Cholangiocyte Organoids: The New Frontier in Regenerative Medicine for the Study and Treatment of Cholangiopathies.

Cholangiopathies include a group of chronic progressive disorders, affecting the cholangiocytes, the epithelial cells that line the biliary tree, leading to liver parenchymal fibrosis and eventually end-stage liver disease necessitating transplantation. Experimental modeling of these multifactorial cholestatic diseases faces challenges due to the lack of adequate experimental in vitro and in vivo models. A novel approach employs three-dimensional organoid systems that offer several advantages for modeling disease and testing drug response in vitro. Organoids mimic intercellular communication, replicate the architecture of organs, and maintain the cell's original phenotype. Cholangiocyte organoids provide an in vitro model to study the pathogenesis and pharmacotherapeutic treatment of cholangiopathies and show great promise for regenerative therapies. In particular, patient-derived organoids allow personalized medicine approaches and the study of individual disease characteristics. This review highlights the significance of cholangiocyte organoid models in advancing our understanding of cholangiopathies and driving advancements in regenerative medicine strategies.

Two pumps or one pump? A comparison of human liver normothermic machine perfusion devices for transplantation.

BACKGROUND: Normothermic machine perfusion provides continuous perfusion to ex situ hepatic grafts through the portal vein and the hepatic artery. Because the portal vein has high flow with low pressure and the hepatic artery has low flow with high pressure, different types of perfusion machines have been employed to match the two vessels' infusion hemodynamics. METHODS: We compared transplanted human livers perfused through a 2-pump (n = 9) versus a 1-pump perfusion system (n = 6) where a C-clamp is used as a tubing constrictor to regulate hemodynamics. RESULTS: There was no significant difference between groups in portal vein or hepatic artery flow rate. The 1-pump group had more hemoglobin in the perfusate. However, there was no significant difference in plasma hemoglobin between the 2-pump and 1-pump groups at each time point or in the change in levels, proving no hemolysis occurred due to C-clamp tube constriction. After transplantation, the 2-pump group had two cases of early allograft dysfunction (EAD), whereas the 1-pump group had no EAD. There was no graft failure or patient death in either group during follow-up ranging from 20-52 months. CONCLUSIONS: Our data show that the 1-pump design provided the same hemodynamic output as the 2-pump design, with no additional hemolytic risk, but with the benefits of lower costs, easier transport and faster and simpler setting.

Sequential Use of Normothermic Regional and Ex Situ Machine Perfusion in Donation After Circulatory Death Liver Transplant.

In Italy, 20 minutes of a continuous flat line on an electrocardiogram are required for declaration of death. In the setting of donation after circulatory death (DCD), prolonged warm ischemia time prompted the introduction of abdominal normothermic regional perfusion (NRP) followed by postprocurement ex situ machine perfusion (MP). This is a retrospective review of DCD liver transplantations (LTs) performed at 2 centers using sequential NRP and ex situ MP. From January 2018 to April 2019, 34 DCD donors were evaluated. Three (8.8%) were discarded before NRP, and 11 (32.4%) were discarded based on NRP parameters (n = 1, 3.0%), liver macroscopic appearance at procurement and/or biopsy results (n = 9, 26.5%), or severe macroangiopathy at back-table evaluation (n = 1, 3.0%). A total of 20 grafts (58.8%; 11 uncontrolled DCDs, 9 controlled DCDs) were considered eligible for LT, procured and perfused ex situ (9 normothermic and 11 dual hypothermic MPs). In total, 18 (52.9%; 11 uncontrolled) livers were eventually transplanted. Median (interquartile range) no-flow time was 32.5 (30-39) minutes, whereas median functional warm ischemia time was 52.5 (47-74) minutes (controlled DCD), and median low-flow time was 112 minutes (105-129 minutes; uncontrolled DCD). There was no primary nonfunction, while postreperfusion syndrome occurred in 8 (44%) recipients. Early allograft dysfunction happened in 5 (28%) patients, while acute kidney injury occurred in 5 (28%). After a median follow-up of 15.1 (9.5-22.3) months, 1 case of ischemic-type biliary lesions and 1 patient death were reported. DCD LT is feasible even with the 20-minute no-touch rule. Strict NRP and ex situ MP selection criteria are needed to optimize postoperative results.

Machine Perfusions in Liver Transplantation: The Evidence-Based Position Paper of the Italian Society of Organ and Tissue Transplantation.

The use of machine perfusion (MP) in liver transplantation (LT) is spreading worldwide. However, its efficacy has not been demonstrated, and its proper clinical use has far to go to be widely implemented. The Società Italiana Trapianti d'Organo (SITO) promoted the development of an evidence-based position paper. A 3-step approach has been adopted to develop this position paper. First, SITO appointed a chair and a cochair who then assembled a working group with specific experience of MP in LT. The Guideline Development Group framed the clinical questions into a patient, intervention, control, and outcome (PICO) format, extracted and analyzed the available literature, ranked the quality of the evidence, and prepared and graded the recommendations. Recommendations were then discussed by all the members of the SITO and were voted on via the Delphi method by an institutional review board. Finally, they were evaluated and scored by a panel of external reviewers. All available literature was analyzed, and its quality was ranked. A total of 18 recommendations regarding the use and the efficacy of ex situ hypothermic and normothermic machine perfusion and sequential normothermic regional perfusion and ex situ MP were prepared and graded according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method. A critical and scientific approach is required for the safe implementation of this new technology.

