Cold Storage Of Livers Research Articles

Gradual rewarming with a hemoglobin-based oxygen carrier improves viability of donation after circulatory death in rat livers.

Donation after circulatory death (DCD) grafts are vital for increasing available donor organs. Gradual rewarming during machine perfusion has proven effective in mitigating reperfusion injury and enhancing graft quality. Limited data exist on artificial oxygen carriers as an effective solution to meet the increasing metabolic demand with temperature changes. The aim of the present study was to assess the efficacy and safety of utilizing a hemoglobin-based oxygen carrier (HBOC) during the gradual rewarming of DCD rat livers. Liver grafts were procured after 30 min of warm ischemia. The effect of 90 min of oxygenated rewarming perfusion from ice cold temperatures (4 °C) to 37 °C with HBOC after cold storage was evaluated and the results were compared with cold storage alone. Reperfusion at 37 °C was performed to assess the post-preservation recovery. Gradual rewarming with HBOC significantly enhanced recovery, demonstrated by markedly lower lactate levels and reduced vascular resistance compared to cold-stored liver grafts. Increased bile production in the HBOC group was noted, indicating improved liver function and bile synthesis capacity. Histological examination showed reduced cellular damage and better tissue preservation in the HBOC-treated livers compared to those subjected to cold storage alone. This study suggests the safety of using HBOC during rewarming perfusion of rat livers as no harmful effect was detected. Furthermore, the viability assessment indicated improvement in graft function.

SIRT1 regulates hepatocyte programmed cell death via GSDME - IL18 axis in human and mouse liver transplantation

Sirtuin 1 (SIRT1) is a histone/protein deacetylase in the cellular response to inflammatory, metabolic, and oxidative stressors. We previously reported that myeloid SIRT1 regulates the inflamed liver’s canonical pyroptosis cell death pathway. However, whether/how hepatocyte SIRT1 is engaged in programmed cell death in the cold-stressed liver remains uncertain. Here, we undertook translational studies in human and mouse orthotopic liver transplantation (OLT) to interrogate the significance of hepatocyte-specific SIRT1 in cold-stored donor livers and liver grafts after reperfusion. In the clinical arm of sixty human OLT patients, hepatic SIRT1 levels in cold-preserved donor livers correlated with the anti-apoptotic Bcl-2 expression. After reperfusion, improved OLT function was accompanied by hepatic SIRT1 levels negatively associated with cleaved caspase-3 expression. In the experimental arm, we compared FLOX-control with hepatocyte-specific SIRT1-KO livers after orthotopic transplantation into WT mouse recipients, parallel with primary murine hepatocyte cultures subjected to cold activation with/without knockdown of SIRT1, GSDME, and IL18Rβ. Indeed, hepatocyte SIRT1 deficiency upregulated apoptosis and GSDME-mediated programmed cell death, deteriorating hepatocellular function and shortening OLT survival. Augmented GSDME processing, accompanied by increased secretion of IL18 by stressed hepatocytes, was prominent in SIRT1-deficient, cold-stored livers. Hepatocyte SIRT1 expression regulated anti-apoptotic Bcl-2/XIAP proteins, suppressed cold stress-triggered apoptosis, and mitigated GSDME licensing to release IL18. Notably, consistent with the ability of IL18 to depress hepatocyte SIRT1 and Bcl-2/XIAP in vitro, IL18 neutralization in vivo prevented hepatocellular damage and restored the anti-apoptotic phenotype in otherwise injury-prone SIRT1-deficient OLTs. In conclusion, this translational study identifies a novel hepatocyte SIRT1-IL18 molecular circuit as a therapeutic target in the mechanism underpinning hepatocyte death pathways in human and mouse liver transplantation.

Targeting Cellular Senescence in Organ Transplantation.

Targeting Cellular Senescence in Organ Transplantation.

CD4+ T Cell NRF2 Signaling Improves Liver Transplantation Outcomes by Modulating T Cell Activation and Differentiation.

