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JOURNALOFBIOSCIENCEANDBIOENGINEERING 2005, The Society for Biotechnology, Japan

Vol. 99, No. 4, 311319. 2005

DOI: 10.1263/jbb.99.311

Bioartificial Liver Systems:Current Status and Future Perspective

JUNG-KEUG PARK1*ANDDOO-HOON LEE

1

Department of Chemical and Biochemical Engineering, Dongguk University,3-26 Pil-dong, Choong-gu, Seoul 100-715, Korea1

Received 26 January 2005/Accepted 12 February 2005

Because the liver is a multifunctional and a vital organ for survival, the management of acuteliver failure requires the support of a huge number of metabolic functions performed by the or-

gan. Many early detoxification-based artificial liver techniques failed to treat the patients owingto the inadequate support of the many essential hepatic functions. For this reason, a bioartificialliver (BAL) comprising of viable hepatocytes on a mechanical support is believed to more likelyprovide these essential functions than a purely mechanical device. From 1990, nine clinical studiesof various BAL systems have been reported, most of which utilize a hollow fiber technology, and amuch larger number of various BAL systems have been suggested to show an enhanced perfor-mance. Safety issues such as immunological reactions, zoonosis and tumorgenicity have been suc-cessfully addressed for regulatory approval, but a recent report from a large-scale, randomized,and controlled phase III trial of a leading BAL system (HepatAssist) failed to meet our expecta-tion of efficacy in terms of the overall survival rate. In this paper, we review the current BAL sys-tems actively studied and discuss critical issues such as the hepatocyte bioreactor configurationand the hepatocyte source. On the basis of the insights gained from previously developed BALsystems and the rapid progress in stem cell technology, the short-term and long-term future per-

spectives of BAL systems are suggested.

[Keywords: bioartificial liver, liver support, hepatocyte bioreactor, fulminant hepatic failure, liver transplantation]

Although both acute and chronic liver failure patients canbe treated effectively by whole-organ transplantation, thereare only approximately 4000 donor livers available in theUnited States annually, while approximately 30,000 patientsdie from liver failure (13), and almost 20,000 patients arecurrently awaiting an available donor liver (liver waitinglist, United Network for Organ Sharing, 2003, available atwww.unos.org).Furthermore, despite the recent advances insupportive therapies, patients with severe fulminant hepatic

failure (FHF) have a mortality rate of approximately 90%(4). If essential liver functions can be restored during thecritical phase of liver failure, either by artificial or auxiliarymethods, it should be possible to improve the survival rateof these patients. Therefore, more donor livers will be avail-able and costly transplantation procedures can be avoided(5). Various nonbiological approaches, such as hemodialy-sis, hemoperfusion, plasmapheresis and plasma exchange,have had a limited success because of the insufficient re-

placement of the synthetic and metabolic functions of theliver in these systems (69). On the other hand, extracor-poreal biological treatments including whole-liver perfusion,liver slice perfusion and cross hemodialysis, have shownsome beneficial results, but they are difficult to implementin a clinical setting (1012).

For these reasons, many investigators have attempted todevelop various extracorporeal bioartificial liver (BAL) sys-tems. Typically, a BAL system incorporates a hepatoma cell

line or isolated primary hepatocytes into a bioreactor inwhich the cells are immobilized, cultured and induced toperform the hepatic functions by processing the blood orplasma of liver failure patients (13, 14). The BAL system isexpected to act as a bridge that provides patients with moretime until a donor organ can become available for transplan-tation or until their own liver can be regenerated (15).

There are many available reviews that describe the his-tory, general bioreactor and system designs, the preclinicaland clinical results of current BAL systems (1620). There-fore, in this paper, we focus on the developmental trends ofthe elements of the BAL system including cell sources, thecell mass, perfusion type, and culture methods adopted in abioreactor. Finally, on the basis of the insight gained from

current BAL systems and the rapid progress in stem cell

* Corresponding author. e-mail:[email protected]: +82-2-2260-3365 fax: +82-2-2271-3489Abbreviations: BAL, bioartificial liver; FHF, fulminant hepatic fail-

ure; OLT, orthotopic liver transplantation; PERV, porcine endogenousretrovirus.
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PARK AND LEE J. BIOSCI. BIOENG.,312

technology, the short-term and long-term future perspec-tives of the BAL systems will be given.

