REVIEW
Cell choice for bioartiﬁcial livers
Naoya Kobayashi, Teru Okitsu and Noriaki Tanaka
Department of Gastroenterological Surgery, Transplantation, and Surgical Oncology, Okayama University
Graduate School of Medicine and Dentistry, Okayama, Japan(Received for publication on January 31, 2003)

Abstract. It is unlikely that human hepatocytes can be isolated on a scale sufﬁcient to treat more thana fraction of the patients who need bioartiﬁcial liver (BAL) treatment. The use of animal cells results inthe concerns related to the transmission of infectious pathogens and immunologic and physiologic
incompatibilities between the donor and humans. Human embryonic stem cells and bone marrowmultipotent adult progenitor cells have received great attention as a possible source for BALs. The useof tightly regulated clonal hepatocyte cell lines would be attractive. Such cell lines grow economicallyin tissue culture and provide the advantage of uniformity, sterility, and freedom of pathogens. In thispaper, the authors review the choice of cells for BALs and discuss our reversible immortalization system of human liver cells using a retroviral transfer of immortalizing genes and subsequent Cre/ loxPmediated site-speciﬁc recombination. (Keio J Med 52 (3): 151–157, September 2003)
Key words: bioartiﬁcial liver, porcine hepatocytes, stem cells, reversibly immortalized humanhepatocytes

Introduction
Acute liver failure (ALF) is often life threatening anddramatically diminishes the quality of life of patients.1
Orthotopic liver transplantation has become a successful therapy for ALF, but this procedure is highly
costly, limited by the scarcity of donor livers and associated with high morbidity and mortality.2 There is acompelling need for developing effective alternatives
for patients with ALF. Considering the potential of theliver to regenerate, temporary support with bioartiﬁcial
livers (BALs) is an attractive approach.3,4 Since technologies of tissue cell culture and biomaterials havebeen greatly advanced, many designs of BALs, including (1) a biological component, (2) a bioreactor, and (3)a whole blood or plasma perfusion system are currently
under investigation. In the present review, we focus oncell choice for developing BALs.
In vitro and in vivo experiments for BAL development
As shown in Tables 1 and 2, many researchers havemade great efforts to develop BALs using various typesof cells in different modules.5–43 For in vitro experiments rat hepatocytes were favorably used and pig
hepatocytes were often utilized for in vivo BAL studies.
Considering BALs developed for humans, possible cellchoice is limited, including normal human adult andfetal hepatocytes, in vivo transformed human hepatocytes, porcine hepatocytes, human-derived stem cells,
and reversibly immortalized human hepatocytes.
Normal human hepatocytes
Although normal human livers are an ideal sourceof cells for BAL therapy, donor liver shortage issevere world-wide and the availability of the liver forhepatocyte isolation is unfortunately limited by competition for the use in whole organ transplantation. Stromet al. demonstrated the usefulness of human fetal hepatocytes for cell therapies because of their proliferative
capacity and differentiation potential, but the use of the
fetal hepatocytes raises an ethical issue.44 On the basisof results from liver surgery in humans, it is estimatedthat approximately 10% to 30% of residual liver
parenchyma would be required to support the life of

Presented in part at the 2002 Keio Medical Science Prize Symposium, November 29, 2002.
Reprint requests to: Dr. Naoya Kobayashi, Department of Gastroenterological Surgery, Transplantation, and Surgical Oncology, Okayama
University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan, facsimile (þ81) 86-221-8775, e-mail:immortal@md.okayama-u.ac.jp
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Table 1

Kobayashi N, et al: Cell choice for bioartiﬁcial livers
In Vitro BAL Experiments

Investigator
Wolf and Munkelt5)
Hager6)
Kasai7)
Demetriou8)
Jauregui9)
Yanagi10)
Moscioni11)
Shatford12)
Sussman and Kelly13)
Nyberg14)
Li15)
Rozga16)
Fremond17)
Kong18)Bader19)
Gerlach20)
Morsiani21)
Kobayashi22)
Linti C23)

Table 2Cell typeEvaluation

Reuber hepatoma cells
Mouse
Dog
Rat
Rat
Rat and rabbit
Human
Rat
C3A cells
Rat
Rat
Rat
Rat
Pig
Rat
Pig
Pig
Immortalized human hepatocytes
Pig

Bilirubin conjugation
Ureagenesis, protein synthesis, diazepam metabolism
Maintenance of ATP
Bilirubin synthesis and conjugation, protein synthesis, diazepam metabolism
Ammonia removal, urea synthesis, cyclosporine metabolism
Albumin synthesis, amino acid and lidocaine clearance
Glucose utilization, albumin synthesis
Synthesis of albumin and urea, lidocaine metabolism, arginine clearance
Urea, albumin synthesis
Cyclosporine and 19-nor-testosterone metabolism, bilirubin conjugation
Lidocaine, ethoxyreso-ruﬁn metabolism
Cyclosporine and rapamycin metabolism
Amino acid and keto-acid metabolism
Cholate metabolism
Clearance of NH3
P450-dependent metabolic function (MEGX test)

patients.45 Thus, it seems to be difﬁcult to provide thefetal hepatocytes in such a large scale.

