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Transplantation remains the preferred treatment for end‐stage kidney disease but is critically limited by the number of available organs. Xenografts from genetically modified pigs have become a promising solution to the loss of life while waiting for transplantation. However, the current clinical model for xenotransplantation will require off‐site procurement, leading to a period of ischemia during transportation. As of today, there is limited understanding regarding the preservation of these organs, including the duration of viability, and the associated molecular changes. Thus, our aim was to evaluate the effects of static cold storage (SCS) on α1,3‐galactosyltransferase knockout (GGTA1 KO) kidney. After SCS, viability was further assessed using acellular sub‐normothermic ex vivo perfusion and simulated transplantation with human blood. Compared to baseline, tubular and glomerular interstitium was preserved after 2 days of SCS in both WT and GGTA1 KO kidneys. Bulk RNA‐sequencing demonstrated that only eight genes were differentially expressed after SCS in GGTA1 KO kidneys. During sub‐normothermic perfusion, kidney function, reflected by oxygen consumption, urine output, and lactate production was adequate in GGTA1 KO grafts. During a simulated transplant with human blood, macroscopic and histological assessment revealed minimal kidney injury. However, GGTA1 KO kidneys exhibited higher arterial resistance, increased lactate production, and reduced oxygen consumption during the simulated transplant. In summary, our study suggests that SCS is feasible for the preservation of porcine GGTA1 KO kidneys. However, alternative preservation methods should be evaluated for extended preservation of porcine grafts. [ABSTRACT FROM AUTHOR]

Access to liver transplantation is limited by a significant organ shortage. The recent introduction of machine perfusion technology allows surgeons to monitor and assess ex situ liver function prior to transplantation. However, many donated organs are of inadequate quality for transplant, though opportunities exist to rehabilitate organ function with adjunct therapeutics during normothermic machine perfusion. In this preclinical study, we targeted the apoptosis pathway as a potential method of improving hepatocellular function. Treatment of discarded human livers during normothermic perfusion with an irreversible pan-caspase inhibitor, emricasan, resulted in significant mitigation of innate immune and pro-inflammatory responses at both the transcriptional and protein level. This was evidenced by significantly decreased circulating levels of the pro-inflammatory cytokines, interleukin-6, interleukin-8, and interferon-gamma, compared to control livers. Compared to emricasan-treated livers, untreated livers demonstrated transcriptional changes notable for enrichment in pathways involved in innate immunity, leukocyte migration, and cytokine-mediated signaling. Targeting of unregulated apoptosis may represent a viable therapeutic intervention for immunomodulation during machine perfusion. [ABSTRACT FROM AUTHOR]

Background: Over 700 donor livers are discarded annually in the United States due to high risk of poor graft function. The objective of this study was to determine the impact of using normothermic machine perfusion to identify transplantable livers among those currently discarded. Study Design: A series of 21 discarded human livers underwent viability assessment during normothermic machine perfusion. Cross‐sectional analysis of the Scientific Registry of Transplant Recipients database and cost analysis was performed to extrapolate the case series to national experience. Results: 21 discarded human livers were included in the perfusion cohort. 11 of 20 (55%) eligible grafts met viability criteria for transplantation. Grafts in the perfusion cohort had a similar donor risk index compared with discarded grafts (n = 1402) outside of New England in 2017 and 2018 (median [IQR]: 2.0 [1.5, 2.4] vs. 2.0 [1.7, 2.3], P =.40). 705 (IQR 677‐741) livers were discarded annually in the United States since 2005, translating to the potential for 398 additional transplants nationally. The median cost to identify a transplantable graft with machine perfusion was $28,099 USD. Conclusions: Normothermic machine perfusion of discarded livers could identify a significant number of transplantable grafts, significantly improving access to liver transplantation. [ABSTRACT FROM AUTHOR]

Ex-vivo liver perfusion (EVLP) is an ideal platform to study liver disease, therapeutic interventions, and pharmacokinetic properties of drugs without any patient risk. Rat livers are an ideal model for EVLP due to less organ quality variability, ease of hepatectomy, well-defined molecular pathways, and relatively low costs compared to large animal or human perfusions. However, the major limitation with rat liver normothermic machine perfusion (NMP) is maintaining physiologic liver function on an ex-vivo machine perfusion system. To address this need, our research demonstrates 24-hour EVLP in rats under normothermic conditions. Early (6 hour) perfusate transaminase levels and oxygen consumption of the liver graft are shown to be good markers of perfusion success and correlate with viable 24-hour post-perfusion histology. Finally, we address overcoming challenges in long-term rat liver perfusions such as rising intrahepatic pressures and contamination, and offer future directions necessary to build upon our work. [ABSTRACT FROM AUTHOR]

Machine perfusion-based organ preservation techniques are prudently transitioning into clinical practice. Although experimental data is compelling, the outcomes in the highly variable clinical donation-transplantation setting are unpredictable. Here, we offer an intermediate tool for pre-clinical assessment of human donor livers. We present a model for ex situ reperfusion of discarded human livers and report on its application in three human livers that have undergone subnormothermic (21°C) machine perfusion as an experimental preservation method. During reperfusion, the livers macroscopically reperfused in the first 15 minutes, and remained visually well-perfused for 3 hours of ex situ reperfusion. Bile production and oxygen consumption were observed throughout ex situ reperfusion. ATP levels increased 4.25-fold during SNMP. Between the end of SNMP and the end of reperfusion ATP levels dropped 45%. ALT levels in blood increased rapidly in the first 30 minutes and ALT release continued to taper off towards the end of perfusion. Release of CRP, TNF-, IL-1, and IL-12, IFN- was sustained during reperfusion. These findings support the use of this model for the evaluation of novel human liver preservation techniques. [ABSTRACT FROM AUTHOR]

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.

