Liver Failure In Pigs Research Articles

An extracorporeal bioartificial liver embedded with 3D-layered human liver progenitor-like cells relieves acute liver failure in pigs.

Clinical advancement of the bioartificial liver is hampered by the lack of expandable human hepatocytes and appropriate bioreactors and carriers to encourage hepatic cells to function during extracorporeal circulation. We have recently developed an efficient approach for derivation of expandable liver progenitor-like cells from human primary hepatocytes (HepLPCs). Here, we generated immortalized and functionally enhanced HepLPCs by introducing FOXA3, a hepatocyte nuclear factor that enables potentially complete hepatic function. When cultured on macroporous carriers in an air-liquid interactive bioartificial liver (Ali-BAL) support device, the integrated cells were alternately exposed to aeration and nutrition and grew to form high-density three-dimensional constructs. This led to highly efficient mass transfer and supported liver functions such as albumin biosynthesis and ammonia detoxification via ureagenesis. In a porcine model of drug overdose-induced acute liver failure (ALF), extracorporeal Ali-BAL treatment for 3 hours prevented hepatic encephalopathy and led to markedly improved survival (83%, n = 6) compared to ALF control (17%, n = 6, P = 0.02) and device-only (no-cell) therapy (0%, n = 6, P = 0.003). The blood ammonia concentrations, as well as the biochemical and coagulation indices, were reduced in Ali-BAL-treated pigs. Ali-BAL treatment attenuated liver damage, ameliorated inflammation, and enhanced liver regeneration in the ALF porcine model and could be considered as a potential therapeutic avenue for patients with ALF.

LBP-520 - Comparison between Li-ALS and Molecular Adsorbents Recirculating System (MARS) for the treatment of acute liver failure in pigs:a randomized controlled study

LBP-520 - Comparison between Li-ALS and Molecular Adsorbents Recirculating System (MARS) for the treatment of acute liver failure in pigs:a randomized controlled study

Evaluation of encapsulated liver cell spheroids in a fluidised-bed bioartificial liver for treatment of ischaemic acute liver failure in pigs in a translational setting.

Liver failure is an increasing problem. Donor-organ shortage results in patients dying before receiving a transplant. Since the liver can regenerate, alternative therapies providing temporary liver-support are sought. A bioartificial-liver would temporarily substitute function in liver failure buying time for liver regeneration/organ-procurement. Our aim: to develop a prototype bioartificial-liver-machine (BAL) comprising a human liver-derived cell-line, cultured to phenotypic competence and deliverable in a clinical setting to sites distant from its preparation. The objective of this study was to determine whether its use would improve functional parameters of liver failure in pigs with acute liver failure, to provide proof-of-principle. HepG2cells encapsulated in alginate-beads, proliferated in a fluidised-bed-bioreactor providing a biomass of 4–6×1010cells, were transported from preparation-laboratory to point-of-use operating theatre (6000miles) under perfluorodecalin at ambient temperature. Irreversible ischaemic liver failure was induced in anaesthetised pigs, after portal-systemic-shunt, by hepatic-artery-ligation. Biochemical parameters, intracranial pressure, and functional-clotting were measured in animals connected in an extracorporeal bioartificial-liver circuit. Efficacy was demonstrated comparing outcomes between animals connected to a circuit containing alginate-encapsulated cells (Cell-bead BAL), and those connected to circuit containing alginate capsules without cells (Empty-bead BAL). Cells of the biomass met regulatory standards for sterility and provenance. All animals developed progressive liver-failure after ischaemia induction. Efficacy of BAL was demonstrated since animals connected to a functional biomass (+ cells) had significantly smaller rises in intracranial pressure, lower ammonia levels, more bilirubin conjugation, improved acidosis and clotting restoration compared to animals connected to the circuit without cells. In the +cell group, human proteins accumulated in pigs' plasma. Delivery of biomass using a short-term cold-chain enabled transport and use without loss of function over 3days. Thus, a fluidised-bed bioreactor containing alginate-encapsulated HepG2cell-spheroids improved important parameters of acute liver failure in pigs. The system can readily be up-scaled and transported to point-of-use justifying development at clinical scale.

