Yonago Acta medica 1999;42:103–112

Biochemical Functions and mRNA Expression of Rat Hepatocytes in a
Bioartificial Liver Module
Shinichi Okada, Yumi Kono and Kazuo Shiraki
Department of Pediatrics, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
Evaluation of hepatic functions of the bioartificial liver (BAL) module is essential fordeveloping an optimal BAL system which can be applied to a patient with fulminant
hepatic failure. Although hepatic function in BALs has been evaluated only in biochemical activities in previous reports, we assessed a newly designed BAL from a hollow
fiber type not only in biochemical activities such as gluconeogenesis, ureogenesis andalbumin synthesis but also in the expressions of albumin messenger RNA (mRNA) andcytochrome P450 2B1 mRNA by Northern blot analysis. One to 3 × 108 hepatocytes
isolated from adult rats were suspended in hydrated collagen, loaded into a hollow
fiber module and perfused with L-15 medium supplemented with various hormonesfor 2 to 13 days. Both production rates of glucose and urea nitrogen were highest on
day 1 (610 µg glucose/107 cells/h and 76.1 µg urea nitrogen/107 cells/h, respectively).
Ureogenesis was maintained for 8 days in BAL, but gluconeogenesis decreased after 7
days. Albumin production rate increased to day 3, reached its maximum (11.0 µg/107
cells/h) and then gradually decreased. The expression of albumin mRNA was highest
on day 2 and detectable until day 11. Cytochrome P450 2B1 mRNA was detected only
on day 2. Northern blot analysis showed that the maintenance period in each hepaticfunction was different. These results suggested that assessment of the functions at amolecular level was invaluable in the development of a potential BAL for clinical use.
It might be necessary to reconstruct the lobular system as well as a 3-dimensional
environment for hepatocytes in the BAL to maintain the ability of detoxification suchas cytochrome P450s.
Key words: albumin mRNA; bioartificial liver; cytochrome P450 2B1 mRNA; hepatic function

Despite recent advances in intensive medical
treatment, a patient with fulminant hepatic
failure has a poor prognosis (Pappas, 1988).
Liver transplantation is an effective therapy, but
is not always available. As another strategy forthe treatment of severe hepatic failure, several
cases using a bioartificial liver (BAL) system
mainly for bridging to a transplant liver havebeen reported (Chen et al., 1997). There are,however, only a few cases that have recovered
using only a BAL (Matsumura et al., 1987;
Sussman and Kelly, 1993; Chen et al., 1997). It
is mainly due to the reason why the ability of

BAL as a “biochemical factory” as the liver invivo is not completely maintained for a
prolonged period. Therefore, accurate andspecific assessment of hepatic functions ofbioartificial modules, such as gluconeogenesis,
ureogenesis, albumin synthesis and also abilityof detoxification, is essential for developing an
optimal BAL system.
In previous reports, the hepatic functions of
BALs were evaluated only in the biochemical
activities of glucose, urea nitrogen or albumin
production (Shatford et al., 1992; Takeshita etal., 1995; Naruse et al., 1996a, 1996b; Sielaff etAbbreviations: BAL, bioartificial liver; CYP 2B1, cytochrome P450 2B1; ELISA, enzyme-linked
immunosorbent assay; FCS, fetal calf serum; hEGF, human epidermal growth factor; L-15, Leibovitz’s L-15
medium; PBS, phosphate buffered saline; PVLA, poly-N-p-vinylbenzyl-D-lactonamide; WE, Williams’ E
medium103

S. Okada et al.

al., 1997) but not on the gene expression level,although hepatocytes of conventional cultures
are investigated extensively using molecularmethods (Kocarek et al., 1993; Kimball et al.,
1995; Nagaki et al., 1995; Tamura et al., 1995).
In this study, we designed a potential BAL of ahollow fiber type loading rat hepatocytes at ahigh density which maintained hepatic functions. The medium for perfusion and basement
matrix of a BAL were selected based on resultsfrom hepatocytes cultured on dishes. Evaluation of maintenance of the liver functions of
BALs over 13 days was performed not only bybiochemical assays but also by Northern blot
analysis of albumin messenger RNA (mRNA)and cytochrome P450 2B1 (CYP 2B1) mRNA.

Materials and Methods

Reagents
Leibovitz’s L-15 medium (L-15), Williams’ E
medium (WE) and fetal calf serum (FCS) wereobtained from Gibco BRL (Rockville, MD).
Collagenase was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Prolin, insulin,
dexamethasone and glucagon were from Sigma(St. Louis, MO). Human epidermal growth
factor (hEGF) was from Wakunaga Pharmaceutical Co. (Hiroshima, Japan). [α-32P]dCTP
was from Amersham (Amersham, United
Kingdom). Other reagents except describedabove were of analytical grade.