BIOREACTOR CONFIGURATIONS OF

BAL SYSTEMS IN CLINICAL TRIALFrom 1990, nine BAL systems have been clinically tested,

most of which utilize a hollow fiber technology, and a muchlarger number of BAL systems in preclinical tests have beensuggested to show an enhanced performance. Among thenine clinically tested BAL systems, three (2123) have abioreactor configuration similar to that of HepatAssist (CirceBiomedical, Lexington, MA, USA) (24). Therefore, in thispaper, we review the bioreactors of the other six BAL sys-tems. The characteristics and major clinical findings arelisted inTable 1.

The extracorporeal liver assist device (ELAD) is theonly BAL system that uses the human hepatocyte cell line

C3A, which is derived from the hepatoma cell line HepG2(2527). C3A cells express normal liver specific pathwayssuch as ureogenesis, gluconeogenesis and cytochrome P-450activities, and produce albumin and -fetoprotein (28). Thecells are grown in the extracapillary space of cartridges andblood flows through the lumen of hollow fibers. A portionof the patients plasma is ultrafiltrated through a membrane(70 kD) and is in direct contact with the C3A cells. In an un-controlled clinical trial (26), 6 out of 11 patients died, whileone patient completely recovered without an orthotopic livertransplantation (OLT) and four patients underwent a suc-cessful OLT after a mean treatment of 40.8 h. In a pilot con-

trolled trial on 24 acute liver failure patients, there was nosignificant difference in survival rate, renal function, andbiochemical parameters between the ELAD-treated patientsand the controls (27). In contrast, the plasma ammonia andbilirubin level were higher than those before the ELAD

treatment.The HepatAssist system developed by Demetriou and co-

workers was examined in the largest controlled clinical trial(2934). In this system, microcarrier-attached cryopreservedporcine hepatocytes are placed in the extracapillary space(29). They particularly use 0.2 m-pore hollow fiber mem-branes. This size is sufficiently small to block the passage ofwhole cells. Plasma is separated by continuous centrifu-gation. It first passes through an activated charcoal columnand flows through the lumen of the hollow fibers. In thephase I of a clinical study, 18 patients with FHF were sup-ported with the system (29, 31). Sixteen patients had a suc-cessful liver transplant and were discharged with normal

neurological states and one patient was bridged to recoverywithout requiring a transplant. In another clinical study, theBAL system treatment resulted in some improvement in thepatients neurological states, intracranial pressure (ICP), andbiochemical parameters (35). Both the total bilirubin and am-monia concentrations decreased by 18% of the initial con-centrations. However, porcine endogenous retrovirus (PERV)transmission was confirmed after the BAL system treatmentbut there was no evidence of viral transmission from por-cine hepatocytes to patients (17).

The liver support system (LSS) developed by Gerlach andcoworkers in Berlin consists of a unique bioreactor with

TABLE 1. Characteristics and clinical results of six BAL systems


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four different capillary membranes, which are woven intoa 3-D lattice (36, 37). These capillaries independently andlocally provide oxygen, nutrients and plasma perfusate in-flow and outflow to hepatocytes. The bioreactor enables thespontaneous aggregation of parenchymal cells in a cocul-

tured with nonparenchymal cells, forming tissue-like struc-tures. The formation of bile canaliculi and bile duct-likestructures and a neospace of Disse with a biomatrix pro-duced was demonstrated. The LSS is the only system thatuses primary human hepatocytes isolated from discardeddonor livers as well as porcine hepatocytes (38). In phase Iof a study using human hepatocytes, the LSS was combinedwith a single-pass albumin dialysis device (MARS), calledthe modular extracorporeal liver support (MELS) (38). BothBAL systems, LSS and MELS, successfully supported eachsix patients, who were all transplant candidates, until anOLT and improved their neurological and coagulation states.