Aims: Innate and adaptive immune responses regulate hepatic ischemia-reperfusion injury (IRI) in orthotopic liver transplantation (OLT). While the mechanism of how nuclear factor erythroid 2-related factor 2 (NRF2) plays a role in liver IRI has been studied, the contribution of T cell-specific NRF2 in OLT remains unknown. In the current translational study, we investigated whether and how CD4+ T cell-specific NRF2 signaling affects liver transplant outcomes in mice and humans. Results: In the experimental arm, cold-stored (4°C/18 h) wild-type (WT) mouse livers transplanted to NRF2-deficient (NRF2-knockout [NRF2-KO]) recipients experienced greater hepatocellular damage than those in Nrf2-proficient (WT) counterparts, evidenced by Suzuki's histological scores, frequency of TdT-mediated dUTP nick end labeling (TUNEL)+ cells, and elevated serum aspartate aminotransferase/alanine aminotransferase (AST/ALT) levels. In vitro studies showed that NRF2 signaling suppressed CD4+ T cell differentiation to a proinflammatory phenotype (Th1, Th17) while promoting the regulatory (Foxp3+) T cell lineage. Furthermore, OLT injury deteriorated in immune-compromised RAG2-KO test recipients repopulated with CD4+ T cells from NRF2-KO compared with WT donor mice. In the clinical arm of 45 human liver transplant patients, the perioperative increase of NRF2 expression in donor livers negatively regulated innate and adaptive immune activation, resulting in reduced hepatocellular injury in NRF2-proficient OLT. Innovation and Conclusion: CD4+ T cell population expressing NRF2 attenuated ischemia and reperfusion (IR)-triggered hepatocellular damage in a clinically relevant mouse model of extended donor liver cold storage, followed by OLT, whereas the perioperative increase of NRF2 expression reduced hepatic injury in human liver transplant recipients. Thus, CD4+ T cell NRF2 may be a novel cytoprotective sentinel against IR stress in OLT recipients. Antioxid. Redox Signal. 38, 670-683.

313.9: Perfusate IL-6 Levels During Liver NMP Might Be Predictive For Hemodynamic Response and Catecholamine Demand After Reperfusion in the Recipient

Introduction: Normothermic liver preservation (NMP) has become a clinical routine at several transplant centres. Reperfusion-syndrome occurs less often in recipients of NMP-livers compared to cold stored livers. We hypothesized that perfusate interleukin (IL)-6 during liver NMP correlate with recipient hemodynamics in the post-reperfusion period. Method: Consecutive NMP-liver transplants at a single-centre were prospectively analysed. Perfusate samples were collected at 1 and 6 hours of NMP and at the end of perfusion and analysed for IL-6 levels. Median arterial pressure (MAP) and catecholamine need during surgery were recorded. The anhepatic phase was defined as baseline for MAP and catecholamine requirements. Results: Over a period of 36 months, IL-6 perfusate measurements were assessed in 77 livers undergoing NMP and transplantation; 15/77 (19.5%) were DCD organs. The median donor age was 61 (15-87) years, median recipient age was 60 (19-73) years. Median (IQR) cold ischemia time was 6.2 (2.1) hrs, NMP-time and overall preservation time were 17.6 (10.4) hrs and 23.6 (10.6) hrs. Median (IQR) IL-6 levels (ng/L) after 1, 6 hrs and NMP-end were 52 (175), 278 (674) and 174 (2171). Neither duration of CIT nor NMP correlated with IL-6 levels over time. NMP-livers were stratified for the median of the last IL-6 measurement. Recipients receiving NMP-livers with perfusate IL-6 levels above the median developed significantly lower post-reperfusion MAP (dropping 20% from baseline) and displayed a significant higher demand of catecholamines (increase of 25% from baseline) up to 30 minutes after reperfusion. Perfusate IL-6 did not correlate with the occurrence of early allograft dysfunction.Conclusion: Perfusate IL-6 levels during liver NMP are clinically relevant as they help to predict the post-reperfusion hemodynamics in recipients.

Quantitative Metabolomics of Tissue, Perfusate, and Bile from Rat Livers Subjected to Normothermic Machine Perfusion.

