Bioartificial liver devices: Perspectives on the state of the art

Abstract

Acute liver failure remains a significant cause of morbidity and mortality. Bioartificial liver (BAL) devices have been in development for more than 20 years. Such devices aim to temporarily take over the metabolic and excretory functions of the liver until the patients’ own liver has recovered or a donor liver becomes available for transplant. The important issues include the choice of cell materials and the design of the bioreactor. Ideal BAL cell materials should be of good viability and functionality, easy to access, and exclude immunoreactive and tumorigenic cell materials. Unfortunately, the current cells in use in BAL do not meet these requirements. One of the challenges in BAL development is the improvement of current materials; another key point concerning cell materials is the coculture of different cells. The bioreactor is an important component of BAL, because it determines the viability and function of the hepatocytes within it. From the perspective of bioengineering, a successful and clinically effective bioreactor should mimic the structure of the liver and provide an in vivo-like microenvironment for the growth of hepatocytes, thereby maintaining the cells’ viability and function to the maximum extent. One future trend in the development of the bioreactor is to improve the oxygen supply system. Another direction for future research on bioreactors is the application of biomedical materials. In conclusion, BAL is, in principle, an important therapeutic strategy for patients with acute liver failure, and may also be a bridge to liver transplantation. It requires further research and development, however, before it can enter clinical practice.

Keywords

acute liver failure / bioartificial livers / hepatocyte / bioreactor

Introduction

The liver has a number of crucial functions, principally carried out by hepatocytes. These cells synthesize many proteins, including clotting factors. They produce bile and regulate carbohydrate, fat, and protein metabolism, and they detoxify the ammonia product of nitrogen metabolism. However, once the liver has been damaged by viral hepatitis, drug- or toxin-induced liver disease, metabolic errors, or ischemia, acute liver failure (ALF) could result [1]. ALF remains a significant cause of morbidity and mortality. Bioartificial liver (BAL) support devices have been in development for more than 20 years. Such devices aim to temporarily take over the metabolic and excretory functions of the liver until the patients’ own liver has recovered or a donor liver becomes available for transplant [2].

BAL typically incorporates isolated, cultured hepatocytes in bioreactors [3]. The important issues are the choice of cell materials and the design of the bioreactor. The cell materials provide liver-specific functions, such as detoxification, drug metabolism, and protein synthesis, while the bioreactors maintain the viability and function of cell materials. More efforts are now underway in search for the best cell resource and best design of bioreactors.

Cell sources

Primary human hepatocytes are the most suitable resource, but there are insufficient good-quality human hepatocytes available for use in a BAL device. Human hepatocytes are difficult to replicate in vitro, and daughter cells have not been shown to express sufficient levels of liver-specific functions [4,5]. One alternative is to use hepatocytes from other species. However, there is an ongoing debate about the risk of zoonoses, animal infections transmitted by xenogeneic cells [6]. Hepatocyte cultures derived from human hepatoblastoma cell lines have been used in BAL, but these have a lower efficiency of ammonia removal and amino acid metabolism compared with actual human hepatocytes [7]. A concern with the use of hepatoblastoma and immortalized hepatocytes is the risk for transmission of potentially tumorigenic cells [8]. Recently, stem cells were differentiated into hepatocytes in vitro; however, it is difficult to achieve such a large number of cells on time and on demand [9,10]. Therefore, a reliable and indefinite cell source is still needed.

One of the developing directions for cell materials within the BAL is to improve current materials. Immortalized hepatocyte lines are a hot topic, one of the best candidates for BAL cell materials. However, while solving the problem of hepatocyte proliferation, it increases the risk of tumor occurrence. Therefore, some have proposed using a reversible immortalized hepatocyte line. While the immortalized hepatocytes have the ability to proliferate, the reversible lines use site-specific recombination to remove the immortalized gene so as to revert the hepatocytes to their pre-immortalized state. Thus, theoretically speaking, the cells could not only proliferate but also do so without increasing the risk of tumor growth. Kobayashi has made an important contribution to this respect [11-13]. Another problem concerning immortalized hepatocyte lines is the insertion of the immortalized gene, which makes the cell proliferate continually, but also leads the cells to gradually lose their expression of hepatocyte-specific functions as less mRNA is made from the genes of differentiated liver cells and more is made from the genes of dedifferentiated liver cells. In this respect, we could take advantage of genetic engineering that transfects the genes for liver-specific functions into the immortalized liver cells, thus maintaining the primary hepatocytic biological functions and characteristics [14].