The bioartificial liver support system (BLSS), which was

developed in Pittsburgh and commercialized by Excorp Med-ical (Minneapolis, MN, USA), uses cellulose acetate hollowfibers with a 100 kD-molecular weight cut-off (MWCO)(39, 40). Primary porcine hepatocytes (70120 g) are mixedwith 20% vol/wt of a 3.1% collagen solution and the mix-ture is infused into the extracapillary space. The blood am-monia levels decreased by 33% compared with the initiallevels. However, the patientsneurological states are not im-proved significantly (40).

The radial flow bioreactor (RFB) BAL system was de-veloped in the University of Ferrara, Italy (41, 42). In thissystem, the patientsplasma passes from the center to theperiphery of the module. Oxygen consumption rate is mea-sured to evaluate the function of the bioreactor; when the

rate decreases during the treatment of a patient, it indicatesthe necrosis of hepatocytes. The RFB-BAL system de-creased the mean ammonia and bilirubin levels by 33% and11%, respectively (42).

The Academic Medical Center (AMC) BAL system de-veloped by Chamuleau and coworkers in the University ofAmsterdam, the Netherlands, consists of a nonwoven hydro-philic polyester matrix for cell attachment and hollow fibersfor oxygen supply and a spacer between the matrix (4346).The matrix, with a thickness of 4 mm and a total surfacearea of 5610 cm2, is spirally wound with longitudinal hol-low fibers. In the AMC-BAL reactor, the plasma of the pa-tient and small hepatocytes aggregates are in direct contact,

resulting in an optimal mass transfer and an optimal oxygensupply is achieved by hollow fibers in the bioreactor. Sixout of seven patients were successfully supported until anOLT. An improved urine output was noted in patients withrenal insufficiency. After the AMC-BAL treatment, theplasma ammonia and bilirubin concentrations decreased by44% and 31%, respectively (46).

In the aspect of hepatic function performance, theHepatAssist system has three important disadvantages. First,it uses a relatively small amount of cryopreserved hepato-cytes, 5% of the liver mass. Second, treatment time is veryshort, 68 h. In most other BAL systems, patients are sup-ported for at least 12 to 24 h. Third, the mass transfer be-tween hepatocytes and plasma occurs by only diffusion and

back filtration through the hollow fiber membrane in the

bioreactor. The other five systems use more than 10 billionhepatocytes (10% of the liver mass), and in the RFB-BAL,MELS, and AMC-BAL systems, the patientsplasma is indirect contact with hepatocytes. The major factor that influ-ences mass transfer efficiency between the BAL systems

and liver failure patients is plasma or blood exchange rates,ranging from 22 to 50 ml min1and 150 to 250 ml min1forplasma and blood, respectively. The plasma exchange ratesof the RFB-BAL (22 ml min1) and MELS (31 ml min1)systems are low when their cell mass is considered.

It is very difficult to compare their performance or choosethe best one among the six BAL systems, due to the com-plexity of patient groups, the different perfusion types, andother conditions in a clinical setting. However, consideringonly the performance-specific parameters, such as cell massand mass transfer rate, the RFB, MELS, and AMC-BALsystems might have a higher performance than the others.On the other hand, the others can be evaluated as safer than

these three high-performance systems.The cell density of a bioreactor in a BAL system is ex-tremely high. In addition, hepatocytes are one of high oxy-gen-consuming cells in the culture (47). Therefore, oxygensupply into the bioreactor is a very important design param-eter. Only the MELS and AMC-BAL systems have an in-tegral oxygenation system in the bioreactor; the others havea separate oxygenator placed immediately before the biore-actor. The integral oxygenator eliminates the hypoxic spaceand reduces dissolved oxygen gradient between the inletand outlet ports. When plasma or blood perfusion flow rateis inadequate, the bioreactor type with an integral oxygen-ator is recommended to prevent hypoxia of the reactor out-let portion.