Machine perfusion (MP) allows the maintenance of liver cells in a metabolically active state ex vivo and can potentially revert metabolic perturbations caused by donor warm ischemia, procurement, and static cold storage (SCS). The present preclinical research investigated the metabolic outcome of the MP procedure by analyzing rat liver tissue, bile, and perfusate samples by means of high-field (600 MHz) nuclear magnetic resonance (NMR) spectroscopy. An established rat model of normothermic MP (NMP) was used. Experiments were carried out with the addition of an oxygen carrier (OxC) to the perfusion fluid (OxC-NMP, n = 5) or without (h-NMP, n = 5). Bile and perfusate samples were collected throughout the procedure, while biopsies were only taken at the end of NMP. Two additional groups were: (1) Native, in which tissue or bile specimens were collected from rats in resting conditions; and (2) SCS, in which biopsies were taken from cold-stored livers. Generally, NMP groups showed a distinctive metabolomic signature in all the analyzed biological matrices. In particular, many of the differentially expressed metabolites were involved in mitochondrial biochemical pathways. Succinate, acetate, 3-hydroxybutyrate, creatine, and O-phosphocholine were deeply modulated in ex vivo perfused livers compared to both the Native and SCS groups. These novel results demonstrate a broad modulation of mitochondrial metabolism during NMP that exceeds energy production and redox balance maintenance.

Transcriptomic profiles of human livers undergoing rewarming machine perfusion before transplantation\u2014first insights

Machine perfusion by controlled oxygenated rewarming (COR) is feasible and safe in clinical application and result in a promising outcome. This study utilizes next-generation sequencing (NGS) to investigate the transcriptome of human liver tissue undergoing COR before liver transplantation. Cold-stored livers were subjected to machine-assisted slow COR for ~120 min before transplantation. Biopsies were taken before (preCOR) and after COR (postCOR) and 1 h after reperfusion (postRep). The samples were sequenced, using RNA-seq to analyze differential transcriptional changes between the different stages and treatments of the grafts. Comparison of differential gene expression preCOR and postCOR demonstrated 10 upregulated genes. postRep 97 and 178 genes were upregulated and 7 and 13 downregulated compared to preCOR and postCOR, respectively. A shift of gene expressions by machine perfusion to the TGF-beta pathway was observed. The present study demonstrates distinct transcriptome profiles associated with machine perfusion by COR and transplantation of human livers. Such data provide a deeper understanding of the molecular mechanisms of machine perfusion technology in human liver transplantation.

Long-term Outcomes After Controlled Oxygenated Rewarming of Human Livers Before Transplantation.

Background.Controlled oxygenated rewarming (COR) has been shown to be a feasible and safe method in clinical practice and to reduce peak serum transaminases after liver transplantation. This study aimed to demonstrate further clinical experience of this method of now 18 clinical liver transplantations utilizing COR and demonstrate the long-term results.Methods.In this extended series of 18 patients, cold-stored livers were subjected to machine-assisted slow COR for ≈120 minutes before transplantation. A cohort of 178 patients transplanted during the same period with similar clinical characteristics were used for comparison of key outcomes.Results.All livers were perfused in accordance to the COR protocol without incidences and transplanted successfully. Early allograft dysfunction was observed in 2 (11.1%) cases after COR. Liver elasticity measurements indicated normal healthy liver parenchyma at the last follow-up. Graft survival demonstrated excellent outcomes after COR. The 1-, 3-, and 5-year patient survival rates were 100%, 100%, and 93.8% compared with 84.5%, 82.0%, and 75.8% in the control group (P = 0.12).Conclusions.The present study demonstrates excellent clinical outcomes after COR before liver transplantation. Comparison with a control cohort shows superiority of graft survival. Further evidence is needed to assess this promising method to improve organ preservation, finally.

Antibiotic pretreatment alleviates liver transplant damage in mice and humans.

