of preservation.
【Results】The PVR of Group 3 was not increased in comparison to Group 2. During 1
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preservation, the oxygen consumption of Group 3 was higher than that of Group 2. However, the
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level of AST did not differ between Groups 2 and 3.
【Conclusion】The present study revealed that perfusate with HbV increased the oxygen
AC C
EP
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consumption of the donor liver during SNMP.
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Introduction
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Despite strategies to expand the organ donor pool, the number of liver transplantations
performed over the past decade has not increased [1,2]. The utilization of grafts from donors
after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool.
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ranging from 20% to 40% overall, and >50% for DCD [3].
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However, liver utilization remains suboptimal, with organ discard rates in the United States
Ischemic reperfusion injury after liver transplantation is a major problem in liver transplantation.
Several studies have demonstrated that subnormothermic machine perfusion (SNMP) is an
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effective method for reducing hepatocellular injury during organ preservation [4–6]. However,
SNMP relies on physically dissolved oxygen [7, 8]. In recent years, some studies have shown
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that the use of perfusate with a hemoglobin-based oxygen carrier during the preservation of the
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liver graft improves the graft function during machine perfusion [9, 10].
The aim of this study was to evaluate and the effectiveness of perfusate with human-derived
hemoglobin vesicles (HbV) under SNMP in a pig model of donation after cardiac death.
Materials and Methods 1
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Domestic female cross-bred Large-Yorkshire, Landrace, and Duroc pigs (approximately 30 kg, 2
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or 3 months of age) were purchased from Sankyo Labo Service Corporation, Inc (Sapporo,
Japan). Ten pigs were used in the present study. The animal care was in accordance with the
Declaration of Helsinki. The study was approved by the Asahikawa Medical University Animal
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Care and Use Committee.
Perfusion Preservation Machine (Fig. 1)
We developed an organ preservation system with temperature-controlled machine perfusion.
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The system has 2 circulating perfusion circuits for the portal vein and hepatic artery. Each circuit
included a roller pump, flow meter, and pressure sensor. The circuits for the hepatic artery
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provided pulsatile flow and installed an oxygenator; the circuits for the portal vein provided no
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pulsatile flow. Both circuits were connected via plastic connectors to the hepatic vessels. This
system has waterproof thermocouples, which measure the solution and the organ temperatures.
A computer included in this system records data and controls the flow conditions and
temperatures of the preservation solution (Fig.1). The temperature in the organ chamber is
controlled by a heat exchanger and ice-cold water. The portal vein and hepatic artery rate are 2
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controlled. The oxygen supply was 300 mmHg during preservation and 380 mmHg during
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reperfusion.
Human-derived HbV as hemoglobin-based oxygen carriers (HBOCs)
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Sakai et al. demonstrated HBOCs as artificial oxygen carriers, which encapsulate a purified and
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concentrated hemoglobin solution in phospholipid vesicles, mimicking the cellular structure of
red blood cells (RBC) [11]. HbV were prepared from out-of-date nucleic-acid amplification
testing (NAT)-inspected RBC provided by the Japanese Red Cross. The carbonyl hemoglobin
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(HbCO) purification procedure includes pasteurization and nanofiltration for the utmost safety
from infection. Liposome encapsulation protects against the toxic effects of molecular
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hemoglobin. It is encapsulated with liposomes composed of four lipids. These lipids were
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selected based on their stability, encapsulation efficiency, and biocompatibility [12]. Organ
preservation would likely be improved if the oxygen metabolism can be supported by perfusion
with a fluid such as HbV [13, 14]. Human-derived HbV were used as hemoglobin (Hb)-based