Another key point for BAL concerning cell materials is the co-culture of multiple types of cells. As is well-known, the liver is a sophisticated organ consisting of abundant hepatocytes and non-parenchymal cells, which interact with each other fulfilling the many complex functions of the liver. This implies that it is impossible to substitute the whole function of liver with just one liver cell material. A so-called “co-culture” means culturing hepatocytes and hepatic non-parenchymal cells or non-liver mesenchymal cells together. These cells mainly include sinusoidal endothelial cells, hepatic stellate cells, bone marrow stem cells, and islet cells. Such cells interacting with each other could better simulate an internal environment that maintains the liver cells’ specific functions. Our investigations indicated that co-cultivation could promote the activities and functions of hepatocytes by forming spherical aggregates, increasing direct contact between the liver cells and other cells; co-cultured non-parenchymal cells can promote the liver cells’ function. The component of the extracellular matrix secreted by non-parenchymal cells could provide a favorable growth microenvironment for hepatocytes [15-19].

Bioreactor design

The bioreactor is another key component of BAL, because it determines the viability and functions of the hepatocytes within it. From the perspective of bioengineering, a successful and clinically effective bioreactor should mimic the structure of the liver and provide an in vivo-like microenvironment for the growth of hepatocytes, thereby maintaining the cells’ viability and function to the maximum possible extent. There are four main types of bioreactors, each with inherent advantages and disadvantages [20,21]. The first type is the hollow fiber bioreactor, which comprises many capillary hollow fibers within a rigid housing. The hollow fibers divide the housing into two luminal compartments, the blood or nutrient medium is circulated in the intraluminal compartment, and the hepatocytes are cultured on the surface of the hollow fiber membranes in the extraluminal compartment. The capillary membrane is semipermeable, which provides selectivity for the size of biological molecules that can be exchanged between the patient and the device. The problems with this bioreactor are the nonuniform cell distribution and the transport barrier of the membranes.

The second type is the perfused bed or scaffold bioreactor, which involves the culture of hepatocytes in a porous scaffold. The disadvantage of this type is that cells are directly exposed to shear forces and would block the pores easily. The third type is the flat plate bioreactor, on which the hepatocytes are seeded uniformly on a flat plate. The advantage of this bioreactor is its uniform cell distribution and microenvironment, while the disadvantages are its complexity of scaling-up and low surface-area-to-volume ratio. The last type is the encapsulation and suspension bioreactor, which comprises hepatocytes in microcapsules. The microcapsules are also semipermeable, which could afford material exchanges. This kind of bioreactor is easy to scale up and has a uniform microenvironment, but the cells in the microcapsules have poor stability in suspension and transport is limited due to the encapsulation. So, there is as of yet no ideal bioreactor, and more efforts need to be made to find a suitable bioreactor for BAL devices.

One future trend in the development of bioreactors is to improve the system of oxygen supply. The hepatocyte consumes oxygen at a high rate, and there is a high density of hepatocytes in a bioreactor, so the oxygen supply of a bioreactor is very important. Some experiments confirmed that the viability and function of hepatocytes in a bioreactor with good oxygen supply are better than those in a bioreactor with poor oxygen supply [22,23]. More and more designs of bioreactors have taken this problem into account, and there are various approaches to integration. There are independent hollow fibers used for oxygen supply in modular extracorporeal liver support (MELS) [24] and Academic Medical Center University of Amsterdam-Bioartificial liver (AMC-BAL) [25]; these are cross-woven with other fibers so as to ensure the uniform distribution of the oxygen supply. The flat-plate-type bioreactor FMB-BAL affords an oxygen region in the surface layer, so that the cells on the surface have enough oxygen [26]. In addition, Sullivan found that adding bovine red blood cells to the circulating culture medium could effectively improve the oxygen supply, thereby improving the viability and function of C3A cell lines in the bioreactor [27-29].

Another trend for the future development of bioreactors is the application of biomaterials. The biocompatibility and physicochemical properties of scaffold materials in bioreactors directly influence cellular activity because of their direct contact with the cells. In the traditional concept, the physicochemical properties of the scaffold were thought to be the key factor affecting cell function. Therefore, the physiochemical properties of various biomaterials were changed in all manner of ways, for example, by modifying and grafting. In recent years, the topology and especially the nanotopography structure of scaffolds were found to affect cell function to a great extent, because they mimicked the extracellular matrix and thereby offered the required microenvironment for cell growth [30-32].

