Bioartificial liver: current status

Liver failure remains a life-threatening syndrome. With the growing disparity between the number of suitable donor organs and the number of patients awaiting transplantation, efforts have been made to optimize the allocation of organs, to find alternatives to cadaveric liver transplantation, and to develop extracorporeal methods to support or replace the function of the failing organ. An extracorporeal liver support system has to provide the main functions of the liver: detoxification, synthesis, and regulation. The understanding that the critical issue of the clinical syndrome in liver failure is the accumulation of toxins not cleared by the failing liver led to the development of artificial filtration and adsorption devices (artificial liver support). Based on this hypothesis, the removal of lipophilic, albumin-bound substances, such as bilirubin, bile acids, metabolites of aromatic amino acids, medium-chain fatty acids, and cytokines, should be beneficial to the clinical course of a patient in liver failure. Artificial detoxification devices currently under clinical evaluation include the Molecular Adsorbent Recirculating System (MARS), Single-Pass Albumin Dialysis (SPAD), and the Prometheus system. The complex tasks of regulation and synthesis remain to be addressed by the use of liver cells (bioartificial liver support). The Extracorporeal Liver Assist Device (ELAD), HepatAssist, Modular Extracorporeal Liver Support system (MELS), and the Amsterdam Medical Center Bioartificial Liver (AMC-BAL) are bioartificial systems. This article gives a brief overview on these artificial and bioartificial devices and discusses remaining obstacles.     

Favorable Effect of New Artificial Liver Support on Survival of Patients with Fulminant Hepatic Failure

Abstract: The two most serious symptoms of fulminant hepatic failure are bleeding and hepatic coma. To overcome these problems, we developed an artificial liver support system comprising a combination of plasma exchange and hemodiafiltration using a high performance membrane. We treated 67 patients with fulminant hepatic failure. Of these, 65 patients (97.0%) regained normal consciousness, and 55 patients (80.9%) were kept alert as long as we continued to apply this system. All 7 patients (100%) with fulminant hepatitis caused by hepatitis A virus infection and 9 of 12 patients (75%) with fulminant hepatitis caused by acute hepatitis B (HB) virus infection survived. In addition, 7 of 15 HB virus carriers (46.7%) who developed fulminant hepatitis and 11 of 29 patients (37.9%) with fulminant hepatitis caused by non-A, non-B hepatitis viruses survived. The overall survival rate was 37 of 67 patients (55.2%). Our artificial liver support system allows as high a survival rate as liver transplantation.     

Hepatic Assist: Present and Future

Fulminant hepatic failure due to acute massive liver cell necrosis is a complex pathophysiological entity, and treatment is still unsatisfactory. Artificial liver supports such as hemodialysis, hemoperfusion, and plasmapheresis have recently been used clinically to treat fulminant hepatic failure. However, survival rate has not improved as expected, although the consciousness of the patient has improved frequently. In this article the present status of clinical artificial liver support and basic research of hybrid artificial liver will be discussed. Moreover, the future aspects of total artificial liver support and hepatocyte transplantation for chronic liver failure will be introduced.     

Nonbiological Liver Support: Historic Overview

Abstract: An effective hepatic assist system could serve as a bridge to transplantation or to treat acute or chronic hepatic failure. Early nonbiological approaches focused on the removal of low molecular weight toxins by dialysis or hemoperfusion, such as over charcoals or resins. This approach led to clinical trials that showed varying degrees of success. Introduction of more porous membranes and blood separation technologies stimulated the development of plasma exchange, on-line plasma fractionation technologies with sorbents and membranes, and other schemes of sorbent-blood interactions based on the principles of dialysis and hemofiltration with sorbent perfusion. Although detoxification of blood has improved the prognosis for acute liver failure, key issues of when to initiate treatment and by which method need to be resolved. In chronic liver disease, blood detoxification can be applied in patients intractable to conventional therapies and for some awaiting transplantation to relieve disease symptoms such as pruritus, jaundice, elevated bile acids, hyperbilirubinemia, endotoxemia, and hypercholesterolemia. Although biological support is considered the ideal, nonbiological techniques can be useful because hepatocytes possess a regenerative capacity and temporary support is helpful. Available nonbiological liver support technologies can substitute for select liver functions in acute and chronic disease.     

