Ex Vivo Normothermic Perfusion, a Novel Method to Assess Pancreases after Preservation

Julien Branchereau1,2* and Etohan Ogbemudia1*, Kaithlyn Rozenberg1, Fungai Dengu1, Gabriella Hakim1, Flavia Neri1, Georg Ebeling1, Letizia Lo Faro1, James Hunter1, Rutger Ploeg1* and Peter Friend1* * "Authors contributed equally to this work" 1-Nuffield Department of Surgical Sciences - University of Oxford 2-Nantes Hospital University - France Introduction Static cold storage (SCS) of pancreases remains the current standard method for preservation prior to transplantation. Pulsatile hypothermic machine perfusion (HMP) is an emerging method that could potentially improve the preservation of pancreases to enhance graft function. This is based on personal extensive experience with HMP of pancreases in human, porcine allotransplantation and large non-human primate models. All models consistently showed preservation of pancreatic parenchyma on histological assessment for up to 24hours. To provide reassurance of organ viability prior to transplantation further investigations are necessary. We propose that normothermic perfusion (NMP) of pancreases after cold preservation allows necessary functional and physiological assessments. Method Porcine pancreases (3) were retrieved from the slaughterhouse after 30 minutes of warm ischaemia and were flushed. After 3 hours of cold ischaemia two pancreases were perfused by HMP (Wave machine; Waters Medical Systems) for 6 hours followed by 1 hour of NMP. One pancreas after 3 hours of CIT was placed on NMP for 2.5hours, this was the first pancreas NMP to assess feasibility of this technique.NMP was achieved by modification of the kidney assist device. Oxygenation was with 21% oxygen. The pancreases were cannulat ed via the aorta with free drainage of perfusate from the portal vein. NMP parameters for all pancreases were a pressure of 40mmHg and temperature of 37C. Perfusate was composed of red blood cells, plasma to provide a haematocrit of 25%, with additives of co-amoxiclav and 25,000IU of heparin. During the perfusions we collected serial perfusate samples for blood gas analysis and for insulin enzyme-linked immunosorbent assay (ELISA). Results The macroscopic appearance of the pancreases and the attached duodenum at the end of NMP appeared viable. Average resistance index during NMP was 0.62 ru (range 0.30 to 0.90 ru). Average flow rate was 77mls/min (range 53 to 100 mls/min). Throughout, the duration of NMP in the two pancreases perfused for one-hour lactate remain stable with no increase from baseline. In the one pancreas perfused for 2.5hours lactate was also stable in the first hour then increased by 50% during the last 1.5hours of perfusion. ELISA confirmed the presence of insulin in the perfusate for all three perfusions. Conclusion Normothermic perfusion is a feasible method to allow physiological and functional assessment of pancreases after cold preservation techniques encouraging us to further develop this model.

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Development of ex vivo normothermic perfusion as an innovative method to assess pancreases after preservation

Journal of the Nuffield Department of Surgical Sciences ◽

10.37707/jnds.v2i4.198 ◽

2021 ◽

Vol 2 (4) ◽

Author(s):

Ann Ogbemudia

Keyword(s):