Because the viability and functional activities of hepato-cytes vary considerably after cell loading and during theBAL treatment, it is very important to monitor their viabil-ity and various functions to estimate their microenviron-ment and to determine the possible treatment time. How-ever, the monitoring results of cellular activities have notbeen reported or have been often neglected. Therefore, de-creases in the bioreactor activities during the treatment of apatient must be checked and the activity information is con-sidered to improve the bioreactor because it was reportedthat plasma from liver failure patients is very harmful tocultured hepatocytes (48, 49).

BAL SYSTEMS IN PRECLINICALORIN VITROTEST

In addition to the above-mentioned BAL systems in clini-cal studies, various different BAL systems have been inves-tigated and examined for their in vitroand in vivo perfor-mance. Here, eight noteworthy BAL systems are consid-ered. Their unique bioreactor configurations are demon-strated schematically in Table 2. The LIVERx2000 systemshave already entered the clinical study phase. However, be-cause their clinical results have not been reported until todate, the BAL system is described in this section.

The LIVERx2000 system was designed and developedby Hu etal. in the Minnesota University and commercial-

ized by Algenix (St. Paul, MN, USA) (50, 51). The hepato-


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cytes suspended in a collagen gel are injected into the lumenof a hollow fiber with an MWCO of 100 kD and the extra-

capillary compartment is perfused with a recirculating me-dium. After 24 h, the gel contracts, creating a third space,which is now perfused with the medium, and the extrafiberspace is perfused with whole blood.

The LIVERAID BAL system was developed by ArbiosSystem (Los Angeles, CA, USA) founded by the principalinvestigator of the HepatAssist system (the information wasavailableatthewebofArbiosSystemInc.,http://www.arbios.com). This BAL system utilizes a multicompartmenthollow fiber module with a unique fiber-within-fiber geo-metry. This geometry allows for the integration of liver celltherapy and blood detoxification within a single module.

The oxygenating hollow fiber bioreactor (OXY-HFB)

BAL system was developed by Jasmund etal., in EberhardKarls University, Germany (52). The OXY-HFB BAL sys-tem consists exclusively of oxygenating and integral heatexchange fibers with a simple and effective design. Primaryliver cells are seeded on the surface of the fibers in the ex-trafiber space. Oxygen requirements are supplied and tem-perature is controlled via the fibers. Plasma from patients isperfused through an extrafiber space and brought into directhepatocellular contact.

The liver lobule-like structure module (LLS) BAL sys-tem, which was designed by Mizumoto and Funatsu inKyushu University, has many hollow fibers that act as ablood capillary and are regularly arranged close to eachother (53). Hepatocytes are inoculated by a centrifugal force,

at a high density in the outer space of the hollow fibers.

They form an organoid and perform many liver-specificfunctions.

The UCLA-BAL system developed by Dixit and Gitnick,in UCLA, involves the direct hemoperfusion of microen-capsulated hepatocytes in an extracorporeal chamber (54).The hepatocytes are located in biocompatible and semiper-meable calcium alginateploylysinssodium alginate com-posite membranes where they can freely interact in variousbiological reactions. Simultaneously, the microencapsulatedhepatocytes are also immunoisolated from the componentsof blood elements, thereby preventing any adverse immuno-logical reactions. Microcapsules have a high surface area, ahigh capacity for hepatocytes, and a high membrane perme-ability. All these characteristics should result in a more ef-fective and efficient metabolic exchange of metabolites with

hepatocytes.We have developed an alginate-entrapped hepatocytespheroid (AHS) BAL system, which has a configurationsimilar to that of the UCLA-BAL system (3). However, theporcine hepatocytes are suspension cultured for 20 h to formspherical aggregates, called spheroids, before entrapment.In previous in vitro studies, hepatocyte spheroids have ahigher performance of liver-specific functions than dispersedsingle hepatocytes, particularly detoxification in plasma orserum (55). A large-scale and a high-yield preparation ofhepatocytes spheroids were achieved using special mediaand a culture vessel. These hepatocyte spheroids were im-mobilized in Ca-alginate beads using a high-capacity solu-tion dropping apparatus within 20 min. The performance of

the AHS-BAL system was actively evaluated at anhepatic

TABLE 2. BAL systems in in vitroand preclinical tests
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tocytes is considered to be an optimal candidate for use inthe investigation of the biological safety of porcine tissuesunder very controlled conditions. This idea is based on thefact that the contact with xenogenic cells will occur tempo-rarily and without direct cellcell contact. Therefore, patho-

gen-free and low immunogenic pigs may offer a good andsafe hepatocytes source for a BAL system in the near future.