The Application of Immobilized Enzymes in an Artificial Liver Support System

A novel method of extracorporeal support for fulminant liver failure is reported whereby the most important detoxification processes of the liver are reproduced in an enzyme reactor. Most of the endogenous toxins involved in hepatic coma can be deactivated directly by conjugation with a hydrophilic residue such as glucuronic acid or glutathione, or by the neutralization of active groups through structural modification by methyl transfer. The enzymes responsible for these processes have been isolated and purified from rabbit liver, and covalently bound onto a hemocompatible form of agarose matrix. This system has been shown to be capable of catalyzing the desired reactions with endogenous toxins such as phenols and mercaptans in vitro, and phenols in rabbits in vivo.     

Extracorporeal blood detoxification by sorbents in treatment of hepatic encephalopathy

Extracorporeal blood detoxification by sorbent therapy long has been applied in treatment of hepatic failure and encephalopathy, starting with hemoperfusion columns and more recently with the currently marketed Liver Dialysis Unit. Liver Dialysis employs hemodiabsorption (dialysis of blood against powdered sorbents including charcoal and cation exchanger) to remove selectively numerous small-molecular-weight toxins of hepatic failure. Liver Dialysis is used in treatment of acute hepatic encephalopathy (AHE) because of decompensation of chronic liver disease (A-on-C) or fulminant hepatic failure (FHF). Controlled, prospective and randomized studies of daily 6-hour Liver Dialysis have shown physiologic and neurologic improvement of patients with AHE, regardless of etiology. Liver dialysis significantly improved the incidence of positive outcomes (recovery of hepatic function or improvement for transplant) of A-on-C patients versus controls (71.5% treated, and 35.7% control, P =.036), but had an insignificant improvement in outcome of patients with FHF as compared with the control group. Other extracorporeal sorbent devices are now in clinical testing phase. The molecular adsorbent regenerating system (MARS) device employs a polysulfone high-permeability dialyzer with albumin on the dialysate side to aid transfer of protein-bound toxins such as bilirubin and bile acids across the membranes. Sorbent columns of charcoal and an anion exchanger remove hepatic toxins from the albumin dialysate, and a second dialyzer removes water-soluble toxins, such as ammonium. Clinical results of daily MARS treatments of patients with hepatic failure are similar to that of Liver Dialysis, with neurologic and physical improvement occurs in most patients with AHE, and improved outcome for patients with A-on-C. The system extends the life of patients with hepatorenal syndrome. PF-Liver Dialysis is an experimental device combining hemodiabsorption with push-pull sorbent-based pheresis with powdered sorbent surrounding plasmafilters. PF-Liver Dialysis (Hemocleanse, Inc, W. Lafayette, IN) has been tested in a few patients with hepatic failure, grade 3-4 encephalopathy, and respiratory and kidney insufficiency. Treatments appeared to be safe and resulted in marked decreases in plasma levels of bilirubin, aromatic amino acids, ammonium, creatinine, and interleukin-1beta (IL-1beta). The PF add-on module adds the capability to Liver Dialysis to remove bilirubin, bile acids, and other strongly protein-bound toxins from treated patients and may be of clinical benefit in management of patients with the most severe hepatic failure and encephalopathy, including patients with FHF or concomitant sepsis.     

An ideal liver support system and xenotransplantation

Currently, the rapid increase in the number of candidates for orthotopic liver transplantation has resulted in a shortage of donor organs and, especially for fulminant hepatic failure, an urgent need for transplantations. Thus the need for a liver support system as a "bridge" before liver transplantation is also urgent, as is the need for treatment of reversible acute liver disease. Various liver support systems have been proposed, for example, cross-circulation systems, extracorporeal liver support systems, hemodialysis, and hemadsorption, but these are not considered to function sufficiently. Demetriou's system, Sussman's system, and Gerlach's system have reached clinical trials. The ideal liver support system should have significant metabolic capacity, the ability to be used in continuous treatment, simplicity of use, biocompatibility, ready availability, consistency, economy, and safety from viral infection, but no system meets all of these criteria.     

