Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage,Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.     

Preservation methods for kidney and liver

With the successful testing of the immunosuppressive effects of cyclosporine in transplant patients in 1978, the field of organ transplants began an exponential growth. With that, the field of organ preservation became increasingly important as the need to increase preservation time and improve graft function became paramount. However, for every patient that receives a transplanted organ, there are 4 more on the waiting list. In addition, a patient dies from the lack of a transplant almost every 1½ hour. To alleviate this donor crisis, there is a need to expand the donor pool to marginal donor organs. The main reason these organs are underutilized is because the current method of static preservation, simple cold storage, is ineffective. This article will provide a general review of the methods of preservation including simple cold storage, hypothermic machine perfusion, normothermic machine perfusion, and oxygen persufflation. In addition, the article will provide a review of how these dynamic preservation methods have improved the recovery and preservation of marginal donor organs including donation after cardiac death and fatty livers.     

Preservation methods for kidney and liver

With the successful testing of the immunosuppressive effects of cyclosporine in transplant patients in 1978, the field of organ transplants began an exponential growth. With that, the field of organ preservation became increasingly important as the need to increase preservation time and improve graft function became paramount. However, for every patient that receives a transplanted organ, there are four more on the waiting list. In addition, a patient dies from the lack of a transplant almost every 1½ hour. To alleviate this donor crisis, there is a need to expand the donor pool to marginal donor organs. The main reason these organs are underutilized is because the current method of static preservation, simple cold storage, is ineffective. This article will provide a general review of the methods of preservation including simple cold storage, hypothermic machine perfusion, normothermic machine perfusion, and oxygen persufflation. In addition, the article will provide a review of how these dynamic preservation methods have improved the recovery and preservation of marginal donor organs including Donation after Cardiac Death and Fatty livers.     

Single cell electrophoresis in determining cell death: potential for use in organ transplant research

Viability of donor tissues is essential for the success of organ transplantation. Although much work has been done in the field of organ preservation, currently there are few objective methods for evaluating transplant organ viability, and thus preservation efficiency. In the field of cancer biology, single-cell gel electrophoresis (SCGE) is a technique commonly used to measure the efficacy of anti-tumor treatments by measuring the breakdown of tumor cell deoxyribonucleic acid (DNA). This assay has recently been applied to various organs from a postmortem porcine animal model, and cells were found to undergo postmortem breakdown in a way similar to apoptosis-induced DNA fragmentation. Collections of cells from each organ reached levels indicative of non-viability as the postmortem interval (PMI) progressed. The rates of cellular DNA degradation were found to be specific to each organ type at a given ambient temperature. We believe that following the application of various preservation techniques, SCGE assay has the potential to provide a clear indication of cell viability in an organ destined for transplant. As a readily available viability assay, this technique could provide transplant researchers with a useful tool to quantify the efficacy of their experimental organ preservation techniques.     

Machine Perfusion versus Cold Storage of Kidneys Derived from Donation after Cardiac Death: A Meta-Analysis

Background

In response to the increased organ shortage, organs derived from donation after cardiac death (DCD) donors are becoming an acceptable option once again for clinical use in transplantation. However, transplant outcomes in cases where DCD organs are used are not as favorable as those from donation after brain death or living donors. Different methods of organ preservation are a key factor that may influence the outcomes of DCD kidney transplantation.

Methods

We compared the transplant outcomes in patients receiving DCD kidneys preserved by machine perfusion (MP) or by static cold storage (CS) preservation by conducting a meta-analysis. The MEDLINE, EMBASE and Cochrane Library databases were searched. All studies reporting outcomes for MP versus CS preserved DCD kidneys were further considered for inclusion in this meta-analysis. Odds ratios and 95% confidence intervals (CI) were calculated to compare the pooled data between groups that were transplanted with kidneys that were preserved by MP or CS.

Results

Four prospective, randomized, controlled trials, involving 175 MP and 176 CS preserved DCD kidney transplant recipients, were included. MP preserved DCD kidney transplant recipients had a decreased incidence of delayed graft function (DGF) with an odd ration of 0.56 (95% CI = 0.36–0.86, P = 0.008) compared to CS. However, no significant differences were seen between the two technologies in incidence of primary non-function, one year graft survival, or one year patient survival.

