- Email: [email protected]
Bioartificial Liver: Current Status
Bioartificial Liver: Current Status G. Pless and I.M. Sauer ABSTRACT 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 (AMCBAL) are bioartificial systems. This article gives a brief overview on these artificial and bioartificial devices and discusses remaining obstacles.
A
CUTE liver failure is a life-threatening state, marked by loss of detoxification (eg, degradation of ammonia, bilirubin), regulation, and synthetic function (eg, clotting factors, albumin) of the liver, resulting in coagulation and blood circulation dysfunction, encephalopathy, and often followed by multi-organ failure. There are a variety of causes, including viral infections (hepatits B or C) and intoxication by pharmaceuticals such as acetaminophen or by other substances (Amanita phalloides, ecstasy). So far, the only effective long-term treatment is orthotopic liver transplantation. Because organ availability is limited and contraindications for liver transplantation may exist, it is necessary to find alternative ways to temporarily assist the diseased organ until a suitable graft arrives (“bridging-totransplantation”) or, ideally, until the liver is able to regenerate and regain its full function (“bridging-to-regeneration”). A liver assist device should be able to detoxify and regulate as well as synthesize molecules in the fashion of the liver. Accumulating toxins, water-soluble (eg, ammonia, mercaptanes) as well as water-insoluble (eg, bile acids,
bilirubin, short chain fatty acids, aromatic amino acids), which are believed to be involved in the pathophysiology of hepatic encephalopathy and organ failure, have to be removed. This task can be addressed by artificial systems, mainly based on the principles of dialysis and adsorbent techniques. To achieve synthetic and optimal regulatory functions, the use of biological components, ie, liver cells, is necessary. ARTIFICIAL LIVER ASSIST DEVICES
Various techniques for artificial detoxification have been evaluated: hemodiafiltration, hemoperfusion, plasmapheresis, and hemodialysis have been used to remove toxins from patient blood via filtration or adsorption. So far, no survival From the Charité, Campus Virchow, General, Visceral and Transplantation Surgery, Berlin, Germany. Address reprint requests to Gesine Pless, Charité, Campus Virchow, General, Visceral and Transplantation Surgery, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: gesine. [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.113
Transplantation Proceedings, 37, 3893–3895 (2005)
3893
3894
benefit has been proven for most of these techniques.1 To clear the blood of albumin-bound, hydrophobic substances with dialysis techniques, adsorbent or acceptor substances are necessary to enhance mass exchange. Albumin is one of the potential acceptor substances. The Molecular Adsorbents Recirculation System (MARS; Gambro Hospal GmbH, Planegg-Martinsried, Germany) is a detoxification system based on albumin dialysis. Separated from patient blood by a high-flux hemodialysis filter, an albumin solution is circulated in a closed circuit. The albumin acts as the acceptor for the toxins. It is partly regenerated by passing an anion exchanger and a charcoal adsorber in a closed circuit, which is itself dialyzed.2 Single-Pass Albumin Dialysis (SPAD) is a noncommercial simple method of albumin dialysis using standard renal replacement therapy machines without an additional perfusion pump system. The patient’s blood flows through a circuit with a high- flux hollow fiber hemodiafilter, identical to that used in the MARS system. The other side of this membrane is cleansed with an albumin solution in counterdirectional flow, which is, instead of being regenerated as in the MARS concept, discarded after passing the filter. Continuous veno-venous hemodiafiltration (CVVHDF) can additionally be performed using the same filter. SPAD and MARS were compared in vitro with regard to detoxification capacity. SPAD showed significantly greater reduction of ammonia compared with MARS. No significant differences were observed between SPAD and MARS concerning other water-soluble substances. SPAD enabled a significantly greater bilirubin reduction than MARS. Concerning the reduction of bile acids, no significant differences between SPAD and MARS were seen.3 The Prometheus system (Fresenius Medical Care AG, Bad Homburg, Germany) is a new device based on the combination of direct albumin adsorption with high-flux hemodialysis after selective filtration of the albumin fraction through a specific polysulfon filter.4 BIOARTIFICIAL LIVER ASSIST DEVICES