HBOCs. We also used HbV that had been created 18-20 months previously to confirm the
molecular stability. 3
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The Preparation and Preservation of the DCD Liver
Under inhalation anesthesia with isoflurane (Forane, Abbott, Japan), cardiac arrest was induced
by the intravenous injection of potassium chloride and the removal of ventilation. The donor
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livers were exposed to a warm ischemic time (WIT) of 60 minutes. After donor liver extirpation,
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we took 240 minutes for preservation. During preservation, all livers were assigned to the
following three groups: Group 1 (N=3), cold storage (4 ) with University of Wisconsin (UW)
solution; Group 2 (N=4), SNMP (20-22 ) with UW perfusate only; Group 3 (N=3), SNMP with
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HbV and UW perfusate. The perfusate of Group 2 consisted of 1500 ml of UW solution. The
perfusate of Group 3 consisted of 1400 ml of UW solution and 100 ml of HbV. The concentration
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of hemoglobin was 0.6-0.7mg/dl in the perfusate. To assess organ viability and
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ischemia–reperfusion injury (IRI) after preservation, all livers were reperfused with autologous
diluted blood for 120 minutes. We assessed the aspartate aminotransferase (AST) level (a
marker of hepatocellular injury). Portal vein (PV) resistance was calculated as the portal vein
pressure and flow. The PV flow divided by the PV pressure was called the PV resistance (PVR,
per 100g liver, wet liver weight). It measures the oxygen pressure between the portal vein and 4
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perfusate. After correcting the temperature, the difference in oxygen pressure was determined
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to calculate the oxygen consumption. We evaluated the PVR and oxygen consumption as
hemodynamic parameters (per 100g liver, wet liver weight). A histological analysis
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(Hematoxylin-Eosin [HE] staining) was performed after 240 minutes of preservation.
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Statistical analysis
All measures were expressed as the mean± SD within each group. All analyses were performed
using the R software program (version 3.1.2) and the EZR software program [15]. Two group
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comparisons of continuous measures between each group were conducted using a two-sample
t-test at a given time point. Repeated measures were used for data with more than three time
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points. When the comparison between groups were performed a univariate Type
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repeated-measures ANOVA was performed with the assumption of sphericity. P values of <0.05
were considered to indicate statistical significance.
Results
Fig. 2 shows the portal vein resistance (PVR) during preservation. During preservation, the PVR 5
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gradually increased in Group 2. However, there was no significant difference between Groups 2
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and 3. The PVR values were 0.010±0.009 (Group 1), 0.008±0.007 (Group 2), 0.008±0.005
(Group 3) mmHg/ml/min/100g liver after 120 minutes of reperfusion. The PVR value of Group 1
was slightly higher than the values of Groups 2 and 3. However, the differences among the
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groups were not statistically significant.
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Fig.3 shows the oxygen consumption during preservation. The oxygen consumption of Group 3
was significantly higher during preservation (P=0.03). The oxygen consumption in Groups 1, 2,
and 3 was 0.65±0.09, 0.68±0.05, and 0.63±0.14 mg/min/100g liver, respectively after 120
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minutes of reperfusion. The differences were not statistically significant.
The AST enzyme levels of Groups 2 and Group 3 did not differ to a statistically significant extent
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during preservation. Fig.4 shows the AST level after 120 minutes of reperfusion. The level of
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AST in the perfusate of Groups 1, 2, and 3 was 4016.7±1598.9, 1388±198.7, and 1598.9±288.0
IU/L, respectively. The level of AST in Group 1 was higher than that in Groups 2 and 3; however,
the difference was not statistically significant. Hepatocellular reperfusion injury was not
prevented in this model. A histological examination revealed was no evidence of HbV or venous
thrombosis at the end of the preservation phase. 6
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Discussion
The present study revealed that perfusate with human-derived HbV increased the oxygen
dose of HbV did not improve hepatocellular injury.