Conclusions

MP preservation of DCD kidneys is superior to CS in terms of reducing DGF rate post-transplant. However, primary non-function, one year graft survival, and one year patient survival were not affected by the use of MP or CS for preservation.     

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.     

The application of fluidics technology for organ preservation

A prototype design of a portable, pulsatile, perfusion preservation device based on a novel application of fluidics technology was tested to evaluate its ability to oxygenate preservation solution and to examine the relationship between organ resistance, perfusion pressure, and perfusion flow characteristics. The effects of organ resistance on pulse rate, perfusion pressure, and perfusion flow were modeled. Interstitial PO2 in canine hearts stored at 4 degrees C for 12 hours in the fluidics device (n = 5) and in static hypothermic storage (n = 5) was also compared. Increasing outflow resistance did not have an effect on operating frequency of the fluidics actuator. Perfusion pressure rose as outflow resistance was increased, and the flow of preservation solution decreased proportionately. The PO2 of the preservation solution increased to 300 mm Hg in two hours and reached a plateau that exceeded 400 mm Hg within six hours. The aortic flow profile during pulsatile perfusion resembled a square wave function with a mean pulse duration of 0.30 +/- 0.05 seconds. Oxygen delivery by the fluidics perfusion device exceeded the oxygen requirements of the hypothermically preserved organs at all resistance levels. Initial interstitial PO2 in the hearts of both groups was greater than 150 mm Hg. In perfused hearts, PO2 declined 30% by the 12th hour, whereas complete depletion of oxygen was noted in the static storage group within six hours. The fluidics organ perfusion/transport apparatus weighs less than 18 kg, uses no electrical power, and can operate continuously for 10 to 12 hours expending 780 L of oxygen.     

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 °C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 °C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 °C and 37 °C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.     

Harmonic analysis of perfusion pumps

The controversy over the use of nonpulsatile versus pulsatile pumps for maintenance of normal organ function during ex vivo perfusion has continued for many years, but resolution has been limited by lack of a congruent mathematical definition of pulsatility. We hypothesized that the waveform frequency and amplitude, as well as the underlying mean distending pressure are all key parameters controlling vascular function. Using discrete Fourier Analysis, our data demonstrate the complexity of the pulmonary arterial pressure waveform in vivo and the failure of commonly available perfusion pumps to mimic in vivo dynamics. In addition, our data show that the key harmonic signatures are intrinsic to the perfusion pumps, are similar for flow and pressure waveforms, and are unchanged by characteristics of the downstream perfusion circuit or perfusate viscosity.     

Studies in cryoprotection: I. A simple method for the controlled introduction and removal of cryoprotective agents during organ perfusion

A method is presented for the introduction and removal of cyroprotective agents from kidneys, utilizing the principle of gradual addition and elution during continuous perfusion. The method differs from those described previously in that it utilizes a standard perfusion machine (Water MOX 100) and a motorized syringe pump; both of which are widely available in laboratories involved in clinical and experimental organ preservation. In addition, a simple method for measuring vascular resistance is described which relies on a comparison of flow rates through the perfused organs to that through a fixed resistance. The utility of this approach is illustrated by studies of organ function and vascular resistance.     

Lysosomal enzyme release in hypothermically perfused dog kidneys

This study investigated lysosomal disruption during hypothermic perfusion preservation of kidneys and its possible relationship to viability. The percentage of free and bound enzyme activity was analyzed for three lysosomal enzymes in homogenates made from perfused canine kidney cortex tissue, including beta-glucuronidase, cathepsin-D, and aryl sulfatase. All three enzymes displayed characteristic increases in free enzyme activity (47-68%) throughout 5 days of perfusion preservation. The increased activity obtained at 5 days of preservation was found to indicate "severe" tissue damage, as shown by a similar increase obtained in renal cortex tissue exposed to warm ischemia (37 degrees C) for 4 hr or longer. Aryl sulfatase was found to be the most sensitive indicator of severe damage. Pretreatment of kidney donors with methylprednisolone, a lysosomal stabilizer, was also studied in kidneys exposed to 5 days of perfusion. Pretreatment was found to reduce the percentage of free lysosomal enzyme activity following 5 days (nonviable) of perfusion to those levels normally obtained following 3-day (viable) perfusion. This indicates that methylprednisolone may be useful in modulating the severe disruption of lysosomes induced by long-term preservation. It is concluded that extensive disruption of lysosomes occurs during hypothermic perfusion preservation and may represent one cause for loss of organ viability.     

