Maria-Louisa Izamis

Subnormothermic Machine Perfusion for Ex Vivo Preservation and Recovery of the Human Liver for Transplantation

To reduce widespread shortages, attempts are made to use more marginal livers for transplantation. Many of these grafts are discarded for fear of inferior survival rates or biliary complications. Recent advances in organ preservation have shown that ex vivo subnormothermic machine perfusion has the potential to improve preservation and recover marginal livers pretransplantation. To determine the feasibility in human livers, we assessed the effect of 3 h of oxygenated subnormothermic machine perfusion (21°C) on seven livers discarded for transplantation. Biochemical and microscopic assessment revealed minimal injury sustained during perfusion. Improved oxygen uptake (1.30 [1.11–1.94] to 6.74 [4.15–8.16] mL O2/min kg liver), lactate levels (4.04 [3.70–5.99] to 2.29 [1.20–3.43] mmol/L) and adenosine triphosphate content (45.0 [70.6–87.5] pmol/mg preperfusion to 167.5 [151.5–237.2] pmol/mg after perfusion) were observed. Liver function, reflected by urea, albumin and bile production, was seen during perfusion. Bile production increased and the composition of bile (bile salts/phospholipid ratio, pH and bicarbonate concentration) became more favorable. In conclusion, ex vivo subnormothermic machine perfusion effectively maintains liver function with minimal injury and sustains or improves various hepatobiliary parameters postischemia.

Subnormothermic Machine Perfusion at Both 20°C and 30°C Recovers Ischemic Rat Livers for Successful Transplantation

BACKGROUND Utilizing livers from donors after cardiac death could significantly expand the donor pool. We have previously shown that normothermic (37°C) extracorporeal liver perfusion significantly improves transplantation outcomes of ischemic rat livers. Here we investigate whether recovery of ischemic livers is possible using sub-normothermic machine perfusion at 20°C and 30°C. METHODS Livers from male Lewis rats were divided into five groups after 1 h of warm ischemia (WI): (1) WI only, (2) 5 h of static cold storage (SCS), or 5 h of MP at (3) 20°C, (4) 30°C, and (5) 37°C. Long-term graft performance was evaluated for 28 d post-transplantation. Acute graft performance was evaluated during a 2 h normothermic sanguineous reperfusion ex vivo. Fresh livers with 5 h of SCS were positive transplant controls while fresh livers were positive reperfusion controls. RESULTS Following machine perfusion (MP) (Groups 3, 4, and 5), ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 100% survival (N ≥ 4) after 4 wk. On the other hand, animals from WI only, or WI + SCS groups all died within 24 h of transplantation. Fresh livers preserved using SCS had the highest alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the lowest bile production during reperfusion, while at 28 d post-transplantation, livers preserved at 20°C and 30°C had the highest total bilirubin values. CONCLUSIONS MP at both 20°C and 30°C eliminated temperature control in perfusion systems and recovered ischemically damaged rat livers. Postoperatively, low transaminases suggest a beneficial effect of sub-normothermic perfusion, while rising total bilirubin levels suggest inadequate prevention of ischemia- or hypothermia-induced biliary damage.

Recovery of Warm Ischemic Rat Liver Grafts by Normothermic Extracorporeal Perfusion

Liver transplantation is currently the only established treatment of end-stage liver disease, but it is limited by a severe shortage of viable donor livers. Donors after cardiac death (DCD) are an untapped source that could significantly increase the pool of available livers. Preservation of these DCD livers by conventional static cold storage (SCS) is associated with an unacceptable risk of primary nonfunction and delayed graft failure. Normothermic extracorporeal liver perfusion (NELP) has been suggested as an improvement over SCS. Livers recovered from male Lewis rats were subjected to 1 hr of warm ischemia and preserved with 5 hr of SCS or NELP, and transplanted into syngeneic recipients. As additional controls, non-ischemic livers preserved with 6 hr of SCS or NELP and unpreserved ischemic livers were transplanted. After NELP, ischemically damaged livers could be orthotopically transplanted into syngeneic recipients with 92% survival (n=13) after 4 weeks, which was comparable with control animals that received healthy livers preserved by SCS (n=9) or NELP (n=11) for 6 hr. On the other hand, animals from ischemia/SCS control group all died within 12 hr postoperatively (n=6). Similarly, animals that received ischemic livers without preservation all died within 24 hr after transplantation (n=6). These results suggest that NELP has the potential to reclaim warm ischemic livers that would not be transplantable otherwise. The rat model in this study is a useful platform to further optimize NELP as a method of recovery and preservation of DCD livers.