In addition, there have been many other novel-designed bioreactors, such as the bioreactor with microchannels [33, 34]. In this bioreactor, microchannels perpendicular to the flow direction were carved on the plates, which protected the hepatocytes from the impact of shear stress and provided enough oxygen. In another Innsbruck BAL (IBAL), the culture vessel rotated around its longitudinal axis, and the organoid hepatocyte aggregates were formed under simulated microgravity. Reportedly, the survival time of pigs with liver failure was prolonged with this BAL [35,36]. Shinohara developed a new bioreactor based on titanium dioxide, which was able to degrade bilirubin, IL-6, IL-8 and IL-10 using its photocatalysis without impacting albumin expression [37]. At present, American scientists are studying a new three-dimensional bio-printer that is able to print cells into a three-dimensional structure while keeping their functions. If this new technique is successful, the current situation of BAL would be changed thoroughly by this completely new concept.

Current application of BAL

As we have seen, BAL has a long way to go both in the cell materials and in the design of bioreactors. Presently, several BAL systems have already undergone phase I clinical trials, and some have even undergone phase II/III clinical trials, but all without satisfactory results [24,25,38-41].

A developing direction in BAL is to use them in conjunction with other blood treatments, such as hemodialysis, albumin dialysis, plasmapheresis, charcoal resin columns, and so on, since these abiological devices have been certified effective in removing the toxic ingredients in patients’ blood. This could be called a “hybrid liver support system,” and it has shown preferable effects in its phase I clinical trial [24].

No obvious complications have yet been reported in these clinical trials. No transmission of malignancy lines or infection of porcine endogenous retroviruses (PERVs) were observed in the application of BALs using C3A cells and porcine hepatocytes, respectively [2,24]. At present, the safeguards to prevent their transmission have mainly focused on the adoption of semipermeable membranes with small pore sizes; this seems to be effective according to the present research results. However, a pivotal solution for avoiding these transmissions would be to choose, develop, or discover a safe and effective cell material.

The research on BAL in China has just begun, and there are still big gaps compared with the techniques abroad. In 2001, the affiliated Drum Tower Hospital of Nanjing University Medical School took the lead in developing a hybrid BAL (HBAL) and performed a phase I clinical trial. About 12 patients with liver failure were involved in this research, and the rate of cure and improvement was up to 80% [42,43]. In recent years, based on this original system, the Drum Tower Hospital constructed a new multi-layer, radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and a co-culture system of porcine hepatocytes and mesenchymal stem cells (MSCs). In vitro evaluation of the new system has completed, in which the culture medium RPMI 1640 was perfused and circulated in the bioreactor at 37°C and 5% CO₂ for dynamic detection of BAL efficacy. The results indicated high hepatocytic activity, good substance exchange, and satisfactory functions of biosynthesis and biotransformation [32]. For the moment, animal experiments are in progress.

In conclusion, as an important therapeutic strategy for patients with ALF, as well as a bridge to liver transplantation, the BAL displays good prospects for future application. It requires further research and development, however, before it can enter clinical practice. We believe that the BAL will lead to revolutionary change in the therapy for liver failure after gradual consummation and combination with non-BAL treatments.

References

[1] Pathikonda M, Munoz S J. Acute liver failure. Ann Hepatol, 2010, 9(1): 7-14

[2] Gerlach J C, Zeilinger K, Patzer Ii J F. Bioartificial liver systems: why, what, whither? Regen Med, 2008, 3(4): 575-595

[3] Chamuleau R A, Deurholt T, Hoekstra R. Which are the right cells to be used in a bioartificial liver? Metab Brain Dis, 2005, 20(4): 327-335

[4] Poyck P P, Hoekstra R, van Wijk A C, Attanasio C, Calise F, Chamuleau R A, van Gulik T M. Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver. Liver Transpl, 2007, 13(4): 589-598

[5] Sauer I M, Zeilinger K, Obermayer N, Pless G, Grünwald A, Pascher A, Mieder T, Roth S, Goetz M, Kardassis D, Mas A, Neuhaus P, Gerlach J C. Primary human liver cells as source for modular extracorporeal liver support—a preliminary report. Int J Artif Organs, 2002, 25(10): 1001-1005

[6] Wang H H, Wang Y J, Liu H L, Liu J, Huang Y P, Guo H T, Wang Y M. Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system. World J Gastroenterol, 2006, 12(8): 1287-1291

[7] Hoekstra R, Chamuleau R A. Recent developments on human cell lines for the bioartificial liver. Int J Artif Organs, 2002, 25(3): 182-191

[8] Kobayashi N, Okitsu T, Nakaji S, Tanaka N. Hybrid bioartificial liver: establishing a reversibly immortalized human hepatocyte line and developing a bioartificial liver for practical use. J Artif Organs, 2003, 6(4): 236-244

[9] Fonsato V, Herrera M B, Buttiglieri S, Gatti S, Camussi G, Tetta C. Use of a rotary bioartificial liver in the differentiation of human liver stem cells. Tissue Eng Part C Methods, 2010, 16(1): 123-132