Isolation and dish culture of hepatocytes
Hepatocytes were isolated from adult male
Wistar rats (140–170 g) by in situ collagenase
perfusion method modified in our laboratory as
described previously (Kohno et al., 1991). In
preliminary experiments, to assess maintenanceof the liver-specific functions in a long-term
culture, 2 different types of media and 3 typesof extracellular matrices were examined with
hepatocytes cultured on dishes. The media
were based on WE or L-15 supplemented with
5% FCS, 5000 KIU/L aprotinin, 10 –7 mol/L
insulin, 10–7 mol/L dexamethasone, 10–8 mol/L

glucagon and 10 ng/mL hEGF. Proline (30 mg/
L) was added only to L-15. Three types ofmatrices tested were non-coated plastic (noncoated), hydrated collagen gel (collagen gel)(Enami et al., 1987) and poly-N-p-vinylbenzylD -lactonamide (PVLA) (Akaike et al., 1993).
The culture media were changed every 24 h andthe hepatocytes were cultured for 24 h to 96 h ina humidified incubator at 37˚C under 5% CO2
in air, and were evaluated by biochemical
assays.

Bioartificial liver module
Hepatocytes were loaded into a hollow fiber
cartridge manufactured for clinical hemodialysis (SULFLUX FS-03, from Kaneka Co.,
Osaka) as a bioartificial liver module. The cartridge had 2 separate chambers: the spacearound the hollow fibers (extracapillary) andthe space within the hollow fibers (lumen). The
fiber-surface area was 0.3 m2 and volumes ofthe lumen and extracapillary space were 35 mL
and 55 mL, respectively. The device consistedof polysulfone fibers of a 340 µm inside diameter, a 50 µm membrane thickness and a 0.2µm porous size. The extracapillary surfaces ofhollow fibers were precoated with 0.015% type
I collagen solution (cellmatrix, from Nitta
Geratin Co., Osaka) before the inoculation ofhepatocytes. Isolated hepatocytes with a viability more than 80% by trypan blue exclusion test
were suspended in 0.1% type I collagen solution with concerted L-15 medium (Enami et al.,1987). In each experiment, 1.0 to 3.0 × 108
hepatocytes in 40 mL of the collagen gel solution were loaded into the extracapillary space ofthe cartridge. After 4 h incubation to accelerate
gel formation in a humidified atmosphere of
95% air and 5% CO2 at 37˚C, perfusion of theluminal space was started with approximately
200 mL of perfusion medium consisting of L15 supplemented with 5% FCS, 5000 KIU/L
aprotinin, 30 mg/L prolin, 10–7 mol/L insulin,
10–7 mol/L dexamethasone, 10 –8 mol/L glucagon and 10 ng/mL hEGF. The perfusion
medium was preincubated at 37˚C, given oxygen at 500 mL/min and circulated at approximately 20 mL/min. The perfusion medium was

104

The mRNA expression of hepatocytes in a BALchanged every 24 h and continued to perfuse for
2 to 13 days under a sterilized hood.

Biochemical assay of metabolic activities

Gluconeogenesis
After removal of the perfusion medium, modules were perfused in the luminal space for 2 hwith glucose-free Hanks’ solution (pH 7.4),
supplemented with 10 mmol/L HEPES, 10 -5
mol/L glucagon, 2 mmol/L L-alanine, 2 mmol/L
DL -lactic acid. Glucose in the perfusate wasmeasured by a Glucose C II-test Wako kit(Wako) using the mutarotase-glucose oxidase
method (Miwa et al., 1972).
Ureogenesis
After the gluconeogenesis assay, Hanks’ solution (pH 7.4) with 10 mmol/L HEPES, 5 mmol/
L NH4Cl was perfused in the luminal space for
2 or 3 h. Urea nitrogen in the perfusate wasmeasured using a Urea-Nitrogen test kit (Wako)
by the diacetylmonoxime method (Coulombeand Favreau, 1963).
Albumin assay
After the Hanks’ solution was perfused forassaying gluconeogenesis and ureogenesis, theluminal space was perfused with a fresh L-15
medium supplemented with 5% FCS, 5000 KIU/
L aprotinin, 30 mg/L prolin, 10–7 mol/L insulin,
10–7 mol/L dexamethasone, 10–8 mol/L glucagon and 10 ng/mL hEGF for 12 to 25 h. The albumin concentration in the perfusate was measured by enzyme-linked immunosorbent assay(ELISA) method (Dunn et al., 1992). Purified
rat albumin and peroxide-conjugated rabbit
anti-rat albumin antibody was obtained fromICN Pharmaceuticals (Costa Mesa, CA). The
antibody was preincubated with an excess ofbovine albumin (30 mg/mL in 0.5% Tween-20
in PBS) before the sample addition in order toblock any cross reaction (Shatford et al., 1992).

added to the perfusate for 24 h before the day of
harvesting hepatocytes.