Cold Storage ◽

Ex Vivo ◽

Gas Analysis ◽

Machine Perfusion ◽

Assessment Strategy ◽

Hypothermic Machine Perfusion ◽

Normothermic Perfusion ◽

Pancreas Preservation ◽

Normothermic Machine Perfusion ◽

Lactate Levels

Ann Ogbemudia, Julien Branchereau (Joint first authors), Gabriella Hakim, Fungai Dengu, FaysalEl-Gilani, John Mulvey, Kaithlyn Rozenberg, Thomas Prudhomme, Letizia Lo Faro, James Hunter,Paul Johnson, Rutger Ploeg and Peter Friend Objective Static cold storage (SCS) is the standard method for pancreas preservation but does not facilitate objective organ assessment prior to transplantation. Normothermic machine perfusion (NMP) has been used to test other abdominal and thoracic organs’ function and viability in transplantation settings. Our aim was to develop a NMP protocol specific for pancreases and then investigate its potential as an organ assessment strategy. Method 8 porcine pancreases were procured in conditions replicating donation after circulatory death with warm ischaemia time of 25 minutes. After 3 hours of static cold storage (SCS) the pancreases were divided into 3 experimental groups 1) the feasibility group (n=2) that underwent 2.5 hours of NMP 2) the SCS group (n = 2) that underwent an additional 6 hours of SCS prior to assessment on NMP for an hour and 3) the Oxygenated Hypothermic Machine Perfusion (oxyHMP) group (n = 4) that underwent 6 hours of oxyHMP followed by 1-hour assessment on NMP. The NMP protocol used autologous, leucodepleted blood delivered at a mean arterial pressure of 40mmHg with a temperature of 37oC. At timed intervals during NMP, perfusate samples were collected for gas analysis and perfusion parameters were recorded. Results The feasibility group was used to develop the NMP protocol and demonstrated stable perfusion parameters throughout NMP. Compared to the SCS group the oxyHMP group demonstrated better average perfusion characteristics with lower resistances, higher flow rates, lower mean lactate levels and physiological pH. The oxyHMP group maintained normal macroscopic appearances during NMP. At the end of NMP the SCS group had an average 32% weight increase compared to the oxyHMP group that were found to have a 17% weight reduction. Conclusion Normothermic machine perfusion of whole pancreases is feasible after cold preservation and potentially useful as an assessment strategy. Furthermore, it demonstrated that oxygenated HMP may be beneficial for pancreas preservation compared to SCS.

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Ischemia-Reperfusion Injuries Assessment during Pancreas Preservation

International Journal of Molecular Sciences ◽

10.3390/ijms22105172 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5172

Author(s):

Thomas Prudhomme ◽

John F. Mulvey ◽

Liam A. J. Young ◽

Benoit Mesnard ◽

Maria Letizia Lo Faro ◽

...

Keyword(s):

Ex Vivo ◽

Ischemia Reperfusion ◽

Graft Function ◽

Impedance Analysis ◽

Experimental Models ◽

Caspase Activation ◽

Pancreas Perfusion ◽

Number Of Factors ◽

Pancreas Preservation ◽

Metabolite Concentrations

Maintaining organ viability between donation and transplantation is of critical importance for optimal graft function and survival. To date in pancreas transplantation, static cold storage (SCS) is the most widely practiced method of organ preservation. The first experiments in ex vivo perfusion of the pancreas were performed at the beginning of the 20th century. These perfusions led to organ oedema, hemorrhage, and venous congestion after revascularization. Despite these early hurdles, a number of factors now favor the use of perfusion during preservation: the encouraging results of HMP in kidney transplantation, the development of new perfusion solutions, and the development of organ perfusion machines for the lung, heart, kidneys and liver. This has led to a resurgence of research in machine perfusion for whole organ pancreas preservation. This review highlights the ischemia-reperfusion injuries assessment during ex vivo pancreas perfusion, both for assessment in pre-clinical experimental models as well for future use in the clinic. We evaluated perfusion dynamics, oedema assessment, especially by impedance analysis and MRI, whole organ oxygen consumption, tissue oxygen tension, metabolite concentrations in tissue and perfusate, mitochondrial respiration, cell death, especially by histology, total cell free DNA, caspase activation, and exocrine and endocrine assessment.

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Re: Renal Transplantation after Ex Vivo Normothermic Perfusion: The First Clinical Study

The Journal of Urology ◽

10.1016/j.juro.2013.05.086 ◽

2018 ◽

Author(s):

David A. Goldfarb

Keyword(s):

Renal Transplantation ◽

Clinical Study ◽

Ex Vivo ◽

Normothermic Perfusion

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Temperature Controlled and Monitored Ex Vivo Lung Perfusion System for Research and Training Purposes

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2019-0074 ◽

2019 ◽

Vol 5 (1) ◽

pp. 293-295

Author(s):

Christina Pongratz ◽

Jens Ziegle ◽

Axel Boese ◽

Michael Friebe ◽

Helena Linge ◽

...