[10] Sharma R, Greenhough S, Medine C N, Hay D C. Three-dimensional culture of human embryonic stem cell derived hepatic endoderm and its role in bioartificial liver construction. J Biomed Biotechnol, 2010, doi:10.1155/2010/236147

[11] Kobayashi N, Westerman K A, Tanaka N, Fox I J, Leboulch P. A reversibly immortalized human hepatocyte cell line as a source of hepatocyte-based biological support. Addict Biol, 2001, 6(4): 293-300

[12] Watanabe T, Shibata N, Westerman K A, Okitsu T, Allain J E, Sakaguchi M, Totsugawa T, Maruyama M, Matsumura T, Noguchi H, Yamamoto S, Hikida M, Ohmori A, Reth M, Weber A, Tanaka N, Leboulch P, Kobayashi N. Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers. Transplantation, 2003, 75(11): 1873-1880

[13] Totsugawa T, Yong C, Rivas-Carrillo J D, Soto-Gutierrez A, Navarro-Alvarez N, Noguchi H, Okitsu T, Westerman K A, Kohara M, Reth M, Tanaka N, Leboulch P, Kobayashi N. Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination. J Hepatol, 2007, 47(1): 74-82

[14] Enosawa S, Miyashita T, Saito T, Omasa T, Matsumura T. The significant improvement of survival times and pathological parameters by bioartificial liver with recombinant HepG2 in porcine liver failure model. Cell Transplant, 2006, 15(10): 873-880

[15] Thomas R J, Bennett A, Thomson B, Shakesheff K M. Hepatic stellate cells on poly(DL-lactic acid) surfaces control the formation of 3D hepatocyte co-culture aggregates in vitro. Eur Cell Mater, 2006, 11: 16-26, discussion 26

[16] Nedredal G I, Elvevold K, Ytrebø L M, Fuskevåg O M, Pettersen I, Bertheussen K, Langbakk B, Smedsrød B, Revhaug A. Significant contribution of liver nonparenchymal cells to metabolism of ammonia and lactate and cocultivation augments the functions of a bioartificial liver. Am J Physiol Gastrointest Liver Physiol, 2007, 293(1): G75-G83

[17] Gu J, Shi X, Zhang Y, Ding Y. Heterotypic interactions in the preservation of morphology and functionality of porcine hepatocytes by bone marrow mesenchymal stem cells in vitro. J Cell Physiol, 2009, 219(1): 100-108

[18] Gu J, Shi X, Chu X, Zhang Y, Ding Y. Contribution of bone marrow mesenchymal stem cells to porcine hepatocyte culture in vitro. Biochem Cell Biol, 2009, 87(4): 595-604

[19] Gu J, Shi X, Zhang Y, Chu X, Hang H, Ding Y. Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro. Hepatol Res, 2009, 39(4): 398-407

[20] Allen J W, Hassanein T, Bhatia S N. Advances in bioartificial liver devices. Hepatology, 2001, 34(3): 447-455

[21] Park J K, Lee D H. Bioartificial liver systems: current status and future perspective. J Biosci Bioeng, 2005, 99(4): 311-319

[22] Park J, Li Y, Berthiaume F, Toner M, Yarmush M L, Tilles A W. Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates. Biotechnol Bioeng, 2008, 99(2): 455-467

[23] Kinasiewicz A, Smietanka A, Gajkowska B, Werynski A. Impact of oxygenation of bioartificial liver using perfluorocarbon emulsion perftoran on metabolism of human hepatoma C3A cells. Artif Cells Blood Substit Immobil Biotechnol, 2008, 36(6): 525-534

[24] Sauer I M, Kardassis D, Zeillinger K, Pascher A, Gruenwald A, Pless G, Irgang M, Kraemer M, Puhl G, Frank J, Müller A R, Steinmüller T, Denner J, Neuhaus P, Gerlach J C. Clinical extracorporeal hybrid liver support—phase I study with primary porcine liver cells. Xenotransplantation, 2003, 10(5): 460-469

[25] Poyck P P, Mareels G, Hoekstra R, van Wijk A C, van der Hoeven T V, van Gulik T M, Verdonck P R, Chamuleau R A. Enhanced oxygen availability improves liver-specific functions of the AMC bioartificial liver. Artif Organs, 2008, 32(2): 116-126

[26] De Bartolo L, Jarosch-Von Schweder G, Haverich A, Bader A. A novel full-scale flat membrane bioreactor utilizing porcine hepatocytes: cell viability and tissue-specific functions. Biotechnol Prog, 2000, 16(1): 102-108

