Metformin Preconditioning Improves Hepatobiliary Function and Reduces Injury in a Rat Model of Normothermic Machine Perfusion and Orthotopic Transplantation.

BACKGROUND: Preconditioning of donor livers before organ retrieval may improve organ quality after transplantation. We investigated whether preconditioning with metformin reduces preservation injury and improves hepatobiliary function in rat donor livers during ex situ normothermic machine perfusion (NMP) and after orthotopic liver transplantation. METHODS: Lewis rats were administered metformin via oral gavage, after which a donor hepatectomy was performed followed by a standardized cold storage period of 4 hours. Graft assessment was performed using NMP via double perfusion of the hepatic artery and portal vein. In an additional experiment, rat donor livers preconditioned with metformin were stored on ice for 4 hours and transplanted to confirm postoperative liver function and survival. Data were analyzed and compared with sham-fed controls. RESULTS: Graft assessment using NMP confirmed that preconditioning significantly improved ATP production, markers for hepatobiliary function (total bile production, biliary bilirubin, and bicarbonate), and significantly lowered levels of lactate, glucose, and apoptosis. After orthotopic liver transplantation, metformin preconditioning significantly reduced transaminase levels. CONCLUSIONS: Preconditioning with metformin lowers hepatobiliary injury and improves hepatobiliary function in an in situ and ex situ model of rat donor liver transplantation.

Transplantation of high-risk donor livers after resuscitation and viability assessment using a combined protocol of oxygenated hypothermic, rewarming and normothermic machine perfusion: study protocol for a prospective, single-arm study (DHOPE-COR-NMP trial).

INTRODUCTION: Extended criteria donor (ECD) livers are increasingly accepted for transplantation in an attempt to reduce the gap between the number of patients on the waiting list and the available number of donor livers. ECD livers; however, carry an increased risk of developing primary non-function (PNF), early allograft dysfunction (EAD) or post-transplant cholangiopathy. Ischaemia-reperfusion injury (IRI) plays an important role in the development of these complications. Machine perfusion reduces IRI and allows for reconditioning and subsequent evaluation of liver grafts. Single or dual hypothermic oxygenated machine perfusion (DHOPE) (4°C-12°C) decreases IRI by resuscitation of mitochondria. Controlled oxygenated rewarming (COR) may further reduce IRI by preventing sudden temperature shifts. Subsequent normothermic machine perfusion (NMP) (37°C) allows for ex situ viability assessment to facilitate the selection of ECD livers with a low risk of PNF, EAD or post-transplant cholangiopathy. METHODS AND ANALYSIS: This prospective, single-arm study is designed to resuscitate and evaluate initially nationwide declined ECD livers. End-ischaemic DHOPE will be performed for the initial mitochondrial and graft resuscitation, followed by COR of the donor liver to a normothermic temperature. Subsequently, NMP will be continued to assess viability of the liver. Transplantation into eligible recipients will proceed if all predetermined viability criteria are met within the first 150 min of NMP. To facilitate machine perfusion at different temperatures, a perfusion solution containing a haemoglobin-based oxygen carrier will be used. With this protocol, we aim to transplant extra livers. The primary endpoint is graft survival at 3 months after transplantation. ETHICS AND DISSEMINATION: This protocol was approved by the medical ethical committee of Groningen, METc2016.281 in August 2016 and registered in the Dutch Trial registration number TRIAL REGISTRATION NUMBER: NTR5972, NCT02584283.

Effect of New Zealand Blackcurrant Extract on Cycling Performance and Substrate Oxidation in Normobaric Hypoxia in Trained Cyclists.