Bioartificial liver assist devices use liver cells to address specific metabolic tasks. In extracorporeal liver perfusion (ECLP), an explanted liver of human or xenogeneic origin is connected to the patients’ blood circulation outside the body.5 Because the liver has to be freshly explanted for this purpose, the concept is logistically unsustainable. Therefore, different types of bioreactors have been designed to keep isolated liver cells in culture for prolonged periods, making them available for extracorporeal liver support. They commonly consist of bundles of hollow fibers that can be perfused by liquid or gas via external ports. For bioreactor cultures, various cell sources have been evaluated. Porcine hepatocytes are easily available in large quantities, but bear the risk of zoonoses (for example porcine endogenous retrovirus [PERV] or herpes species) and metabolic incompatibility. Human tumor cell lines can be easily expanded to large quantities, but have the disadvantage of
PLESS AND SAUER
poor metabolic performance and potential metastatic ability. Primary human cells meet all the demands of compatibility but are not available in appropriate quantities and originate from histologically impaired organs. The extracorporeal liver assist device (ELAD) uses about 200 g of cells of the human hepatoblastoma cell line C3A (derived from HepG2) in modified dialysis cartridges. The cells are located in the extracapillary space separated from plasma by the capillary membranes. Prior to entering the bioreactor, the plasma passes a charcoal adsorber and a membrane oxygenator. First clinical applications were performed, to demonstrate the safety of the system.6,7 HepatAssist is a system that uses 5–7 ⫻ 109 cryopreserved porcine hepatocytes in a similar setting. A large randomized, controlled multicenter study with a total of 171 patients (86 in the control group and 85 in the bioartificial liver treatment group) was conducted by Demetriou et al.8 Patients with fulminant/subfulminant hepatic failure and primary nonfunction following liver transplantation were included in the study, demonstrating the safety of the system and an improved 30-day survival in a subgroup. The modular extracorporeal liver support (MELS) system is based on the Cell Module,9 a unit consisting of 3 interwoven capillary bundles in a polyurethane housing. One of the bundles serves as decentralized oxygenation; 2 bundles are used for perfusion with patient plasma. It is operated with primary porcine hepatocytes as well as human hepatocytes isolated from discarded donor organs.10 For MELS, the CellModule is combined with SPAD and continuous veno-venous hemodiafiltration (CVVHDF). A phase 1 clinical study was performed with the CellModule charged with porcine hepatocytes11 and 12 patients were treated with human hepatocytes.12 Within the Amsterdam Medical Center Bioartificial Liver (AMC-BAL), in contrast to all other mentioned systems, the capillary membranes exclusively serve oxygenation. The cell compartment of the device, which has a polyester matrix, is loaded with about 200 g of primary porcine hepatocytes. During therapy, the matrix is directly perfused by patient plasma.13 A phase 1 study showed the safety of the treatment.14 FURTHER DEVELOPMENT IN BIOARTIFICIAL LIVER ASSIST DEVICES
Bioartificial liver assist devices are limited in performance mainly by 3 factors: liver cell mass, mass exchange over the membranes, and plasma/blood flow rate in the system.15 Currently, cell mass ranges from about 50 –500 g, which is at most about a third of the average liver mass of an adult. Experience from living donor liver transplantation shows that a minimum cell mass of about 40% of the patient’s ideal liver mass (approximately 1500 g) is necessary to assure proper graft function. Furthermore, cells in most devices are not in direct contact with patient plasma or blood, but separated by 1 or even 2 capillary membranes. Thus, the mass exchange is additionally limited by the
BIOARTIFICIAL LIVER
