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Background: Liver grafts are frequently declined due to high donor age or age mismatch with the recipient. To improve the outcome of marginal grafts, we aimed to characterize the performance of elderly vs. young liver grafts in a standardized rat model of normothermic ex vivo liver machine perfusion (NMP)., Methods: Livers from Sprague-Dawley rats aged 3 or 12 months were procured and perfused for 6 h using a rat NMP system or collected as a reference group (n = 6/group). Tissue, bile, and perfusate samples were used for biochemical, and proteomic analyses., Results: All livers cleared lactate during perfusion and continued to produce bile after 6 h of perfusion (614 mg/h). Peak urea levels in 12-month-old animals were higher than in younger animals. Arterial and portal venous pressure, bile production and pH did not differ between groups. Proteomic analysis identified a total of 1477 proteins with oxidoreductase and catalytic activity dominating the gene ontology analysis. Proteins such as aldehyde dehydrogenase 1A1 and 2-Hydroxyacid oxidase 2 were significantly more present in livers of older age., Conclusions: Young and elderly liver grafts exhibited similar viability during NMP, though proteomic analyses indicated that older grafts are less resilient to oxidative stress. Our study is limited by the elderly animal age, which corresponds to mature but not elderly human age typically seen in marginal human livers. Nevertheless, reducing oxidative stress could be a promising therapeutic target in the future., (© 2024. The Author(s).)

Background: Considering the expected increase in the elderly population and the growing emphasis on aging-related biomedical research, the demand for aged laboratory animals has surged, challenging established husbandry practices. Our objective was to establish a cost-effective method for environmental enrichment, utilizing the liver as a representative organ to assess potential metabolic changes in response to differing enrichment levels., Methods: We conducted a six-month study involving 24 male Sprague Dawley rats, randomly assigned to four environmental enrichment groups. Two groups were housed in standard cages, while the others were placed in modified rabbit cages. Half of the groups received weekly playtime in an activity focused rat housing unit. We evaluated hormone levels, playtime behavior, and subjective handling experience. Additionally, liver tissue proteomic analysis was performed., Results: Initial corticosterone levels and those after 3 and 6 months showed no significant differences. Yet, testosterone levels were lower in the control group by the end of the study (p = 0.007). We observed 1871 distinct proteins in liver tissue, with 77% being common across groups. In gene ontology analysis, no specific pathways were overexpressed. In semiquantitative analysis, we observed differences in proteins associated in lipid metabolism such as Apolipoprotein A-I and Acyl-CoA 6-desaturase, which were lower in the control group (p = 0.024 and p = 0.009). Rats in the intervention groups with weekly playtime displayed the least amount of reported distress during inspection or upon room entry and were less prone to accepting treats. Removing animals from their enclosure was most effortless for those in the large cage group. Over time, there was a decrease in conflicts among rats that interacted only twice weekly during playpen time., Discussion: In summary, refining husbandry practices for aging rats is both simple and budget-friendly, with no apparent adverse effects on stress levels, animal development, or relevant metabolic changes in the liver., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Roschke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Tissue engineered bioscaffolds based on decellularized composites have gained increasing interest for treatment of various diaphragmatic impairments, including muscular atrophies and diaphragmatic hernias. Detergent-enzymatic treatment (DET) constitutes a standard strategy for diaphragmatic decellularization. However, there is scarce data on comparing DET protocols with different substances in distinct application models in their ability to maximize cellular removal while minimizing extracellular matrix (ECM) damage., Methods: We decellularized diaphragms of male Sprague Dawley rats with 1 % or 0.1 % sodium dodecyl sulfate (SDS) and 4 % sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. We evaluated decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semiquantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy., Results: All protocols produced decellularized matrices with micro- and ultramorphologically intact architecture and adequate biomechanical performance with gradual differences. The proteomic profile of decellularized matrices contained a broad range of primal core and ECM-associated proteins similar to native muscle. While no outstanding preference for one singular protocol was determinable, SDS-treated samples showed slightly beneficial properties in comparison to SDC-processed counterparts. Both application modalities proved suitable for DET., Conclusion: DET with SDS or SDC via orbital shaking or retrograde perfusion constitute suitable methods to produce adequately decellularized matrices with characteristically preserved proteomic composition. Exposing compositional and functional specifics of variously treated grafts may enable establishing an ideal processing strategy to sustain valuable tissue characteristics and optimize consecutive recellularization. This aims to design an optimal bioscaffold for future transplantation in quantitative and qualitative diaphragmatic defects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Normothermic ex vivo liver machine perfusion (NEVLP) has been developed to address the increasing organ shortage in liver transplantation, through optimal preservation, assessment, and conditioning of grafts from extended criteria donors. There remains a need to establish simple and standardized animal models that simulate clinical NEVLP to test novel therapies. Liver grafts from 36 Sprague-Dawley rats were perfused for 6 h in a dual-vessel NEVLP system with a Dulbecco's modified Eagles medium-based perfusate supplemented with rat plasma and erythrocytes. Varying doses of the clinically used vasodilator epoprostenol, Kupffer cell inhibitor glycine, and a Steen™-based perfusate were assessed. Perfusion pressures and bile production were recorded, and perfusate was analyzed for transaminase secretion. Tissue samples were evaluated histologically, and levels of cytokines and 8-Isoprostane were measured. Increasing levels of epoprostenol and the addition of glycine resulted in a stepwise decrease of transaminase secretion and improved bile production. Steen further decreased transaminase release and interleukin 1 beta levels. Liver grafts perfused with the optimized Steen-based protocol exhibited lowest levels of oxidative stress and best-preserved liver integrity. In conclusion, epoprostenol seemed to ameliorate liver function and prevent cellular damage beyond its vasodilatory effect, with glycine acting synergistically. The anti-inflammatory and antioxidative properties of Steen further improved the outcome of perfusion. Our rodent NEVLP system may be used to rapidly test new agents for the pharmacologic conditioning of livers and help translate findings from bench-to-bedside.