As another source of hepatocytes, established cell linesthat can be sufficiently expanded have been approached bydevelopment tumor-derived cell lines (28) and immortalizedcelllines, and their gene manipulations (67, 68). The C3Asubclone of HepG2 has already been used in clinical trialsdespite its poor liver functions. To enhance their weak orlost liver functions that are important for liver support, suchas ammonia removal activity, the glutamine synthetase genewas transfected and transfected cells demonstrated a highammonia removal activity in a bioreactor (68).

Cells as promising sources of human hepatocytes for a

BAL system in the future are classified into two types ac-cording to the site of origin. One is intrahepatic stem cellsand the other is extrahepatic stem cells. Intrahepatic stemcells include mature hepatocytes (69, 70), oval cells (71),small hepatocytes (72,73), hepatoblasts (74), and hepato-cyte-colony-forming units in culture (H-CFU-C) (75, 76),whereas extrahepatic stem cells include hematopoietic stemcells (HSCs) (77, 78) and mesenchymal stem cells (MSCs)(79, 80). It is believed that there is a close relationship be-tween cell types in terms of their origin (e.g., oval cell vsHSCs).

A BAL system should be custom-made for emergencysituations. Therefore, to use hepatic stem cells as a hepato-cyte source, it will be necessary to cultivate them in large

quantities and differentiate them into functional maturehepatocytes in vitro. Although the hepatic stem cells men-tioned above may have differentiation potential from a basicscientific point of view, they must satisfy three requirementsfor applications. First, they must have a proliferative capac-ity of up to at least 10 billion cells (the minimal cell numberfor a BAL system) without a loss of differentiation poten-tial. For this, it is believed that the bioreactor technologywill be involved. Second, they must differentiate to func-tional mature hepatocytes with a maximal yield based onthe precise lineage-specific differentiation protocol. Finally,the high cost arising from the use of expensive culture me-dia, growth factors, and cytokines must be solved.

DISCUSSION

Recently, the first prospective, multicenter, randomized,controlled clinical trial of a HepatAssist system has beenconducted on 171 patients in 11 U.S. and 9 European medi-cal centers (81). This trial did not achieve its primary end-point (30-d survival rate) in the overall study population.For the entire patient population, the 30 d survival rate was71% for the BAL treatment vs 62% for the control (P0.26).However, when adjusting for the impact of the disease etiol-ogy and liver transplantation on the patientssurvival, pa-tients with fulminant and subfulminant hepatic failure treatedwith the BAL system had a statistically significant (P0.048)

survival advantage compared to the controls receiving stan-

dard medical care. This clinical trial was a milestone in thehistory of BAL technology and gave a chance to carefullyconsider the requirements of a BAL system to support liverfunctions.

Recently, the membrane of the bioreactor and the reac-

tor flow rate of the ELAD system has been modified from70 kD to 120 kD and 200 ml min1to 500 ml min1, respec-tively (82). More recently, the principal developers of theHepatAssist system fabricated an improved version of theHepatAssist system, the HepatAssist-2 system, which hasspecifications similar to those of their early BAL system ex-cept for the increased cell mass (http://www.arbios.com).The HepatAssist-2 system can accommodate 15 billionhepatocytes; 3 times that of the former BAL system. More-over, the AMC-BAL system developed in Amsterdam Med-ical Center, which is the most novel noteworthy BAL sys-tem that has shown impressive phase I clinical results (46),uses 10 billion fresh (not cryopreserved) hepatocytes (two-

fold of the HepatAssist system), and the hepatocytes loadedin the bioreactor form small aggregates in the porous ma-trix. The hepatocyte aggregates are directly perfused withplasma without any immunological barriers. Gerlach in Ger-many designed the bioreactor of the LSS or MELS in theearly 1990s (36), similar to the HepatAssist system. Thisreactor can contain up to 500 g of hepatocytes, these cellsform in vivoliver-like sinusoidal structures and plasma canbe perfused via two independent compartment hollow fiberbundles. In addition, this BAL system is the only systemthat has been subjected to clinical studies using primaryhuman hepatocytes derived from discarded donor livers.Therefore, their experience might provide useful informa-tion that may assist in the development of next-generation