Keyword(s):

Storage Temperature ◽

Ex Vivo ◽

Culture Conditions ◽

Lung Perfusion ◽

Tissue Storage ◽

Ex Vivo Lung Perfusion ◽

Device Development ◽

Preservation Method ◽

Normothermic Perfusion ◽

Short Period

AbstractEx vivo lung perfusion (EVLP) is a preservation method for donor lungs, which keep lungs viable in a physiological environment outside of a body for a short period of time. EVLP is established clinically for lung transplantation. Experimental applications for EVLP are e.g. lung cancer research or medical device development and testing. For preservation, a lung is ventilated artificially in an organ chamber and perfused antegrade through the pulmonary artery. Here we introduce a thermoregulation system for an experimental EVLP system to be used for translational research approaches as well as for training medical staff. To implement physiological culture conditions that are a prerequisite for lung preservation and tissue homeostasis, a thermoregulation is needed to rewarm the explanted lung tissue (storage temperature 4°C). Technically, the EVLP system must be thermally insulated, so loss of caloric is avoided. For monitoring, temperature sensors are integrated within the lung, in the organ chamber and in the afferent perfusate tube, whereby the measured values determine the thermoregulation. Initial tests using thermal packs (cooled to 4-6°C) placed on a heating mat, as a part of the perfusion circuit, showed that the perfusate temperature falls to 34°C, but restores after approximately 60 minutes (36.5°C), whereby the thermal pack is warmed. With this setup longer perfusion times should be obtained rather than without thermoregulation due to normothermic perfusion of the lung.

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26-hour Storage of a Declined Liver Before Successful Transplantation Using Ex Vivo Normothermic Perfusion

Annals of Surgery ◽

10.1097/sla.0000000000001834 ◽

2017 ◽

Vol 265 (1) ◽

pp. e1-e2 ◽

Cited By ~ 31

Author(s):

Christopher J. E. Watson ◽

Lucy V. Randle ◽

Vasilis Kosmoliaptsis ◽

Paul Gibbs ◽

Michael Allison ◽

...

Keyword(s):

Ex Vivo ◽

Normothermic Perfusion ◽

Successful Transplantation

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Parvovirus infection in children and pregnant women

Infekcionnye bolezni ◽

10.20953/1729-9225-2021-2-119-125 ◽

2021 ◽

Vol 19 (2) ◽

pp. 119-125

Author(s):

E.V. Mikhailova ◽

◽

T.K. Chudakova ◽

D.Yu. Levin ◽

A.V. Romanovskaya ◽

...

Keyword(s):

Pregnant Women ◽

Ex Vivo ◽

Parvovirus B19 ◽

Virus Transmission ◽

Intrauterine Infection ◽

Enzyme Linked Immunosorbent Assay ◽

Minute Virus Of Mice ◽

Gestation Age ◽

B19 Parvovirus ◽

Minute Virus

Parvovirus (PV) is a widespread infection, despite the fact that this pathogen was discovered only recently. The therapeutic effect of PV, in particular its oncolytic activity, is being actively studied now. Notably, PVs causing infections in animals, such as rat PV H-1, caninae PV, and rodent protoparvovirus (minute virus of mice) suppress oncogenesis in these animals. There is an ex vivo evidence of rat glioblastoma and gliosarcoma sensitivity to PV. The affinity of PV B19 to P-antigen located primarily on the membranes of erythroid cells is crucial for the disease pathogenesis. The teratogenic effect of PV B19 is associated with its ability to infect placental cells (P-antigen is present on the cells of chorionic villi and surface of the trophoblast). PV infection can be acquired or congenital, typical or atypical. The outcome of intrauterine infection with PV B19 largely depends on the gestation age when the infection occurred. Women infected during the second trimester are at higher risk of vertical transmission and severe intrauterine pathology with a poor outcome than those infected during the third trimester. Constant contact with young children significantly increases the risk of PV B19 infection among pregnant women with no immunity to this virus. Serum is the most convenient biomaterial for detecting both PV DNA and virus-specific antibodies. One test for anti-PV IgG using enzyme-linked immunosorbent assay is sufficient to determine the immune status of a patient. Polymerase chain reaction with amniotic fluid is used to diagnose intrauterine infection with PV B19. Blood components and products should be checked for PV B19. High frequency of PV B19 detection in the blood of donors necessitates the development of special measures aimed at prevention of virus transmission. Key words: pregnant women, children, parvovirus B19, parvovirus infection