Primary human hepatocytes isolated from surgically resected liver tissue are an essential resource for pharmaceutical and toxicological studies. Patients undergoing partial liver resections have often received preoperative chemotherapy. The aim of our study was to investigate whether preoperative chemotherapy has effects on the outcome of cell isolation or the metabolic function of cultured hepatocytes. Liver specimens from 48 patients were used for hepatocyte isolation. Out of these, 21 patients had prior chemotherapy, with fluoropyrimidine-based regimen in 14 patients. Viability and cell yield as parameter for the outcome of isolation, as well as transaminase levels, urea or albumin secretion to the culture medium were not different between hepatocytes from pretreated and untreated donor. Furthermore, the transcription levels of cytochrome P450 (CYP) 1A2, CYP 2B6, and CYP 3A4 of cultured hepatocytes were not affected by prior chemotherapy of the tissue donors. In conclusion, hepatocytes from tissue donors that underwent fluoropyrimidine-based chemotherapy regimens before isolation seem to perform as well as hepatocytes without preoperative chemotherapy exposure. Our results suggest that hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies. Impact statement Isolated primary human hepatocytes are an essential resource for pharmacological and toxicological studies. Our results present further evidence that isolated hepatocytes from patients who received combination chemotherapy before liver resection are an uncompromised resource for pharmacological and toxicological studies-especially when fluoropyrimidine-based regimens are used.

The most common preservation technique for liver grafts is static cold storage. Due to the organ shortage for liver transplantation (LT), extended criteria donor (ECD) allografts are increasingly used-despite the higher risk of inferior outcome after transplantation. Ex vivo liver machine perfusion (MP) has been developed to improve the outcome of transplantation, especially with ECD grafts, and is currently under evaluation in clinical trials. We performed a literature search on PubMed and ISI Web of Science to assemble an overview of rodent and porcine animal models of ex vivo liver MP for transplantation, which is essential for the present and future development of clinical liver MP. Hypothermic, subnormothermic, and normothermic MP systems have been successfully used for rat and pig LT. In comparison with hypothermic systems, normothermic perfusion often incorporates a dialysis unit. Moreover, it enables metabolic assessment of liver grafts. Allografts experiencing warm ischemic time have a superior survival rate after MP compared with cold storage alone, irrespective of the temperature used for perfusion. Furthermore, ex vivo MP improves the outcome of regular and ECD liver grafts in animal models. Small and large animal models of ex vivo liver MP are available to foster the further development of this new technology. Impact Statement Ex vivo machine perfusion is an important part of current research in the field of liver transplantation. While evidence for improve storage is constantly rising, the development of future applications such as quality assessment and therapeutic interventions necessitates robust animal models. This review is intended to provide an overview of this technology in common large and small animal models and to give an outlook on future applications. Moreover, we describe developmental steps that can be followed by others, and which can help to decrease the number of animals used for experiments based on the replace, reduce, refine concept.

