Liver Harvesting Research Articles

Early bifurcation of the common hepatic artery: A pitfall that should be known and recognized

Early bifurcation of the common hepatic artery (EBCHA) is a rare anatomical variation (1%), that is often overlooked but can lead to accidental ligation of the right branch of the hepatic artery with consequent arterial ischemia of the right liver and potentially very serious complications during pancreaticoduodenectomy, partial hepatectomy, or liver harvesting for transplantation. It may be difficult to diagnose EBCHA using transverse imaging sections. However, on standard CT sections with intravenous contrast injection, three warning signs should allow the image reader to suspect it: presence of two hepatic arteries to the right of the celiac trunk, presence of a retro-portal hepatic artery, and absence of a right hepatic artery arising from the superior mesenteric artery. Analysis of the CT with reconstruction then allows for definitive diagnosis and limits the risk of accidental arterial injury or ligation.

Formation of CYP3A-specific metabolites of ibrutinib in vitro is correlated with hepatic CYP3A activity and 4β-hydroxycholesterol/cholesterol ratio.

Ibrutinib is an orally administered Bruton's tyrosine kinase inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia. Ibrutinib is metabolized primarily via oxidation by cytochrome P450 (CYP) 3A4/5 to M37 (the primary active metabolite), M34, and M25. The objectives of this study were to assess the relationship between formation of the major CYP3A-specific ibrutinib metabolites in vitro and hepatic CYP3A activity and protein abundance, and to evaluate the utility of the endogenous CYP3A biomarker, plasma 4β-hydroxycholesterol (4β-HC) to cholesterol ratio, to predict ibrutinib metabolite formation in individual cadaveric donors with matching hepatocytes. Ibrutinib (5μM) was incubated with single-donor human liver microsomes (n=20) and primary human hepatocytes (n=15), and metabolites (M37, M34, and M25) were measured by liquid chromatography-tandem mass spectrometry analysis. CYP3A4/5 protein concentrations were measured by quantitative targeted absolute proteomics, and CYP3A activity was measured by midazolam 1'-hydroxylation. Ibrutinib metabolite formation positively correlated with midazolam 1'-hydroxylation in human liver microsomes and hepatocytes. Plasma 4β-HC and cholesterol concentrations were measured in plasma samples obtained at the time of liver harvest from the same 15 donors with matching hepatocytes. Midazolam 1'-hydroxylation in hepatocytes correlated with plasma 4β-HC/cholesterol ratio. When an infant donor (1 year old) was excluded based on previous ontogeny studies, M37 and M25 formation correlated with plasma 4β-HC/cholesterol ratio in the remaining 14 donors (Spearman correlation coefficients [r] 0.62 and 0.67, respectively). Collectively, these data indicate a positive association among formation of CYP3A-specific ibrutinib metabolites in human hepatocytes, hepatic CYP3A activity, and plasma 4β-HC/cholesterol ratio in the same non-infant donors.

P11.07: BMSCs Combined With Normothermic Machine Perfusion Resuscitate Aged Donor Liver With Long Warm Ischemia Time in Rats

Background: Both aged and donation after cardiac death (DCD) donor liver, considered as marginal organs, were used for transplantation due to huge organ shortage in the world. Organs combined with both danger factors would impair the donor liver viability. Using normothermic machine perfusion (NMP), we investigated the possibility of mesenchymal stem cells (MSCs) to revive the aged DCD donor liver in a rat model. Methods: The SD rats used for NMP were all over 14 months of age. BMSCs were isolated from the bone marrow of SD rats. Rats were anesthetized and cardiac arrest was induced by compression of the heart for 30 minutes as warm ischemia prior to donor liver harvesting. The liver was connected to a rat liver perfusion machine for NMP with or without 10^6 BMSCs infusion. Results: The weight of the aged liver in the experimental and control groups was 23.03 ± 0.25 g and 21.40 ± 0.87 g. the donor liver appeared to multiple bruised foci in both prior- and post- perfusion, while liver with BMSCs perfusion had fewer bruised foci (Figure 1A, B). The pH was maintained between 7.35 and 7.45 in both groups during perfusion. Lactate in the BMSCs group decreased after 1 hour during perfusion and fell below 1 gradually within 3 hours, while in the control group it decreased more slowly and remained around 4 even after 4 hours of perfusion (Figure 1C). Pathological appearance showed that the pathological Suzuki‘s sores of liver in the control group were higher than MSCs group, with bruised liver sinusoids, apoptosis and focal necrosis of hepatocytes. In contrast, pathological change in the liver of the BMSC group showed significant improvement with no obvious necrosis and structural integrity of the liver lobules (Figure 1D). The climbing rate and peak value of AST and ALT in the BMSCs group were lower than that in the control group, indicating that BMSCs protected liver from damage during perfusion (Figure 1E, 1F). Conclusion: Our results demonstrate that the use of MSCs during normothermic machine perfusion is a feasible strategy to resuscitate the aged DCD donor liver.National Natural Science Foundation of China (81401324 and 81770410).