Harvest of hepatocytes
After removing the perfusion medium, themodule was perfused in the extracapillary spacewith RNase free 0.05% collagenase solution for
1 h followed by PBS for 1 h to harvest hepatocytes. The collected cell pellets by centrifugation (50 × g, 2 min, 4˚C) were stored at –80˚Cuntil RNA extraction.
RNA extraction and cDNA probes
Total RNA of hepatocytes was extracted using
an acid guanidinium thiocyanate phenol chloroform method (Chomczynski and Sacchi, 1987)(ISOGEN, from Nippon Gene Co., Ltd., Toyama,
Japan). The liver RNA was extracted by themodified guanidine ultracentrifugation method
using Beckman SW41Ti roter (25,000 rpm, 20
h, 20˚C) (Ullrich et al., 1977) from normal liver
specimens and liver specimens of adult Wistar
rats, which were induced CYP 2B1 by intraperitoneal injections of phenobarbital at a doseof 150 mg/kg weight at 48 h and 24 h before
sacrifice. The rat albumin complementary
DNA (cDNA) probe (HG293: pAct-Alb) was
obtained from the Health Science Research
Resources Bank (Osaka), the rat CYP2B1
cDNA probe (rat pcP-450pb1) (Fujii-Kuriyama
et al., 1982) was from the Riken Gene Bank
(Ibaragi, Japan) and the rat β-actin cDNA probe
was a kind gift from Dr. Ueta (The Gene Research Center, Tottori University).
Northern blot analysis
Total RNA (20 µg) of each sample was hybridized with [32P]-labeled cDNA probes of rat albumin and CYP 2B1. The same blots were rehybridized with rat β-actin cDNA probe to assess
equal loading of the blots. Autoradiograms
were quantified by densitometric scanning
using a Phosphor Image Scanner (Molecular
Imager system GS-525, BioRad, Hercules, CA).

Northern blot analysis

Induction of CYP 2B1
To induce CYP 2B1, 1 mmol/L phenobarbital(Marco Seiyaku Co., Ltd., Nagoya, Japan) was

Statistical analysis
Experimental dates were expressed as mean ±SD (Tables 1 and 2) and mean ± SE (Figs. 1–3).

105

S. Okada et al.

Table 1. Metabolic activities of hepatocytes cultured on non-coated dishes with WE or L-15
medium for 24 h or 72 h
Medium†Glucose24 h

72 h

Production rate‡ (µg/107 cells/h)
Urea nitrogen
24 h
72 h

Albumin
24 h

72 h

WE
466 ± 30.4
156 ± 9.60
8.40 ± 1.60
6.20 ± 1.60
11.2 ± 5.00
5.30 ± 1.40
L-15
275 ± 22.8
200 ± 8.10
27.8 ± 2.20
15.9 ± 2.60
22.0 ± 11.2
11.0 ± 3.00
P value
0.01030.00380.01010.00390.07680.0004
FCS, fetal calf serum; hEGF, human epidermal growth factor; L-15, Leibovitz’s L-15 medium; WE,
Williams’ E medium.
† Both media were supplemented with 5% FCS, 5000 KIU/L aprotinin, 10–7 mol/L insulin, 10–7 mol/L
dexamethasone, 10–8 mol/L glucagon and 10 ng/mL hEGF. the L-15 was added 20 mmol/L NaHCO3 and 30
mg/L proline also.
‡ Production rates were calculated as µg products/107 cells/h and expressed as mean ± SD of 6 dishes in one
experiment.
Mann-Whitney’s U test was performed between WE and L-15 medium.

The differences between any 2 groups wereanalyzed by Mann-Whitney’s U test. Values of
P < 0.05 were considered significant.