Artificial liver

An artificial liver should in fact be called an artificial liver assist device or system because at this point in its development it is unable to prolong the life of an ahepatic animal, whereas, an artificial heart or an artificial kidney enables the animal to live without a heart or kidneys for a long period of time. The hepatic assist devices are classified into three types: Artificial (charcoal hemoperfusion, PAN membrane dialysis or filtration); biological (baboon liver perfusion, cross dialysis between pig liver and patients systemic circulation); and hybrid (combined form of artificial and biological). Our hepatic support system is composed of a membrane plasma separator, blood and plasma pumps, hemodialyzer and controller. Using this system, the patients plasma is replaced with fresh donor plasma in amount of 5,000 ml daily. This procedure are taken place in the intensive care unit, until the patient recovers consciousness or his cerebral death is confirmed. A national survey of the patients with fulminant hepatic failure, revealed that the survival rate of the patients treated with plasma exchange was 34.1% (15/45), while that of the patient untreated with plasma exchange was 14.3% (5/35). The difference is statistically significant. However, plasma exchange requires a large amount of fresh plasma which occasionally induce hepatitis or allergy and its detoxication of the patients plasma was insufficient in severe cases. To overcome these problems, specific adsorpton of hepatic toxins and a combined therapy of blood purification with plasma exchange will be studied further.     

The molecular adsorbents recycling system as a liver support system based on albumin dialysis: a summary of preclinical investigations, prospective, randomized, controlled clinical trial, and clinical experience from 19 centers

Artificial liver support aims to prolong survival time of patients with liver failure by detoxification. Albumin as a molecular adsorbent in dialysis solution is capable of attracting even tightly albumin-bound toxins from blood into the dialysate if a specific dialysis membrane is used and if the albumin's binding sites are on-line-purified by a sorbent/dialysis-based recycling system (i.e., molecular adsorbents recycling system, or MARS). The MARS technology has been shown to remove water-soluble and albumin-bound toxins and to provide renal support in case of renal failure. Fourteen centers have reported that MARS treatment improved mental status of patients with liver failure and hepatic encephalopathy. In treating liver failure and cholestasis, MARS was associated with hemodynamic stabilization, improvement of hepatic and kidney function, and disappearance of pruritus. In hepatic failure and hepatorenal syndrome, a prospective, randomized, controlled trial of MARS treatment was able to prolong survival time significantly. MARS has been used in 26 patients with acute liver failure or primary graft dysfunction. Nineteen centers reporting on 103 patients have shown that MARS treatment is safe, easy to handle, feasible, and effective.     

Hemodynamic improvement as an additional parameter to evaluate the safety and tolerability of the molecular adsorbent recirculating system in liver failure patients

BACKGROUND: The molecular adsorbent recirculating system (MARS) is an extracorporeal acute liver failure (ALF) support system method using albumin-enriched dialysate to remove albumin-bound toxins. PATIENTS AND METHODS: Since 1999 we performed 2027 MARS treatments in 191 patients: 39 fulminant hepatic failure (FHF), 16 primary nonfunction (PNF), 21 delayed function (DF), 94 acute-on-chronic liver failure (AoCHF), 7 post-hepatic resection, and 14 intractable pruritus. RESULTS: We divided the complications by the AoCHF versus the ALF populations. Among 83 ALF patients, we observed worsening of hemodynamic parameters in 16 patients: 3 with PNF, 2 with DF without retransplantation, 9 with FHF, and 2 after hepatic resection. Among 94 AoCHF patients, 42 showed hemodynamic instability requiring intensive care unit support. Our study did not note significant adverse effects (1.8%), except for infections and hemorrhage from the central venous catheter not due to MARS treatment. The thrombocytopenia was controlled through administration of platelets before the start of treatment when a patient showed a level under 30,000 mm(3). CONCLUSION: Our results confirmed that nonbiological hepatic support by MARS was safe and tolerable.     

