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African Journal of Pig Farming ISSN 2375-0731 Vol. 7 (2), pp. 001-011, February, 2019. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 
 

Full Length Research Paper 

 

Evaluation of a reversibly immortalized human 
hepatocyte line in bioartificial liver in pigs 

 
Lifu Zhao1, Jianzhou Li1, Guoliang Lv1, Anye Zhang 1, Pengcheng Zhou1, Ying Yang1, 

Xiaoping Pan1, Xiaopeng Yu1, Yimin Zhang1, Shusen Zheng2, Yu Chen1, Yuemei Chen1, 
Chengbo Yu1, Weibo Du1, Tao Song3, Jiansheng Xu3, Yang Yu3 and Lanjuan Li1* 

 
1
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of 

Medicine, Zhejiang University, Hangzhou 310003, China. 
2
Department of Surgery, State Key Laboratory of 

Combined Multi-organ Transplantation, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 

310003, China. 
3
Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190, China. 

 
Accepted 25 October, 2018 

 
An appropriate cell source is essential for the clinical application of bioartificial liver (BAL) system. This study 
aimed to test a reversibly immortalized human hepatocyte line (HepLi-4) in our newly validated choanoid fluidized 
bed bioreactor based BAL in pigs with fulminant hepatic failure (FHF). 15 FHF pigs were allocated to three 
groups: a BAL group receiving BAL treatment with HepLi-4 cells; a sham BAL group receiving cell-free BAL 
treatment; and a FHF group receiving intensive care only. Expression of liver-specific genes in HepLi-4 cells 
before and after BAL was analyzed, adult human hepatocytes acted as a reference. In BAL group, Fischer index 
was higher and serum indirect bilirubin level was lower compared with two control groups. Survival time in BAL 
group was longer than that in two control groups, but the difference was not statistically significant. Gene 
expression analysis showed that the transcript levels of liver-specific genes in HepLi-4 were retained after BAL, 
but significant variations were observed between HepLi-4 and human hepatocytes. HepLi-4 showed beneficial 
metabolic effects on FHF pigs in BAL, but is still not an appropriate cell source for BAL. More insights into 
interpreting the conditions for hepatocyte differentiation are needed. 

 

Key words: Reversible immortalization, hepatocytes, bioartificial liver, fulminant hepatic failure. 
 
 
INTRODUCTION 

 
Facing the world-wide donor organ shortage, bioartificial 
liver (BAL) system has been anticipated to be a bridge to  
 
 

 
*Corresponding author. E-mail: ljli@zju.edu.cn. Tel/Fax: 86-571-
87236759. 

 
Abbreviations: BAL, Bioartificial liver; FHF, fulminant hepatic 
failure; AC, alginate-chitosan; PERV, porcine endogenous 
retroviruses; SV40 LT, simian virus 40 large T antigen; BCAAs, 
branched chain amino acids; AAAs, aromatic amino acids; GS, 
glutamine synthetase; UGT1A1, uridine diphosphate 
glucuronyltransferase; ALB, albumin; GST-P, glutathione S-
transferase P; β-actin, human beta-actin; HE, hepatic 
encephalopathy; RT-PCR, reverse transcription-polymerase 
chain reaction. 

 
 
 
 

 
liver transplantation or to provide a chance for the native 
liver to recover in case of liver failure (Strain and 
Neuberger, 2002; Carpentier et al., 2009). Bioreactor and 
cell source are two main issues of BAL (Pless, 2010). 
Fluidized bed bioreactor is promising for liver support 
(Dore and Legallais, 1999; Coward et al., 2009; Desille et 
al., 2001; David et al., 2004). We recently designed a 
BAL system based on a choanoid fluidized bed bioreactor 
filled with alginate-chitosan (AC) encapsulated primary 
porcine hepatocytes and proved it could prolong the 
survival of pigs with fulminant hepatic failure (FHF) 
induced by D-galactosamine injection (Lv et al., 2011). 
The potential risk of zoonotic transmission, however, 
might exist, which limits its clinical application (Patience 
et al., 1997). Researchers in our group also proved that 
porcine endogenous retroviruses (PERV) can pass 



 
 
 

 

through the immune barrier in AC microbeads (Yang et 
al., 2010). Therefore, developing a safe cell source for 
BAL is necessary.  