Results

Metabolic activities of hepatocytes
cultured on dishes
Glucose, urea nitrogen and albumin production
rates in hepatocytes cultured on plastic dishes
compared to WE and L-15 medium are summarized in Table 1. After 24 h in culture, urea

nitrogen production was higher in hepatocytes
cultured with L-15 based medium than thehepatocytes cultured with WE based medium(P < 0.05) although gluconeogenesis was higher in hepatocytes cultured with WE medium (P< 0.05). All production rates, glucose, urea
nitrogen and albumin in hepatocytes cultured
with L-15 were significantly higher than with
WE after 72 h in culture (P < 0.01, P < 0.01, P <0.001, respectively). Marked differences inthese activities were observed in hepatocytes
cultured on different types of extracellularmatrices (Table 2). Production rates of glucose
and urea nitrogen at 48 h (P < 0.001, P < 0.001,

Table 2. Metabolic activities of hepatocytes cultured on 3 types of matrix-coated dishes†
Matrixcoated

Glucose48 h

96 h

Production rate‡ (µg/107 cells/h)
Urea nitrogen
48 h
96 h

Albumin48 h

96 h

Non-coated
158 ± 17.1
151 ± 27.5
78.8 ± 25.5
66.2 ± 55.4 21.0 ± 5.10
37.7 ± 21.9
Collagen gel 1780 ± 238*** 914 ± 239*** 329 ± 57.5*** 245 ± 153* 19.0 ± 6.00NS 53.9 ± 26.9*
PVLA
159 ± 8.70NS 140 ± 22.8NS 66.9 ± 27.0NS 55.4 ±41.0NS 26.9 ± 8.20* 39.5 ± 19.7NS
FCS, fetal calf serum; hEGF, human epidermal growth factor; L-15, Leibovitz’s L-15 medium; PVLA,
poly-N-p-vinylbenzyl-D-lactonamide.
† Hepatocytes were cultured with the L-15 medium supplemented with 20 mmol/L NaHCO3, 5% FCS, 5000
KIU/L aprotinin, 30 mg/L proline, 10–7 mol/L insulin, 10–7 mol/L dexamethasone, 10–8 mol/L glucagon and
10 ng/mL hEGF for 48 h or 96 h.
‡ Production rates were calculated as µg products/107 cells/h and expressed as mean ± SD of 6 dishes in 2
separate experiment.
Mann-Whitney’s U test was performed between non-coated and collagen gel, between non-coated and
PVLA. *P < 0.05, **P < 0.005, ***P < 0.001; NS, not significant.

106The mRNA expression of hepatocytes in a BAL(µg/107 cells/h)

Urea nitrogen production(µg/107 cells/h)

Glucose production

1000
800
600
400
200

0

80

60
40

20
0

1

2

3

4

5

6

7

8

9

1

10 11 12 13

Day

2

3

4

5

6

7

8

9

10 11 12 13Day

Fig. 1. Gluconeogenesis of hepatocytes in the bioartificial modules. Production rates were calculated
as µg glucose/107 cells/h and expressed as mean ±SE of 10 samples in 5 separate experiments. The
highest glucose production rate was observed on day
1 (610 µg/107 cells/h).

respectively) and all the rates of glucose, urea
nitrogen and albumin at 96 h (P < 0.001, P <0.05, P < 0.05, respectively) of hepatocytes entrapped in collagen gel were significantly higher than those of the hepatocytes on non-coateddishes or on PVLA coated dishes. According tothese results, L-15 based medium and collagengel were selected as the perfusion medium andthe extracellular matrix for a bioartificial liver
module.
(µg/107 cells/h)
20
18Albumin production

100

16
14
12
10
8

Fig. 2. Ureogenesis of hepatocytes in the bioartificial modules. Production rates were calculated as µg
urea nitrogen/107 cells/h and expressed as mean ±SE of 10 samples in 5 separate experiments. The production of urea nitrogen was highest on day 1 (76. 1µg/107 cells/h).

Metabolic activities of hepatocytes ofthe bioartificial liver module
To evaluate metabolic activities of hepatocytes
in the long-term culture of all modules, the production rate was calculated from total cell numbers at the time of inoculation and expressed asµg products/107 cells/h. The highest glucose
production rate was observed on day 1 (610 µg/
107 cells/h), gradually decreased to 70.2 µg/107
cells/h on day 8 and was maintained until day
10 on the same level (Fig. 1). The production of
urea nitrogen was highest on day 1 (76.1 µg/107
cells/h) and was maintained to day 8 (32.8 µg/
107 cells/h) as shown in Fig. 2. Albumin production rate was at its maximum on day 3 (11.0 µg/
107 cells/h), then gradually decreased and wasmeasurable until day 13 in the module (Fig. 3).

6
4

Northern blot analysis of hepatocytes ofthe bioartificial liver module

2
0
1

2

3

4

5

6

7

8

9

10 11 12 13Day
Fig. 3. Albumin production rate of hepatocytes inthe bioartificial modules. Production rates were
calculated as µg albumin/107 cells/h and expressed
as mean ± SE of 18 samples in 6 separate experiments. The albumin production rate was at its maximum on day 3 (11.0 µg/107 cells/h).