Although modifications of gut microbiota with antibiotics (Abx) influence mouse skin and cardiac allografts, its role in orthotopic liver transplantation (OLT) remains unknown. We aimed to determine whether and how recipient Abx pretreatment may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. Mice (C57BL/6) with or without Abx treatment (10 days) were transplanted with allogeneic (BALB/c) cold-stored (18 hours) livers, followed by liver and blood sampling (6 hours). We divided 264 human OLT recipients on the basis of duration of pre-OLT Abx treatment into control (Abx-free/Abx <10 days; n = 108) and Abx treatment (Abx ≥10days; n = 156) groups; OLT biopsy (Bx) samples were collected 2 hours after OLT (n = 52). Abx in mice mitigated IRI-stressed OLT (IRI-OLT), decreased CCAAT/enhancer-binding protein homologous protein (CHOP) (endoplasmic reticulum [ER] stress), enhanced LC3B (autophagy), and inhibited inflammation, whereas it increased serum prostaglandin E2 (PGE2) and hepatic PGE2 receptor 4 (EP4) expression. PGE2 increased EP4, suppressed CHOP, and induced autophagosome formation in hepatocyte cultures in an EP4-dependent manner. An EP4 antagonist restored CHOP, suppressed LC3B, and recreated IRI-OLT. Remarkably, human recipients of Abx treatment plus OLT (Abx-OLT), despite severe pretransplantation clinical acuity, had higher EP4 and LC3B levels but lower CHOP levels, which coincided with improved hepatocellular function (serum aspartate aminotransferase/serum aspartate aminotransferase [sALT/sAST]) and a decreased incidence of early allograft dysfunction (EAD). Multivariate analysis identified "Abx-free/Abx <10 days" as a predictive factor of EAD. This study documents the benefits of Abx pretreatment in liver transplant recipients, identifies ER stress and autophagy regulation by the PGE2/EP4 axis as a homeostatic underpinning, and points to the microbiome as a therapeutic target in OLT.

Efficacy and Quality of Flush-Out Prior to Cold Storage of Liver and Kidney in Donation after Circulatory Death

Background and Aims There is a general perception that viscous solutions reduce the rate at which blood is washed out of organs during the cold flush during procurement, inhibit efficient cool-down, and prohibit optimal cortical perfusion of donor organs. Actual data on this topic are scarce but opinions are strong. To study perfusion characteristics we compared four hypothermic preservation solutions for abdominal organs i.e. UW SCS, HTK, IGL-1 and UW-MPS in a large animal model simulating donation after circulatory death (DCD). Materials and Methods Twenty 70kg female pigs were terminated [UW (6) and HTK (6), IGL-1 (4), UW-MPS (4)] followed by aortic cold flush-out and addition of slush-ice after 40min warm ischaemia. Companies’ instructions for volumes were used. During wash-out at pre-defined time points perfusate samples were obtained for further analysis and to determine viscosity. Organ temperature was measured continuously and cortical perfusion of kidney and liver was recorded using contrast-enhanced ultrasound. Biopsies were taken at start and end for histology and EM, including assessment of wash-out of blood. Results All solutions decreased the temperature of liver and kidney, although no solution reached temperatures lower than 18°C and 15°C resp. No significant difference in end liver temperatures was observed between different solutions (p=0.63), however end temperatures of kidneys were significantly different when comparing UW to HTK (15.1°C vs 20.3°C) (p=0.04). Cortical perfusion of livers was equally good between solutions (p=0.28), while in kidneys UW and IGL-1 penetrated better compared to HTK (p=0.02 and 0.03, resp.). No significant differences in histology or EM were seen in kidneys at the beginning or the end of the procedure, reflecting adequate intravascular wash-out, irrespective of viscosity. Discussion This study contradicts a popular perception and provides evidence that increased viscosity of a preservation solution does not negatively affect cooling and quality of organ perfusion. In fact, we found that UW SCS may be better at flushing out blood and cooling DCD kidneys. This study also provides interesting physiological data about the interaction between cold flush-out solutions and kidney and liver tissue at time of retrieval and start of preservation. Conclusion A higher viscosity in a preservation solution does not negatively affect cooling during flush-out at time of retrieval and does not have any detrimental effect on the quality of organ perfusion and preservation.

Controlled oxygenated rewarming up to normothermia for pretransplant reconditioning of liver grafts.

Controlled oxygenated rewarming (COR) up to 20°C during ex vivo machine perfusion limits reperfusion-induced tissue injury upon graft implantation. Rewarming up to normothermia might add further benefits and provide better prediction of post-transplantation organ function. The effect of 90minutes of oxygenated machine perfusion with Aqix RS-I after cold storage combined with gentle rewarming up to 20°C (COR20) or 35°C (COR35) was studied in rat livers and compared with cold storage alone (CS, n=6, resp). Postpreservation recovery was evaluated upon warm reperfusion using an established in vitro system. COR generally resulted in significantly improved energetic recovery, increased bile flow, less activities alanine aminotransferase (ALT) release, and improved histopathology upon reperfusion as compared to only cold-stored livers, without significant differences between COR20 and COR35. Parameters obtained during COR, especially during COR35, also allowed for prediction of hepatic recovery upon reperfusion. For instance, ulterior bile production upon reperfusion was found closely correlated to bile flow observed already during COR35 (R2 =0.91). COR significantly improved liver quality after static cold storage. Elevation of machine perfusion temperature up to 35°C may prove promising to refine ex vivo evaluation of the graft prior to transplantation.