Widespread clinical use of primary human hepatocytes 
in BAL is almost impossible because healthy donor livers 
are so scarce that only organ or tissue discarded from 
transplantation are available. Among non-primary cell 
sources, several human liver tumor derived cell lines, 
including GS-HepG2 (glutamine synthetase, GS), 
HepG2-GS-3A4 and FLC-4, were used in BAL in large 
animal models. Prolongations of survival were achieved 
in these studies (Kanai et al., 2007; Enosawa et al., 2001, 
2006; Wang et al., 2005). However, none of them has 
been so far applied to clinical trials. Poor differentiation 
and the potential risk of tumor transmigration might be the 
main hurdles (Nyberg et al., 1994). Only the C3A cell line, 
a subclone of HepG2, was used in a pilot-controlled 
clinical trial (Ellis et al., 1996). Unfortunately, no improve-
ment in either survival or biochemical parameters is 
demonstrated.  

For several years, we have been dedicated to esta-
blishing immortalized hepatocyte line (Pan et al., 2010; Li 
et al., 2005). Although these cell types have unlimited 
expansion capabilities in vitro, continuous expression of 
simian virus 40 large T antigen (SV40 LT) might be 
tumorigenic (Kobayashi et al., 2001; Hahn et al., 1999; 
Woodworth et al., 1988). To solve this problem, resear-
chers in Japan established a reversibly immortalized 
human hepatocyte line (NKNT-3) in the year 2000 
(Kobayashi et al., 2000). It shows that the immortalizing 
oncogene, e.g., SV40 LT, can be excised by a Cre/LoxP 
site-specific recombination. Then, liver-specific genes 
expression can be increased later. However, obtaining a 
large number of reverted hepatocytes was hindered by 
the secondary gene transfer until the drug-mediated 
Cre/LoxP recombination was applied (Totsugawa et al., 
2007). With the drug-medicated Cre/LoxP recombination, 
we established another reversibly immortalized cell line 
(HepLi-4) by transfection of primary human hepatocytes 
with the retrovirus containing SV40 LT flanked by a pair 
of LoxP recombination targets. Thus, enough number of 
reversibly immortalized hepatocytes can be achieved to 
equip our newly validated choanoid fluidized bed bio-
reactor based BAL system. In order to demonstrate the 
clinical potential of our hepatocytes in BAL, we carried 
out experiments in large animal models. 
 

 
MATERIALS AND METHODS 

 
This study protocol was ratified by the Animal Ethics Committee of 
Zhejiang University and the Ethics Committee of the First Affiliated 
Hospital, Zhejiang University School of Medicine. 
 

 
Large-scale cultivation and encapsulation of HepLi-4 cells 
 
The HepLi-4 cell line was developed by immortalization of primary 
human hepatocytes with the retrovirus containing SV40LT flanked 

 
 

  
 
 

 
by a pair of LoxP recombination targets, and the HepLi-4 cells were 
subsequently transfected with the recombinant vector containing 
Cre-ERT2 fusion protein gene derived from pCAG-CreERT2 
plasmid (kindly gifted by Addgene, USA). Reverted HepLi-4 cells 
were capable of expressing liver-specific genes in vitro and 
prolonging the survival of common bile duct ligated mice after 
intrasplenic transplantation (Pan et al., 2009). There was also no 
evidence for tumorigenesis of reverted HepLi-4 cells in nude mice 
within three months after subcutaneous transplantation.  

