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Bio-artificial liver devices – Tentative, but promising progress

2007; Elsevier BV; Volume: 48; Issue: 2 Linguagem: Inglês

10.1016/j.jhep.2007.11.008

1600-0641

Konrad L. Streetz,

Tissue Engineering and Regenerative Medicine

Acute and chronic liver injury often lead to organ deterioration and fibrosis requiring orthotopic liver transplantation as the only available permanent treatment option. Worldwide numerous liver transplantations are performed annually. However, there are only enough available livers for about twenty percent of the patients in need of an organ replacement. Thus, new treatment options are needed. Bio-artificial liver systems (BAL) function as bridging devices to bypass the time-frame until a donor organ becomes available or until the acute injured liver can eventually regenerate itself. The successful development of a widely useable system to provide liver specific organ functions, like detoxification, drug metabolism and protein synthesis could potentially rescue thousands of patients with acute liver failure and if used intermittently improve the quality of lives of chronically injured patients.Up to now numerous attempts have been undertaken to generate alternative cell sources to establish bio-artificial liver support systems using iso- and xenogenic hepatocytes or other hepatocyte-like cells. Although several animal studies could demonstrate an improved outcome in models of acute liver failure [[13]van de Kerkhove M.P. Hoekstra R. van Gulik T.M. Chamuleau R.A. Large animal models of fulminant hepatic failure in artificial and bioartificial liver support research.Biomaterials. 2004; 25: 1613-1625Crossref PubMed Scopus (44) Google Scholar] after using bio-artificial liver devices, the proof of a clinical success in a controlled human trial for BALs is still lacking. However, several phase I studies using either porcine or human hepatocytes have demonstrated the safety, feasibility, and an improvement of biochemical, neurological, and hemodynamic parameters in human subjects [6Miwa Y. Ellis A.J. Hughes R.D. Langley P.G. Wendon J.A. Williams R. Effect of ELAD liver support on plasma HGF and TGF-beta 1 in acute liver failure.Int J Artif Organs. 1996; 19: 240-244PubMed Google Scholar, 3Demetriou A.A. Brown Jr., R.S. Busuttil R.W. Fair J. McGuire B.M. Rosenthal P. et al.Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure.Ann Surg. 2004; 239: 660-667Crossref PubMed Scopus (527) Google Scholar, 12van de Kerkhove M.P. Hoekstra R. Chamuleau R.A. van Gulik T.M. Clinical application of bioartificial liver support systems.Ann Surg. 2004; 240: 216-230Crossref PubMed Scopus (144) Google Scholar].Bio-artificial liver devices have improved on many levels in recent years. Therefore two major aspects have to be considered. First, technical changes, which are related to the construction of the bioreactor itself and second the use of the right cell source to charge BALs. Modern bioreactors, like the one used in the study presented here, consist of a three-dimensional system of capillaries for oxygen supply and carbon dioxide removal. The cells will then be loaded into the matrix (usually polypropylene) of the BAL. Emphasis is currently paid to modifications of the matrix by coating it with different materials to enhance attachment, survival and growth of the seeded cells [[9]Poyck P.P. Pless G. Hoekstra R. Roth S. Van Wijk A.C. Schwartlander R. et al.In vitro comparison of two bioartificial liver support systems: MELS CellModule and AMC-BAL.Int J Artif Organs. 2007; 30: 183-191PubMed Google Scholar].Most efforts are now being made in the search for the best cell to be loaded in the BAL device, as this is clearly the most important factor, if such bioreactors might be used clinically to assist hepatic functions of patients who are in need of metabolic liver support. This is challenging as the minimum quantity of cells was determined to be at least 1010 hepatocytes in total (about 150 g of cells, corresponding to 10% of the normal liver mass) to provide enough metabolic and detoxification capacity for an injured human liver. In order to provide viable liver cells – enough for the efficient treatment of a human patient – hepatocytes or their substitutes need to be grown on large scales and maintained and cultured in multi-dimensional devices at a preferential high density. Therefore it is important to construct the culture system in a liver organoid structure. To provide such a large number of cells in time and on demand a reliable and indefinite cell source is needed. Most potent candidate cells are currently adult