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

BackgroundLiver 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.MethodsRat 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).ResultsOne-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.ConclusionThis 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.

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.The realization of long–term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a novel technique based on sub–zero non–freezing tissue preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to 4 days, thereby tripling the viable preservation duration.

Hepatocyte viability and adenosine triphosphate content decrease linearly over time during conventional cold storage of rat liver grafts.

INTRODUCTION The gold standard in organ preservation is static cold storage (SCS) using University of Wisconsin solution (UW). Although it is well-known that there is a finite limit to SCS preservation, and that there is a correlation between the adenosine triphosphate (ATP) levels and organ function post-preservation, a quantitative relationship has not been established, which is important in understanding the fundamental limitations to preservation, minimizing cold ischemic injury, and hence maximizing use of the donor organ pool. AIM This study determines the time limits of cellular viability and metabolic function during SCS, and characterizes the relationship between cellular viability and energetic state using clinically relevant techniques in organ preservation. METHODS Rat livers were procured and stored using conventional storage in UW solution at 4 °C. Viability was assessed by determining the amount of viable hepatocytes and intracellular ATP content after 0, 24, 48, 72, and 120 hours of storage. RESULTS Numbers of viable hepatocytes that were isolated from these livers decreased steadily during SCS. After 5 days, viable hepatocytes decreased from 25.95 × 10(6) to 0.87 × 10(6) cells/gram tissue. Intracellular ATP content decreased from 9.63 to 0.93 moles/g tissue. Statistical analysis of variance established a linear relation for both parameters as a function of time (P < .05). CONCLUSION The linear correlation between hepatocyte viability, ATP content, and storage time suggests a shared physiological foundation. These findings confirm ATP as direct predictor for organ quality in the context of liver preservation, which will aid quantitative assessment of donor organs for various applications.

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.

A Metabolic Index of Ischemic Injury for Perfusion-Recovery of Cadaveric Rat Livers

With over 110,000 patients waiting for organ transplantation, the current crisis in organ transplantation is based on a lack of donors after brain-death (DBD). A very large alternative pool of donor organs that remain untapped are the donors after cardiac death (DCD), recovered after cardiac activity has ceased and therefore sustained some ischemic injury. Machine perfusion has been proposed as a novel modality of organ preservation and treatment to render such cadaveric organs, and in particular livers, transplantable. Two key issues that remain unaddressed are how to assess whether a DCD liver is damaged beyond repair, and whether machine perfusion has rendered an injured organ sufficiently viable for transplantation. In this work, we present a metabolic analysis of the transient responses of cadaveric rat livers during normothermic machine perfusion (NMP), and develop an index of ischemia that enables evaluation of the organ ischemic injury level. Further, we perform a discriminant analysis to construct a classification algorithm with >0.98 specificity to identify whether a given perfused liver is ischemic or fresh, in effect a precursor for an index of transplantability and a basis for the use of statistical process control measures for automated feedback control of treatment of ischemic injury in DCD livers. The analyses yield an index based on squared prediction error (SPE) as log(SPE) >1.35 indicating ischemia. The differences between metabolic functions of fresh and ischemic livers during perfusion are outlined and the metabolites that varied significantly for ischemic livers are identified as ornithine, arginine, albumin and tyrosine.

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.M 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.

In situ metabolic flux analysis to quantify the liver metabolic response to experimental burn injury

Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post‐burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady‐state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham‐burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic‐like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose‐dependent metabolic response, but rather leads to qualitatively different responses. Biotechnol. Bioeng. 2011; 108:839–852.