Ex Situ Liver Machine Perfusion: The Impact of Fresh Frozen Plasma.

The primary aim of this single-center, phase 1 exploratory study was to investigate the safety, feasibility, and impact on intrahepatic hemodynamics of a fresh frozen plasma (FFP)-based perfusate in ex situ liver normothermic machine perfusion (NMP) preservation. Using an institutionally developed perfusion device, 21 livers (13 donations after brain death and 8 donations after circulatory death) were perfused for 3 hours 21 minutes to 7 hours 52 minutes and successfully transplanted. Outcomes were compared in a 1:4 ratio to historical control patients matched according to donor and recipient characteristics and preservation time. Perfused livers presented a very low resistance state with high flow during ex situ perfusion (arterial and portal flows 340 ± 150 and 890 ± 70 mL/minute/kg liver, respectively). This hemodynamic state was maintained even after reperfusion as demonstrated by higher arterial flow observed in the NMP group compared with control patients (220 ± 120 versus 160 ± 80 mL/minute/kg liver, P = 0.03). The early allograft dysfunction (EAD) rate, peak alanine aminotransferase (ALT), and peak aspartate aminotransferase (AST) levels within 7 days after transplantation were lower in the NMP group compared with the control patients (EAD 19% versus 46%, P = 0.02; peak ALT 363 ± 318 versus 1021 ± 999 U/L, P = 0.001; peak AST 1357 ± 1492 versus 2615 ± 2541 U/L, P = 0.001 of the NMP and control groups, respectively). No patient developed ischemic type biliary stricture. One patient died, and all other patients are alive and well at a follow-up of 12-35 months. No device-related adverse events were recorded. In conclusion, with this study, we showed that ex situ NMP of human livers can be performed safely and effectively using a noncommercial device and an FFP-based preservation solution. Future studies should further investigate the impact of an FFP-based perfusion solution on liver hemodynamics during ex situ normothermic machine preservation.

Comorbidities and Age in Brain-Dead Donors and Liver Transplantation: A 15-Year Retrospective Investigation.

OBJECTIVES: Although livers from older donors (> 70 y) have been shown to be increasingly more efficiently used for transplant, donor comorbidities are considered additional risk factors. This is quite intriguing as comorbidities are known to increase with advancing age in the donor population. MATERIALS AND METHODS: We assessed whether age and donor comorbidities influenced liver procurement over a 15-year period in a cohort of 1702 brain-dead donors in Tuscany, Italy. RESULTS: Over the study period, age of potential donors significantly increased (P = .02) as well as the proportion of patients who were > 55 years old. The incidence of hypertension, diabetes mellitus, and previously known coronary artery disease also significantly increased. We observed a progressive increase in the number of transplanted livers from donors with advancing age despite an increase in comorbidities. The highest incidences of traumatic brain injury and anoxic brain injury were observed in the youngest donors. Transaminase levels and use of vasoactive drugs were lower in donors who were ≥ 72 years old. CONCLUSIONS: According to our results, criteria for liver donors have already changed. Although age does not seem to be a limiting factor, older donors deserve a more accurate donor selection due to the higher incidence of risk factors (primarily diabetes mellitus).

Successful Transplant of a Nonagenarian Liver Graft With Fully Replaced Right Hepatic Artery Reconstruction.

Organ shortage and increasing donor age in liver transplant are stimulating transplant centers to accept otherwise discarded grafts due to donor age or vascular abnormalities; nevertheless, the use of nonagenarian donor grafts is uncommon because advanced age is associated with a higher risk of ischemic-type biliary lesions and worse long-term graft survival. We herein report the case of a 90-year-old donor with fully replaced right hepatic artery. After back-table vascular assessment, the donor right hepatic artery was anastomosed end-to-end with the gastroduodenal artery with 2 polypropylene 8/0 running sutures. Even if the back-table reconstruction of a replaced right hepatic artery is not associated with an enhanced risk of posttransplant vascular complications, vascular abnormalities might discourage the use of otherwise acceptable elderly grafts. The present case underscores that elderly liver grafts should not be discarded per se even in the presence of vascular variants.

Pilot, Open, Randomized, Prospective Trial for Normothermic Machine Perfusion Evaluation in Liver Transplantation From Older Donors.