The cultivation and reversion procedures were previously 
described in detail (Totsugawa et al., 2007). Briefly, HepLi-4 cells at 
the 30th passage (Figure 1A) were expanded in roller bottles 
(Bellco, USA) with Dulbecco Modified Eagle Medium (DMEM; 
Gibco, USA) supplemented with 10% (v/v) fetal calf serum (Gibco, 
USA) using a cell production roller bottle apparatus (Bellco, USA) 
(Figure 1B). After achieving the required number of cells, SV40 LT 
genes were removed by keeping the cells in culture media 
containing 500 nM 4-hydroxytamoxifen (4-OHT; Sigma-Aldrich, 
USA) for five to seven days. To verify the efficiency of excision of 
the SV40 LT gene, the mRNA expression of SV40 LT in 
immortalized and reverted HepLi-4 cells was analyzed by reverse 
transcription-polymerase chain reaction (RT-PCR). The primer 
sequence and amplification condition were described in our 
previous study (Pan et al., 2010). As shown in Figure 1C, SV40 LT 
gene was almost not detected in reverted HepLi-4 cells.  

Thereafter, reverted HepLi-4 cells were trypsinized. The cell 
viability assessed by trypan blue exclusion was above 95%. We still 
adopted the single-stage AC encapsulation procedure which had 
previously been reported by us (Lv et al., 2011). Briefly, about 3.0 × 

10
9
 hepatocytes were resuspended in 300 ml 1.7% sodium alginate 

(Sigma-Aldrich, USA) solution. The mixture was extruded through 
an electrostatic microencapsulator (300 µM nozzle; Nisco, 
Switzerland) into a 0.7% chitosan solution (Jinan Haidebei Marine 
Bioengineering Co. Ltd, China). The gelation process lasted for 30 
min, of which microbeads of 600 to 1000 µM in diameter were 
obtained (Figure 1D). 

 

Experimental animals 

 
Male Chinese experimental miniature pigs weighting 10 to 15kg 
were purchased from China Agriculture University. All pigs were 
kept in separate cages under standard conditions and fed standard 
laboratory chow. 

 

Induction of FHF 
 
D-galactosamine (Shanghai Hanhong Chemical Co., Ltd, China) 
induced pig model of FHF was used in this experiment. The 
experiment work flow is shown in Figure 2. Catheterization, which 
had been previously described (Li et al., 2006), was carried out 
under general anesthesia achieved by continuous intravenous 
injection of Diprivan (AstraZeneca, Italy) at a rate of 2.5 mg/kg/h. 
Then, pigs awakened from anesthesia were sent back to their 
cages. Twenty-four hours after the catheterization, without 
anesthesia, D-galactosamine was delivered to pigs via the venous 
catheter at a dose of 1.5 g/kg body weight. 

 

Experimental groups 
 
As shown in Figure 2, 15 FHF pigs were allocated to three groups: 
a BAL group (n = 5), receiving BAL treatment with HepLi-4 cells; a 
sham BAL group (device control, n = 5), receiving cell-free BAL 
treatment; and a FHF group (baseline control, n = 5), only receiving 
intensive care under general anesthesia. These three types of 
interventions were initiated 18 h after D-gal injection and lasted for 



          
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 1. Large-scale cultivation and encapsulation of HepLi-4 cells. (A) Morphology of HepLi-4 
cells under optical microscopy. (B) Large-scale cultivation of HepLi-4 cells in roller bottles. (C) The 
mRNA expression of SV40 LT in HepLi-4 cells before and after reversion. SV40 LT mRNA was 
detected in immortalized HepLi-4 cells, whereas almost not detected in the reverted cells by RT-
PCR analysis. (D) Optical micrograph of alginate–chitosan (AC) microbeads containing HepLi-4 

cells. Cell density was approximately 1×10
7
/ml. Scale bar = 500 µM.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Figure 2. Experimental flow chart. 
 

 
6 h according to previous studies (Lv et al., 2011; Li et al., 2006). 