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consumption of the donor liver during SNMP. However, it also revealed that perfusate with a low
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Perfusate with HbV increased the oxygen consumption of the donor liver during SNMP. Burisma
et al. suggested the performance of SNMP for ex vivo preservation and the recovery of the
human liver for transplantation [16] and investigated the perfusion outcomes with a
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metabolomics analysis [17]. Oxygenated perfusion enables any changes in the tissue energy of
the graft to be restored [18, 19]. Compagnon et al. reported that an oxygenated and
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transportable MP system could fully rescue liver grafts exposed to lethal ischemic damage in a
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pig model of DCD liver transplantation [20]. Thus, we hypothesized that oxygen might be carried
from the perfusate to the donor liver in order to respond to oxygen consumption during perfusion.
P. Fontes et al. [9] reported that a new, cell-free HBOC solution was developed by combining a
stable second-generation bovine-derived hemoglobin compound. They also reported that the
higher hepatic O2 delivery using HBOC solution was associated with an improved graft function. 7
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However, their hemoglobin was derived from bovine, which may have unknown virus infectious
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and will be difficult to use in clinical setting. Liu et al. [10] reported that bile production and
hepatocellular injury were improved by blood donor perfusate normothermic machine perfusion.
In our study, human-derived HbV-containing perfusate was used. It was shown that oxygen
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consumption was significantly higher in the HbV perfusate during perfusion. This indicates that
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oxygen had been transferred from the perfusate to the donor liver by the HbV. We used HbV at
18 to 20 months after its creation. However the HbV was able to transport oxygen. Furthermore,
human-derived hemoglobin and virus removal were performed during the creation of the HbV;
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thus, it would seem easy to use in the clinical setting.
Because of a low dose of HbV, this study could not show to improve the hepatocelular injury.
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The perfusate of Fontes et al. [9] was set to Hb 3.5 g/dl, while that of Liu et al. [10] perfusion was
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set to 21-25% hematocrit. In the present study, our HbV content was 0.6-0.7g/dl hemoglobin,
which was lower in comparison to the 2 previous reports. We hypothesized that HbV carried
oxygen but that the amount of oxygen transported was not sufficient to improve the
hepatocellular injury.
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The present study is associated with some limitations. The study was performed using an
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animal model. This is an experiment on the transplant model, and the result of the actual
transplant is unknown.
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Conclusion
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The present study demonstrated the use of perfusate with human-derived HbV increased the
oxygen consumption of the donor liver during SNMP. However, this perfusate could not prevent
liver injury because the amount of HbV was insufficient. We plan to perform further studies using
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Figure Captions:
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Fig. 1. We developed an organ preservation system with temperature controlled machine
perfusion. The system had 2 circulating perfusion circuits for the portal vein and hepatic artery.
Oxygen was supplied from the hepatic artery and portal vein through an artificial lung. This
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perfusion device quantified the flow rate and the oxygen supply.
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Fig. 2. The portal vein resistance (PVR) in the preservation phase.
Fig. 3. The oxygen consumption in the preservation phase.
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EP
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Fig. 4. The AST level of the perfusate after 120 minutes of reperfusion.
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PV
Pressure Gage
Flow meter
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HA
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Optical Oxy. Sensor
Oxygenator and Heat Exchanger
Air Trap
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EP
Liver
Organ holder
Pump
Pump
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mmHg/ml/min/100g liver
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0.025
Group2
0.02
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Group3
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0.015
EP
0.01
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0.005
0 0
30
60
90
120
150
180
210
240
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mg/min/100g liver
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0.7
P=0.03
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0.6
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0.5
0.3
Group2
EP
0.2
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0.4
AC C
Group3
0.1 0 0
30
60
90
120
150
180
210
240
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6000
IU/L
5000
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P=0.09
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P=0.12
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4000
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3000
1000
AC C
EP
2000
0 Group1
Group2
Group3
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1. Hemoglobin vesicles (HbV) is a human-derived oxygen carrier. 2. We evaluated the effectiveness of perfusate with HbV.
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Highlights
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3. Pig liver after cardiac arrest was preserved under subnormothermic machine perfusion.
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EP
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4. The perfusate with HbV increased the oxygen consumption during machine perfusion.