Backtable preparation after liver harvesting with "en bloc" technique with video.

The aim of this didactical video is to show an easy and standardized technique of liver preparation after "en bloc" extraction and access a young surgeon to perform liver procurement. The technique entails five steps: beginning with the dissection of the vena cava, the superior mesenteric artery, and the coeliac trunk, followed by the common hepatic artery, the bile duct and finally the portal vein. This technique of liver graft preparation has high reproducibility while maintaining the safety of the procedure for young surgeons. The "en bloc" extraction with a standardized liver graft preparation is an easy and a reproducible technique.

Generation of Human Liver Chimeric Mice and Harvesting of Human Hepatocytes from Mouse Livers.

Primary human hepatocytes (PHHs) are widely used as an in vitro model to evaluate various aspects of human hepatic physiology and pathology. However, PHHs isolated from the human liver have very limited ability for ex vivo expansion in culture. Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice are proven to be an ideal bioincubator for repopulation of PHHs. The human liver chimeric FRG mouse is not only a humanized animal model for disease study and drug screening in vivo, but also a potential source of PHHs for cellular therapy. This chapter describes experimental protocols to generate chimeric FRG mice with humanized liver and to isolate PHHs from human liver chimeric FRG mice. Using these methods, PHHs can be expanded to more than 100-fold for harvesting.

Effect of Hyperbaric Oxygen Therapy on Acute Liver Injury and Survival in a Rat Cecal Slurry Peritonitis Model.

Background: The effects of hyperbaric oxygen therapy (HBOT) in sepsis remain unclear. This study evaluated its effects on acute liver injury and survival in a rat model. Methods: Cecal slurry peritonitis was induced in male rats, which were then randomly allocated into the HBOT and control groups. In the survival experiment, six 90 min HBOT sessions (2.6 atmospheres absolute 100% oxygen) were performed over 48 h; the survival rate was determined 14 days after sepsis induction. In the acute liver injury experiment, three HBOT sessions were performed, followed by liver and plasma harvesting, 24 h after sepsis induction. Serum levels of alanine aminotransferase (ALT), interleukin (IL)-6, and IL-10 were measured, and the hepatic injury scores were determined. Reactive oxygen species (ROS) generation was detected by 2′,7′-dihydrodichlorofluorescein diacetate (H2DCF-DA) assay. Western blot assays assessed protein kinase B (Akt), phosphorylated-Akt (p-Akt), glycogen synthase kinase (GSK)-3β, phosphorylated-GSK-3β, and cleaved caspase-3 levels. Results: Survival in the HBOT group (57.1%) was significantly higher than that in the controls (12.5%, p = 0.029), whereas IL-6, IL-10, and ALT levels were significantly lower in the HBOT group. The ROS generation was significantly inhibited to a greater extent in the HBOT group than in the control group. Additionally, in the HBOT group, the p-Akt and p-GSK-3β increased significantly and cleaved caspase-3 levels decreased significantly. Conclusions: HBOT showed a beneficial effect on acute liver injury and rat survival by enhancing the Akt signaling pathway and decreasing apoptosis.

3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals.

Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.

Novel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold.

