Mouse Liver Transplantation Research Articles

Supervised machine learning of outbred mouse genotypes to predict hepatic immunological tolerance of individuals

It is essential to elucidate the molecular mechanisms underlying liver transplant tolerance and rejection. In cases of mouse liver transplantation between inbred strains, immunological rejection of the allograft is reduced with spontaneous apoptosis without immunosuppressive drugs, which differs from the actual clinical result. This may be because inbred strains are genetically homogeneous and less heterogeneous than others. We exploited outbred CD1 mice, which show highly heterogeneous genotypes among individuals, to search for biomarkers related to immune responses and to construct a model for predicting the outcome of liver allografting. Of the 36 mice examined, 18 died within 3 weeks after transplantation, while the others survived for more than 6 weeks. Whole-exome sequencing of the 36 donors revealed more than 9 million variants relative to the C57BL/6 J reference. We selected 6517 single-nucleotide and indel variants and performed machine learning to determine whether or not we could predict the prognosis of each genotype. Models were built by both deep learning with a one-dimensional convolutional neural network and linear classification and evaluated by leave-one-out cross-validation. Given that one short-lived mouse died early in an accident, the models perfectly predicted the outcome of all individuals, suggesting the importance of genotype collection. In addition, linear classification models provided a list of loci potentially responsible for these responses. The present methods as well as results is likely to be applicable to liver transplantation in humans.

P.413: Mass cytometry and single cell RNA sequencing analysis of mouse liver transplantation.

P.413: Mass cytometry and single cell RNA sequencing analysis of mouse liver transplantation.

MSR1-dependent efferocytosis improved ischemia-reperfusion injury following aged-donor liver transplantation in mice by regulating the pro-resolving polarisation of macrophages

Compared with young liver donors, aged liver donors are more susceptible to ischemia-reperfusion injury (IRI) following transplantation, which may be related to excessive inflammatory response and macrophage dysfunction, but the specific mechanism is unclear. Macrophage scavenger receptor 1 (MSR1) is a member of the scavenger receptor family, and plays an important regulatory role in inflammation response and macrophage function regulation. But its role in IRI following aged-donor liver transplantation is still unclear. This study demonstrates that MSR1 expression is decreased in macrophages from aged donor livers, inhibiting their efferocytosis and pro-resolving polarisation. Decreased MSR1 is responsible for the more severe IRI suffered by aged donor livers. Overexpression of MSR1 using F4/80-labelled AAV9 improved intrahepatic macrophage efferocytosis and promoted pro-resolving polarisation, ultimately ameliorating IRI following aged-donor liver transplantation. In vitro co-culture experiments further showed that overexpression of MSR1 promoted an increase in calcium concentration, which further activated the PI3K-AKT-GSK3β pathway, and induced the upregulation of β-catenin. Overall, MSR1-dependent efferocytosis promoted the pro-resolving polarisation of macrophages through the PI3K-AKT-GSK3β pathway-induced up-regulating of β-catenin leading to improved IRI following aged-donor liver transplantation.

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.

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Extracellular vesicles from bone marrow mesenchymal stem cells alleviate acute rejection injury after liver transplantation by carrying miR-22-3p and inducing M2 polarization of Kupffer cells.

Acute rejection (AR) is a major problem following liver transplantation. Extracellular vesicles (EVs) are involved in various pathological processes including liver disease. The present study investigated the effect of EVs derived from bone marrow mesenchymal stem cells (BMSCs) on AR injury after orthotopic liver transplantation (OLT) in mice. BMSCs and EVs were isolated and identified. The OLT mouse model was established using Kamada's two-cuff method and injection with EVs, followed by liver function detection and measurement of inflammatory cytokines (interleukin-10, interferon-γ, and tumor necrosis factor-α), M1 and M2 markers (tumor necrosis factor-α, inducible nitric oxide synthase, resistin like alpha, and Arg1) were detected. Kupffer cells (KCs) were cultured and treated with lipopolysaccharides. miR-22-3p expression was detected. The effect of EVs-shuttled miR-22-3p on Kupffer cell polarization was studied. Binding relation of miR-22-3p and interferon regulatory factor 8 (IRF8) was verified. The effect of IRF8 on KC polarization was verified. BMSC-EV treatment enhanced liver function of OLT mice and alleviated AR and apoptosis, which were annulled after removing KCs. EVs induced KC M2 polarization. Mechanically, EVs carried miR-22-3p into KCs, upregulated miR-22-3p in KCs, and inhibited IRF8 expression. Upregulation of IRF8 in KCs inhibited EV-induced KC M2 polarization. BMSCs-EVs carry miR-22-3p into KCs and upregulate miR-22-3p, inhibit IRF8 expression, induce KC M2 polarization, and attenuate AR injury after liver transplantation.