New Zealand blackcurrant (NZBC) extract enhanced exercise-induced fat oxidation and 16.1 km cycling time trial (TT) in normobaric normoxia. The effect of NZBC extract on physiological and metabolic responses was examined during steady state cycling and a 16.1 km TT in normobaric hypoxia. This study used a randomized, double-blind, crossover design. Eleven healthy male cyclists (age: 38 ± 11 y, height: 179 ± 4 cm, body mass: 76 ± 8 kg, V ˙ O2max: 47 ± 5 mL·kg-1·min-1, mean ± SD) ingested NZBC extract (600 mg·day-1 CurraNZ® containing 210 mg anthocyanins) or a placebo (600 mg microcrystalline cellulose M102) for seven days (washout 14 days) and performed a steady state cycling test (3 × 10 min at 45%, 55% and 65% V ˙ O2max) followed by a 16.1 km TT at a simulated altitude of ~2500 meters (~15% of O2). Indirect calorimetry was used to measure substrate oxidation during steady state cycling. Intake of NZBC extract had no effect on blood glucose and lactate, heart rate, substrate oxidation, and respiratory exchange ratio during steady state cycling at 45%, 55% and 65% V ˙ O2max, and on 16.1 km TT performance (placebo: 1685 ± 92 s, NZBC extract: 1685 ± 99 s, P = 0.97). Seven days intake of New Zealand blackcurrant extract does not change exercise-induced metabolic responses and 16.1 km cycling time trial performance for moderately endurance-trained men in normobaric hypoxia.

Supercooling preservation and transplantation of the rat liver.

The current standard for liver preservation involves cooling of the organ on ice (0-4 °C). Although it is successful for shorter durations, this method of preservation does not allow long-term storage of the liver. The gradual loss of hepatic viability during preservation puts pressure on organ sharing and allocation, may limit the use of suboptimal grafts and necessitates rushed transplantation to achieve desirable post-transplantation outcomes. In an attempt to improve and prolong liver viability during storage, alternative preservation methods are under investigation. For instance, ex vivo machine perfusion systems aim to sustain and even improve viability by supporting hepatic function at warm temperatures, rather than simply slowing down deterioration by cooling. Here we describe a novel subzero preservation technique that combines ex vivo machine perfusion with cryoprotectants to facilitate long-term supercooled preservation. The technique improves the preservation of rat livers to prolong storage times as much as threefold, which is validated by successful long-term recipient survival after orthotopic transplantation. This protocol describes how to load rat livers with cryoprotectants to prevent both intracellular and extracellular ice formation and to protect against hypothermic injury. Cryoprotectants are loaded ex vivo using subnormothermic machine perfusion (SNMP), after which livers can be cooled to -6 °C without freezing and kept viable for up to 96 h. Cooling to a supercooled state is controlled, followed by 3 h of SNMP recovery and orthotopic liver transplantation.

Multipolar radiofrequency ablation for colorectal liver metastases close to major hepatic vessels.

BACKGROUND: Resection of colorectal liver metastases (CRLM) is often hindered by their location close to the major hepatic vessels. So far, radiofrequency ablation for perivascular tumours was thought to be ineffective and unsafe due to either the heat sink effect or vascular thrombosis. The aim of this study was to examine whether RFA using multipolar probes could be a safe and effective option for CRLM adjacent to major hepatic vessels. METHODS: Patients were treated with multipolar RFA during an open procedure using 3 simultaneously placed electrodes. In 52 consecutive patients with CRLM, 144 tumours were ablated with RFA. In 16 out of 52 (31%) patients, metastases were abutting major hepatic vessels. We examined whether perivascular location was a risk factor for local tumour progression. The relation between perivascular location and time to local tumour progression and recurrence free survival was assessed using cox-regression analysis. RESULTS: All patients were followed for at least 3 years after RFA unless they deceased before this time. Local tumour progression following RFA occurred in 17 out of 144 tumours (12%), of which 4 out of 21 were perivascular tumours. Tumour size was the only risk factor for local tumour progression in this study. Proximity to large vessels was neither a risk factor for local local tumour progression, nor for time to local tumour progression or recurrence free survival. DISCUSSION: This study indicates that patients with CRLM abutting any of the large hepatic vessels can be safe and effectively treated with RFA when using a multipolar system.

Liver Transplantation in Groningen, The Netherlands: A Single Center Status Report.