 

Bioreactor 
 
The bioreactor (Figure 3A; Chinese Patent no: ZL 200710070279.0) 
has a funnel-shaped structure with filters of 200 and 600 mesh/inch 
fixed to the bottom and top, respectively. To stop potential cell chips 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
from entering the body, a 3 µM strainer was installed in the outlet. 
The volume inside the bioreactor was about 500 ml. Other 
parameters were detailed in our previous report (Lv et al., 2011). 

 

BAL treatment 
 
The BAL system (Figures  3B  and  C)  which  had  previously  been 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. Bioreactor and BAL treatment. (A) Schematic diagram of the choanoid fluidized bed bioreactor loaded with microbeads. 1, 

Plasma inlet; 2, plasma outlet; 3, microbeads inlet; 4, microbeads outlet; 5, filters; 6, strainer (pore size: 3 µM). The solid arrows and 

the hollow arrows show the direction of plasma flow and the trajectory of microbeads in the bioreactor, respectively. (B) Photograph 

of the BAL treatment. (C) Structural diagram of the BAL system. BAL, Bioartificial liver. 
 
 
 
reported by us consisted of a plasma separation unit and a 
bioreactor unit (Lv et al., 2011). Both were driven by a series of 
roller pumps. Under general anesthesia, arterial blood was drawn 
from pig’s body at 20 to 25 ml/min and pumped into the plasma 
separator (OP-02W, Asahi-Kasei, Japan) through which plasma 
was separated from the cellular components at 8 to 10 ml/min into 
the reservoir. In the bioreactor unit, plasma was perfused through 

the bioreactor containing 3.0 × 10
9
 encapsulated HepLi-4 cells at 25 

to 35 ml/min. At the same time, the purified plasma was returned to 
the pig’s body at a rate equal to plasma separation. To prevent 
blood clotting, the first dose of heparin (100 U/kg) was administered 
intravenously and followed by sustained injection of heparin into the 
extracorporeal circulation at a rate of 40 U/kg per hour. In sham 
BAL treatment, bioreactor contained cell-free microbeads with the 
same volume. 

 
 
 
 
observed respectively. Trypan blue exclusion test was used to 
assess the viability of encapsulated hepatocytes before and after 
BAL treatment. Microbeads were lysed by a solution composed of 

0.2 mol/L NaHCO3 and 0.06 mol/L Na3C6H5O7•2H2O (pH = 7.8) 
(Xue et al., 2004), then we used 0.4% trypan blue solution to dye 
dead cells according to the method described previously (David et 
al., 2004). Also, comparison between the viability of encapsulated 
HepLi-4 cells before and after BAL was performed by a previously 
reported MTT method with some modifications (Haque et al., 2005). 
In brief, 15 microbeads were incubated in a 96-well plate with 100 
µL media and 25 µL MTT per well for 24 h. Thereafter, the 
supernatants were substituted for 100 µL dimethyl sulfoxide 
(DMSO). Within 30 min, the absorbance of light at 595 nm was 
measured using a DTX 800 multi-mode detector (Beckman Coulter, 
USA). We repeated these experiments three times. 
 

 
Test of microbead integrity and cell viability 

 
The percentage of intact beads before and after BAL treatment was 
determined under light microscope. 100 beads in five samples were 

 
 
Characterization of liver-specific genes 

 
Microbeads containing HepLi-4 cells before and after BAL treatment 
were lysed by the solution mentioned above. Total RNA was 



 
 
 
 

 
Table 1. Microbead integrity and cell viability before and after BAL treatment.  

 
Paramater Pre-BAL Post-BAL P value   

Integrity of microbeads (percentage of intact beads, %, n = 5) 

Viability of encapsulated HepLi-4 cells (trypan blue 

exclusion, percentage of live cells, %, n = 5)  
Viability of encapsulated HepLi-4 cells (MTT test, OD Value, n = 5)  

  
 

99.2 ± 0.84 98.4 ± 1.14 0.24 

92.2 ± 1.79 90.8 ± 1.92 0.27 

0.73 ± 0.08 0.69 ± 0.08 0.45 
 

Values are expressed as mean ± standard deviation. Difference between groups was analyzed using Student's t-test. The experiments were 
repeated three times. 