Establishment of vein bypass assisted porcine liver auto transplantation model

Objective To establish the vein bypass assisted porcine liver auto transplantation model and study the methods of liver cold storage during the operation, in order to provide experience for the clinical application of liver auto transplantation and experimental evidence for liver machine perfusion. Methods A total of 5 pigs were operated to establish the portal-vein bypass by inserting cannula into vena jugularis interna, femoral vein and splenic vein, with liver being hypothermic perfused and stored in situ by 4℃ UW solution through hepatic artery and portal vein. Vital signs and clinical outcomes were monitored during the operation, and the changes of liver function indexes such as aminotransferase, total bilirubin and albumin were detected at different time points. Results During the operation, the vital signs kept steady and liver were perfused well, achieving fully blood supply after reperfusion. All animals survived over 120 h. Both alanine aminotransferase and aspartate transaminase peaked at 24 h after blood reperfusion (69.8±16.2 U/L and 457.7±50.1 U/L), and then dropped within normal range at 96 ho after reperfusion. Total bilirubin peaked at 48 h after blood reperfusion (7.1±3.3 μmol/L), and then decreased rapidly. The plasma albumin kept falling after blood reperfusion, reaching the minimum of 11.1±2.7 g/L at 4 h after reperfusion, and then rose to normal level steadily at 16 h. Conclusions The vein bypass assisted liver auto transplantation could guarantee the steady vital signs and survival rate, which may supply an experimental basis for developing new efficient liver preservation and restoration methods. Key words: Liver auto transplantation; Bypass; Animal model

Controlled Oxygenated Rewarming of Cold Stored Livers Prior to Transplantation: First Clinical Application of a New Concept.

Abrupt temperature shift from hypothermia to normothermia incurred on reperfusion of organ grafts has been delineated as a genuine factor contributing to reperfusion injury and graft dysfunction after transplantation. In a first clinical series of 6 patients, cold-stored livers, all allocated by the rescue offer mechanism by Eurotransplant, were subjected to machine-assisted slow controlled oxygenated rewarming (COR) for 90 minutes before engrafting. A historical cohort of 106 patients basically similar in graft (all rescue offer organs) and recipient factors was used for comparison. The clinical benefit of COR was documented by a significant reduction by approximately 50% in peak serum transaminases after transplantation compared to untreated controls (AST 563.5 vs. 1204 U/L, P = 0.023). After 6 months graft survival was 100% in the COR group and 80.9% in the controls (P = 0.24). Respective patient survival was 100% and 84.7% (P = 0.28). Real-time assessment of glucose concentration in the perfusion solution correlated well with postoperative synthetic graft function (r = 0.78; P < 0.02). All treated recipients had normal liver function after a 6-month follow-up and are well and alive. This first clinical application suggests that controlled graft rewarming after cold storage is a feasible and safe method in clinical praxis and might become an adjunct in organ preservation.

The effect of a hydrogen sulfide releasing molecule (Na2S) on the cold storage of livers from cardiac dead donor rats. A study in an ex vivo model