Current techniques and the future of lung preservation

Although lung transplant remains the only option for patients suffering from end-stage lung failure, donor supply is insufficient to meet demand. Static cold preservation is the most common method to preserve lungs in transport to the recipient; however, this method does not improve lung quality and only allows for 8 h of storage. This results in lungs which become available for donation but cannot be used due to failure to meet physiologic criteria or an inability to store them for a sufficient time to find a suitable recipient. Therefore, lungs lost due to failure to meet physiological or compatibility criteria may be mitigated through preservation methods which improve lung function and storage durations. Ex situ lung perfusion (ESLP) is a recently developed method which allows for longer storage times and has been demonstrated to improve lung function such that rejected lungs can be accepted for donation. Although greater use of ESLP will help to improve donor lung utilization, the ability to cryopreserve lungs would allow for organ banking to better utilize donor lungs. However, lung cryopreservation research remains underrepresented in the literature despite its unique advantages for cryopreservation over other organs. Therefore, this review will discuss the current techniques for lung preservation, static cold preservation and ESLP, and provide a review of the cryopreservation challenges and advantages unique to lungs.     

Hypothermic perfusion preservation: the future of organ preservation revisited?

Hypothermic perfusion preservation (HPP) was an integral step in the development of early clinical transplantation programmes, and considerable progress was made in understanding the basic principles underlying the technique. In subsequent years, the development of better preservation solutions for cold hypoxic storage, along with pragmatic choices made on grounds of costs and logistics, saw a fall in the application of HPP. More recently, the acute shortage of suitable organ donors and the inevitable pressure to use organs from sub-optimal (or expanded criteria) donors, has forced a re-evaluation of HPP, and the development of a new generation of HPP machines and associated perfusion solutions. This review sets out the historical development of HPP across the range of organs in which the method was originally investigated, describes the biological benefits and drawbacks associated with HPP, and sets out the most recent literature on the topic (including comments on the interest in use of higher temperatures in organ perfusion).     

细根与根系总质量比的相似性导致了共存的本土植物和入侵植物之间植物-土壤反馈的对比

三维土壤异质性对种子萌发影响的实验研究 土壤生物通过植物-土壤的反馈作用调控植物间相互作用和外来植物入侵。因此,探明植物-土壤反馈作用种间差异的形成原因,对于预测土壤生物在植物入侵过程中的作用具有重要价值。近期的研究发现,植物性状可以用于预测植物-土壤反馈作用。同时,研究发现植物入侵也与植物的一些性状相关联,暗示植物-土壤反馈作用通过植物性状与外来植物入侵之间存在关联,但尚缺乏实验证明。鉴于此,本研究选取了3对近缘入侵和本土植物为对象,比较了其植物-土壤反馈作用,探讨了植物-土壤反馈作用与植物根部性状的关联性。首先,通过种植实验植物3个月,驯化采自于田间的土壤。之后,将实验植物种植于对照和灭菌处理的同种或近缘(同科或同属)种驯化过的土壤中,评价同种或近缘种根际土壤生物对植物生长的净反馈作用(与灭菌土比较),并比较了两类土壤生物对植物的反馈作用。总体而言,同种或近缘种根际土壤生物对入侵与本土植物的净反馈作用无显著差异,两类土壤生物对入侵和本土植物的反馈作用亦无显著差异。土壤反馈作用的强度和种植于对照土壤中植物细根生物量比存在正相关关系,且入侵与本土植物细根生物量比无显著差异。这一发现表明：相似的细根生物量比可能是导致入侵与本土植物间土壤反馈作用无差异的一个重要原因。为提升人们对土壤生物在植物入侵过程中 作用的认识,亟需选取更多入侵与本土植物(尤其是亲缘关系较远的物种)开展实验研究,进一步探明植物性状、土壤反馈作用和外来植物入侵之间的关联性。     

Resuscitation of Ischemic Donor Livers with Normothermic Machine Perfusion: A Metabolic Flux Analysis of Treatment in Rats

Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use.     