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Twenty-Four-Hour Normothermic Perfusion of Isolated Ex Vivo Hearts Using Plasma Exchange

Journal of Thoracic and Cardiovascular Surgery ◽

10.1016/j.jtcvs.2020.11.158 ◽

2020 ◽

Author(s):

Lise Tchouta ◽

Daniel Drake ◽

Mark Hoenerhoff ◽

Alvaro Rojas-Pena ◽

Jonathan Haft ◽

...

Keyword(s):

Plasma Exchange ◽

Ex Vivo ◽

Normothermic Perfusion

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Case series: Transplantation of kidneys from donors with renal artery aneurysm

Canadian Urological Association Journal ◽

10.5489/cuaj.4244 ◽

2017 ◽

Vol 11 (7) ◽

pp. E307-10 ◽

Cited By ~ 3

Author(s):

Mahmoud Alameddine ◽

Zhobin Moghadamyeghaneh ◽

Giselle Guerra ◽

Mahmoud Morsi ◽

Mohammed Osman ◽

...

Keyword(s):

Renal Function ◽

Renal Artery ◽

Ex Vivo ◽

Average Length ◽

Graft Function ◽

Case Series ◽

Artery Aneurysm ◽

Renal Artery Aneurysm ◽

Antibody Mediated Rejection ◽

The Mean

Introduction: With the present disparity between organ availability and recipient demands, we reported our experience in transplanting kidneys with renal artery aneurysm after back-table reconstruction.Methods: Four patients were identified. The repair consisted of excision of the aneurysm with ostial closure, and for one of the cases, an ovarian vein patch was used. We reviewed the safety and outcomes of this procedure. All donors were asymptomatic before surgery and were diagnosed incidentally during living donor evaluation. The nephrectomies performed were hand-assisted laparoscopic approaches. All recipients had followup renal function and ultrasound duplex of renal artery at six and 12 months and then annually.Results: The mean age of the recipients was 28.7 years (range 3‒45). The mean size of the aneurysm was 7.4 ± 2.7 mm. All patients had immediate graft function with median serum creatinine of 1.9 ± 1.5 mg/dL at discharge. The average length of hospital stay was 6.25 ± 2.6 days. They also maintained good renal function with an average estimated glomerular filtration rate (eGFR) of 102.8 mL/min/1.73m2 (range 53.4‒199 mL/min/1.73m2) and patent vessels at one year. One patient suffered from acute antibody-mediated rejection and lost his graft (medication non-compliance). One patient had two simultaneous benign renal cysts that were resected. Three of the kidneys were right-sided and one left. Mean cold ischemia time was 86 ± 18 minutes. No deaths have been recorded.Conclusions: Transplanting kidneys with a renal artery aneurysm after ex-vivo repair is safe and the outcomes are encouraging. Also, it may play an important role in

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Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis

10.1101/2020.08.17.253252 ◽

2020 ◽

Author(s):

Caitriona M. McEvoy ◽

Sergi Clotet-Freixas ◽

Tomas Tokar ◽

Chiara Pastrello ◽

Shelby Reid ◽

...

Keyword(s):

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Graft Function ◽

Kidney Injury ◽

Tca Cycle ◽

Proteomics Analysis ◽

Beneficial Effects ◽

Kidney Perfusion

AbstractNormothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of DCD injury compared to static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at 3 time points from pig kidneys subjected to 30-minutes of warm ischemia, followed by 8 hours of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (FDR<0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the TCA-cycle and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion, and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (ETFB, CPT2) by immunoblotting. Transcription factor databases identified PPARGC1A, PPARA/G/D and RXRA/B as the upstream regulators of our dataset, and we confirmed their increased expression in NEVKP with RT-PCR. The proteome-level changes observed in NEVKP mediate critical metabolic pathways that may explain the improved graft function observed. These effects may be coordinated by PPAR-family transcription factors, and may represent novel therapeutic targets in ischemia-reperfusion injury.

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