A growing number of clinical risk scores have been proposed to predict allograft failure after liver transplantation. However, validation of currently available scores in the Eurotransplant region is still lacking. We aimed to analyze all clinically relevant donor and recipient risk scores on a large German liver transplantation data set and performed a retrospective cohort analysis of liver transplantations performed at the Charité-Universitätsmedizin Berlin from January 2007 until December 2021 with organs from donation after brain death. We analyzed 9 previously published scores in 906 liver transplantations [Eurotransplant donor risk index (ET-DRI/DRI), donor age and model for end-stage liver disease (D-MELD), balance of risk (BAR), early allograft dysfunction (EAD), model for early allograft function (MEAF), liver graft assessment following transplantation (L-GrAFT7), early allograft failure simplified estimation (EASE), and a score by Rhu and colleagues). The EASE score had the best predictive value for 3-month, 6-month, and 12-month graft survival with a c-statistic of 0.8, 0.77, and 0.78, respectively. In subgroup analyses, the EASE score was suited best for male recipients with a high-MELD (>25) and an EAD organ. Scores only based on pretransplant data performed worse compared to scores including postoperative data (eg, ET-DRI vs. EAD, p<0.001 at 3-month graft survival). Out of these, the BAR score performed best with a c-statistic of 0.6. This a comprehensive comparison of the clinical utility of risk scores after liver transplantation. The EASE score sufficiently predicted 12-month graft and patient survival. Despite a relatively complex calculation, the EASE score provides significant prognostic value for patients and health care professionals in the Eurotransplant region., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Background: Liver transplantation is the only curative treatment option for end-stage liver disease; however, its use remains limited due to a shortage of suitable organs. In recent years, ex vivo liver machine perfusion has been introduced to liver transplantation, as a means to expand the donor organ pool., Purpose: To present a systematic review of prospective clinical studies on ex vivo liver machine perfusion, in order to assess current applications and highlight future directions., Methods: A systematic literature search of both PubMed and ISI web of science databases as well as the ClinicalTrials.gov registry was performed., Results: Twenty-one articles on prospective clinical trials on ex vivo liver machine perfusion were identified. Out of these, eight reported on hypothermic, eleven on normothermic, and two on sequential perfusion. These trials have demonstrated the safety and feasibility of ex vivo liver machine perfusion in both standard and expanded criteria donors. Currently, there are twelve studies enrolled in the clinicaltrials.gov  registry, and these focus on use of ex vivo perfusion in extended criteria donors and declined organs., Conclusion: Ex vivo liver machine perfusion seems to be a suitable strategy to expand the donor pool for liver transplantation and holds promise as a platform for reconditioning diseased organs.

Concepts to ameliorate the continued mismatch between demand for liver allografts and supply include the acceptance of allografts that meet extended donor criteria (ECD). ECD grafts are generally associated with an increased rate of complications such as early allograft dysfunction (EAD). The costs of liver transplantation for the health care system with respect to specific risk factors remain unclear and are subject to change. We analyzed 317 liver transplant recipients from 2013 to 2018 for outcome after liver transplantation and hospital costs in a German transplant center. In our study period, 1-year survival after transplantation was 80.1% (95% confidence interval: 75.8%-84.6%) and median hospital stay was 33 days (interquartile rage: 24), with mean hospital costs of €115,924 (SD €113,347). There was a positive correlation between costs and laboratory Model for End-Stage Liver Disease score (r s  = 0.48, P  < 0.001), and the development of EAD increased hospital costs by €26,229. ECD grafts were not associated with a higher risk of EAD in our cohort. When adjusting for recipient-associated risk factors such as laboratory Model for End-Stage Liver Disease score, recipient age, and split liver transplantation with propensity score matching, only EAD and cold ischemia increased total costs. Conclusion: Our data show that EAD leads to significantly higher hospital costs for liver transplantation, which are primarily attributed to recipient health status. Strategies to reduce the incidence of EAD are needed to control costs in liver transplantation., (© 2020 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of the American Association for the Study of Liver Diseases.)