Complement inhibition alleviates donor brain death-induced liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase signaling

Brain death (BD) donors are the primary source of donor organs for liver transplantation. However, the effects of BD on donor livers and outcomes after liver transplantation remain unclear. Here, we explored the role of complement and the therapeutic effect of complement inhibition in BD-induced liver injury and posttransplantation injury in a mouse BD and liver transplantation model. For complement inhibition, we used complement receptor 2 (CR2)-Crry, a murine inhibitor of C3 activation that specifically targets sites of complement activation. In the mouse model, BD resulted in complement activation and liver injury in donor livers and a cascade liver injury posttransplantation, mediated in part through the C3a-C3aR (C3a receptor) signaling pathway, which was ameliorated by treatment with CR2-Crry. Treatment of BD donors with CR2-Crry improved graft survival, which was further improved when recipients received an additional dose of CR2-Crry posttransplantation. Mechanistically, we determined that complement inhibition alleviated BD-induced donor liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase (PI3K) signaling pathways. Together, BD induced donor liver injury and cascade injury post-transplantation, which was mediated by complement activation products acting on PI3K signaling pathways. Our study provides an experimental basis for developing strategies to improve the survival of BD donor grafts in liver transplantation.

α-Connexin Carboxyl Terminal Peptide 1 Attenuates Ischemia-Reperfusion Injury in Liver Transplantation With Extended Cold Preservation by Stabilizing Cell Junctions in Mice

• ⚫Peptide α-connexin carboxyl terminal peptide (ACT1) protects graft livers during cold storage by steadying cell junctions. • ⚫ACT1 reduces hepatic ischemia-reperfusion injury following transplantation. • ⚫ACT1 inhibits acute and chronic inflammation after transplantation. • ⚫The effect of ACT1 on cell junctions is long term. Ischemia-reperfusion injury is an inevitable process that the liver must go through after liver transplantation. Because of the worldwide shortage of grafts, reliable intervention strategies should be explored to protect grafts. α-Connexin carboxyl terminal peptide (ACT1), which is a synthetic peptide containing 25 amino acids, has the effect of stabilizing cell junctions. The purpose of this study was to investigate the role of ACT1 on ischemia-reperfusion injury after liver transplantation in mice. Experimental results showed that injury to livers that underwent extended cold preservation was reduced by ACT1. After 6 hours of reperfusion, ACT1-treated livers showed less necrosis and acute inflammation and the cell junctions were strengthened. Moreover, after 28 days of reperfusion, the application of ACT1 distinctly alleviated hepatic damage and later cholangiopathy as well as conspicuously consolidated the cell junctions. This study indicated that ACT1 produced affirmative effects in suppressing hepatic ischemia-reperfusion injury in liver transplantation with extended cold preservation in mice by protecting cell junctions and may be a potential strategy to extend the cold preservation time of grafts for liver transplantation.

Characteristics of changes in double positive CD4+CD8+ T cells in liver transplantation

Although double positive CD4+CD8+ T (DPT) cells has been reported to be involved in some diseases, their trajectory and function as associated with liver transplantation (LT) remain unclear. In the present study, we found that the number of DPT cells was increased in the blood and liver tissue of LT patients. Meanwhile, we compared the distribution of DPT cells in peripheral blood samples and in penetrating liver tissue between liver rejection versus non-rejection patients, as well as the proportion of DPT cells as a function of the extent of liver rejection. The number of DPT cells in the rejection group was significantly increased. An analysis of the spatial distance and correlations between DPT and Treg cells, revealed that these cells showed a high degree of contiguity. In a mouse liver transplant model, the number of DPT cells were significantly increased in liver tissue, and the number of CD8+ T cells gradually increased, while CD4+ T cells decreased as a function of time post-transplantation. Expression level of PD-1 in DPT cells also increased in a temporally-dependent manner post liver transplantation and the changes of PD-1+ DPT cells were related to the degree of liver transplant rejection. In DPT cells interacting with Treg, there was an increased expression of PD-1, which enhanced cellular exhaustion. In conclusion, the capacity for DPT cells to induce immune tolerance may represent a new and important protocol for use in targeting treatments for the prevention of liver transplant rejection.