Development of a Standardized Model for Liver Failure in Pigs: Anatomopathophysiologic Findings After Extended Liver Resection

Eighteen pigs weighing a mean 19 ± 4 kg, were divided into group 1 (n = 2), that underwent resection of the left lateral lobe, group 2 (n = 2), resection of the left median and right median lobes; and group 3 (n = 18), resection of the left lateral, left median, right median, and right lateral lobes. All animals were followed for 5 days. Liver failure (n = 8) leading to animal death within 3 days after surgery was observed in 65% of group 3, whereas no group 1 or 2 animal experienced liver insufficiency. Multivariate analysis revealed that the extent of liver resection expressed as a percentage of total body weight <2.3%, international normalized ratio > 1.6 as postoperative day 2, serum bilirubin > 4.2 on postoperative day 2, and serum lactates > 9 mmol/L after resection were independent predictors of liver failure (P < .05). The number of resected liver lobes was not a good predictor of liver failure in swine, whereas the extent of resection expressed as a percentage of total body weight was an independent predictor of early liver failure. A resected liver-to-body weight ratio >2.3% was associated with a 65% probability of developing liver insufficiency. This parameter may be useful when developing a model of liver failure after extended liver resection in swine.

Early myocardial injury is an integral component of experimental acute liver failure - a study in two porcine models.

IntroductionThere is accumulating clinical evidence that acute liver failure may be regularly associated with myocardial injury. To test this hypothesis in a standardized experimental setting, we used two porcine models of ALF.Material and methodsIn 14 domestic pigs ALF was induced by either a) surgical devascularization of the liver (DV group, n = 7), or b) partial (70-75%) hepatectomy and ischaemia/reperfusion of the liver remnant for 150 min (I/R group, n = 7). Four additional animals constituted the sham operation group. All animals were monitored for a 12-h period, at the end of which their hearts were harvested. Plasma troponin I (cTnI) and malondialdehyde (MDA) were measured before the operation (baseline) and at 6 h and 12 h postoperatively. The harvested hearts were histologically analysed, appointing a score from 0 (no injury) to 3 (maximum injury) to selected injury indicators.ResultsIn the sham group, all cTnI measurements and total myocardial injury score were zero in all animals. In both ALF groups, plasma cTnI levels increased by the 6th and remained elevated up to the 12th postoperative hour (p < 0.01 vs. sham animals). Total myocardial injury score and total histological score revealed some extent of myocardial injury. The rise of MDA levels suggests an underlying oxidative mechanism.ConclusionsOur study provides direct evidence of early myocardial injury in the setting of acute liver failure in pigs. The mechanism of injury remains to be elucidated.

Laparoscopy-Assisted Creation of a Liver Failure Model in Pigs

We created a hepatic failure pig model that was suitable for the assessment of cell therapies, such as hepatocyte transplantation and bioartificial livers, using a laparoscopic surgical technique. In our model, all of three hepatic arteries were resected, 5, 7.5, or 10 ml of carbon tetrachloride (CCL4) was injected into the liver through the portal vein, and subsequently the portal vein was mechanically occluded for 30 min. After the portal occlusion was released, a liver biopsy was performed, and then the surgery was completed. Blood samples were regularly taken during the surgery in order to perform biochemical examinations. All of five pigs in which 5 ml of CCL4 was infused recovered spontaneously and survived; in contrast, all of five pigs that received 10 ml CCL4 died within 1.5 h after surgery. The pigs in which 7.5 ml CCL4 was administered developed liver failure and survived for 6.4 h on average (+/-1.4 SD). Induction of liver failure with the use of 7.5 ml CCL4 and 30-min hepatic ischemia fulfilled five of the six criteria that were proposed by Terblanche and Hickman: reversibility, reproducibility, death from liver failure, a therapeutic window, and a large-animal model. We believe that our model is the first report on creation of a reliable model for liver failure in pigs to assess the efficacy of liver-targeted cell therapies.