The liver transplantation program of the University Medical Center Groningen in the Netherlands was started in 1979, making it one of the first programs worldwide. During the past 36 years, a total of 1478 liver transplantations have been performed, 459 of which were in children. One of the first patients transplanted in 1979 is still alive and is one of the longest surviving patients after liver transplantation worldwide. During the last decade, an increasing number of donation after circulatory death (DCD) donor livers have been accepted for transplantation. Over 30% of the livers transplanted in Groningen come from DCD donors. These livers have an increased risk of developing biliary complications, such as non-anastomotic biliary strictures (NAS). One of the main research topics in Groningen has been the pathogenesis and prevention of NAS. In an attempt to reduce the incidence of NAS after liver transplantation, machine perfusion technology has been developed as an alternative to the traditional method of static cold storage. Researchers of the Groningen liver transplant team were the first in the world to report a method of ex situ normothermic machine perfusion of human donor livers. The efficacy and safety of various types of machine perfusion are currently studied in both animal models and clinical trials. A second line of research in Groningen focuses on alterations in the blood coagulation system in patients with liver disease and undergoing liver transplantation. Groningen researchers were the first to describe a 'rebalanced state' of the coagulation system in patients with liver disease, making them prone to both bleeding and thrombo-embolic complications. Clinicians and researchers at the Groningen liver transplant program will continue to collaborate with a shared focus and the aim to provide innovation and the highest level of care to patients with endstage liver disease.

Layer-by-layer heparinization of decellularized liver matrices to reduce thrombogenicity of tissue engineered grafts.

BACKGROUND: Tissue-engineered liver grafts may offer a viable alternative to orthotopic liver transplantation and help overcome the donor organ shortage. Decellularized liver matrices (DLM) have a preserved vasculature and sustain hepatocellular function in culture, but graft survival after transplantation remains limited due to thrombogenicity of the matrix. AIM: To evaluate the effect of heparin immobilization on DLM thrombogenicity. METHODS: Heparin was immobilized on DLMs by means of layer-by-layer deposition. Grafts with 4 or 8 bilayers and 2 or 4 g/L of heparin were recellularized with primary rat hepatocytes and maintained in culture for 5 days. Hemocompatibility of the graft was assessed by ex vivo diluted whole-blood perfusion and heterotopic transplantation. RESULTS: Heparin was deposited throughout the matrix and the heparin content in the graft was higher with increasing number of bilayers and concentration of heparin. Recellularization and in vitro albumin and urea production were unaffected by heparinization. Resistance to blood flow during ex vivo perfusion was lower with increased heparinization and, macroscopically, no clots were visible in grafts with 8 bilayers. Following transplantation, flow through the graft was limited in all groups. Histological evidence of thrombosis was lower in heparinized DLMs, but transplantation of DLM grafts was not improved. CONCLUSIONS: Layer-by-layer deposition of heparin on a DLM is an effective method of immobilizing heparin throughout the graft and does not impede recellularization or hepatocellular function in vitro. Thrombogenicity during ex vivo blood perfusion was reduced in heparinized grafts and optimal with 8 bilayers, but transplantation remained unsuccessful with this method. RELEVANCE FOR PATIENTS: Tissue engineered liver grafts may offer a viable solution to dramatic shortages in donor organs.

Supercooling enables long-term transplantation survival following 4 days of liver preservation.

The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.

Determination and extension of the limits to static cold storage using subnormothermic machine perfusion.

BACKGROUND/AIMS: Static cold storage (SCS) of the liver for transplantation is limited by time. Continuation of metabolic activity leads to depletion of energy stores and loss of cellular function, which results in poor post-transplant function. Machine perfusion (MP) applied at the end of preservation may improve the viability of marginal liver grafts and provides information on the quality of the organ. We attempt to define the limits to SCS in terms of easily measurable perfusion parameters and investigate whether MP can improve liver viability. 
 METHODS: Rat livers were cold-stored for 0, 24, 48, 72, and 120 h, after which they were treated with subnormothermic machine perfusion (SNMP). Livers cold-stored for 48 and 72 h were transplanted orthotopically with or without SNMP. During SNMP easily measurable parameters were monitored and adenosine triphosphate (ATP) content was measured following preservation and SNMP. 
 RESULTS: ATP increased significantly during SNMP, but the recovered ATP content deteriorated with increased duration of SCS, with minimal improvement after 72 h of SCS. Vascular resistance during SNMP increased with extended preservation. After 48 h of SCS, orthotopic transplantation survival increased significantly from 50% to 100% with SNMP, but did not improve after 72 h. 
 CONCLUSIONS: Vascular resistance and ATP recovery suggest a decrease in viability after 48 h of SCS. Survival data confirms the loss of post-transplant graft function and supports the use of ATP and vascular resistance as useful indicators. Further, we show that the recoverability of a liver using SNMP is limited to 48 h of SCS.