Background:  The lack of suitable allografts limits the availability of liver transplantation in Germany. The quality of potentially available German donor livers has to date not been analyzed., Methods:  Analysis of all donors for potential liver transplantations reported to the Eurotransplant by the German Organ Transplantation Foundation from 2010 to 2018. Categorization of transplanted and discarded organs utilizing available histopathological reports and predefined extended criteria for organ donation., Results:  A total of 8594 livers were offered for transplantation, of which 15.2 % were discarded. During the analysis period the proportion of donor livers from extended criteria donors increased from 65 % to 70 % (p = 0.005). In 2018, 21.3 % of offered donor livers were discarded, more than half (56.4 %) of these organs came from donors meeting multiple extended criteria. Livers were significantly more likely to be not transplanted, when from donors of older age (> 65 years; 41 vs. 28 %), BMI > 30 kg/m 2 (29 vs. 14 %) or elevated transaminase levels (all p < 0,001)., Conclusion:  Despite the consistent organ scarcity in Germany, a relevant amount of livers cannot be transplanted due to a multitude of organ quality limitations. This should stimulate the search for concepts such as normothermic ex vivo machine perfusion to evaluate, protect and potentially improve organ quality., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Background: Normothermic ex vivo liver perfusion (NEVLP) is a promising strategy to increase the donor pool in liver transplantation. Small animal models are essential to further investigate questions regarding organ preservation and reconditioning by NEVLP. A dual vessel small animal NEVLP (dNEVLP) model was developed using metamizole as a vasodilator and compared to conventional portovenous single vessel NEVLP (sNEVLP)., Methods: Livers of male Wistar rats were perfused with erythrocyte-supplemented culture medium for six hours by either dNEVLP via hepatic artery and portal vein or portovenous sNEVLP. dNEVLP was performed either with or without metamizole treatment. Perfusion pressure and flow rates were constantly monitored. Transaminase levels were determined in the perfusate at the start and after three and six hours of perfusion. Bile secretion was monitored and bile LDH and GGT levels were measured hourly. Histopathological analysis was performed using liver and bile duct tissue samples after perfusion., Results: Hepatic artery pressure was significantly lower in dNEVLP with metamizole administration. Compared to sNEVLP, dNEVLP with metamizole treatment showed higher bile production, lower levels of transaminases during and after perfusion as well as significantly lower necrosis in liver and bile duct tissue. Biochemical markers of bile duct injury showed the same trend., Conclusion: Our miniaturized dNEVLP system enables normothermic dual vessel rat liver perfusion. The administration of metamizole effectively ameliorates arterial vasospasm allowing for six hours of dNEVLP, with superior outcome compared to sNEVLP., Competing Interests: The authors have declared that no competing interests exist.

Ex vivo liver machine perfusion (MP) is a promising alternative for preservation of liver grafts from extended criteria donors. Small animal models can be used to evaluate different perfusion conditions. We here describe the development of a miniaturized ex vivo MP system for rat liver grafts, evaluating cell-free and erythrocyte-based perfusion solutions, subnormothermic and normothermic temperatures, and dialysis. A perfusion chamber was designed after a suitable liver position was identified. Normothermic ex vivo liver perfusion (NEVLP) required supplementation of erythrocytes to reduce cell damage. Perfusion with erythrocytes led to rising potassium levels after 12 h (NEVLP, 16.2 mM, interquartile range [IQR]: 5.7 and subnormothermic ex vivo liver perfusion [SNEVLP], 12.8 mM, IQR: 3.5), which were normalized by dialysis using a laboratory dialysis membrane (NEVLP, 6.2 mM, IQR: 0.5 and SNEVLP, 5.3 mM, IQR: 0.1; p  = 0.004). Livers treated with NEVLP conditions showed higher bile production (18.52 mg/h/g, IQR: 8.2) compared to livers perfused under SNEVLP conditions (0.4 mg/h/g, IQR: 1.2, p  = 0.01). Reducing the perfusion volume from 100 to 50 mL allowed for higher erythrocyte concentrations, leading to significantly lower transaminases (15.75 U/L/mL, IQR: 2.29 vs. 5.97 U/L/mL, IQR: 18.07, p  = 0.002). In conclusion, a well-designed perfusion system, appropriate oxygen carriers, dialysis, and miniaturization of the perfusion volume are critical features for successful miniaturized ex vivo liver MP. Impact Statement Ex vivo liver machine perfusion (MP) is an emerging preservation alternative to static cold storage. Even though clinical studies have shown benefits for extended criteria donor grafts, standardized systems for perfusion of rat liver grafts are not available, which are inevitable for large-scaled studies on liver reconditioning by ex vivo MP. We here comprehensively describe the development of an ex vivo rat liver perfusion system that can be used as modular setting in various approaches of liver MP. We describe pitfalls and techniques that might be of interest when establishing such perfusion systems for basic and translational research.