IntroductionLiver transplantation is currently the only curative therapy for end-stage liver failure; however, establishment of alternative treatments is required owing to the serious donor organ shortage. Here, we propose a novel model of hybrid three-dimensional artificial livers using both human induced pluripotent stem cells (hiPSCs) and a rat decellularized liver serving as a scaffold.MethodsRat liver harvesting and decellularization were performed as reported in our previous studies. The decellularized liver scaffold was recellularized with hiPSC-derived hepatocyte-like cells (hiPSC-HLCs) through the biliary duct. The recellularized liver graft was continuously perfused with the culture medium using a pump at a flow rate of 0.5 mL/min in a standard CO2 (5%) cell incubator at 37 °C.ResultsAfter 48 h of continuous perfusion culture, the hiPSC-HLCs of the recellularized liver distributed into the parenchymal space. Furthermore, the recellularized liver expressed the albumin (ALB) and CYP3A4 genes, and secreted human ALB into the culture medium.ConclusionNovel hybrid artificial livers using hiPSCs and rat decellularized liver scaffolds were successfully generated, which possessed human hepatic functions.

03:09 PM Abstract No. 180 Liver radiofrequency ablation affects the infiltrated cellular dynamics in the periablational rim and induces aggressiveness in the distant subcutaneous R3230 breast adenocarcinoma tumor

To assess the effect of normal non-tumor liver thermal ablation on periablation rim and distant R3230 breast adenocarcinoma tumor in F344 rats while using atorvastatin as an adjuvant tumor inhibitor. To this end, we used F344 rat (150g, n=65), R3230 breast adenocarcinoma tumor, sham (needle placement without energy for 5 minutes) or RFA (70°C for 5 minutes) with or without atorvastatin IP administration (1.15mg per rat) were used for this study. The rats were grouped into; sham, sham + 24 h post-ablation treatment, sham + daily (0, 24 and 48 h) post-ablation treatment, RFA only, RFA+24 h post-ablation treatment and RFA + daily (0, 24 and 48 h) post-ablation treatment. Tumors were measured daily post-R3230 transplantation until liver harvest at day 3 and 7 post-ablation for IHC and Elisa. We assessed the levels myofibroblast and macrophage infiltration (SMA and CD68 respectively), Ki-67 proliferative indexes, CD34 microvascular density and hepatic growth factor (HGF) in the harvested liver samples. From day 3 to day 7 post-ablation, RFA group significantly increased the distant tumor size when compared to the rest of the treatment groups. RFA + daily (0, 24 and 48 h) atorvastatin treatment group had the least distant tumor growth effect relative to the ablated groups. While there was no significant difference in the amount of SMA expressed in all the sham groups with or without atorvastatin, more than 2-fold increase in the levels of SMA, KI-67, CD38 and CD68 were observed in the periablational rim of the RFA group unlike RFA + 24 h atorvastatin treatment and RFA + daily (0, 24 and 48 h) atorvastatin treatment groups. Also, HGF was least expressed in RFA + daily (0, 24 and 48 h) atorvastatin treatment but not RFA group. RFA + 24 h atorvastatin treatment group showed a moderate level of HGF while there was generally no significant difference among the sham groups (with or without atorvastatin). Our results suggest that the ablation of normal non-tumor bearing liver affects distant subcutaneous R3230 breast tumor growth and alters the infiltrated cellular dynamics of the periablational rim when treated with or without atorvastatin after ablation.

Human-Scale Liver Harvest and Decellularization for Preclinical Research.

Decellularized scaffolds are promising for organ regeneration; to facilitate analyses of decellularization and preclinical research, experiments using large animals are indispensable. An efficient protocol using large animals requires the modification of current methods to account for differences in anatomy, size, stiffness, and animal handling. This chapter describes a method for the decellularization of livers from large animals (especially porcine livers), including anesthesia, organ harvest, organ decellularization, and storage, with a summary of potential pitfalls. This methodology can hypothetically be applied to large animals for preclinical studies and subsequent clinical applications.