 
 
 
extracted from HepLi-4 cells using Trizol reagent (Invitrogen, USA). 
To provide a reference, adult human liver samples obtained during 
the course of graft reduction for liver transplantation were included 
in the analysis. RT-PCR containing 1 µg total RNA was carried out 
using two-step RT-PCR kit (QIAGEN, USA) according to 
manufacturer's protocols. Primer sequences for amplification of 
liver-specific genes and internal control gene, including GS (535 
bp), uridine diphosphate glucuronyltransferase (UGT1A 1,495 bp), 
albumin (ALB, 576 bp), glutathione S-transferase P (GST-P, 496 
bp) and human beta-actin (β-actin, 610 bp), were detailed in 
previous report (Totsugawa et al., 2007). The thermal cycle 
involved one cycle of initial denaturation at 95°C for 2 min, followed 
by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 
30 s and elongation at 72°C for 40 s. Amplification was concluded 
with a one cycle of extension program at 72°C for 5 min. The 
amplification products were run on 1% agarose gels stained with 
ethidium bromide. 

 

Measurement of parameters 
 
The three blood sampling time points were the time just prior to the 
D-gal injection, before and after the 6-h intervention, respectively (0,  
18 and 24 h after D-gal injection). Biochemical parameters, 
including serum bilirubin, albumin, aminotransferases, total cho-
lesterol, lactate, glucose, creatinine, urea nitrogen and prothrombin 
time, were examined in clinical laboratories. Analysis of serum 
amino acids was performed by high-performance liquid 
chromatographic (HPLC, HITACHI, Japan). The moment of D-gal 
injection was the starting point for survival time. Death of animal 
was defined as the cessation of breathing, cardiac arrest and fixed 
pupil dilation. 

 

Statistics 

 
Values are expressed as mean ± standard deviation. Group 
differences were analyzed using one-way analysis of variance 
(ANOVA) and Student's t-test. Survival time was compared by 
Kaplan-Meier analysis (log-rank significance test). P value <0.05 
was defined as significant. All data were analyzed using SPSS for 
Windows version 15.0. 
 
 
RESULTS 
 
Microbead integrity and cell viability 
 
As shown in Table 1, having experienced the entire BAL 
treatment, few beads lost its integrity (percentage of 
intact beads reduced from 99.2 ± 0.84 to 98.4 ± 1.14%, n  
= 5) (P>0.05). Though the viability of encapsulated 
HepLi-4 cells showed a slight downward trend by both 

 
 

 

trypan blue exclusion (%, from 92.2±1.79 to 90.8±1.92, n 

= 5) and MTT test (OD, from 0.73 ± 0.08 to 0.69 ± 0.08, n  
= 5) after BAL treatment, the differences were not 
statistically significant (P>0.05). 
 

 

Expression of liver-specific genes 

 

As shown in Figure 4, HepLi-4 cells that had experienced 
the BAL treatment still retained liver-specific gene 
expression. β-actin was an internal control, while the 
mRNA levels of mature human hepatocytes acted as a 
reference. In HepLi-4 cells, the mRNA level of GS was 
not less than that in adult human liver, while the mRNA 
level of UGT1A1 was somewhat lower. Moreover, the 
ALB mRNA level was extremely lower and GST-P mRNA 
level was extremely higher in HepLi-4 cells compared 
with those in human liver. 
 

 

Treatment process and survival 

 

Throughout the 6-h BAL or sham BAL treatment process, 
no hemodynamic instability and bleeding episodes 
occurred in pig; no blood coagulation in extracorporeal 
circulation was observed. All pigs died of liver failure. As 
shown in Figure 5, survival time in BAL group (69.4 ± 
16.5 h) was longer than that in FHF group (56.2 ± 13.3 h) 
and sham BAL group (56.8 ± 18.2 h), although the 
difference was not statistically significant (P>0.05). 
 