Liver transplantation for fulminant hepatitis

Liver transplantation has been recognized as an effective therapeutic method for end-stage liver disease in Japan. Fulminant hepatic failure is also an indication for liver transplantation, and the number of patients undergoing liver transplantation has been increasing. Reversibility and urgency are characteristics of fulminant hepatitis. If given appropriate critical support, many patients recover spontaneously. However, many patients develop cerebral edema or multiorgan failure before the liver can regenerate. Indications, operative procedures, and outcome of liver transplantation for fulminant hepatitis are discussed here. At Shinshu University, 23 of 169 cases of liver transplantation were for fulminant hepatitis. One transplantation was from a cadaveric donor and 22 from living donors. The actuarial 5-year patient and graft survival rate was 85.4%. Although some problems remain in liver transplantation for fulminant hepatitis, the results are better than those of conventional therapy. Therefore patients with fulminant hepatic failure should be listed for liver transplantation when grade 2 hepatic encephalopathy develops. Moreover, in cases of severe acute hepatitis, i.e., before patients develop grade 2 encephalopathy, liver transplantation should be considered among choices of therapy in the near future.     

Galactosamine-induced fulminant liver failure--observation in a porcine model

Fulminant hepatic failure can only be treated successfully by liver transplantation, which, however, is not always available. To "bridge" the patient with fulminant hepatic failure until a liver graft is available, various forms of liver support devices had been designed but they were not uniformly successful. To prove the efficacy of a liver support device for fulminant hepatic failure, testing in an animal model is necessary. We attempted to induce a pig model with fulminant hepatic failure by administering galactosamine into pigs and reported the observation. Three pigs were given a dose of 0.5 gm/kg of galactosamine and five pigs were given 1 gm/kg of galactosamine. One pig receiving 0.5 gm/kg galactosamine survived after manifestation of liver failure, while all the other pigs died. The two pigs receiving 0.5 gm/kg galactosamine survived longer than the five pigs receiving 1 gm/kg galactosamine. Before death, a significant elevation of parenchymal liver enzymes, lactate dehydrogenase, bilirubin, bile acid, ammonia, tumour necrosis factor-alpha, activated clotting time, a decrease of platelet concentration, ketone bodies ratio, blood glucose and plasma albumin, and serious impairment of indocyanine green clearance were indicated. At post-mortem, severe liver necrosis was observed. The model may be suitable for testing the efficacy of liver support device for fulminant hepatic failure, preferably 24-48 hours after administration of galactosamine.     

Liver support technology--an update

BACKGROUND: Currently, there is no direct treatment for hepatic failure, and patients must receive a transplant or endure prolonged hospitalization, with significant morbidity and mortality. Because of the scarcity of donor organs, liver support strategies are being developed with the aim of either supporting patients with borderline functional liver cell mass until an appropriate organ becomes available for transplantation or until their livers recover from injury. METHODS: A literature review was performed using MEDLINE and library searches. Only major blood detoxification/purification devices and cell-based techniques are included in this review. RESULTS: Currently, a number of blood purification systems and devices utilizing viable liver cells are in various stages of clinical development. Non-biological systems include plasma exchange, albumin dialysis, hemo(dia)filtration, and sorbent-based devices (charcoal, resin). These systems are able to remove toxins of hepatic failure, and their utility is limited by their inability to provide missing liver-specific functions. In contrast, hepatocyte-based devices are able to provide whole liver functions, including detoxification, biosynthesis, and biotransformation. Molecular adsorbent recycling system (MARS) blood detoxification system has been tested in thousands of patients, but additional well-conducted controlled studies are warranted to better define the role of MARS in the treatment of patients with acute hepatic failure and acute exacerbation of chronic liver disease. HepatAssist was tested in a phase II/III controlled clinical trial that demonstrated safety and proof of concept for use of biological liver support systems to improve patient survival in acute hepatic failure. CONCLUSIONS: Developing an effective liver assist technology has proven difficult, because of the complexity of liver functions that must be replaced, as well as heterogeneity of the patient population. Non-biological systems may have a role in the treatment of specific forms of liver failure where the primary goal is to provide blood detoxification/purification. Biological systems appear to be useful in treating liver failure where the primary objective is to provide whole liver functions which are impaired or lost. It is suggested that there will be a role for hybrid liver support systems that offer liver cell therapy and various forms of blood purification (sorption, hemofiltration and diafiltration) to treat patients with specific forms of liver failure at various stages of their illness.     