In Vivo BAL Experiments

Investigator

Cell typeMatsumura24)
Olumide25)
Kasai7)
Uchino26)
Yanagi10)
Arnaout27)
Shnyra28)
Nyberg14)
Sussman29)
Takahashi30)
Fremond17)
Rozga31,32)
Gerlach33)
Jauregui34)

Rat
Pig
Dog
Pig
RabbitRat
Rat
Rat
C3A cells
Pig
Rat
Dog, Pig
Pig
RabbitDixit35)
Chen36)
Suh37)

Pig
Pig
Rat

Stevens38)
Mazariegous39)

Pig
Pig

Millis40)
Abrahames41)

C3A cells
Pig

Ambrosino42,43)

Pig, Matrix

Evaluation
Albumin synthesis
Neurologic improvement
Improved survival
Improved survival
Clearance of NH3
Bilirubin conjugation
Improved survival
Albumin synthesis
Improved survival
Improved survival
Bilirubin conjugation
Clearance of NH3 and lactate
Clearance of NH3
Clearance of diazepam andlidocaine clearance, improved
survival
Improved survival
Improved survival
Improved coagulopathy andsurvival
Improved survival
Hemodynamics (transienthypotension andthrombocytopenia)
Elimination of phenylephrine,
reduction of dopamine andrespiratory support
LDH leakage, NH3 clearance,urea synthesis, 7-ethoxycoumarinO-deethylase (ECOD) activity
and pseudocholine esterase

In vivo transformed human hepatocytes
Human hepatocytes should be ideally the optimal
biological component in BALs, however, this approachis impractical due to the shortage of human livers.
Sussman et al. used a human differentiated hepatoblastoma cell line C3A as a source of the ELAD.3 In
the literature, the C3A retains differentiated hepaticfunctions while showing a short population doublingtime and contact inhibition. Patients underwent ELAD
with whole-blood perfusion continuously for relatively
long periods of time.3 In the initial group of 10 patients,
no signiﬁcant effect on the outcome in patients with
ALF was noted, with only one survivor. The ELAD
therapy was further tested at King’s College Hospitalin London, with no signiﬁcant evidence of metabolic
support and no beneﬁcial effect on patient survival.13
However, leakage of these tumor-derived cells into thepatient’s circulation on unexpected device failure remains a major concern in such a compromised host.
In fact, the extracapillary passage of HepG2 cells,
which are similar to C3A, from a hollow ﬁber membrane was observed in vitro BAL experiments.39
Porcine hepatocytes
In 1994, Nyberg et al. reported that primary hepatocytes outperformed all available liver cell lines inKeio J Med 2003; 52 (3): 151–157

terms of bio-transforming functions.46 The result
prompted investigators to use porcine hepatocytes todevelop BALs. In addition, pigs have similar physiology to humans and one porcine liver can provide
enough hepatocytes for several BAL treatments.
Demetriou et al. started Federal Drug Administration(FDA) approved clinical trials using a porcine hepatocyte-based BAL system (HepatAssist, Circe Biomedical Inc., Lexington, MA, USA) since 1994.47 They
have used plasma separation and BAL perfusion for 6hours and patients tolerated the treatments well.
They have mentioned that porcine hepatocytes havea tendency to form cell aggregates, resulting in themaintenance of differentiated hepatic functions.47 Regarding the clinical studies using xenogenic cells, thepotential hazard of pathogens from porcine-derived
organs raises a new issue of xenozoonosis. Concern has
particulary increased regarding xenozoonotic infection,
since porcine endogenous retroviruses (PERV) werereported to infect human cells.48–50 The phase II/III
trial using HepatAssist in 171 patients was conducted
and the 30-day survival was reported to be 70% fora BAL-treated group and 60% for a control group
receiving standard medical therapy (personal communication with Dr. Demetriou AA, Cedars-Sinai Medical
Center, LA, USA). Surviving patients were retrospectively examined for the presence of PERV and there
was no evidence of viral transmission from pigs tohumans. The data were encouraging in developing
clinical therapies using porcine hepatocytes, however,needless to say, we should collect pertinent results regarding the safety of xenogenic porcine tissues and cells
in the future.
Human-derived stem cells
Regenerative medicine using stem cell biology is
attracting great attention. Strategies for regenerative
therapy using a stem cell system are roughly partitioned
into: 1) the use of organ (or tissue) stem cells, 2)
the use of embryonic stem (ES) cells, 3) the use of
dedifferentiation/transdifferentiation of differentiatedcells. Therapeutic designs should be determined basedon a comprehensive consideration of the available information. Differentiation of human ES cells and multipotent adult progenitor cells (MAPCs) in the humanbone marrow into hepatocytic cells are reviewed here.
ES cells are undifferentiated stem cell lines establishedfrom the epiblasts that are present in the inner cell massof an early embryo at the blastocyst stage. Epiblasts
differentiate into the developmental process and they
are capable of differentiating into the three germ layers.
Thus, ES cells can be induced to differentiate into various types of cells under speciﬁc culture conditions. Human ES cell lines were established by Thomson et al. in