Comparison between bioartificial and artificial liver for the treatment of acute liver failure in pigs

To characterize and evaluate the therapeutic efficacy of bioartificial liver (BAL) as compared to that of continuous hemodiafiltration (CHDF) with plasma exchange (PE), which is the current standard therapy for fulminant hepatic failure (FHF) in Japan. Pigs with hepatic devascularization were divided into three groups: (1) a non-treatment group (NT; n = 4); (2) a BAL treatment group (BAL; n = 4), (3) a PE + CHDF treatment group using 1.5 L of normal porcine plasma with CHDF (PE + CHDF, n = 4). Our BAL system consisted of a hollow fiber module with 0.2 mum pores and 1 multiply 10(10) of microcarrier-attached hepatocytes inoculated into the extra-fiber space. Each treatment was initiated 4 h after hepatic devascularization. The pigs in the BAL and the PE + CHDF groups survived longer than those in the NT group. The elimination capacity of blood ammonia by both BAL and PE + CHDF was significantly higher than that in NT. Aromatic amino acids (AAA) were selectively eliminated by BAL, whereas both AAA and branched chain amino acids, which are beneficial for life, were eliminated by PE + CHDF. Electrolytes maintenance and acid-base balance were better in the CPE + CHDF group than that in the BAL group. Our results suggest that PE + CHDF eliminate all factors regardless of benefits, whereas BAL selectively metabolizes toxic factors such as AAA. However since PE + CHDF maintain electrolytes and acid-base balance, a combination therapy of BAL plus CPE + CHDF might be more effective for FHF.

Standardization of ischemic hepatic failure in pigs as a model for bioartificial liver assessment.

A standard protocol of ischemic liver failure in pigs was examined to establish a system for assessing the efficacy of a bioartificial liver, based on clinical practice. The portal blood flow was extracorporeally bypassed into the cervical jugular vein, using a centrifugal blood pump. The portal vein and hepatic artery were then ligated. The maintenance protocol was established as follows: (1) the concentration of the inhaled anesthetic was decreased by 0.2% when the systolic blood pressure was <100 mmHg; (2) the volume of an infusion containing 5% glucose was increased to 10 ml/kg per hour when central venous pressure was <5 mmHg; (3) 20 ml of 50% glucose was injected intravenously when the blood glucose was <50 mg/dl; (4) 2000 units of heparin was injected intravenously when the activated clotting time was <150 s; (5) sodium bicarbonate was given when the blood pH was <7.3; (6) tidal volume was increased by 1 ml/kg when the pCO(2) was >80 mmHg; (7) oxygen was increased by 25% when the pO(2) was <100 mmHg. No vasopressors were used in the experiment. Our protocol reduced the operating time and minimized the risk of data deviation that can arise from variations in operating techniques and individual animal conditions. This experimental model is also easy to use as a bridge to transplantation.

Development of a new bioartificial liver using a porcine autologous biomatrix as hepatocyte support.

Long-term maintenance of hepatocyte viability and differentiated function expression is crucial for bioartificial liver support. The maintenance of hepatocyte function in a bioreactor is still a problem. A major advance was the recognition that hepatocytes in attachment cultures can maintain their differentiation longer. To restore hepatocyte polarity and prolong their function, we developed a new bioreactor with a cross-flow geometry configuration and an original hepatocyte extracellular autologous biomatrix (Porcine Bio-Matrix) support. To test this new bioreactor, we compared it with a standard bioartificial liver cartridge in a suitable surgical model of acute liver failure in pigs. In our model, we performed a total hepatectomy, followed by partial liver transplantation after an 18 hour anhepatic phase. The results showed that the bioreactor containing the biomatrix was able to bridge the animal to transplantation and to sustain the transplanted liver until all function recovered (80% of animals survived, p = 0.0027). No animal survived more than 24 hours after liver transplantation in the group treated with the traditional bioartificial liver, whereas hepatocyte viability on the Porcine Bio-Matrix was 65% after 12 hours of treatment. The results suggest that our biomatrix is a suitable cell support and guarantees long-term maintenance of metabolic activity of hepatocytes. Further studies are needed, but the results obtained with this new three-dimensional bioreactor are promising, and its potential is attractive.

Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system.

Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration. In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center. Pigs (40-60 kg) were anaesthetised and a total hepatectomy was performed. Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system. Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed. Subsequently, two groups were studied. In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours. In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control. After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated. The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production. However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor.

Hepatocyte transplantation: an experimental study to treat acute liver failure in pigs

Hepatocyte transplantation: an experimental study to treat acute liver failure in pigs

Experimental discordant hepatic xenotransplantation in the recipient with liver failure: implications for clinical bridging trials

Background: Clinical xenotransplantation might start with bridge-to-bridge trials. Situations where hyperacute rejection is avoided would provide opportunities for the initiation of bridging trials. Patients with liver failure have a diminished capacity to initiate antibody and complement-induced injury of xenogeneic endothelium. Hyperacute rejection of a liver xenograft manifests as a coagulopathy. We examined the ability of a recipient with liver failure to hyperacutely reject a liver xenograft in the dog-to-pig model in the immediate postoperative period. Study Design: Liver failure in pigs was induced with galactosamine. Canine livers were transplanted into pigs with liver failure and into healthy pigs. The postoperative course was monitored for 1 hour for histologic changes in the xenograft, changes in platelet counts, and whole blood clotting with Sonoclot analysis. In vitro assays with pig serum and canine hepatic sinusoidal endothelial cells were used to assess the effect of liver failure on serum cytotoxicity and xenoreactive antibody levels. Results: All untreated pig recipients of liver xenografts died from a coagulopathy. Recipients with liver failure manifested no signs of coagulopathy, and had minimal change in platelet counts or Sonoclot (Sienco Inc., Morrison, CO) tracings. Liver xenograft biopsies from recipients with liver failure showed no evidence of the tissue injury that characterized the biopsies of control recipients. Serum from pigs was less cytotoxic to the canine hepatic sinusoidal endothelium after induction of liver failure. The xenoreactive antibody levels and repertoire were similar in the pig serum before and after liver failure was induced. CH 50 (total complement) levels were diminished in pigs after the induction of liver failure. Conclusions: Liver xenotransplantation used in bridging trials in recipients with liver failure might not face the barrier of hyperacute rejection. (J Am Coll Surg 2000;191:54-64. © 2000 by the American College of Surgeons)

Efficacy of a bioreactor filled with porcine hepatocytes immobilized on nonwoven fabric for ex vivo direct hemoperfusion treatment of liver failure in pigs.

We developed a new bioreactor for a bioartificial liver filled with porcine hepatocytes immobilized on polyester nonwoven fabric (NWF) and in our previous study showed that this NWF bioreactor has promising in vitro efficiency. In the present study, we investigated the efficacy of the NWF bioreactor in a direct hemoperfusion experiment conducted to treat pigs with liver failure. Porcine hepatocytes were isolated from the whole liver of a Sangen strain pig. They were immobilized in a 200 ml column containing NWF via perfusion in a closed circuit for 24 h to prepare a NWF bioreactor. The following day an operative liver failure model was produced by creating a portocaval shunt and ligating the entire hepatoduodenal ligament in the porta hepatis. Perfusion treatment was initiated 4 h after operative induction of liver failure and continued for about 1 h. The pigs which underwent perfusion treatment showed significant improvements in survival and blood data, including ammonia, total bile acid, glucose, and prothrombin time, attributed to significant improvements in the post- as compared to the prebioreactor levels in the perfused blood of the treated pigs. These beneficial effects of the NWF bioreactor were based on its excellent composition which allows the accommodation of adequate numbers of hepatocytes and direct contact between hepatocytes and perfused blood.

Treatment of acute liver failure in pigs by auxiliary partial liver transplantation

Treatment of acute liver failure in pigs by auxiliary partial liver transplantation