The albumin mRNA was expressed in thehepatocytes cultured in the bioartificial liver
modules from day 2 to day 11 (Fig. 4A). To
assess quantitative expression of mRNAs, theratio of albumin mRNA/β-actin mRNA andCYP 2B1 mRNA/β-actin mRNA were calculated (Fig. 5). It was most strongly expressed107

S. Okada et al.

Hepatocytes

No

rm

al
Ind
uc
ed
Da
y2
Da
y5
Da
y1
1

Liver

A
Albumin

Discussion
The development of hybrid artificial liver devices has been exhaustively investigated for decades (Matsumura et al., 1987; Takahashi et al.,
1992; Fremond et al., 1993; Li et al., 1993;
Naruse et al., 1996a; Chen et al., 1997), and yetnone of the studies has been established as astandard clinical treatment for liver failure. The
complexity of functional activities of the liver
makes it difficult to design such systems. In
order to develop modules that can support andimprove patients with liver failure, it is necessary to evaluate the functional capacity ofhepatocytes in the systems over periods. Suitable markers for following quantitative specificfunctions of the device are required (Lazar etal., 1995). We designed a hollow fiber typeBAL inoculating primary hepatocytes from rats
cultured with L-15 medium entrapped in collagen gel which was expected to maintain hepatic function for a prolonged period. In addition to assays of biochemical activities, such as
gluconeogenesis, ureogenesis, and albumin
synthesis, the expression of albumin mRNA

Albumin/β-Actin mRNA ratio

B

CYP 2B1

C

10

1.0

Albumin
CYP 2B1

8

6

0.6

4

0.4

2

0.2

0

0

1
y1

y5

Da

Da

Fig. 4. Northern blot analysis of hepatocytes in thebioartificial modules. A) Albumin mRNA. B)
Cytochrome P450 2B1 (CYP 2B1) mRNA. C) βActin mRNA. Each lane shows control RNA from anormal adult rat liver (Normal), RNA from an adultrat liver induced with phenobarbital (Induced),
RNAs on days 2, 5 and 11 of hepatocytes inducedwith phenobarbital from bioartificial liver modules.

y2

al

Liver

Da

rm

ed
uc
Ind

No

β-Actin

0.8

CYP 2B1/β-Actin mRNA ratio

on day 2 in the ratio of albumin mRNA/β-actinmRNA and then gradually decreased. The
expression of CYP 2B1 mRNA by induction
with phenobarbital was detected at day 2 but onvery low level (Fig. 4B). No inducibility ofmRNA of CYP 2B1 was found on day 5 or day
11. The expression of rat β-actin mRNA, used
as an internal control, was the same level during
culturing in the device (Fig. 4C).

Hepatocytes

Fig. 5. The ratio of albumin mRNA/β-actin mRNA
expression and cytochrome P450 2B1 (CYP 2B1)
mRNA/β-actin mRNA expression. Each lane shows
control RNA from a normal adult rat liver (Normal),RNA from an adult rat liver induced withphenobarbital (Induced), RNAs on days 2, 5 and 11
of hepatocytes induced with phenobarbital frombioartificial liver modules.