153

1998 and, therefore, have been a focus of regenerative
medicine and received attention as a potential sourcefor BAL.51 Investigators at Geron Corporation (Menlo
Park, CA, USA) have currently shown that human ES
cells can be differentiated into hepatocytic cells in vitro
under the presence of sodium butyrate in the culture
medium.52 The expression of albumin, cytokeratin 8,
anti-alpha trypsin, and glycogen is positive in such differentiated cells. Catherine et al. have identiﬁed thepresence of MAPCs in the mouse, rat, and human bonemarrows and demonstrated that MAPCs differentiatedinto the functional hepatocytic cells with producing albumin and urea in the hepatocyte growth factor (HGF)and basic ﬁbroblast growth factor (bFGF) based culture
medium.53 These cells would be a candidate for developing BALs, however, one should keep in mind thatneither transdifferentiation or dedifferentiation of suchcells in BAL modules can be guaranteed. Stem cell biology should be well characterized in the near future tofacilitate BAL therapies.
Reversibly immortalized human hepatocytes
Concerns about porcine hepatocytes include xenozoonosis and immunologic and physiologic incompatibility with the human host, while human cell-lines expose patients to the potential risk of releasing tumor
cells or tumorigenic products from the BAL device into
their circulation.54,55 The utilization of stem cells inhumans will need some time to clearly address thestem cell biological system. To overcome these problems, we have focused on reversible immortalizationof human hepatocytes to intentionally control the population expansion. A tightly regulated system for cell
growth should be considered to generate immortalized
hepatocyte cell lines that are suitable for clinical use.
Thus, we applied a Cre/loxP site-speciﬁc recombination
system. DNA sequences intervened by loxP recombination targets can be excised after expression of Cre
recombinase.56 The Cre/loxP system has been widelyused to control gene expression in transgenic mice.
In order to provide stringent control over expressionof transforming genes, we immortalized human hepatocytes with retroviral vectors SSR#69 (for expressing
SV40T)57 and SSR#197 (for expressing human telomerase reverse transcriptase (hTERT))58 with selectable positive and negative markers, which were intervened by a pair of loxP recombination targets andsubsequently excised by Cre/loxP site-speciﬁc recombination, as illustrated in Fig. 1. Using SSR#69, we established a reversibly immortalized human hepatocyte cellline NKNT-3.59
The ability to measure in vitro differentiated functions is extremely dependent on culture conditions and,for oncogene-transformed cells, there is evidence that

154

Fig. 1 Illustration demonstrating reversible cell immortalization. We
utilized retroviral vectors SSR#69 and SSR#197 to immortalize humanliver cells. After Cre/loxP site-speciﬁc recombination, the intervening
DNA segment between the two loxP recombination targets can be
excised (LTR, long terminal repeat; SV40T, simian virus 40 large T
antigen; hTERT; human telomerase reverse transcriptase; IRES, internal ribosomal entry site).

an improvement in differentiated cellular responses
can be accomplished simply by serially transferring
the cells into animals.60,61 Thus, we performed intrasplenic transplantation of NKNT-3 cells treated withCre recombinase, reverted NKNT-3 cells, in a rat modelof liver failure. Transplantation of the cells signiﬁcantly
prolonged the survival of the rats. Extension of thisprocedure to other cell types presenting in the humanliver allows studies of cell-cell interaction and further
contributes to the development of cell therapies andBALs.62 One of our long-term goals is the development of BAL systems that closely mimic the function of
the normal liver in vivo. Pure cultures of hepatocytes
recapitulate several key liver functions but fail to provide adequate levels of a few important detoxyfying
enzymes that include the cytochrome p450 associatedenzymes (CYPs). Among the known crosstalk between
hepatocytes and other liver cells, hepatic stellate cell
(HSCs) are believed to play an essential role.63,64 Previous attempts to develop BAL have focused on thehepatocyte biosynthetic function, ignoring the reticuloendothelial role performed by liver sinusoidal lining
cells. Recently, heterotypic cell interactions between
parenchymal cells and nonparenchymal neighbors havebeen recognized to be central to the function of manyorgan systems. In both the developing and mature
adult liver, cell-to-cell interactions are imperative forcoordinating the sophisticated liver functions.65 There-