membrane cutoff. In situ, 1.5 L of blood are processed per minute by the liver. The flow in a bioartificial liver device, however, is limited to about 100 –300 mL/min for technical and rheological reasons, eg, plasma separation rates and shear stress. The most urgent problem is an appropriate cell source. Before establishing a reliable, safe, highly metabolically active, and easily expandable human cell source, clinical success of a bioartificial liver support systems is unlikely. Research on human hepatic progenitor cells, embryonic stem cells, or fetal stem cells might offer the necessary expandable and metabolically active cell sources. Bearing in mind the technical limitations, the combination of artificial and bioartificial support concepts appears to be promising. To date, scientific efforts concentrate on either artificial detoxification systems (such as albumin dialysis, adsorber suspension with activated charcoal particles, and ion exchange resin) or biological systems (bioreactors with liver cells). To address the conceptual limitations, the HepatAssist system (bioreactor ⫹ charcoal adsorber) and MELS (bioreactor ⫹ SPAD) combine biological components with detoxification techniques. None of the liver cell– based systems has been clinically evaluated in as extensive a way as Demetriou’s HepatAssist system. Therefore, no conclusions concerning their clinical efficiency and benefit for patients can be drawn based on existing data. Twelve trials on artificial or bioartificial support systems versus standard medical therapy (483 patients) and 2 trials comparing different artificial support systems (105 patients) were included into the latest Cochrane Review concerning artificial and bioartificial liver support systems. Their analysis indicates that artificial support systems may reduce mortality in acute-on-chronic liver failure. Artificial and bioartificial support systems did not appear to affect mortality in acute liver failure. Designers of clinical studies are asked to compare the therapeutic potential of one support system with standard medical therapy before such systems are introduced as control interventions to more sophisticated support systems. Such trials should be adequately designed, including centralized randomization (using stratification for important prognostic factors) as well as blinded outcome assessment.16
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REFERENCES 1. Rifai K, Bahr MJ: Akutes Leberversagen. Internist 44:585, 2003 2. Heemann U, Treichel U, Loock J, et al: Albumin dialysis in cirrhosis with superimposed acute liver injury: a prospective, controlled study. Hepatology 36:949, 2002 3. Sauer IM, Goetz M, Steffen I, et al: In vitro comparison of the molecular adsorbent recirculation system (MARS) and single-pass albumin dialysis (SPAD). Hepatology 39:1408, 2004 4. Rifai K, Ernst T, Kretschmer U, et al: Prometheus—a new extracorporeal system for the treatment of liver failure. J Hepatol 39:984, 2003 5. Pascher A, Sauer IM, Hammer C, et al: Extracorporal liver perfusion as hepatic assist in acute liver failure: a review of world experience. Xenotransplantation 9:309, 2002 6. Sussman NL, Gislason GT, Conlin CA, et al: The Hepatix extracorporeal liver assist device: initial clinical experience. Artif Organs. 18:390, 1994 7. Millis JM, Cronin DC, Johnson R, et al: Initial experience with the modified extracorporeal liver-assist device for patients with fulminant hepatic failure: system modifications and clinical impact. Transplantation 74:1735, 2002 8. Demetriou AA, Brown RS Jr, Busuttil RW, et al: Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure. Ann Surg 239:660, 2004 9. Gerlach JC, Encke J, Hole O, et al: Bioreactor for a larger scale hepatocyte in vitro perfusion. Transplantation 58:984, 1994 10. Sauer IM, Zeilinger K, Obermayer N, et al: Primary human liver cells as source for modular extracorporeal liver support—a preliminary report. Int J Artif Org 25:1001, 2002 11. Sauer IM, Kardassis D, Zeillinger K, et al: Clinical extracorporeal hybrid liver support—phase I study with primary porcine liver cells. Xenotransplantation 10:460, 2003 12. Sauer IM, Zeilinger K, Pless G, et al: Extracorporeal liver support based on primary human liver cells and albumin dialysis— treatment of a patient with primary graft non-function. J Hepatol 39:649, 2003 13. Flendrig LM, la Soe JW, Jorning GG, et al: In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates. J Hepatol 26:1379, 1997 14. van de Kerkhove MP, Di Florio E, Scuderi V, et al: Phase I clinical trial with the AMC-bioartificial liver. Int J Artif Organs 25:950, 2002 15. Iwata H, Ueda Y: Pharmacokinetic considerations in development of a bioartificial liver. Clin Pharmacokinet 43:211, 2004 16. Liu JP, Gluud LL, Als-Nielsen B, et al: Artificial and bioartificial support systems for liver failure. Cochrane Database Systematic Reviews:CD003628.pub2, 2004