108

The mRNA expression of hepatocytes in a BAL

and CYP 2B1 mRNA by Northern blot analysis
was achieved to clarify whether or not thesefunctions were present in sufficient amounts.
We selected the L-15 medium as the perfusion medium for the module, but the WE medium had been used in many previous reports(Shatford et al., 1992; Takeshita et al., 1995;
Naruse et al., 1996a). The differences between
the L-15 medium and the WE are as follows:
the concentration of amino acids in L-15 ishigher than in WE, galactose is used instead of
glucose in L-15, and the pH of L-15 medium isstable in an atmosphere without 5% of CO2.
Tanaka and others (1979) reported that hepatocytes cultured at a high amino acid concentration were consuming amino acids and forming alarge amount of urea under an anabolic state.
Although both media contain high concentrations of amino acids, there was a significant difference in ureogenesis in the experiments ofhepatocytes cultured in dishes. The stability ofthe pH of the medium was also an importantfactor in selecting the type of medium, because
the L-15 medium can be recirculated for a longtime without CO2 supplementation. As well as
the type of medium, the hormonal addition of
10–7 mol/L insulin, 10–7 mol/L dexamethasone,
10–8 mol/L glucagon and 10 ng/mL hEGF was
crucial for the maintenance of hepatic functions(data not shown). The levels of biochemical
activities in BAL were found to be lower thanthose in the dish-cultures. It could be because
the amounts of the perfusion medium were notsufficient and hepatocytes in BAL could notexert full hepatic functions. Culturing hepatocytes in an atomosphere without CO2 might beanother cause of low hepatic functions of
BALs. Further studies are needed on these aspects.
It was also important to choose an appropriate basement matrix for hepatocytes in thehollow fiber module. The data of hepatocytes
cultured on dishes showed marked differencesin the production rates of glucose, urea nitrogenand albumin between the collagen gel and noncoated dishes. Many investigations on the effects of the extracellular matrix revealed thatthe constituents of the extracellular matrix for
hepatocytes in vitro strongly influence expres-sion and maintenance of liver specific genes(Kocarek et al., 1993; Nagaki et al., 1995; Tamura
et al., 1995). Two different types of matrices,collagen gel and PVLA, both providing a 3dimensional configuration for hepatocytes,
were studied (Akaike et al., 1993; Lazar et al.,1995). Hepatocytes entrapped in collagen gel
were found maintaining much higher levels of
biochemical activities consistant with several
reports of BAL systems using collagen gel as acellular matrix in the hollow fiber cartridge(Shatford et al., 1992; Sielaff et al., 1995a;
Takeshita et al., 1995). To obtain a firm attachment of hepatocytes in the extracapillary membrane of fibers, we modified the method of
hepatocyte inoculation; firstly, we coated theextracapillary membrane with a low concentration (0.015%) of collagen solution, thenloaded the hepatocytes in 0.1% hydrated collagen and then incubated them at 37˚C allowing
them to gel rapidly to avoid a disproportional
distribution of collagen gel in the extracapillary
space.
Determination of albumin production in
perfusates has been supposed to be an optimal
index of the metabolic activities of hepatocytes
because albumin is stable in media and only
hepatocytes synthesize it (Rothschild et al.,1972). But the biochemical assay of albumin in
perfusates does not completely reflect the newly synthesized albumin. Even if the perfusion
medium was changed every day and there werebe no stored albumin in the medium being usedin our experiments, the albumin stored in theextracapillary space of the hollow-fiber module
could not be neglected. Thus, we performed analbumin mRNA analysis in addition to a biochemical assay by ELISA. Northern blot
analysis reflects the transcriptional activity of
the albumin gene at the time of cell harvest sothat the existence of stored albumin in theextracapillary space or cross-reaction to FCS in
the medium can be neglected. There are no previous reports evaluating the functional activityof BALs in mRNA levels, mainly because
hepatocytes must be collected from devices,which means the end of perfusion experiments.
The albumin mRNA in our module was maintained for 11 days. The change of expression

109

S. Okada et al.

levels of mRNA (Fig. 4) was parallel to thechange of albumin production rates measured
by ELISA in the media (Fig. 3). This result suggests that the measurement of albumin production in perfusates correctly reflects the newly
synthesized albumin in hepatocytes of a BAL.
We also performed Northern blot analysis of
CYP 2B1 mRNA as an index of the ability ofdetoxification of the module. In previous reports, the drug metabolizing activity of a BAL
was assessed by measuring concentrations ofcertain drugs, for example lidocaine, and their
metabolites by high performance liquid chromatography (Shatford et al., 1992; Nyberg etal., 1993; Rozga et al., 1993). The increased
concentration of metabolites shows the activity
of cytochrome P450 related drug enzymes and apart of the drug metabolic pathway, but does
not show general levels or the inducibility ofcytochrome P450s. It was, therefore, thought tobe valuable to perform Northern blot analysis.
In the present study, the inducibility andexpression of CYP 2B1 mRNA was maintained
for a shorter term than the expression ofalbumin mRNA. Although albumin synthesis
might be a useful marker of hepatocyte viability(Sielaff et al., 1995b), in the sense of potential
therapeutic value for patients with liver failure,cytochrome P450 activity is suggested to be thecritical function that must be provided by successful liver assistance devices (Nyberg et al.,1993). A number of researchers (Kocarek et al.,
1993; Sidhu et al., 1993; Donato et al., 1994)
have demonstrated that hepatocytes in culture
expressed constitutive cytochrome P450 activity only in a differentiated environment. It is
known that their distribution of the expressionof cytochrome P450s in the hepatocytes in vivo
depends on the location of the hepatocytes in
the lobules, for example Zones 1, 2 and 3(Wojcik et al., 1988). In order to maintain highlevels of the expression of cytochrome P450s,
the reconstruction of the lobular system in the
BAL seemed necessary. Hepatocytes in thisstudy were entrapped in collagen gel and loaded
into the device evenly so that hepatocytes
should have been under a 3-dimensional configuration (Lazar et al., 1995). Even under these
conditions, hepatocytes maintained CYP 2B1mRNA only for 2 days in the BAL, which is not
a completely suitable environment for hepatocytes. It is necessary for hepatocytes to haveproper polarity and microcirculation likely invivo, including the presence of vessels, nonparenchymal cells and bile duct systems.
In summary, we evaluated the maintenanceof hepatic functions of a potential hollow fiber
type BAL by biochemical activities and mRNAlevels. The albumin mRNA was detected for 11days parallel to albumin production measured
by ELISA. The expression of CYP 2B1
mRNA, however, was maintained only for ashort term even though it was expected to maintain itself for as long a period as albumin. Itshould be necessary to reconstitute a microenvironment for hepatocytes in a BAL as invivo, probably retaining zone lobulation, toachieve the total functions of the liver.
Acknowledgments: The authors thank Prof.
Hironaka Kawasaki (Second Dept. of Internal
Medicine, Faculty of Medicine, Tottori University)and Prof. Kazuo Yamada (Dept. of Biochemistry,
Faculty of Medicine, Tottori University) for theirvaluable suggestions and critical readings of themanuscript. We also thank Dr. Eiji Nanba and Dr.
Etsuko Ueta (The Gene Research Center, Tottori
University) for assistance with the Northern blot
analysis.