Laparoscopic right hepatectomy for living donation

Living donor liver transplantation has become a widely accepted alternative to cadaveric transplantation. The laparoscopic approach has demonstrated its safety and reproducibility for the left lateral section harvesting in the last decade, but remains controversial for right liver harvesting due to concerns about donor and graft safety. The selection of an eligible donor means the evaluation of, in one hand, the technical feasibility, and in the other hand, the operative risk for the donor. Such issues imply an extensive workup procedure. The operation is divided in three main steps: right pedicle dissection, parenchymal transection including bile duct division, and graft extraction. To enhance donor safety, these steps are performed in rotation between two senior surgeons in our team. Conversion criteria and technical aspects are precisely defined preoperatively to enhance donor safety.

Laparoscopic right hepatectomy for living donation

Living donor liver transplantation has become a widely accepted alternative to cadaveric transplantation. The laparoscopic approach has demonstrated its safety and reproducibility for the left lateral section harvesting in the last decade, but remains controversial for right liver harvesting due to concerns about donor and graft safety. The selection of an eligible donor means the evaluation of, in one hand, the technical feasibility, and in the other hand, the operative risk for the donor. Such issues imply an extensive workup procedure. The operation is divided in three main steps: right pedicle dissection, parenchymal transection including bile duct division, and graft extraction. To enhance donor safety, these steps are performed in rotation between two senior surgeons in our team. Conversion criteria and technical aspects are precisely defined preoperatively to enhance donor safety.

PREOPERATIVE COMPUTED TOMOGRAPHY VOLUMETRY AND GRAFT WEIGHT ESTIMATION IN ADULT LIVING DONOR LIVER TRANSPLANTATION.

ABSTRACTBackground:Computed tomography volumetry (CTV) is a useful tool for predicting graft weights (GW) for living donor liver transplantation (LDLT). Few studies have examined the correlation between CTV and GW in normal liver parenchyma. Aim:To analyze the correlation between CTV and GW in an adult LDLT population and provide a systematic review of the existing mathematical models to calculate partial liver graft weight. Methods:Between January 2009 and January 2013, 28 consecutive donors undergoing right hepatectomy for LDLT were retrospectively reviewed. All grafts were perfused with HTK solution. Estimated graft volume was estimated by CTV and these values were compared to the actual graft weight, which was measured after liver harvesting and perfusion. Results:Median actual GW was 782.5 g, averaged 791.43±136 g and ranged from 520-1185 g. Median estimated graft volume was 927.5 ml, averaged 944.86±200.74 ml and ranged from 600-1477 ml. Linear regression of estimated graft volume and actual GW was significantly linear (GW=0.82 estimated graft volume, r2=0.98, slope=0.47, standard deviation of 0.024 and p<0.0001). Spearman Linear correlation was 0.65 with 95% CI of 0.45 - 0.99 (p<0.0001). Conclusion:The one-to-one rule did not applied in patients with normal liver parenchyma. A better estimation of graft weight could be reached by multiplying estimated graft volume by 0.82.

Incidental malignancy in cadaver donor: What is the real impact?

Worldwide organ shortage is one of the most common hurdles while treating patients with end-stage liver disease, especially in countries where there is lack of interest in organ donation. Apart from this, cost of this remedy is one reason, which makes this treatment distant dream for many. In such a situation, to lose even a single donor becomes too costly, cost of which is life of some other patients. Extended criteria for liver donation are already in use, though in some situations clinicians feel like trapped between providing best care and managing organ demands. One of such tricky situations is incidentally found malignancy in donor. Recently, we came across such situation during the harvest of liver from a cadaver donor, which made us go through the literature and find the answer. Here, in this review, we share that experience and try to throw light on this enigmatic issue with special focus on incidentally found malignancies in cadaver donor.