A
CUTE liver failure is a life-threatening state, marked by loss of detoxification (eg, degradation of ammonia, bilirubin), regulation, and synthetic function (eg, clotting factors, albumin) of the liver, resulting in coagulation and blood circulation dysfunction, encephalopathy, and often followed by multi-organ failure. There are a variety of causes, including viral infections (hepatits B or C) and intoxication by pharmaceuticals such as acetaminophen or by other substances (Amanita phalloides, ecstasy). So far, the only effective long-term treatment is orthotopic liver transplantation. Because organ availability is limited and contraindications for liver transplantation may exist, it is necessary to find alternative ways to temporarily assist the diseased organ until a suitable graft arrives (“bridging-totransplantation”) or, ideally, until the liver is able to regenerate and regain its full function (“bridging-to-regeneration”). A liver assist device should be able to detoxify and regulate as well as synthesize molecules in the fashion of the liver. Accumulating toxins, water-soluble (eg, ammonia, mercaptanes) as well as water-insoluble (eg, bile acids,
bilirubin, short chain fatty acids, aromatic amino acids), which are believed to be involved in the pathophysiology of hepatic encephalopathy and organ failure, have to be removed. This task can be addressed by artificial systems, mainly based on the principles of dialysis and adsorbent techniques. To achieve synthetic and optimal regulatory functions, the use of biological components, ie, liver cells, is necessary. ARTIFICIAL LIVER ASSIST DEVICES
Various techniques for artificial detoxification have been evaluated: hemodiafiltration, hemoperfusion, plasmapheresis, and hemodialysis have been used to remove toxins from patient blood via filtration or adsorption. So far, no survival From the Charité, Campus Virchow, General, Visceral and Transplantation Surgery, Berlin, Germany. Address reprint requests to Gesine Pless, Charité, Campus Virchow, General, Visceral and Transplantation Surgery, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: gesine. [email protected]
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.113
Transplantation Proceedings, 37, 3893–3895 (2005)
3893
3894
benefit has been proven for most of these techniques.1 To clear the blood of albumin-bound, hydrophobic substances with dialysis techniques, adsorbent or acceptor substances are necessary to enhance mass exchange. Albumin is one of the potential acceptor substances. The Molecular Adsorbents Recirculation System (MARS; Gambro Hospal GmbH, Planegg-Martinsried, Germany) is a detoxification system based on albumin dialysis. Separated from patient blood by a high-flux hemodialysis filter, an albumin solution is circulated in a closed circuit. The albumin acts as the acceptor for the toxins. It is partly regenerated by passing an anion exchanger and a charcoal adsorber in a closed circuit, which is itself dialyzed.2 Single-Pass Albumin Dialysis (SPAD) is a noncommercial simple method of albumin dialysis using standard renal replacement therapy machines without an additional perfusion pump system. The patient’s blood flows through a circuit with a high- flux hollow fiber hemodiafilter, identical to that used in the MARS system. The other side of this membrane is cleansed with an albumin solution in counterdirectional flow, which is, instead of being regenerated as in the MARS concept, discarded after passing the filter. Continuous veno-venous hemodiafiltration (CVVHDF) can additionally be performed using the same filter. SPAD and MARS were compared in vitro with regard to detoxification capacity. SPAD showed significantly greater reduction of ammonia compared with MARS. No significant differences were observed between SPAD and MARS concerning other water-soluble substances. SPAD enabled a significantly greater bilirubin reduction than MARS. Concerning the reduction of bile acids, no significant differences between SPAD and MARS were seen.3 The Prometheus system (Fresenius Medical Care AG, Bad Homburg, Germany) is a new device based on the combination of direct albumin adsorption with high-flux hemodialysis after selective filtration of the albumin fraction through a specific polysulfon filter.4 BIOARTIFICIAL LIVER ASSIST DEVICES