The present status and the future of artificial liver support

Orthotopic liver transplantation, started by Dr. Starzl in 1963, has already become an well established therapeutic method for the treatment of fulminant hepatic failure especially since the introduction of Cyclosporin A. On the other hand, hemoadsorption using charcoal, PAN membrane hemodialysis and plasma exchange were actively applied for clinical cases with fulminant hepatic failure between 1960 and 1970, but the survival rates were not so improved as expected due to the lack of metabolic functions. Hybrid artificial liver has been therefore investigated experimentally to support metabolic functions by using biomaterials such as isolated hepatocytes. Today many efforts are being made to keep hepatocytes highly viable for a long period of time by calcium alginate entrapment, spheroid formation, or by using biomatrix, some polymers, and microcarriers, etc. In the future, xeno-hepatocytes or so-called super cell will be realized and applied for artificial liver support with the development of gene operation techniques.     

Intensive care support therapy

Patients with small-for-size syndrome (SFSS) and acute liver failure share some important clinical features that are paralleled by common approaches to their intensive care unit management. Both are characterized by a period of acute hepatic insufficiency, with clinical features reflecting the impairment of metabolic and immunologic function that results. The basic principles of management of the two conditions remain essentially the same: to support hepatic regeneration, to anticipate and prevent the development of complications, and to identify patients unlikely to survive early in their clinical course so that retransplantation may be considered. Many treatments are available in the intensive care unit to overcome biochemical and metabolic disturbances in acute liver failure. Optimal pharmacologic management of SFSS complicated by portal hypertension and variceal hemorrhage is currently uncertain. Extracorporeal liver support has several theoretical attractions in the critically ill patient with SFSS, through its ability by removal of hepatotoxins to provide an environment more conducive to hepatic regeneration and recovery, or to support and bridge the patient to transplantation. The molecular adsorbent recycling system has been proposed to remove both water-soluble and protein-bound toxins. This system is particularly attractive in the treatment of SFSS, however, despite its current clinical application, there are presently limited published data to support its use.     

Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure

OBJECTIVE: The HepatAssist liver support system is an extracorporeal porcine hepatocyte-based bioartificial liver (BAL). The safety and efficacy of the BAL were evaluated in a prospective, randomized, controlled, multicenter trial in patients with severe acute liver failure. SUMMARY BACKGROUND DATA: In experimental animals with acute liver failure, we demonstrated beneficial effects of the BAL. Similarly, Phase I trials of the BAL in acute liver failure patients yielded promising results. METHODS: A total of 171 patients (86 control and 85 BAL) were enrolled. Patients with fulminant/subfulminant hepatic failure and primary nonfunction following liver transplantation were included. Data were analyzed with and without accounting for the following confounding factors: liver transplantation, time to transplant, disease etiology, disease severity, and treatment site. RESULTS: For the entire patient population, survival at 30 days was 71% for BAL versus 62% for control (P = 0.26). After exclusion of primary nonfunction patients, survival was 73% for BAL versus 59% for control (n = 147; P = 0.12). When survival was analyzed accounting for confounding factors, in the entire patient population, there was no difference between the 2 groups (risk ratio = 0.67; P = 0.13). However, survival in fulminant/subfulminant hepatic failure patients was significantly higher in the BAL compared with the control group (risk ratio = 0.56; P = 0.048). CONCLUSIONS: This is the first prospective, randomized, controlled trial of an extracorporeal liver support system, demonstrating safety and improved survival in patients with fulminant/subfulminant hepatic failure.     

Hepatoprotective Effect of an Immortal Human Fetal Hepatic Cell Transplantation on CCL4-Induced Acute Liver Injury in Mice

Hepatocyte transplantation has been widely confirmed in the animal model experiments as an effective method for treatment of fulminant hepatic failure. However, the lack of donor organs remains a major problem. One solution is the development of transplantable hepatocytes. Herein we have transplanted intraperitoneally an established immortalized human fetal hepatic cell line (HL-7702) into CCl₄-treated mice with acute liver injury to determine whether they provided life-saving metabolic support. The results showed lower levels of blood ammonia and higher content of liver albumin (P < .05) after HL-7702 transplantation versus nontransplanted controls at days 3 and 7. Histologic examination showed the transplantation group to be less affected at day 7 with no difference at day 14. In conclusion, an established immortal human fetal hepatic cell line may be a promising cell source providing life-saving metabolic support as a bioartificial liver device for the treatment of acute liver injury.     