BAL systems that use human hepatocytes.From these developments along with the characteristics

of the modified ELAD, HepatAssist-2, AMC-BAL, MELSand other BAL systems, a large animal study of which hasrecently been completed, the following three trends can beidentified. First, cell mass employed is increasing and mostBAL systems use approximately 10 to 20 billion fresh hepa-tocytes. Second, immunological barriers are not a require-ment for a BAL system. This is because the short-term con-tact with xenogenic cells does not induce any immunologi-cal complications, and the immunological barriers are fre-quently removed for the maximum mass transfer. Third,hepatocytes are cultured as an organoid with appropriate

cellcell interactions, which can enhance and prolong thehepatic functions.Regarding hepatocyte cryopreservation, several cryo-

preservation methods for hepatocytes have been reported(83, 84). However, hepatocyte functional activities afterthawing are still unsatisfactory. The availability and logis-tics of BAL systems will be significantly improved if meth-ods of long-term preservation of hepatocytes without a lossof cellular activity were developed. Therefore, until a pres-ervation method is fully established, centralized regionalliver support centers such as the Charite Center in Germanyare needed to increase the frequency of supporting fulmi-nant and subfulminant hepatic failure patients, up to two tothree cases per week. At this frequency, the continuous

maintenance of ready-to-use BAL systems may be reason-
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able and economical.In developing a new BAL system, the design of bioreac-

tors and evaluation are the most important and difficult pro-cedures. However, there are a number of obstacles at eachdevelopment step awaiting the inventor. The poor yield andviability of isolated hepatocytes, the unstable hepatic failuremodel of small and large animal (85), the absence of a re-versible FHF pig model (86), the high research and utility

cost for experiments, the strict safety regulations for xeno-genic cell therapeutics (Guidance for industry: source ani-mal, product, preclinical and clinical issues concerning theuse of xenotransplantation product in humans. Final guid-ance, U.S. FDA, April 2003), and the difficult controlledclinical trials are the most common difficulties encounteredin BAL system development. Nevertheless, the develop-ment of BAL systems is an interesting research field for cellculture and bioreactor engineers as well as medical practi-tioners.

Although there are still many obstacles to overcome, it isexpected that highly functional human hepatocytes will be-come available within a decade with the development ofstem cell technology. Hepatocytes derived from stem cells

are first utilized for cell transplantation therapy. However,BAL systems also require human hepatocytes generatedfrom stem cells. According to recent clinical results, BALsystems using porcine hepatocytes have proven to be safe,but immunological rejection and zoonosis still remain asmajor problems. If a sufficient number of human hepato-cytes can be used as a cell source for BAL systems, the cor-responding progress in bioreactor development and the bet-ter understanding of cell biology and the physiology of liverfailure will allow the development of next-generation BALsystem. The different characteristics of the present and next-generation BAL systems are listed in Table 4.

If development of the BAL system were to progress and

the system were to reach the level of the artificial kidney, itis expected that nobody will die from acute liver failure ordue to a shortage of donor livers in the near future. More-over, a diseased liver can be supported or substituted withnew tissues generated by liver tissue engineering.

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TABLE 4. Characteristics of present and next-generation BAL systems

Present BAL system Next-generation BAL system

Hepatocyte source Pig or hepatoma Human hepatocytes from stem cells or humanized pigCulture methods (bioreactor) Simple and conventional culture techniques Liver-like structures with optimum cellcell interactions,

microarchitectured 3-D coculture, etc.

Treatment time Less than 24 h Weeks to monthsLiver function Several vital functions Entire liver functions including bile excretionTarget disease Acute liver failure, mainly fulminant

hepatic failureChronic as well as acute liver failure


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