Access to liver transplantation continues to be hindered by the severe organ shortage. Extended-criteria donor livers could be used to expand the donor pool but are prone to ischemia-reperfusion injury (IRI) and post-transplant graft dysfunction. Ex situ machine perfusion may be used as a platform to rehabilitate discarded or extended-criteria livers prior to transplantation, though there is a lack of data guiding the utilization of different perfusion modalities and therapeutics. Since amino acid derivatives involved in inflammatory and antioxidant pathways are critical in IRI, we analyzed differences in amino acid metabolism in seven discarded non-steatotic human livers during normothermic- (NMP) and subnormothermic-machine perfusion (SNMP) using data from untargeted metabolomic profiling. We found notable differences in tryptophan, histamine, and glutathione metabolism. Greater tryptophan metabolism via the kynurenine pathway during NMP was indicated by significantly higher kynurenine and kynurenate tissue concentrations compared to pre-perfusion levels. Livers undergoing SNMP demonstrated impaired glutathione synthesis indicated by depletion of reduced and oxidized glutathione tissue concentrations. Notably, ATP and energy charge ratios were greater in livers during SNMP compared to NMP. Given these findings, several targeted therapeutic interventions are proposed to mitigate IRI during liver machine perfusion and optimize marginal liver grafts during SNMP and NMP. [ABSTRACT FROM AUTHOR]

Ex situ machine perfusion is a promising technology to help improve organ viability prior to transplantation. However, preclinical studies using discarded human livers to evaluate therapeutic interventions and optimize perfusion conditions are limited by significant graft heterogeneity. In order to improve the efficacy and reproducibility of future studies, a split-liver perfusion model was developed to allow simultaneous perfusion of left and right lobes, allowing one lobe to serve as a control for the other. Eleven discarded livers were surgically split, and both lobes perfused simultaneously on separate perfusion devices for 3 h at subnormothermic temperatures. Lobar perfusion parameters were also compared with whole livers undergoing perfusion. Similar to whole-liver perfusions, each lobe in the split-liver model exhibited a progressive decrease in arterial resistance and lactate levels throughout perfusion, which were not significantly different between right and left lobes. Split liver lobes also demonstrated comparable energy charge ratios. Ex situ split-liver perfusion is a novel experimental model that allows each graft to act as its own control. This model is particularly well suited for preclinical studies by avoiding the need for large numbers of enrolled livers necessary due to the heterogenous nature of discarded human liver research. [ABSTRACT FROM AUTHOR]

There continues to be significant debate regarding the most effective mode of ex situ machine perfusion of livers for transplantation. Subnormothermic (SNMP) and normothermic machine perfusion (NMP) are two methods with different benefits. We examined the metabolomic profiles of discarded steatotic human livers during three hours of subnormothermic or normothermic machine perfusion. Steatotic livers regenerate higher stores of ATP during SNMP than NMP. However, there is a significant depletion of available glutathione during SNMP, likely due to an inability to overcome the high energy threshold needed to synthesize glutathione. This highlights the increased oxidative stress apparent in steatotic livers. Rescue of discarded steatotic livers with machine perfusion may require the optimization of redox status through repletion or supplementation of reducing agents. [ABSTRACT FROM AUTHOR]

Multiple perfusion systems have been investigated on vascularized composite allografts, with various temperatures and different preservation solutions, most using continuous flow (CF). However, physiological flow is pulsatile and provides better outcomes in kidney and lung ex vivo perfusions. The objective of this pilot study is to compare pulsatile flow (PF) with CF in our 24-h subnormothermic machine perfusion protocol for swine hindlimbs. Partial hindlimbs were harvested from Yorkshire pigs and perfused with a modified Steen solution at 21°C for 24 h either with CF (n = 3) or with pulsatile flow (PF) at 60 beats/min (n = 3). Perfusion parameters, endothelial markers, and muscle biopsies were assessed at different timepoints. Overall, lactate levels were significantly lower in the PF group (P = 0.001). Glucose uptake and potassium concentration were similar in both groups throughout perfusion. Total nitric oxide levels were significantly higher in the PF group throughout perfusion (P = 0.032). Nitric oxide/endothelin-1 ratio also tends to be higher in the PF group, reflecting a potentially better vasoconductivity with PF, although not reaching statistical significance (P = 0.095). Arterial resistances were higher in the PF group (P < 0.001). Histological assessment did not show significant difference in muscular injury between the two groups. Weight increased quicker in the CF group but reached similar values with the PF after 24 h. This pilot study suggests that PF may provide superior preservation of vascularized composite allografts when perfused for 24 h at subnormothermic temperatures, with potential improvement in endothelial function and decreased ischemic injury. [ABSTRACT FROM AUTHOR]