Bioartificial liver assist devices use liver cells to address specific metabolic tasks. In extracorporeal liver perfusion (ECLP), an explanted liver of human or xenogeneic origin is connected to the patients’ blood circulation outside the body.5 Because the liver has to be freshly explanted for this purpose, the concept is logistically unsustainable. Therefore, different types of bioreactors have been designed to keep isolated liver cells in culture for prolonged periods, making them available for extracorporeal liver support. They commonly consist of bundles of hollow fibers that can be perfused by liquid or gas via external ports. For bioreactor cultures, various cell sources have been evaluated. Porcine hepatocytes are easily available in large quantities, but bear the risk of zoonoses (for example porcine endogenous retrovirus [PERV] or herpes species) and metabolic incompatibility. Human tumor cell lines can be easily expanded to large quantities, but have the disadvantage of
PLESS AND SAUER
poor metabolic performance and potential metastatic ability. Primary human cells meet all the demands of compatibility but are not available in appropriate quantities and originate from histologically impaired organs. The extracorporeal liver assist device (ELAD) uses about 200 g of cells of the human hepatoblastoma cell line C3A (derived from HepG2) in modified dialysis cartridges. The cells are located in the extracapillary space separated from plasma by the capillary membranes. Prior to entering the bioreactor, the plasma passes a charcoal adsorber and a membrane oxygenator. First clinical applications were performed, to demonstrate the safety of the system.6,7 HepatAssist is a system that uses 5–7 ⫻ 109 cryopreserved porcine hepatocytes in a similar setting. A large randomized, controlled multicenter study with a total of 171 patients (86 in the control group and 85 in the bioartificial liver treatment group) was conducted by Demetriou et al.8 Patients with fulminant/subfulminant hepatic failure and primary nonfunction following liver transplantation were included in the study, demonstrating the safety of the system and an improved 30-day survival in a subgroup. The modular extracorporeal liver support (MELS) system is based on the Cell Module,9 a unit consisting of 3 interwoven capillary bundles in a polyurethane housing. One of the bundles serves as decentralized oxygenation; 2 bundles are used for perfusion with patient plasma. It is operated with primary porcine hepatocytes as well as human hepatocytes isolated from discarded donor organs.10 For MELS, the CellModule is combined with SPAD and continuous veno-venous hemodiafiltration (CVVHDF). A phase 1 clinical study was performed with the CellModule charged with porcine hepatocytes11 and 12 patients were treated with human hepatocytes.12 Within the Amsterdam Medical Center Bioartificial Liver (AMC-BAL), in contrast to all other mentioned systems, the capillary membranes exclusively serve oxygenation. The cell compartment of the device, which has a polyester matrix, is loaded with about 200 g of primary porcine hepatocytes. During therapy, the matrix is directly perfused by patient plasma.13 A phase 1 study showed the safety of the treatment.14 FURTHER DEVELOPMENT IN BIOARTIFICIAL LIVER ASSIST DEVICES
Bioartificial liver assist devices are limited in performance mainly by 3 factors: liver cell mass, mass exchange over the membranes, and plasma/blood flow rate in the system.15 Currently, cell mass ranges from about 50 –500 g, which is at most about a third of the average liver mass of an adult. Experience from living donor liver transplantation shows that a minimum cell mass of about 40% of the patient’s ideal liver mass (approximately 1500 g) is necessary to assure proper graft function. Furthermore, cells in most devices are not in direct contact with patient plasma or blood, but separated by 1 or even 2 capillary membranes. Thus, the mass exchange is additionally limited by the
BIOARTIFICIAL LIVER