Tissue engineering: a new frontier in physiological genomics.

Considerable progress has been made in the last decade in the engineering and construction of a number of artificial tissue types. These constructs are typically viewed from the perspective of possible sources for implant and transplant materials in the clinical arena. However, incorporation of engineered tissues, often referred to as three-dimensional (3D) cell culture, also offers the possibility for significant advancements in research for physiological genomics. These 3D systems more readily mimic the in vivo setting than traditional 2D cell culture, and offer distinct advantages over the in vivo setting for some organ systems. As an example, cardiac cells in 3D culture 1) are more accessible for siRNA studies, 2) can be engineered with specific cell types, and 3) offer the potential for high-throughput screening of gene function. Here the state-of-the-art is reviewed and the applications for engineered tissue in genomics research are proposed. The ability to use engineered tissue in combination with genomics creates a bridge between traditional cellular and in vivo studies that is critical to enabling the transition of genetic information into mechanistic understanding of disease processes.     

Preservation of guinea pig hearts by hypothermic gas perfusion

The lack of a satisfactory method for long-term preservation of hearts during transport limits the source of human hearts for transplant to the geographic vicinity of the transplant center. Experimentally, reduction of myocardial oxygen requirements with hypothermia and cardioplegia prolong storage time to 48 h, but always with some evidence of myocardial damage. In this study, the combination of hypothermia with a procedure known to increase oxygen tension in cardiac muscle, gas perfusion, preserved contractile activity in guinea pig hearts for 24 h and did not cause edema. Cardioplegia or gas perfusion at temperatures below 10 degrees C or above 20 degrees C resulted in failure of hearts to contract upon rewarming. Contracture, dehydration, elevation of tissue calcium, reduced perfusate flow, and elevated creatine kinase levels occurred if liquid reperfusion was begun at 15 degrees C but not 25 degrees C. The results suggest that under the appropriate conditions, hypothermic gas perfusion is a potentially useful means of extending storage time of hearts for transplant.     

Hypothermic preservation of hepatocytes : I. Role of cell swelling

Hepatocytes from isolated rat livers were hypothermically incubated (5 degrees C) in an oxygenated environment with continuous shaking (to simulate organ perfusion preservation). The incubation solution was either a tissue culture medium (L-15), an organ preservation perfusate (UW gluconate), or a simple cold-storage solution used for organ preservation (UW lactobionate). Hepatocyte viability was assessed from the release of lactate dehydrogenase (LDH) into the incubation medium. Cell swelling (due to the uptake of water) was also measured. Within 24 hr, hepatocytes hypothermically stored in each of the three incubation solutions became swollen (30 to 40% water gain) and lost a significant amount of LDH (as much as 60%). The addition of polyethylene glycol (PEG; relative molecular mass 8000; 5 g%) to the solutions suppressed cell swelling and allowed the incubated hepatocytes to remain relatively well preserved (30% LDH release) for as long as 120 hr. Adding either dextran (relative molecular mass 10,000 to 78,000; 5 g%) or saccharides (100 mmol/liter) instead of PEG neither prevented cell swelling nor prevented the cells from dying. The results of this study suggest (i) there is a direct correlation (r = 0.873) between hypothermia-induced cell swelling and cell death (i.e., the suppression of cell swelling prevents cell death); (ii) the mechanism by which PEG prevents cell swelling (and thus maintains cell viability) is not related to the osmotic or oncotic properties of the molecule but instead is apparently related to some unknown interaction between PEG and the cell, an interaction that provides stability during hypothermic incubation; and (iii) hypothermia-induced cell swelling must be prevented if isolated hepatocytes are to be used as a model for studying the mechanism by which cell damage occurs during hypothermic organ preservation. By eliminating cell death due to cell swelling, the biochemical mechanisms of cell death can be studied.