Ex situ normothermic machine perfusion (NMP) might minimize ischemia/reperfusion injury (IRI) of liver grafts. In this study, 20 primary liver transplantation recipients of older grafts (≥70 years) were randomized 1:1 to NMP or cold storage (CS) groups. The primary study endpoint was to evaluate graft and patient survival at 6 months posttransplantation. The secondary endpoint was to evaluate liver and bile duct biopsies; IRI by means of peak transaminases within 7 days after surgery; and incidence of biliary complications at month 6. Liver and bile duct biopsies were collected at bench surgery, end of ex situ NMP, and end of transplant surgery. Interleukin (IL) 6, IL10, and tumor necrosis factor α (TNF-α) perfusate concentrations were tested during NMP. All grafts were successfully transplanted. Median (interquartile range) posttransplant aspartate aminotransferase peak was 709 (371-1575) IU/L for NMP and 574 (377-1162) IU/L for CS (P = 0.597). There was 1 hepatic artery thrombosis in the NMP group and 1 death in the CS group. In NMP, we observed high TNF-α perfusate levels, and these were inversely correlated with lactate (P < 0.001). Electron microscopy showed decreased mitochondrial volume density and steatosis and an increased volume density of autophagic vacuoles at the end of transplantation in NMP versus CS patients (P < 0.001). Use of NMP with older liver grafts is associated with histological evidence of reduced IRI, although the clinical benefit remains to be demonstrated.

Lipid metabolism and functional assessment of discarded human livers with steatosis undergoing 24 hours of normothermic machine perfusion.

Normothermic machine perfusion (NMP) is an emerging technology to preserve liver allografts more effectively than cold storage (CS). However, little is known about the effect of NMP on steatosis and the markers indicative of hepatic quality during NMP. To address these points, we perfused 10 discarded human livers with oxygenated NMP for 24 hours after 4-6 hours of CS. All livers had a variable degree of steatosis at baseline. The perfusate consisted of packed red blood cells and fresh frozen plasma. Perfusate analysis showed an increase in triglyceride levels from the 1st hour (median, 127 mg/dL; interquartile range [IQR], 95-149 mg/dL) to 24th hour of perfusion (median, 203 mg/dL; IQR, 171-304 mg/dL; P = 0.004), but tissue steatosis did not decrease. Five livers produced a significant amount of bile (≥5 mL/hour) consistently throughout 24 hours of NMP. Lactate in the perfusate cleared to <3 mmol/L in most livers within 4-8 hours of NMP, which was independent of bile production rate. This is the first study to characterize the lipid profile and functional assessment of discarded human livers at 24 hours of NMP. Liver Transplantation 24 233-245 2018 AASLD.

Use of Elderly Donors in Liver Transplantation: A Paired-match Analysis at a Single Center.

OBJECTIVE: To evaluate the use of elderly donors in liver transplantation (LT) and identify risk factors associated with a worse outcome. SUMMARY BACKGROUND DATA: Use of livers from very old donors could expand the donor pool but is not universally implemented. METHODS: This is a retrospective, single-center medical record review. From January 2001 to December 2014, 1354 LTs were performed. After exclusion of donors <18 years, ABO-incompatible LT, re-LT and UNOS 1 status patients, LT recipients were stratified into 2 groups based on donor age: 18-69 (n=692) vs. ≥70 years (n=515) then matched using a propensity score approach. Two groups were finally matched (young group = 448 cases; old group = 515 cases). RESULTS: The median (interquartile range [IQR]) follow-up was 5.0 (2.0-8.4) years. Comparing the 2 identified groups, no differences were observed regarding early retransplants (1.8 vs. 2.9; P = 0.3), HCV-related death (7.6 vs. 8.7%; P = 0.6), vascular (5.8 vs. 5.0%; P = 0.7), and biliary complications (16.5 vs. 18.6%; P = 0.4). On multivariate analysis, independent risk factors for graft loss were: HCV-positive recipient (HR = 2.1; 95% CI = 1.6-2.7; P < 0.001), donor age (HR = 1.0; 95% CI = 1.0-1.0; P < 0.001), cold ischemia time (HR = 1.0; 95% CI = 1.0-1.0; P = 0.042), and donor history of diabetes mellitus (HR = 1.48; 95% CI = 1.03-2.13; P = 0.036). CONCLUSIONS: Use of elderly donors is not associated per se with an increased risk of vascular and biliary complications. In the presence of cold ischemia time and diabetes mellitus, appropriate donor-to-recipient matching is warranted.

Perfusion machines and hepatocellular carcinoma: a good match between a marginal organ and an advanced disease?

Hepatocellular carcinoma (HCC) accounts for 90% of primary liver cancers, is the second leading cause of cancer-related deaths and the leading cause of death in patients with cirrhosis. Liver transplantation (LT) represents the ideal treatment for selected patients as it removes both the tumor and the underlying cirrhotic liver with 5-year survival rates higher than 70%. Unfortunately, due to tumor characteristics, patient co-morbidities or shortage of organs available for transplant, only 20% of patients can undergo curative treatment. Ex situ machine perfusion (MP) is a technology recently introduced that might potentially improve organ preservation, allow graft assessment and increase the pool of available organs. The purpose of this review is to provide an update on the current role of ex situ liver MP in liver transplantation for HCC patients.