Enzymatic Detoxification Using Lipophilic Hollow-Fiber Membranes: III. Oxidation Reactions of Sulfides

A lipophilic hollow-fiber membrane preparation that was previously described for glucuronidation and sulfation reactions was used for the enzymatic oxidation of sulfides. Endogenous and exogenous toxins in buffer solution or in serum or blood of intoxicated animals were circulated through the internal lumen of lipophilic hollow fibers. Native liver microsomes of the rabbit were circulated on the outside of the hollow fibers. Lipophilic toxins accumulate in and penetrate through the lipophilic membrane and the toxins are oxidized by the mixed function oxygenase system of liver microsomes. The oxidized products cannot rediffuse to the donor side. The endogenous toxin dimethylsulfide (DMS) was converted on the enzyme side to dimethylsulfoxide (DMSO) and small amounts of dimethylsulfone, which are hydrophilic and nontoxic substances. Other sulfide compounds, ethylmethylsulfide (EMS) and s-butylmethylsulfide (BMS), have also been converted to their oxidized forms. The enzymatic clearance of the hollow-fiber module for DMS in in vivo experiments in the rabbit was found to be 1.30 nmol/h/mg protein/cm2 hollow-fiber surface. The transmembranous enzymatic clearance of the in vitro oxidation reactions of DMS, EMS, and BMS in buffer solutions (open circuit) were measured, respectively, as 1.63, 3.45, and 5.16 nmol/h/mg protein/cm2 hollow-fiber surface. This technique allows the enzymatic oxidation of sulfur compounds with liver microsomes in vitro and in vivo without immunological hazards, and it is suited for artificial liver support.     

Combined twice-daily plasma exchange and continuous veno-venous hemodiafiltration for bridging severe acute liver failure

Aiming to remove the toxins produced during the course of severe hepatic failure, we combined hemodiafiltration and plasma exchange (patient plasma replaced by fresh frozen plasma in a twice-daily regimen) for treatment of five patients: two affected by primary nonfunction of a liver graft and three by fulminant hepatic failure. The simultaneous use of the two extracorporeal techniques allowed a rapid reduction in the administration of vasoactive drugs and a rapid, significant decrease in the indices of liver necrosis. Native liver functional recovery occurred in one case, and the wait for a second graft was made possible in the other four. Although it has been reported that the detoxifying efficacy of plasma exchange is optimal when the replaced volume of plasma is high, such a technique requires both long treatment times and high blood flows in the extracorporeal circuit, making it often hemodynamically intolerable. Our approach leads to replacement of smaller volumes, allowing lower blood flows that are better tolerated despite the often unstable hemodynamics of these patients. Liver transplantation and retransplantation remains the definite therapy for severe liver failure or primary nonfunction. However, the organ waiting time is unpredictable and often does not coincide with the patients' clinical needs. Thus alternative strategies must be developed until a suitable donor is found or there is spontaneous recovery. From this point of view, in our albeit limited experience, twice-daily plasma exchange combined with hemodiafiltration has proved to be an effective therapeutic approach.     

Auxiliary Partial Orthotopic Living Donor Liver Transplantation: Kyoto University Experience

Auxiliary partial orthotopic liver transplantation (APOLT) was initially indicated as a potentially reversible fulminant hepatic failure and non-cirrhotic metabolic liver disease to compensate for enzyme deficiency without complete removal of the native liver. We expand our indication of APOLT for small-for-size grafts to support the function of implanted grafts during the early post-operative period, and for ABO-incompatibility to sustain a patient's life if the patient has a graft failure. We retrospectively reviewed 31 patients undergoing APOLT from living donor. The indication of APOLT was fulminant hepatic failure in 6, non-cirrhotic metabolic liver disease in 6, small-for-size grafts in 13 and ABO-incompatible cases in 6. The cumulative survival rate for APOLT at 1 and 5 years was 57.9% and 50.6%, and 78.8% and 73.8% for standard LDLT. None of the patients who underwent transplantation with APOLT for fulminant hepatic failure had long-term patient survival. The incidence of acute cellular rejection was higher in APOLT (58.1%) than standard LDLT (35.0%). Biliary complication was higher and the need for retransplantation was greater in APOLT than standard LDLT (p < 0.01). The results suggest that the indications of APOLT should be reconsidered in view of the risk for complications and retransplantation.