hepatocytes and hepatoblasts originating from fetal liver. Furthermore the development of embryonic stem cells (ES cells) [[2]Carpenter M.K. Rosler E.S. Fisk G.J. Brandenberger R. Ares X. Miura T. et al.Properties of four human embryonic stem cell lines maintained in a feeder-free culture system.Dev Dyn. 2004; 229: 243-258Crossref PubMed Scopus (248) Google Scholar] and the isolation, culture and differentiation of somatic stem cells originating from different tissues and organs (pancreatic duct epithelial cells, cells of hematopoetic origin) are currently a major focus of experimental research activities in the field.In the recent past, multiple approaches using different kinds of cells have been attempted in animal models and – in phase I studies – also in humans. Porcine hepatocytes for instance would be regularly available but their use is restricted by potential immunological problems and the risk of zoonosis transmissions [1Baquerizo A. Mhoyan A. Shirwan H. Swensson J. Busuttil R.W. Demetriou A.A. et al.Xenoantibody response of patients with severe acute liver failure exposed to porcine antigens following treatment with a bioartificial liver.Transplant Proc. 1997; 29: 964-965Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 7Paradis K. Xenotransplantation: a program for development.Pathol Biol (Paris). 2000; 48: 449-450PubMed Google Scholar]. Apart from xenogenic hepatocytes mostly primary human hepatocytes, human hepatoma cell lines, immortalized human fetal and adult hepatocytes have been used in experimental BAL systems with varying results. The application of different kinds of stem cells (embryonic, hematopoetic, and hepatic) has currently to be seen as a rather theoretical option for any use in humans. Although very recent studies in mice using differentiated mouse embryonic stem cells look promising [[11]Soto-Gutierrez A. Kobayashi N. Rivas-Carrillo J.D. Navarro-Alvarez N. Zhao D. Okitsu T. et al.Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes.Nat Biotechnol. 2006; 24: 1412-1419Crossref PubMed Scopus (187) Google Scholar].Primary human hepatocytes are mostly derived from non-transplantable livers and have already been shown to be safe in human trials [5Millis J.M. Cronin D.C. Johnson R. Conjeevaram H. Faust T.W. Trevino S. et al.Bioartificial liver support: report of the longest continuous treatment with human hepatocytes.Transplant Proc. 2001; 33: 1935Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 10Sauer I.M. Zeilinger K. Obermayer N. Pless G. Grunwald A. Pascher A. et al.Primary human liver cells as source for modular extracorporeal liver support – a preliminary report.Int J Artif Organs. 2002; 25: 1001-1005PubMed Google Scholar]. However, the major drawback of using primary human cells relates to their availability, because they do function best if isolated and used freshly. Freezing procedures for primary hepatocytes and subsequent storage have not proven to be efficient as most cells are not viable anymore after thawing, thus limiting their use as an on demand available cell source. Even more critical has been the use of hepatoma cells, as the safety and biological efficiency of these is still a major concern. More recently techniques became available to immortalize primary cells, thus enabling them to proliferate without becoming cancerogenic, especially if immortalization will be reversible, e.g. by the appliance of the Cre-loxP system. Here most frequently SV40 large T-antigen [[4]Kobayashi N. Noguchi H. Westerman K.A. Watanabe T. Matsumura T. Totsugawa T. et al.Successful retroviral gene transfer of simian virus 40 T antigen and herpes simplex virus-thymidine kinase into human hepatocytes.Cell Transplant. 2001; 10: 377-381PubMed Google Scholar] and hTERT (human telomerase reverse transcriptase) [[15]Wege H. Le H.T. Chui M.S. Liu L. Wu J. Giri R. et al.Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.Gastroenterology. 2003; 124: 432-444Abstract Full Text PDF PubMed Scopus (158) Google Scholar] were used as immortalizing genes. As fetal hepatocytes still possess some intrinsic proliferative capacity they might be even more useful to create an immortal cell line for charging bioreactors. In a recent study Wege et al. showed [[15]Wege H. Le H.T. Chui M.S. Liu L. Wu J. Giri R. et al.Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.Gastroenterology. 2003; 124: 432-444Abstract Full Text PDF PubMed Scopus (158) Google Scholar] that hTERT immortalized human fetal hepatocytes displayed more than 300 doublings without becoming senescent. They still appeared to be non-tumorigenic and were capable of expressing hepatocyte specific genes and to show a detoxification capacity believed to be sufficient for BAL application.Human fetal hepatocytes have previously been shown to be a suitable substitute for mature human hepatocytes [[8]Poyck P.P. Hoekstra R. van Wijk A.C. Attanasio C. Calise F. Chamuleau R.A. et al.Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver.Liver Transpl. 2007; 13: 589-598Crossref PubMed Scopus (47) Google Scholar] in terms of their detoxification and metabolic potential. These features make them already today an attractive model for pharmacological studies using bioreactor systems. Based on these considerations a new immortalized human fetal cell line (cBAL111) using hTERT was engineered recently. In this issue of the Journal Poyck et al. from the University of Amsterdam [[16]Poyck P.P.C. van Wijk A.C.W.A. van der Hoeven T.V. de Waart D.R. Chamuleau R.A.F.M. van Gulik T.M. et al.Evaluation of a new immortalized human fetal liver cell line (cBAL111) for application in bioartificial liver.J Hepatol. 2008; 48: 266-275Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar] have now applied cBAL111 in the well-evaluated AMC-bioreactor [[14]van de Kerkhove M.P. Poyck P.P. Deurholt T. Hoekstra R. Chamuleau R.A. van Gulik T.M. Liver support therapy: an overview of the AMC-bioartificial liver research.Dig Surg. 2005; 22: 254-264Crossref PubMed Scopus (45) Google Scholar], which has been used successfully during recent years to grow hepatocytes in a highly developed three-dimensional setting.However, before these immortalized cells can be used safely and effectively in patients many hurdles have yet to be overcome. It must be ensured, that the immortalizing gene can be truly shut off. Proper hepatic functionality must then be given in those cells after reversing the immortalization process. This is not trivial as proliferation and differentiation are especially in hepatocytes, diametric biological functions. Additionally, the aspect of the physiological hepatic zonal gene expression, which is related to the three-dimensional organisation of the liver, has to be taken into account. This could mean that ultimatively more than one cell line, potentially combined with other non-parenchymal cells, has to be used in co-culture to provide the needed full spectrum of hepatic functions. However, within this setting cBAL111 as shown here might be at least one promising component for successful charging of BALs to provide extracorporeal liver support. Acute and chronic liver injury often lead to organ deterioration and fibrosis requiring orthotopic liver transplantation as the only available permanent treatment option. Worldwide numerous liver transplantations are performed annually. However, there are only enough available livers for about twenty percent of the patients in need of an organ replacement. Thus, new treatment options are needed. Bio-artificial liver systems (BAL) function as bridging devices to bypass the time-frame until a donor organ becomes available or until the acute injured liver can eventually regenerate itself. The successful development of a widely useable system to provide liver specific organ functions, like detoxification, drug metabolism and protein synthesis could potentially rescue thousands of patients with acute liver failure and if used intermittently improve the quality of lives of chronically injured patients. Up to now numerous attempts have been undertaken to generate alternative cell sources to establish bio-artificial liver support systems using iso- and xenogenic hepatocytes or other hepatocyte-like cells. Although several animal studies could demonstrate an improved outcome in models of acute liver failure [[13]van de Kerkhove M.P. Hoekstra R. van Gulik T.M. Chamuleau R.A. Large animal models of fulminant hepatic failure in artificial and bioartificial liver support research.Biomaterials. 2004; 25: 1613-1625Crossref PubMed Scopus (44) Google Scholar] after using bio-artificial liver devices, the proof of a clinical success in a controlled human trial for BALs is still lacking. However, several phase I studies using either porcine or human hepatocytes have demonstrated the safety, feasibility, and an improvement of biochemical, neurological, and hemodynamic parameters in human subjects [6Miwa Y. Ellis A.J. Hughes R.D. Langley P.G. Wendon J.A. Williams R. Effect of ELAD liver support on plasma HGF and TGF-beta 1 in acute liver failure.Int J Artif Organs. 1996; 19: 240-244PubMed Google Scholar, 3Demetriou A.A. Brown Jr., R.S. Busuttil R.W. Fair J. McGuire B.M. Rosenthal P. et al.Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure.Ann Surg. 2004; 239: 660-667Crossref PubMed Scopus (527) Google Scholar, 12van de Kerkhove M.P. Hoekstra R. Chamuleau R.A. van Gulik T.M. Clinical application of bioartificial liver support systems.Ann Surg. 2004; 240: 216-230Crossref PubMed Scopus (144) Google Scholar]. Bio-artificial liver devices have improved on many levels in recent years. Therefore two major aspects have to be considered. First, technical changes, which are related to the construction of the bioreactor itself and second the use of the right cell source to charge BALs. Modern bioreactors, like the one used in the study presented here, consist of a three-dimensional system of capillaries for oxygen supply and carbon dioxide removal. The cells will then be loaded into the matrix (usually polypropylene) of the BAL. Emphasis is currently paid to modifications of the matrix by coating it with different materials to enhance attachment, survival and growth of the seeded cells [[9]Poyck P.P. Pless G. Hoekstra R. Roth S. Van Wijk A.C. Schwartlander R. et al.In vitro comparison of two bioartificial liver support systems: MELS CellModule and AMC-BAL.Int J Artif Organs. 2007; 30: 183-191PubMed Google Scholar]. Most efforts are now being made in the search for the best cell to be loaded in the BAL device, as this is clearly the most important factor, if such bioreactors might be used clinically to assist hepatic functions of patients who are in need of metabolic liver support. This is challenging as the minimum quantity of cells was determined to be at least 1010 hepatocytes in total (about 150 g of cells, corresponding to 10% of the normal liver mass) to provide enough metabolic and detoxification capacity for an injured human liver. In order to provide viable liver cells – enough for the efficient treatment of a human patient – hepatocytes or their substitutes need to be grown on large scales and maintained and cultured in multi-dimensional devices at a preferential high density. Therefore it is important to construct the culture system in a liver organoid structure. To provide such a large number of cells in time and on demand a reliable and indefinite cell source is needed. Most potent candidate cells are currently adult hepatocytes and hepatoblasts originating from fetal liver. Furthermore the development of embryonic stem cells (ES cells) [[2]Carpenter M.K. Rosler E.S. Fisk G.J. Brandenberger R. Ares X. Miura T. et al.Properties of four human embryonic stem cell lines maintained in a feeder-free culture system.Dev Dyn. 2004; 229: 243-258Crossref PubMed Scopus (248) Google Scholar] and the isolation, culture and differentiation of somatic stem cells originating from different tissues and organs (pancreatic duct epithelial cells, cells of hematopoetic origin) are currently a major focus of experimental research activities in the field. In the recent past, multiple approaches using different kinds of cells have been attempted in animal models and – in phase I studies – also in humans. Porcine hepatocytes for instance would be regularly available but their use is restricted by potential immunological problems and the risk of zoonosis transmissions [1Baquerizo A. Mhoyan A. Shirwan H. Swensson J. Busuttil R.W. Demetriou A.A. et al.Xenoantibody response of patients with severe acute liver failure exposed to porcine antigens following treatment with a bioartificial liver.Transplant Proc. 1997; 29: 964-965Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 7Paradis K. Xenotransplantation: a program for development.Pathol Biol (Paris). 2000; 48: 449-450PubMed Google Scholar]. Apart from xenogenic hepatocytes mostly primary human hepatocytes, human hepatoma cell lines, immortalized human fetal and adult hepatocytes have been used in experimental BAL systems with varying results. The application of different kinds of stem cells (embryonic, hematopoetic, and hepatic) has currently to be seen as a rather theoretical option for any use in humans. Although very recent studies in mice using differentiated mouse embryonic stem cells look promising [[11]Soto-Gutierrez A. Kobayashi N. Rivas-Carrillo J.D. Navarro-Alvarez N. Zhao D. Okitsu T. et al.Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes.Nat Biotechnol. 2006; 24: 1412-1419Crossref PubMed Scopus (187) Google Scholar]. Primary human hepatocytes are mostly derived from non-transplantable livers and have already been shown to be safe in human trials [5Millis J.M. Cronin D.C. Johnson R. Conjeevaram H. Faust T.W. Trevino S. et al.Bioartificial liver support: report of the longest continuous treatment with human hepatocytes.Transplant Proc. 2001; 33: 1935Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 10Sauer I.M. Zeilinger K. Obermayer N. Pless G. Grunwald A. Pascher A. et al.Primary human liver cells as source for modular extracorporeal liver support – a preliminary report.Int J Artif Organs. 