 

Biochemical parameters 

 

Fischer index is calculated as a ratio of branched chain 
amino acids (BCAAs; valine, isoleucine and leucine) to 
aromatic amino acids (AAAs; /tyrosine and phenyl-
alanine) in plasma. As shown in Figure 6A, after D-galac-
tosamine injection, Fischer index declined progressively 
in all groups. BAL treatment made the trend slow. At the 
end of the 6-h intervention (24 h after D- galactosamine 
injection), Fischer index in BAL group (2.32 ± 0.42) was 
significantly higher than those in sham BAL group (1.46 ± 
0.34) and FHF group (1.49 ± 0.28) (P<0.05). As shown in 
Figure 6B, gradual increase of serum indirect bilirubin 
was observed in two control groups, while BAL treatment 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure  4.  Liver-specific genes expression in encapsulated  
HepLi-4 cells before BAL treatment (1), encapsulated HepLi-  
4 cells after BAL treatment (2) and adult human hepatocytes 
(3). BAL, Bioartificial liver.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 5. Survival curves for the FHF pigs in BAL group, sham BAL group and FHF group. The time of D-gal injection was 
the starting point for survival time. Survival curves did not show significant prolongation of survival in BAL group compared 
with two control groups by Kaplan. 



   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 6. Biochemical parameter changes caused by BAL treatment. (A) Fischer 
index in all groups (*P = 0.005 versus both control groups by one-way ANOVA. 
Fischer index = valine + isoleucine + leucine/tyrosine + phenylalanine. (B) Serum 
indirect bilirubin levels in all groups (*P = 0.025 versus both control groups by one-
way ANOVA). BAL, Bioartificial liver; FHF, fulminant hepatic failure. 

 

 

prevented its further increase. The serum level of indirect 
bilirubin at 24 h after D-galactosamine injection in BAL 
group (8.40 ± 2.97 µmol/L) was significantly lower 
compared with sham BAL group (12.80 ± 1.92 µmol/L) 

 
 

 

and FHF group (12.60 ± 2.41 µmol/L) (P<0.05). 
Biochemical parameters with non-statistically significant 
differences (P>0.05) between three groups are listed in 
Table 2. Among them, after D-galactosamine injection, 



  
 
 

 
Table 2. Biochemical parameters with non-statistically significant differences between three groups.  

 
Parameter Group Baseline (0 h) Pre-BAL (18 h) Post-BAL (24 h) 