Background: Liver transplantation is the only curative treatment option for patients with end-stage liver disease; however, the 40% decline of available organ donors in recent years in Germany necessitates the optimization of available resources and possibly extending the criteria to older donors., Material and Methods: All 2652 livers made available to the Charité Universitätsmedizin Berlin from 2010 to 2016 were retrospectively analyzed and the clinical outcome of 526 liver transplantations during this time frame were evaluated., Results: The median age of donors of transplanted organs increased from 49.3 years in 2010 to 57.3 years in 2016 (p = 0.02). Organs from donors ≥65 years were more frequently discarded than organs from younger donors (n = 344, 18.4% vs. n = 220, 28.1%; p = 0.005). Moreover, the older donors had higher rates of diabetes mellitus and hepatic steatosis. Organs from older donors had a higher donor risk index (2.8 vs. 2.2; p < 0.001) and were transplanted more often in patients with preserved liver function and hepatocellular carcinoma and liver cirrhosis (n = 121, 74.7% of indications). The 3‑year survival after liver transplantation from donors ≥65 and ≥80 years old was not significantly reduced in comparison to younger donors; however, there was an increased retransplantation rate (28.6%; p = 0.005) after transplantation of organs from donors ≥80 years old., Conclusion: Despite conservative organ acceptance there were higher rates of retransplantation after transplantation from very old donors. In the light of an increasing scarcity of suitable organs this mandates caution and highlights the need for adequate assessment instruments for marginal donor organs before transplantation.

Normothermic ex vivo liver machine perfusion might be a superior preservation strategy for liver grafts from extended criteria donors. However, standardized small animal models are not available for basic research on machine perfusion of liver grafts. A laboratory-scaled perfusion system was developed consisting of a custom-made perfusion chamber, a pressure-controlled roller pump, and an oxygenator. Male Wistar rat livers were perfused via the portal vein for 6 hours using oxygenated culture medium supplemented with rat erythrocytes. A separate circuit was connected via a dialysis membrane to the main circuit for plasma volume expansion. Glycine was added to the flush solution, the perfusate, and the perfusion circuit. Portal pressure and transaminase release were stable over the perfusion period. Dialysis significantly decreased the potassium concentration of the perfusate and led to significantly higher bile and total urea production. Hematoxylin-eosin staining and immunostaining for single-stranded DNA and activated caspase 3 showed less sinusoidal dilatation and tissue damage in livers treated with dialysis and glycine. Although Kupffer cells were preserved, tumor necrosis factor α messenger RNA levels were significantly decreased by both treatments. For proof of concept, the optimized perfusion protocol was tested with donation after circulatory death (DCD) grafts, resulting in significantly lower transaminase release into the perfusate and preserved liver architecture compared with baseline perfusion. In conclusion, our laboratory-scaled normothermic portovenous ex vivo liver perfusion system enables rat liver preservation for 6 hours. Both dialysis and glycine treatment were shown to be synergistic for preservation of the integrity of normal and DCD liver grafts., (Copyright © 2018 by the American Association for the Study of Liver Diseases.)

Due to the shortage of liver allografts and the rising prevalence of fatty liver disease in the general population, steatotic liver grafts are considered for transplantation. This condition is an important risk factor for the outcome after transplantation. We here analyze the characteristics of the donor pool offered to the Charité - Universitätsmedizin Berlin from 2010 to 2016 with respect to liver allograft nonacceptance and steatosis hepatis. Of the 2653 organs offered to our center, 19.9% (n=527) were accepted for transplantation, 58.8% (n=1561) were allocated to other centers, and 21.3% (n = 565) were eventually discarded from transplantation. In parallel to an increase of the incidence of steatosis hepatis in the donor pool from 20% in 2010 to 30% in 2016, the acceptance rates for steatotic organs increased in our center from 22.3% to 51.5% in 2016 (p < 0.001), with the majority (86.9%; p > 0.001) having less than 30% macrovesicular steatosis hepatis. However, by 2016, the number of canceled transplantations due to higher grades of steatosis hepatis had significantly increased from 14.7% (n = 15) to 63.6% (42; p < 0.001). The rising prevalence of steatosis hepatis in the donor pool has led to higher acceptance rates of steatotic allografts. Nonetheless, steatosis hepatis remains a predominant phenomenon in discarded organs necessitating future concepts such as organ reconditioning to increase graft utilization.