2002; 25: 1001-1005PubMed Google Scholar]. However, the major drawback of using primary human cells relates to their availability, because they do function best if isolated and used freshly. Freezing procedures for primary hepatocytes and subsequent storage have not proven to be efficient as most cells are not viable anymore after thawing, thus limiting their use as an on demand available cell source. Even more critical has been the use of hepatoma cells, as the safety and biological efficiency of these is still a major concern. More recently techniques became available to immortalize primary cells, thus enabling them to proliferate without becoming cancerogenic, especially if immortalization will be reversible, e.g. by the appliance of the Cre-loxP system. Here most frequently SV40 large T-antigen [[4]Kobayashi N. Noguchi H. Westerman K.A. Watanabe T. Matsumura T. Totsugawa T. et al.Successful retroviral gene transfer of simian virus 40 T antigen and herpes simplex virus-thymidine kinase into human hepatocytes.Cell Transplant. 2001; 10: 377-381PubMed Google Scholar] and hTERT (human telomerase reverse transcriptase) [[15]Wege H. Le H.T. Chui M.S. Liu L. Wu J. Giri R. et al.Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.Gastroenterology. 2003; 124: 432-444Abstract Full Text PDF PubMed Scopus (158) Google Scholar] were used as immortalizing genes. As fetal hepatocytes still possess some intrinsic proliferative capacity they might be even more useful to create an immortal cell line for charging bioreactors. In a recent study Wege et al. showed [[15]Wege H. Le H.T. Chui M.S. Liu L. Wu J. Giri R. et al.Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential.Gastroenterology. 2003; 124: 432-444Abstract Full Text PDF PubMed Scopus (158) Google Scholar] that hTERT immortalized human fetal hepatocytes displayed more than 300 doublings without becoming senescent. They still appeared to be non-tumorigenic and were capable of expressing hepatocyte specific genes and to show a detoxification capacity believed to be sufficient for BAL application. Human fetal hepatocytes have previously been shown to be a suitable substitute for mature human hepatocytes [[8]Poyck P.P. Hoekstra R. van Wijk A.C. Attanasio C. Calise F. Chamuleau R.A. et al.Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver.Liver Transpl. 2007; 13: 589-598Crossref PubMed Scopus (47) Google Scholar] in terms of their detoxification and metabolic potential. These features make them already today an attractive model for pharmacological studies using bioreactor systems. Based on these considerations a new immortalized human fetal cell line (cBAL111) using hTERT was engineered recently. In this issue of the Journal Poyck et al. from the University of Amsterdam [[16]Poyck P.P.C. van Wijk A.C.W.A. van der Hoeven T.V. de Waart D.R. Chamuleau R.A.F.M. van Gulik T.M. et al.Evaluation of a new immortalized human fetal liver cell line (cBAL111) for application in bioartificial liver.J Hepatol. 2008; 48: 266-275Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar] have now applied cBAL111 in the well-evaluated AMC-bioreactor [[14]van de Kerkhove M.P. Poyck P.P. Deurholt T. Hoekstra R. Chamuleau R.A. van Gulik T.M. Liver support therapy: an overview of the AMC-bioartificial liver research.Dig Surg. 2005; 22: 254-264Crossref PubMed Scopus (45) Google Scholar], which has been used successfully during recent years to grow hepatocytes in a highly developed three-dimensional setting. However, before these immortalized cells can be used safely and effectively in patients many hurdles have yet to be overcome. It must be ensured, that the immortalizing gene can be truly shut off. Proper hepatic functionality must then be given in those cells after reversing the immortalization process. This is not trivial as proliferation and differentiation are especially in hepatocytes, diametric biological functions. Additionally, the aspect of the physiological hepatic zonal gene expression, which is related to the three-dimensional organisation of the liver, has to be taken into account. This could mean that ultimatively more than one cell line, potentially combined with other non-parenchymal cells, has to be used in co-culture to provide the needed full spectrum of hepatic functions. However, within this setting cBAL111 as shown here might be at least one promising component for successful charging of BALs to provide extracorporeal liver support.