[27] Sullivan J P, Gordon J E, Palmer A F. Simulation of oxygen carrier mediated oxygen transport to C3A hepatoma cells housed within a hollow fiber bioreactor. Biotechnol Bioeng, 2006, 93(2): 306-317

[28] Sullivan J P, Gordon J E, Bou-Akl T, Matthew H W, Palmer A F. Enhanced oxygen delivery to primary hepatocytes within a hollow fiber bioreactor facilitated via hemoglobin-based oxygen carriers. Artif Cells Blood Substit Immobil Biotechnol, 2007, 35(6): 585-606

[29] Sullivan J P, Harris D R, Palmer A F. Convection and hemoglobin-based oxygen carrier enhanced oxygen transport in a hepatic hollow fiber bioreactor. Artif Cells Blood Substit Immobil Biotechnol, 2008, 36(4): 386-402

[30] Chu X H, Shi X L, Feng Z Q, Gu Z Z, Ding Y T. Chitosan nanofiber scaffold enhances hepatocyte adhesion and function. Biotechnol Lett, 2009, 31(3): 347-352

[31] Feng Z Q, Chu X, Huang N P, Wang T, Wang Y, Shi X, Ding Y, Gu Z Z. The effect of nanofibrous galactosylated chitosan scaffolds on the formation of rat primary hepatocyte aggregates and the maintenance of liver function. Biomaterials, 2009, 30(14): 2753-2763

[32] Chu X H, Shi X L, Feng Z Q, Gu J Y, Xu H Y, Zhang Y, Gu Z Z, Ding Y T. In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds. Biomaterials, 2009, 30(27): 4533-4538

[33] Park J, Berthiaume F, Toner M, Yarmush M L, Tilles A W. Microfabricated grooved substrates as platforms for bioartificial liver reactors. Biotechnol Bioeng, 2005, 90(5): 632-644

[34] Park J, Berthiaume F, Toner M, Yarmush M L, Tilles A W. Microfabricated grooved substrates as platforms for bioartificial liver reactors. Biotechnol Bioeng, 2005, 90(5): 632-644

[35] Hochleitner B, Hengster P, Bucher H, Ladurner R, Schneeberger S, Krismer A, Kleinsasser A, Barnas U, Klima G, Margreiter R. Significant survival prolongation in pigs with fulminant hepatic failure treated with a novel microgravity-based bioartificial liver. Artif Organs, 2006, 30(12): 906-914

[36] Hochleitner B, Hengster P, Duo L, Bucher H, Klima G, Margreiter R. A novel bioartificial liver with culture of porcine hepatocyte aggregates under simulated microgravity. Artif Organs, 2005, 29(1): 58-66

[37] Shinohara H, Shimada M, Ikemoto T, Morine Y, Imura S, Fujii M, Imaizumi T, Murayama M, Aiba Y. New type of artificial liver support system (ALSS) using the photocatalytic effect of titanium oxide. Dig Dis Sci, 2007, 52(9): 2271-2275

[38] Sussman N L, Gislason G T, Conlin C A, Kelly J H. The Hepatix extracorporeal liver assist device: initial clinical experience. Artif Organs, 1994, 18(5): 390-396

[39] Watanabe F D, Mullon C J, Hewitt W R, Arkadopoulos N, Kahaku E, Eguchi S, Khalili T, Arnaout W, Shackleton C R, Rozga J, Solomon B, Demetriou A A. Clinical experience with a bioartificial liver in the treatment of severe liver failure. A phase I clinical trial. Ann Surg, 1997, 225(5): 484-491, discussion 491-494

[40] Patzer J F, Mazariegos G V, Lopez R. Preclinical evaluation of the Excorp Medical, Inc., bioartifical liver support system. Am Coll Surg, 2002, 195: 299-310

[41] Morsiani E, Pazzi P, Puviani A C, Brogli M, Valieri L, Gorini P, Scoletta P, Marangoni E, Ragazzi R, Azzena G, Frazzoli E, Di Luca D, Cassai E, Lombardi G, Cavallari A, Faenza S, Pasetto A, Girardis M, Jovine E, Pinna A D. Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients. Int J Artif Organs, 2002, 25(3): 192-202

[42] Ding Y T, Qiu Y D, Chen Z, Xu Q X, Zhang H Y, Tang Q, Yu D C. The development of a new bioartificial liver and its application in 12 acute liver failure patients. World J Gastroenterol, 2003, 9(4): 829-832

[43] Xu Q, Yu D, Qiu Y, Zhang H, Ding Y. Function of a new internal bioartificial liver: an in vitro study. Ann Clin Lab Sci, 2003, 33(3): 306-312

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