110

References
1 Akaike T, Tobe S, Kobayashi A, Goto M,
Kobayashi K. Design of hepatocyte-specificextracellular matrices for hybrid artificial liver.
Gastroenterol Jpn 1993;28:45–52
2 Chen SC, Mullon C, Kahaku E, Watanabe F,
Hewitt W, Eguchi S, et al. Treatment of severe
liver failure with a bioartificial liver. Ann N Y
Acad Sci 1997;31:350–360.
3 Chomczynski P, Sacchi N. Single-step methodof RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal
Biochem 1987;162:156–159
4 Coulombe JJ, Favreau L. A new simple semimicro method for colorimetric determination of
urea. Clin Chem 1963;9:102–108.
5 Donato MT, Castell JV, Gómez-Lechón MJ.
Cytochrome P450 activities in pure and cocultured rat hepatocytes. Effects of model inducers. In Vitro Cell Dev Biol 1994;30A:825–
832.

The mRNA expression of hepatocytes in a BAL

6 Dunn JCY, Tompkins RG, Yarmush ML. Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation. J Cell
Biol 1992;116:1043–1053.
7 Enami J, Koezuka M, Hata M, Kawamura K,
Tachibana Y, Kusama Y, et al. [Collagen gel
culture method. (I)] Soshiki Baiyo 1987;13:26–
30 (in Japanese).
8 Fremond B, Malandain C, Guyomard C, Chesne
C, Guillouzo A, Campion JP. Correction of bilirubin conjugation in the gunn rat using hepatocytes immobilized in alginate gel beads as an
extracorporeal bioartificial liver. Cell Transplant
1993;2:453–460.
9 Fujii-Kuriyama Y, Mizukami Y, Kawajiri K,
Sogawa K, Muramatsu M. Primary structure of acytochrome P-450: coding nucleotide sequenceof phenobarbital-inducible cytochrome P-450
cDNA from rat liver. Proc Natl Acad Sci USA
1982;79:2793–2797.
10 Kimball SR, Horetsky RL, Jefferson LS. Hormonal regulation of albumin gene expression inprimary cultures of rat hepatocytes. Am J Physiol
1995;268:E6–E14.
11 Kocarek TA, Schuetz EG, Guzelian PS. Expression of multiple forms of cytochrome P450
mRNAs in primary cultures of rat hepatocytes
maintained on matrigel. Mol Pharmacol 1993;
43:328–334.
12 Kohno Y, Shiraki K, Mura T. The effect of human milk on DNA synthesis of neonatal rat
hepatocytes in primary culture. Pediatr Res
1991;29:251–255.
13 Lazar A, Mann HJ, Remmel RP, Shatford RA,
Cerra FB, Hu WS. Extended liver-specificfunctions of porcine hepatocyte spheroids entrapped in collagen gel. In Vitro Cell Dev Biol 1995;
31:340–346.
14 Li AP, Barker G, Beck D, Colburn S, Monsell R,
Pellegrin C. Culturing of primary hepatocytes as
entrapped aggregates in a packed bed bioreactor:
a potential bioartificial liver. In Vitro Cell Dev
Biol 1993;29A:249–254.
15 Matsumura KN, Guevara GR, Huston H,
Hamilton WL, Rikimura M, Yamasaki G, et al.
Hybrid bioaritificilal liver in hepatic failure: preliminary clinical report. Surgery 1987;101:99–
103.
16 Miwa I, Okuda J, Maeda K, Okuda G. Mutarotase effect on colorimetric determination ofblood glucose with β-D-glucose oxidase. Clin
Chim Acta 1972;37:538–540.
17 Nagaki M, Shidoji Y, Yamada Y, Sugiyama A,
Tanaka M, Akaike T, et al. Regulation of hepatic
genes and liver transcription factors in rat
hepatocytes by extracellular matrix. Biochem
Biophys Res Commun 1995;210:38–43.
18 Naruse K, Sakai Y, Nagashima I, Jiang GX,
Suzuki M, Muto T. Comparisons of porcine