TROAP switches DYRK1 activity to drive hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is one of the common malignancy and lacks effective therapeutic targets. Here, we demonstrated that ectopic expression of trophinin-associated protein (TROAP) dramatically drove HCC cell growth assessed by foci formation in monolayer culture, colony formation in soft agar and orthotopic liver transplantation in nude mice. Inversely, silencing TROAP expression with short-hairpin RNA attenuated the malignant proliferation of HCC cells in vitro and in vivo. Next, mechanistic investigation revealed that TROAP directly bound to dual specificity tyrosine phosphorylation regulated kinase 1A/B (DYRK1A/B), resulting in the cytoplasmic retention of proteins DYRK1A/B and promoting cell cycle process via activation of Akt/GSK-3β signaling. Combination of cisplatin with an inhibitor of DYRK1 AZ191 effectively inhibited tumor growth in mouse model for HCC cells with high level of TROAP. Clinically, TROAP was significantly upregulated by miR-142-5p in HCC tissues, which predicted the poor survival of patients with HCC. Therefore, TROAP/DYRK1/Akt axis may be a promising therapeutic target and prognostic indicator for patients with HCC.

Incompatibility between recipient CD47 and donor SIRPα is not a key risk factor for thrombocytopenia or anemia following rat liver xenotransplantation in mice

Liver xenotransplantation (LXT) is greatly impeded by severe thrombocytopenia, anemia, and coagulopathy. Hepatic phagocytic cells are thought to play an important role in LXT-induced thrombocytopenia and anemia. In this study, we investigated whether the lack of recipient CD47-donor SIRPα interaction, which is known to induce xenograft rejection by macrophages, exacerbates platelet and RBC depletion following LXT. We first addressed this question in the absence of anti-donor immune responses using a syngeneic mouse liver transplantation (LT) model. Neither wild-type (WT) nor CD47KO B6 mice developed thrombocytopenia following LT from WT B6 donors. Although a moderate decline in RBCs was detected following LT, there was no significant difference in RBC counts between WT and CD47KO recipients. Because mouse CD47 is cross-reactive with rat SIRPα, we then compared thrombocytopenia and anemia between WT and CD47KO mice following rat LXT. Unlike syngeneic mouse LT, significant thrombocytopenia and anemia were detected following rat LXT. However, the severities of both platelet and RBC depletions were comparable between WT and CD47KO recipients. Furthermore, WT and CD47KO recipients showed a similar extent of early platelet activation. Our results indicate that CD47-SIRPα signaling does not significantly affect the loss of platelets or RBCs following LXT, suggesting that the limited cross-reactivity between recipient CD47 and donor SIRPα is not a significant risk factor for LXT-induced thrombocytopenia and anemia.

AS009 - Pretransplant rifaximin administration reduces posttransplant liver injury and early allograft dysfunction in mouse and human liver transplantation

AS009 - Pretransplant rifaximin administration reduces posttransplant liver injury and early allograft dysfunction in mouse and human liver transplantation

Human Antigen R (HuR): A Regulator of Heme Oxygenase-1 Cytoprotection in Mouse and Human Liver Transplant Injury.

Background and AimsIschemia–reperfusion injury (IRI) represents a risk factor in liver transplantation (LT). We have shown that overexpression of heme oxygenase‐1 (HO‐1) mitigates hepatic IRI in LT recipients. Here, we hypothesized that human antigen R (HuR), the stabilizer of adenylate‐uridylate (AU)‐rich mRNAs, is required for hepatoprotection in LT.Approach and ResultsIn an experimental arm, HuR/HO‐1 protein expression was correlated with hepatic IRI phenotype. In an in vitro inflammation mimic model of hepatic warm IRI, induction of HuR/HO‐1 and cytoplasmic localization following cytokine preconditioning were detected in primary hepatocyte cultures, whereas HuR silencing caused negative regulation of HO‐1, followed by enhanced cytotoxicity. Using the HuR‐inhibitor, we showed that HuR likely regulates HO‐1 through its 3′ untranslated region and causes neutrophil activation (CD69+/lymphocyte antigen 6 complex locus G [Ly6‐G]). HuR silencing in bone marrow–derived macrophages decreased HO‐1 expression, leading to the induction of proinflammatory cytokines/chemokines. RNA sequencing of HuR silenced transcripts under in vitro warm IRI revealed regulation of genes thymus cell antigen 1 (THY1), aconitate decarboxylase 1 (ACOD1), and Prostaglandin E Synthase (PTGES). HuR, but not hypoxia‐inducible protein alpha, positively regulated HO‐1 in warm, but not cold, hypoxia/reoxygenation conditions. HuR modulated HO‐1 in primary hepatocytes, neutrophils, and macrophages under reperfusion. Adjunctive inhibition of HuR diminished microtubule‐associated proteins 1A/1B light chain 3B (LC3B), a marker for autophagosome, under HO‐1 regulation, suggesting a cytoprotective mechanism in hepatic IR. In a clinical arm, hepatic biopsies from 51 patients with LT were analyzed at 2 hours after reperfusion. Graft HuR expression was negatively correlated with macrophage (CD80/CD86) and neutrophil (Cathepsin G) markers. Hepatic IRI increased HuR/HO‐1 expression and inflammatory genes. High HuR–expressing liver grafts showed lower serum alanine aminotransferase/serum aspartate aminotransferase levels and improved LT survival.ConclusionsThis translational study identifies HuR as a regulator of HO‐1–mediated cytoprotection in sterile liver inflammation and a biomarker of ischemic stress resistance in LT.