Kobayashi N, et al: Cell choice for bioartiﬁcial livers

fore, we have applied the Cre/loxP system to humanliver endothelial cells and hepatic stellate cells.58,62 We
have found that the co-culture of NKNT-3 cells with
SSR#197-immortalized hepatic stellate TWNT-1 cells
increased CYP3A4 and CYP2C9 expression. The ﬁnding supports the contention that heterotypic cell interaction is of importance to enhance the production ofliver speciﬁc enzymes by hepatocytes in vitro.66,67 Because newly developed drugs are still screened for theirsafety and efﬁcacy in animal models, BALs of multiple
cell composition should become an attractive platform
as an alternative to animal testing. Provision of the biledrainage system is also an important issue to develop
novel BALs. To address the issue, we have currently
established a human cholangiocyte cell line MMNK-1.
The MMNK-1 cells showed the expression of cytokeratins 9 and 17 and cholangiogenic potential in a Matrigel. Development of a coculture system of hepatocytes
and MMNK-1 cells is now under investigation.68 The
application of BALs that we are currently developing
includes: 1) Bridge use until organ transplantation isavailable or hepatic regeneration is completed, 2) Prevention of hepatic encephalopathy of patients withdecompensated liver cirrhosis, 3) Models for drug testing, and 4) Development of new cellular products. The
use of Cre/loxP-based reversible immortalization represents an important step in the development of a potentially novel strategy for resolving the organ shortage
that currently limits the use of healthy human hepatocytes for BALs. This technology may be applicable
to a variety of somatic cells and could potentially beutilized to treat a large fraction of clinically signiﬁcantpathologic conditions.
Redundant safeguards in the reversibly immortalized
human hepatocytes
A method to protect patients from the possible
migration of cells utilized in BALs is to introduce suicide genes into the cells. The cells modiﬁed to containa herpes simplex virus-thymidine kinase gene (HSVTK) become sensitive to the treatment with an antiviral agent ganciclovir (GCV), whereas normal cells
are unaffected by the drug.69 GCV is converted into
nucleotide-like precursors that kill cells containing
HSV-TK by blocking DNA synthesis. Since GCV does
not interact with human thymidine kinase, it is not toxic
to most human tissues lacking HSV-TK. A cytosine
deaminase gene can be utilized as a suicide material.70
5-ﬂuorocytosine (5-FC) at the therapeutic doses is nottoxic to normal cells, but the cells expressing cytosine
deaminase convert 5-FC to 5-ﬂuorouracil (5-FU). Then,
5-FU is further metabolized to produce suppression ofcell growth and cell death by inhibition of RNA andDNA syntheses.

Keio J Med 2003; 52 (3): 151–157

Prospect of the future of BAL therapy
Kjaergard et al. have currently performed a systematic review to evaluate the effect of artiﬁcial andbioartiﬁcial support systems for acute and acute-onchronic liver failure.71 They have concluded that artiﬁcial support systems reduce mortality by 33% in
acute-on-chronic liver failure compared with standard
medical therapy and that artiﬁcial and bioartiﬁcial support systems did not appear to affect mortality in ALF.
Precipitating factors in ALF include drug toxicity andviral hepatitis, which are difﬁcult to treat. This may
explain why support systems are effective in acute-onchronic liver failure but not in ALF. Based on theirreview, currently available BALs cannot be effective
enough in patients with ALF. Thus, it would be important to treat such patients not only with a new typeof a highly functional BAL in which hepatocytes will
be utilized in conjunction with non-parenchymal liver
cells and extra cellular matrices but also with currently
available viral therapies.
Conclusion
The goal of BAL therapy is to replace whole-livertransplantation in patients with an acutely devastatedliver. Toward this goal, integration of cell culture andgene transfer technology and the cutting wedged-biotechnology is urgently required for the development ofsophisticated BAL systems that mimic the in vivo liver.
Acknowledgements: We thank Dr. Achilles A. Demetriou (Department of Surgery and the Liver Support Unit, Cedars-Sinai Medical
Center, UCLA School of Medicine, Los Angels, CA, USA) for his
creative thinking and discussion on HepatAssist BAL treatments,
Drs. Jane Lebkowski and Choi-Pik (Geron Corporation, Menlo Park,
CA, USA) for providing fruitful information regarding human ES
cell-derived hepatocytic cells and Mrs. Stephen Andre and Shawn P.
Cain (Circe Biomedical, Inc., Lexington, MA, USA) for important
information on HepatAssist. The work presented in this review paper
was supported in part by the Ministry of Education, Science, andCulture, and the Ministry of Economy and Industry, and Life Science
Project of 21st Century, Japan.

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