 BAL 3.26 ± 1.02 22.68 ± 6.48 34.24 ± 8.52 

Total bilirubin (µmol/L) Sham BAL 2.8 ± 0.45 24.6 ± 6.07 38 ± 13.82 

 FHF 2.38 ± 0.43 25.4 ± 9.11 37.88 ± 10.5 

 BAL 11.56 ± 1.15 21.6 ± 2.75 65.98 ± 33.27 

Prothrombin time (s) Sham BAL 12 ± 2.32 23.16 ± 4.56 65.22 ± 32.75 

 FHF 11.62 ± 1.44 22.2 ± 2.32 53.38 ± 7.82 

 BAL 2.44 ± 0.54 5.42 ± 0.8 6.46 ± 1.37 

Lactate (mmol/L) Sham BAL 2.18 ± 0.59 5.06 ± 0.89 6.28 ± 1.28 

 FHF 2.66 ± 0.41 5.88 ± 0.91 7.25 ± 1.25 

 BAL 42.4 ± 9.96 100.6 ± 49.41 171.2 ± 47.52 

Alanine aminotransferase (IU/L) Sham BAL 43.6 ± 3.13 90 ± 11.94 164.4 ± 77.06 

 FHF 46.4 ± 9.56 92 ± 25.44 170.6 ± 40.16 

 BAL 5.88 ± 1.15 4.54 ± 0.58 3.61 ± 1.59 

Glucose (mmol/L) Sham BAL 5.66 ± 0.51 4.7 ± 1.16 2.53 ± 0.4 

 FHF 5.45 ± 0.64 4.15 ± 0.44 2.71 ± 0.91 

 BAL 2.18 ± 0.16 1.37 ± o.39 0.94 ± 0.3 

Total cholesterol (mmol/L) Sham BAL 1.89 ± 0.31 1.24 ± 0.4 0.81 ± 0.24 

 FHF 1.88 ± 0.16 1.27 ± 0.35 0.85 ± 0.18 

 BAL 37.18 ± 5.6 38.12 ± 4.14 33.46 ± 3.5 

Albumin (g/L) Sham BAL 39.02 ± 4.78 37.72 ± 5.67 33.62 ± 5.28 

 FHF 38.76 ± 3.15 39.52 ± 4.36 37.58 ± 4.24 

 BAL 37.2 ± 13.44 46 ± 5.96 44.8 ± 6.38 

Creatinine (µmol/L) Sham BAL 40.4 ± 14.29 41.8 ± 4.27 43 ± 7.84 

 FHF 39.4 ± 5.22 46.2 ± 11.65 44.6 ± 10.97 

 BAL 3.35 ± 0.66 3.01 ± 0.44 2.98 ± 0.52 

Urea nitrogen (mmol/L) Sham BAL 3.91 ± 1.37 3.75 ± 0.52 3.58 ± 0.94 

 FHF 3.35 ± 0.6 3.2 ± 1.13 3.15 ± 0.97 
 

Values are expressed as mean ± standard deviation. Difference between groups was analyzed using one-way ANOVA. 
 

 

the levels of serum total bilirubin, prothrombin time, 
lactate and alanine aminotransferases increased gra-
dually. On the other hand, the levels of glucose and total 
cholesterol decreased gradually, while the levels of 
albumin, creatinine and urea nitrogen did not change 
significantly. 
 

 

DISCUSSION 

 

To test the efficiency of a human derived hepatocyte line, 
we chose pigs as the animal model due to the high 
metabolic similarities between human and pig hepatocytes 

 
 

 

(Donato et al., 1999). Drug-induced FHF was applied to pigs 

because it met the criteria proposed by Terblanche and 

Hickman (1991). Also, it closely resembled human FHF 

(Kalpana et al., 1999). In this study, progressively prolonged 

prothrombin time increa-sed serum total bilirubin and lactate 

levels and decreased serum glucose level, and Fischer 

index were observed in all pigs. Histological examination 

showed massive hepatic necrosis in all of them (data not 

shown). It was therefore confirmed that all pigs died of FHF. 

Studies related to the encapsulation of mammalian cells in 

AC microbeads have shown that AC membrane provides 

mechanical stability, mass transfer capacity and 

immunoisolation in vitro (Baruch and Machluf, 2006;



 
 
 

 

Yu et al., 2009; Haque et al., 2005). In this study, cell 
viability and microbead integrity were maintained at 
acceptable levels after the 6-h perfusion of FHF pig’s 
plasma and this was consistent with our previous study 
(Lv et al., 2011). Also, the mRNA levels of GS, UGT1A1, 
ALB and GST-P in encapsulated HepLi-4 cells were all 
retained after BAL treatment.  