Strategies to rescue steatotic livers before transplantation in clinical and experimental studies.

The shortage of donor livers has led to an increased use of organs from expanded criteria donors. Included are livers with steatosis, a metabolic abnormality that increases the likelihood of graft complications post-transplantation. After a brief introduction on the etiology, pathophysiology, categories and experimental models of hepatic steatosis, we herein review the methods to rescue steatotic donor livers before transplantation applied in clinical and experimental studies. The methods span the spectrum of encouraging donor weight loss, employing drug therapy, heat shock preconditioning, ischemia preconditioning and selective anesthesia on donors, and the treatment on isolated grafts during preservation. These methods work at different stages of transplantation process, although share similar molecular mechanisms including lipid metabolism stimulation through enzymes or nuclear receptor e.g., peroxisomal proliferator-activated receptor, or anti-inflammation through suppressing cytokines e.g., tumor necrosis factor-α, or antioxidant therapies to alleviate oxidative stress. This similarity of molecular mechanisms implies possible future attempts to reinforce each approach by repeating the same treatment approach at several stages of procurement and preservation, as well as utilizing these alternative approaches in tandem.

Supercooling as a viable non-freezing cell preservation method of rat hepatocytes.

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

Excorporeal normothermic machine perfusion resuscitates pig DCD livers with extended warm ischemia.

BACKGROUND: The shortage in donor livers has led to increased use of allografts derived from donation after cardiac death (DCD). The compromised viability in these livers leads to inferior post-transplantation allograft function and survival compared with donation after brain death (DBD) donor grafts. In this study, we reconditioned DCD livers using an optimized normothermic machine perfusion system. METHODS: Livers from 12 Yorkshire pigs (20-30 kg) were subjected to either 0 min (WI-0 group, n = 6) or 60 min (WI-60 group, n = 6) of warm ischemia and 2 h of cold storage in UW solution, followed by 4 h of oxygenated sanguineous normothermic machine perfusion. Liver viability and metabolic function were analyzed hourly. RESULTS: Warm ischemic livers showed elevated transaminase levels and reduced ATP concentration. After the start of machine perfusion, transaminase levels stabilized and there was recovery of tissue ATP, coinciding with an increase in bile production. These parameters reached comparable levels to the control group after 1 h of machine perfusion. Histology and gross morphology confirmed recovery of the ischemic allografts. CONCLUSION: Our data demonstrate that metabolic and functional parameters of livers with extended warm ischemic time (60 min) can be significantly improved using normothermic machine perfusion. We hereby compound the existing body of evidence that machine perfusion is a viable solution for reconditioning marginal organs.

A simplified subnormothermic machine perfusion system restores ischemically damaged liver grafts in a rat model of orthotopic liver transplantation.

BACKGROUND: Liver donor shortages stimulate the development of strategies that incorporate damaged organs into the donor pool. Herein we present a simplified machine perfusion system without the need for oxygen carriers or temperature control, which we validated in a model of orthotopic liver transplantation. METHODS: Rat livers were procured and subnormothermically perfused with supplemented Williams E medium for 3 hours, then transplanted into healthy recipients (Fresh-SNMP group). Outcome was compared with static cold stored organs (UW-Control group). In addition, a rat liver model of donation after cardiac death was adapted using a 60-minute warm ischemic period, after which the grafts were either transplanted directly (WI group) or subnormothermically perfused and transplanted (WI-SNMP group). RESULTS: One-month survival was 100% in the Fresh-SNMP and UW-Control groups, 83.3% in the WI-SNMP group and 0% in the WI group. Clinical parameters, postoperative blood work and histology did not differ significantly between survivors. CONCLUSION: This work demonstrates for the first time in an orthotopic transplantation model that ischemically damaged livers can be regenerated effectively using practical subnormothermic machine perfusion without oxygen carriers.

Decellularization and recellularization of whole livers.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions. Decellularized native tissues such blood vessels and skin on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization. In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.