CD47 blockade alleviates acute rejection of allogeneic mouse liver transplantation by reducing ischemia/reperfusion injury

Despite advances in immunosuppressive therapies, acute rejection response is still a serious concern especially in the early phase after liver transplantation. This study aimed to evaluate whether blocking the TSP1-CD47 signaling pathway could attenuate the acute rejection after liver transplantation. An allogeneic mouse orthotopic liver transplantation model (Balb/c→C3H) with prolonged cold ischemic phase was used to induce severe IRI and lethal acute rejection. CD47mAb or isotype matched-control IgG2a was administered to donor liver during graft perfusion. Recipients were sacrificed at 1d, 3d, 5d and 7d after reperfusion. Blood samples were collected to evaluate serum alanine aminotransferase, total bilirubin, HMGB-1,TNF-α, IL-2 and INF-γ level. Flow cytometric analysis was used to detect the strength of innate and adaptive immune response. Liver tissue was obtained for HE, TUNEL staining and F4/80 immumohistochemical staining. Moreover, we conducted a mixed lymphocyte reaction treated with IgG2a or CD47mAb. Mice in CD47mAb-treated group demonstrated improved survival and significantly lower increase in Suzuki score, apoptosis index, acute rejection index, serum alanine aminotransferase, total bilirubin, HMGB-1, TNF-α, IL-2, INF-γ level and the degree of Kupffer cells' activation especially in the early phase of acute rejection. In addition, Pearson's correlation analysis confirmed significant correlation between Suzuki score/ALT and acute rejection index. The in vitro inhibition assay showed that CD47 blockade couldn’t directly inhibit recipient lymphocyte proliferation. Based on the evidence that TSP1-CD47 signaling blockade with CD47mAb could alleviate acute rejection by reducing the extent of IRI after liver transplantation indirectly, this study provided a basis for new interventions and management methods to support better transplant outcomes.

Bioinformatics analysis of gene chip data in vivo after liver transplantation in mice

Bioinformatics analysis of gene chip data in vivo after liver transplantation in mice

Su1559 – Reconstruction of the Hepatic Artery in Mouse Liver Transplantation

Su1559 – Reconstruction of the Hepatic Artery in Mouse Liver Transplantation

Dual Function of IRF1/IRF4 on Dendritic Cells During Mouse Liver Transplantation

Dual Function of IRF1/IRF4 on Dendritic Cells During Mouse Liver Transplantation

Graft-infiltrating PD-L1hi cross-dressed dendritic cells regulate antidonor T cell responses in mouse liver transplant tolerance.

These findings suggest that graft-infiltrating PD-L1hi CD-DCs may play a key role in the regulation of alloimmunity and in the induction of liver transplant tolerance. (Hepatology 2018;67:1499-1515).

Graft-Infiltrating PD-L1hi Cross-Dressed Dendritic Cells Subvert Donor-Reactive T Cell Responses in Mouse Liver Transplant Tolerance