hepatocyte spheroids and single hepatocytes in
the non-woven fabric bioartificial liver module.
Inter J Artif Organs 1996a;19:605–609.
19 Naruse K, Sakai Y, Nagashima I, Jiang GX,
Suzuki M, Muto T. Development of a new bioartificial liver module filled with porcine hepatocytes immobilized on non-woven fabric. Inter J
Artif Organs 1996b;19:347–352.
20 Nyberg SL, Shirabe K, Peshwa MV, Sielaff TD,
Crotty PL, Mann HJ, et al. Extracorporeal application of a gel-entrapment, bioartificial liver:demonstration of drug metabolism and otherbiochemical functions. Cell Transplant 1993;
2:441–452.
21 Pappas SC. Fulminant hepatic failure and needfor artificial liver support. Mayo Clin Proc 1988;
63:198–200.
22 Rothschild MA, Oratz M, Schreiber SS. Albumin synthesis. 1. N Engl J Med 1972;286:748–
757.
23 Rozga J, Williams F, Ro MS, Neuzil DF, Giorgio
TD, Backfisch G, et al. Development of a bioartificial liver: properties and function of ahollow-fiber module inoculated with liver cells.
Hepatology 1993;17:258–265
24 Shatford RA, Nyberg SL, Meier SJ, White JG,
Payne WD, Hu WS, et al. Hepatocyte function ina hollow fiber bioreactor: a potential bioartificial
liver. J Surg Res 1992;53:549–557.
25 Sidhu JS, Farin FM, Omiecinski CJ. Influence of
extracellular matrix overlay on phenobarbitalmediated induction of CYP2B1, 2B2, and 3A1
genes in primary adult rat hepatocyte culture.
Arch Biochem Biophys 1993;301:103–113.
26 Sielaff TD, Hu MY, Amiot B, Rollins MD, Rao
S, McGuire B, et al. Gel-entrapment bioartificial
liver therapy in galactosamine hepatitis. J Surg
Res 1995a;59:179–184.
27 Sielaff TD, Hu MY, Rao S, Groehler K, Olson D,
Mann HJ, et al. A technique for porcine hepatocyte harvest and description of differentiatedmetabolic functions in static culture. Transplantation 1995b;59:1459–1463.
28 Sielaff TD, Nyberg SL, Rollins MD, Hu MY,
Amiot B, Lee A, et al. Characterization of thethree-compartment gel-entrapment porcine
hepatocyte bioartificial liver. Cell Biol Toxicol
1997;13:357–364.
29 Sussman NL, Kelly JH. Improved liver function
following treatment with an extracorporeal liver
assist device. Artif Organs 1993;17:27–30.
30 Takahashi M, Matsue H, Matsushita M, Sato K,
Nishikawa M, Koike M, et al. Does a porcine
hepatocyte hybrid artificial liver prolong the survival time of anhepatic rabbits?. ASAIO J 1992;
38:M468–M472.
31 Takeshita K, Ishibashi H, Suzuki M, Yamamoto
T, Akaike T, Kodama M. High cell-density culture system of hepatocytes entrapped in a three-111

S. Okada et al.

dimensional hollow fiber module with collagengel. Artif Organs 1995;19:191–193.
32 Tamura T, Koide N, Hada H, Shiraha H, Tsuji T.
Gene expression of liver-specific proteins inhepatocyte spheroids in primary culture. Acta
Med Okayama 1995;49:161–167.
33 Tanaka K, Kishi K, Ichihara A. Biochemical
studies on liver functions in primary cultured
hepatocytes of adult rats. J Biochem (Tokyo)
1979;86:863–870.

34 Ullrich A, Shine J, Chirgwin J, Pictet R, Tischer
E, Rutter WJ, et al. Rat insulin genes: construction of plasmids containing the coding sequences.
Science 1977;196:1313–1319.
35 Wojcik E, Dvorak C, Chianale J, Traber PG,
Keren D, Gumucio JJ. Demonstration by in situ
hybridization of the zonal modulation of rat liver
cytochrome P-450b and P-450e gene expression
after phenobarbital. J Clin Invest 1988;82:658–
666.

(Received December 21, 1998, Accepted January 11, 1999)

112