Liver transplantation is currently the preferred treatment option for end-stage liver disease. Donation after cardiac death was a common practice in the early years of organ donation before brain death criteria were established. Those organs were subjected to variable periods of warm ischemia that might intensify cold ischemia/reperfusion injuries. In the present, shortage of brain dead donors has led to the reassessment of organ donation after cardiac death.Since many cytoprotective roles have been describe for H2S during ischemia/reperfusion on a variety of tissues, we hypothesized that graft exposure to this bioactive gas might improve preservation of non-heart beating donated organs.Therefore, to establish a rat model of donation post-cardiac arrest and using this approach to judge H2S delivery effects on graft hypothermic preservation, were the main objectives of this investigation.Cardiopulmonary arrest was induced in sedated rats by overload of potassium (K+). Livers were surgically removed and subsequently stored in HTK Solution (Histidine–tryptophan–ketoglutarate) at 0–4°C. After 24h of hypothermic preservation, livers were rewarmed in an ex vivo model. Three experimental groups were established as follows: I – Livers procured before cardiac death and cold stored 24h in HTK (BCD); II – Livers procured after cardiac death (45min) and cold stored 24h in HTK (ACD); III – Livers procured after cardiac death (45min) and cold stored 24h in HTK+10μM Sodium Sulfide (Na2S) (ACD-SS). Data suggest that after 45min of warm ischemia, viability parameters assessed during reperfusion in the ex vivo model were significantly impaired. Real time PCR revealed that after ex vivo reperfusion there is an increased expression of HO-1 and TNF-α and a modest drop in Bcl-2 mRNA, which could be interpreted as the cellular response to the hypoxic insult sustained during warm ischemia.On the other hand, warm ischemic livers exposed to H2S during cold storage, improved microcirculation, morphology and viability parameters during ex vivo reperfusion and showed significant modulation of HO-1 mRNA expression.In conclusion, HTK supplementation with Na2S arose as a potential treatment to recover non-heart beating harvested organs. Furthermore, an appropriate model of cardiac dead liver donors was successfully developed.

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.

Functional human liver preservation and recovery by means of subnormothermic machine perfusion.

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 °C.

Strategies for short-term storage of hepatocytes for repeated clinical infusions.

Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1-2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated infusions. However, during cold storage isolated hepatocytes undergo cell death. We investigated if tissue preservation and repeated isolations are better than storage of isolated hepatocytes when cold preserving human hepatocytes. Liver tissue obtained from liver surgery or organ donors was divided into two pieces. Hepatocytes were isolated by collagenase digestion. Hepatocytes were analyzed directly after isolation (fresh) or after storage for 48 h at 4°C in University of Wisconsin solution (UW cells). Liver tissue from the same donor was stored at 4°C in UW and hepatocytes were isolated after 48 h (UW tissue cells). Hepatocyte viability and function was evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism, CYP 1A1/2, 2C9, 3A7, and 3A4 activities, phase II conjugation, and apoptosis evaluation by TUNEL assay and caspase-3/7 activities. Hepatocytes stored in UW showed a significantly lower viability compared to fresh cells or hepatocytes isolated from tissue stored for 48 h (54% vs. 71% vs. 79%). Plating efficiency was significantly decreased for cells stored in UW (40%) compared to fresh and UW tissue cells (63% vs. 55%). No significant differences between UW cells and UW tissue cells could be shown for CYP activities or ammonia metabolism. Hepatocytes stored in UW showed a strong increase in TUNEL-positive cells, whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 h was unchanged. This observation was confirmed by increased caspase-3/7 activities in UW cells. Although preservation of isolated hepatocytes in UW maintains function, cold storage of liver tissue and repeated hepatocyte isolations is superior to cold storage of isolated hepatocytes in preserving hepatocyte viability and function.

The Effect of Preservation Solutions for Storage of Liver Allografts on Transplant Outcomes

The objective of this review was to systematically evaluate the evidence comparing preservation fluids for liver allografts on transplant outcomes. Adequate preservation of liver allografts for transplantation is essential for successful transplant outcomes. There are several preservation fluids available that have been specifically designed for the static cold storage of livers. These fluids differ in composition and cost. literature search was performed using MEDLINE, EMBASE, Cochrane Library, Transplant Library, and the International Clinical Trials Registry Platform. Only randomized controlled trials were included. Studies were assessed for methodological quality. Primary outcomes were the risk of early dysfunction, primary nonfunction, retransplantation, patient survival, and graft survival. Secondary outcomes were serum biochemical parameters in the first week and biliary complications. Summary effects were calculated as relative risk and relative log survival with 95% confidence intervals (95% CIs). Sixteen randomized controlled trials met the full inclusion criteria (1619 livers). There is good evidence that the University of Wisconsin and Celsior solutions are associated with the same rates of early dysfunction (relative risk = 1.08, 95% CI = 0.63-1.86, P = 0.77), primary nonfunction (relative risk = 0.73, 95% CI = 0.22-2.40, P = 0.60), patient survival (relative log survival = 0.86, 95% CI = 0.58-1.28, P = 0.46), and graft survival (relative log survival = 0.85, 95% CI = 0.59-1.23, P = 0.39). There was no good evidence of any difference in outcomes when comparing histidine-tryptophan-ketoglutarate with either of the University of Wisconsin or Celsior solution, although data were limited. Data from included studies suggest that preservation of deceased donor livers with the University of Wisconsin or Celsior solution results in equivalent outcomes.