Introduction: The mechanisms underlying “spontaneous” liver allograft tolerance remain unclear. Given recent reports of the key role of host dendritic cells (DCs) in mouse spleens that express intact donor MHC (cross-dressing) in the instigation of graft rejection[1][2] we investigated the role of cross-dressed (CD) DCs in mouse liver transplantation tolerance. Methods: Liver allografts from C57BL/6 (B6; H-2b) or B6 SJL CD45.1 mice to C3H/HeJ (C3H; H-2 k) recipients were performed. In this combination, unlike heart, skin or kidney allografts, > 90% of liver grafts are accepted without immunosuppressive therapy[3]. Graft non-parenchymal cells (NPC) and splenocytes were examined by flow cytometry and imaging cytometry on post-operative days (POD) 1, 3, 7, 14, 30, and 300. Mixed leukocyte reactions (CFSE-MLR) were also performed to assess DC function. Results: Infiltration by recipient DCs (lineage (−), CD11c (+)) in liver allografts peaked on POD 7, while donor DCs in liver grafts gradually disappeared and could not be detected by POD 7. Interestingly, more than half of the graft-infiltrating recipient DCs at that time displayed donor MHC-I, indicating cross-dressing; these DCs persisted in the graft (approx. 20 % of recipient DC) at least until POD 300. In contrast, only a very minor fraction of cross-dressed DCs (CD-DCs) (0-2%) were detected in the spleen at any time point. Control staining for donor MHC-I using naïve C3H mouse liver NPC or splenocytes was negative. Moreover, especially on POD 7, and persisting until POD 300, CD-DCs isolated from liver grafts expressed higher levels of T cell inhibitory programed death ligand 1 (PD-L1) compared to non CD-DCs (nCD-DCs). Importantly, unlike nCD-DCs, CD-DCs from liver grafts did not stimulate proliferation of allo-reactive donor T cells and markedly suppressed donor-reactive host T cell proliferation in CFSE-MLR. Conclusions: A large proportion of CD recipient DCs with capacity to subvert donor-reactive host T cell responses are evident in liver allografts early post-transplant. Moreover, cross-dressing by graft-infiltrating DCs that also express high levels of PD-L1, persists indefinitely. This suggests that graft-infiltrating host CD-DC may play a key role in regulation of alloimmunity and the promotion of donor-specific liver transplant tolerance.Figure 1: Marked reduction in donor dendritic cells (DCs) and emergence of cross-dressed host DCs in the graft after liver allo-transplantaion. (A) (B) Graft and spleen DCs were analyzed using donor recipient MHC class 1 (A) or CD45.1/ CD 45.2 antibodies (B) after B6 WT graft to C3H recipient transplatation. Data shown are representative data from 3-5 mice.Figure 2: Infiltrating host DCs and CD-DCs peak on 7 POD and PD-L1high CD-DCs persist in the graft for at least 300 POD. (A) Chronological changes in (left) the incidence of total DC and (right) the incidence of CD-DC (as % total DC) in the graft. (B) Expression of PD-L1 by CD-DCs and non (n)CD-DCs in the graft. PD-L1 expression by CD-DCs was significally higher than of nCD-DC, especially on 7POD. 3-6 mice per time point. *: P<0.05, ***: P<0.001Figure 3: Liver CD-DCs markedly sppress donor-reactive host T cell proliferation in CFSE-MLR Spleen (Sp) or liver CD-DCs or nCD-DCs isolated from graft recipients on 7 POD were co- cultured with responder (C3H (host) spleen T cells) and stimulators (B6 (donor) spleen DC) in CFSE-MLR (T cells: DC (each) =10:1). Liver CD-DCs, markedly suppressed host T cell proliferation. % T cell proliferation in indicated. Data are representative of 3 experiments that gave similar results.

Efficient nonarterialized mouse liver transplantation using 3‐dimensional–printed instruments

Because of the wide availability of genetically modified animals, mouse orthotopic liver transplantation is often preferred over rat liver transplantation. We present a simplified mouse liver transplantation technique and compare transplantation outcomes with versus without hepatic artery anastomosis. Instruments for liver implantation were designed and printed with a 3-dimensional (3D) printer. The suprahepatic vena cava anastomosis was performed with a 10-0 running suture. The vena porta and infrahepatic vena cava were joined on extraluminal cuffs, using the 3D-printed device for spatial alignment and stabilization. The hepatic artery was reconstructed in half of the recipients using intraluminal stents. Liver function tests (3, 7, and 28 days) and histology (7 and 28 days) were assessed after transplantation. We performed 22 consecutive syngeneic C57BL/6 mouse orthotopic liver transplantations. The median portal clamping time was 12.5 ± 1.5 minutes. The survival rate at 4 weeks was 100% for both arterialized and nonarterialized recipients (n = 7, 4 recipients of each group being killed for early histology at day 7). Liver function tests at 3, 7, and 28 days were similar between arterialized versus nonarterialized groups. Liver parenchyma demonstrated only irrelevant abnormalities in both groups. The proposed device allows for a shorter clamping time compared with the published literature. Using this technique, the artery does not need to be anastomosed, with no impact on graft and recipient outcomes. The device is available for 3D printing. Liver Transplantation 22 1688-1696 2016 AASLD.