Living Liver Donor Selection and Resection at the University of Tokyo Hospital

ObjectivesDonor selection and operative procedures for adult-to-adult living donor liver transplantation at the University of Tokyo are presented. MethodsDonor selection criteria are as follows: age between 20 and 65 years, within 3 degrees of consanguinity, without coercion, free from any major comorbidities, body mass index (BMI) < 30, and ABO blood type identical or compatible. Liver biopsy is indicated for BMI > 25 kg/m2 or any liver function abnormality, and those with macroscopic steatosis >10% are rejected. Thereafter, an indocyanine green retention test and dynamic computed tomography are evaluated. Graft type is determined based on computed tomography volumetry. An estimated graft volume of 40% to recipient standard liver volume ratio is the lower limit. For donor safety, the left liver is the first choice, provided that it satisfies the lower limit. Otherwise, right liver harvesting is indicated, providing that the estimated remnant liver volume is >30% of the donor's total liver volume. A posterior sector graft is a possible option. ResultsBetween 1996 and 2014, 462 donor hepatectomies were performed, with 257 right livers, 179 left livers, and 26 posterior sectors. There was no mortality, and the incidence of morbidity grades I, II, IIIa, and IIIb was 16%, 5%, 5%, and 3%, respectively, without a difference between right and left liver grafts. The left liver was used without impairing recipient outcome. Two aborted hepatectomies (0.4%) and 3 near-miss events (0.6%) were encountered. ConclusionsMaximal effort should be applied to donor selection and operation for donor safety.

How to avoid injuries of a replaced right hepatic artery during liver harvesting in marginal and/or non-beating hearth donors

How to avoid injuries of a replaced right hepatic artery during liver harvesting in marginal and/or non-beating hearth donors

Cavernous transformation of the portal vein

A male patient with an unknown medical history sustained injuries that were deemed incompatible with life. He became an organ donor. At the time of donor liver harvest, periportal collaterals were seen around the liver (A), with webs visualized inside the portal vein (B) consistent with cavernous transformation of the portal vein (CTPV). This made the liver unsuitable for transplantation, and the procedure was aborted.

Liver harvesting from the donor to the transplantee: A proposed protocol for nurses

Objective: To propose a clinical protocol model for nurses in liver harvesting for transplant. Methods: A descriptive study, qualitative in nature, held in Fortaleza, in the period from June to August 2014. Interviews were carried out and observing the work of nurses in a referral hospital in Ceara. The data was processed through the thematic analysis and provided the construction of the proposed Protocol pilot, analysed by nurses for the final version. Results: The study allowed the construction of a protocol, with specific actions that permeate the harvesting procedure, specifically the stage of organ removal, streamlining interventions and making them safer and more uniform in the liver harvest-transplantation process. Conclusions: The proposal of a protocol concerning liver harvesting for transplant is a systematization assistance model with the potential to codify the actions of the nurse in the harvesting and transplantation process.

Laparoscopic liver resection: Experience based guidelines.

Laparoscopic liver resection (LLR) has been progressively developed along the past two decades. Despite initial skepticism, improved operative results made laparoscopic approach incorporated to surgical practice and operations increased in frequency and complexity. Evidence supporting LLR comes from case-series, comparative studies and meta-analysis. Despite lack of level 1 evidence, the body of literature is stronger and existing data confirms the safety, feasibility and benefits of laparoscopic approach when compared to open resection. Indications for LLR do not differ from those for open surgery. They include benign and malignant (both primary and metastatic) tumors and living donor liver harvesting. Currently, resection of lesions located on anterolateral segments and left lateral sectionectomy are performed systematically by laparoscopy in hepatobiliary specialized centers. Resection of lesions located on posterosuperior segments (1, 4a, 7, 8) and major liver resections were shown to be feasible but remain technically demanding procedures, which should be reserved to experienced surgeons. Hand-assisted and laparoscopy-assisted procedures appeared to increase the indications of minimally invasive liver surgery and are useful strategies applied to difficult and major resections. LLR proved to be safe for malignant lesions and offers some short-term advantages over open resection. Oncological results including resection margin status and long-term survival were not inferior to open resection. At present, surgical community expects high quality studies to base the already perceived better outcomes achieved by laparoscopy in major centers' practice. Continuous surgical training, as well as new technologies should augment the application of laparoscopic liver surgery. Future applicability of new technologies such as robot assistance and image-guided surgery is still under investigation.

Dissection technique for selective liver harvesting

Dissection technique for selective liver harvesting