Controlled Oxygenated Rewarming of Cold Stored Liver Grafts by Thermally Graduated Machine Perfusion Prior to Reperfusion

The quality of cold-stored livers declines with the extension of ischemic time, increasing the risk of primary dys or nonfunction. A new concept to rescue preserved marginal liver grafts by gentle oxygenated warming-up prior to blood reperfusion was investigated. Porcine livers were preserved by cold storage (CS) in modified HTK-solution for 18 h. Some grafts were subsequently subjected to 90 min of controlled oxygenated rewarming (COR) by machine perfusion with gradual increase of perfusate temperature up to 20°C or simple oxygenated machine perfusion in hypothermia (HMP) or subnormothermia (SNP). Graft viability was assessed thereafter by 4 h of normothermic blood reperfusion ex vivo. Endischemic tissue energetics were significantly improved by COR or SNP and to a notably lesser extent by HMP. COR significantly reduced cellular enzyme loss, gene expression and perfusate activities of TNF-alpha, radical mediated lipid peroxidation (LPO) and increase of portal vascular perfusion resistance upon reperfusion, while HMP or SNP were less protective. Only COR resulted in significantly more bile production than after CS. Histological injury score and caspase 3-activation were significantly lower after COR than after CS. Oxygenated rewarming prior to reperfusion seems to be a promising technique to improve subsequent organ recovery upon reperfusion of long preserved liver grafts.

Impact of venous systemic oxygen persufflation supplemented with nitric oxide gas on cold-stored, warm ischemia-damaged experimental liver grafts

The increasing shortage of donor organs has led to the increasing use of organs from non-heart-beating donors. We aimed to assess the impact of venous systemic oxygen persufflation (VSOP) supplemented with nitric oxide (NO) gas during the cold storage (CS) of warm ischemia (WI)-damaged experimental liver grafts. Rat livers (n = 5 per group) were retrieved after 30 minutes of WI induced by cardiac arrest (the WI group) and were thereafter preserved for 24 hours by CS in histidine tryptophan ketoglutarate solution. During CS, gaseous oxygen was insufflated via the caval vein with 40 ppm NO (the VSOP-NO group) or without NO (the VSOP group). Cold-stored livers without WI served as controls. Liver viability was assessed after the preservation period by normothermic isolated reperfusion for 45 minutes with oxygenated Krebs-Henseleit buffer. After 45 minutes of reperfusion, the VSOP-NO-treated livers showed significantly lower alanine aminotransferase values than the WI-damaged livers (10.2 ± 0.2 versus 78.2 ± 14.6 IU/L), whereas the control livers showed no differences from the VSOP-NO-treated livers. The mitochondrial enzyme release was lower in the VSOP-NO group (4.0 ± 0.7 IU/L) versus the WI group (18.2 ± 4.9 IU/L). An increased portal vein pressure was observed throughout reperfusion (45 minutes) in the WI group (21.7 ± 0.2 mm Hg) versus the VSOP-NO group (12.2 ± 0.8 mm Hg) and the control group (19.9 ± 0.4 mm Hg). Furthermore, the NO concentration in the perfusate after 5 minutes of reperfusion was highest in the VSOP-NO group. The release of malondialdehyde into the perfusate was significantly reduced in the VSOP-NO group (0.9 ± 0.1 nmol/mL) versus the WI group (31.3 ± 5.3 nmol/mL). In conclusion, the resuscitation of livers after 30 minutes of WI to a level comparable to that of nonischemically damaged livers is possible with VSOP supplemented with NO gas. Moreover, the application of VSOP with NO minimizes the extent of injuries caused by oxygen free radicals during preservation.