membrane cutoff. In situ, 1.5 L of blood are processed per minute by the liver. The flow in a bioartificial liver device, however, is limited to about 100 –300 mL/min for technical and rheological reasons, eg, plasma separation rates and shear stress. The most urgent problem is an appropriate cell source. Before establishing a reliable, safe, highly metabolically active, and easily expandable human cell source, clinical success of a bioartificial liver support systems is unlikely. Research on human hepatic progenitor cells, embryonic stem cells, or fetal stem cells might offer the necessary expandable and metabolically active cell sources. Bearing in mind the technical limitations, the combination of artificial and bioartificial support concepts appears to be promising. To date, scientific efforts concentrate on either artificial detoxification systems (such as albumin dialysis, adsorber suspension with activated charcoal particles, and ion exchange resin) or biological systems (bioreactors with liver cells). To address the conceptual limitations, the HepatAssist system (bioreactor ⫹ charcoal adsorber) and MELS (bioreactor ⫹ SPAD) combine biological components with detoxification techniques. None of the liver cell– based systems has been clinically evaluated in as extensive a way as Demetriou’s HepatAssist system. Therefore, no conclusions concerning their clinical efficiency and benefit for patients can be drawn based on existing data. Twelve trials on artificial or bioartificial support systems versus standard medical therapy (483 patients) and 2 trials comparing different artificial support systems (105 patients) were included into the latest Cochrane Review concerning artificial and bioartificial liver support systems. Their analysis indicates that artificial support systems may reduce mortality in acute-on-chronic liver failure. Artificial and bioartificial support systems did not appear to affect mortality in acute liver failure. Designers of clinical studies are asked to compare the therapeutic potential of one support system with standard medical therapy before such systems are introduced as control interventions to more sophisticated support systems. Such trials should be adequately designed, including centralized randomization (using stratification for important prognostic factors) as well as blinded outcome assessment.16
3895
REFERENCES 1. Rifai K, Bahr MJ: Akutes Leberversagen. Internist 44:585, 2003 2. Heemann U, Treichel U, Loock J, et al: Albumin dialysis in cirrhosis with superimposed acute liver injury: a prospective, controlled study. Hepatology 36:949, 2002 3. Sauer IM, Goetz M, Steffen I, et al: In vitro comparison of the molecular adsorbent recirculation system (MARS) and single-pass albumin dialysis (SPAD). Hepatology 39:1408, 2004 4. Rifai K, Ernst T, Kretschmer U, et al: Prometheus—a new extracorporeal system for the treatment of liver failure. J Hepatol 39:984, 2003 5. Pascher A, Sauer IM, Hammer C, et al: Extracorporal liver perfusion as hepatic assist in acute liver failure: a review of world experience. Xenotransplantation 9:309, 2002 6. Sussman NL, Gislason GT, Conlin CA, et al: The Hepatix extracorporeal liver assist device: initial clinical experience. Artif Organs. 18:390, 1994 7. Millis JM, Cronin DC, Johnson R, et al: Initial experience with the modified extracorporeal liver-assist device for patients with fulminant hepatic failure: system modifications and clinical impact. Transplantation 74:1735, 2002 8. Demetriou AA, Brown RS Jr, Busuttil RW, et al: Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure. Ann Surg 239:660, 2004 9. Gerlach JC, Encke J, Hole O, et al: Bioreactor for a larger scale hepatocyte in vitro perfusion. Transplantation 58:984, 1994 10. Sauer IM, Zeilinger K, Obermayer N, et al: Primary human liver cells as source for modular extracorporeal liver support—a preliminary report. Int J Artif Org 25:1001, 2002 11. Sauer IM, Kardassis D, Zeillinger K, et al: Clinical extracorporeal hybrid liver support—phase I study with primary porcine liver cells. Xenotransplantation 10:460, 2003 12. Sauer IM, Zeilinger K, Pless G, et al: Extracorporeal liver support based on primary human liver cells and albumin dialysis— treatment of a patient with primary graft non-function. J Hepatol 39:649, 2003 13. Flendrig LM, la Soe JW, Jorning GG, et al: In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates. J Hepatol 26:1379, 1997 14. van de Kerkhove MP, Di Florio E, Scuderi V, et al: Phase I clinical trial with the AMC-bioartificial liver. Int J Artif Organs 25:950, 2002 15. Iwata H, Ueda Y: Pharmacokinetic considerations in development of a bioartificial liver. Clin Pharmacokinet 43:211, 2004 16. Liu JP, Gluud LL, Als-Nielsen B, et al: Artificial and bioartificial support systems for liver failure. Cochrane Database Systematic Reviews:CD003628.pub2, 2004