In this study, 3×10
9
 encapsulated immortal hepatocytes 

were used to treat an FHF pig weighing 10 to 15 kg. The 
cell mass should be adequate assuming the cells were 
fully-functional (Hoekstra and Chamuleau, 2002). How-
ever, it was disappointing to note that the prolon-gation of 
survival by BAL treatment was not statistically significant. 
This problem might lie mainly in cell functions. The results 
from RT-PCR analysis could explain partially. Though we 
only adopted a semi-quantitative analysis, it could be 
intuitively seen in Figure 4 that the expression of liver-
specific genes of HepLi-4 significantly varied with adult 
human hepatocytes, especially since the HepLi-4 had an 
extremely lower transcript level of mature hepatocytes 
marker (ALB) but a higher level of immature hepatocytes 
marker (GST-P) than human liver. Thus, we concluded 
that HepLi-4 cells were not fully differentiated after 
reversion, which was also found by other studies 
(Deurholt et al., 2009; Chamuleau et al., 2005). It was 
demonstrated by quantitative RT-PCR in these studies 
that the transcript levels of some mature hepatocytes 
markers (albumin, α-1-antitrypsin, and transferrin) in 
reverted NKNT-3 cells were only equivalent to 0.1 to 1% 
of those in human liver, while the mRNA level of GST-P 
was much higher. Another reversibly immortalized human 
hepatocyte line, 16-T3, has never been compared with 
mature human hepatocytes at genetic level (Totsugawa 
et al., 2007). In our previous studies, BALs charged with 
primary porcine hepatocytes could reduce serum lactate 
and stabilize blood glucose in FHF pigs (Lv et al., 2011; 
Li et al., 2006). However, this phenomenon was not 
observed in the current experiment. This revealed that 
there are some other functional deficiencies in HepLi-4 
cells.  

Although obvious defects existed, the relatively appro-
priate expression of detoxification-related genes (GS and 
UGT1A1) in HepLi-4 cells prompted us to continue the 
study. GS, an enzyme responsible for ammonia elimi-
nation, was considered essential for liver function 
(Kobayashi et al., 2000). Some researchers had modified 
HepG2 cells to realize high expression of GS (Tang et al., 
2008; Enosawa et al., 2000). Although the expression of 
GS in HepLi-4 cells was not weaker than that in human 
liver, we failed to reveal their ammonia removal 
efficiencies in FHF pigs due to the shortage of test 
equipments. UGT1A1 is the only physiological enzyme 
that can transform water-insoluble indirect bilirubin into 
water-soluble direct bilirubin by conjugating it with 
glucuronic acid (Strassburg et al., 2008). Thus, bilirubin 
can be excreted into bile duct and eliminated from the 
body. Though the mRNA level of UGT1A1 in HepLi-4 

 
 
 
 

 

cells was somewhat lower than human liver, we did 
observe a decrease of serum indirect bilirubin after BAL 
treatment compared with two control groups. However, 
the levels of serum total bilirubin at 24 h after D-
galactosamine injection showed no significant difference 
between three groups. This hurdle cannot be conquered 
because all of the current bioreactors lack a biliary 
system capable of collecting bile and moving it out of 
extracorporeal circulation. Nevertheless, we proved that 
encapsulated HepLi-4 cells could fulfill the function of 
bilirubin glucuronidation in our BAL system. Also, it can 
be inferred that AC membrane is permeable to pig serum 
albumin for bilirubin is an albumin-bound toxin 
(Falkenhagen et al., 1999). In addition to bilirubin, 
UGT1A1 has several other substrates, such as hormones 
and drugs (Strassburg et al., 2008). Perhaps these 
functions benefited on correcting the metabolic disorders 
in FHF pigs, but they were not reflected in routine 
biochemical tests and survival.  

Studies have shown that hepatic encephalopathy (HE) 
is associated with an imbalance in plasma BCAAs and 
AAAs; Fischer index and the grade of HE are negatively 
correlated (Koivusalo et al., 2008; Fischer et al., 1976). In 
this study, BAL treatment tended to normalize the Fischer 
index in pigs with FHF. However, it is difficult to evaluate 
the mental status of an animal accurately, especially in 
the early stage of HE. The method of reversible 
immortalization was once encouraging (Kobayashi, 2009, 
2000; Totsugawa et al., 2007). However, our studies 
showed that hepatocytes generated by this method were 
poorly differentiated in vitro. The newly established 
immortalized human fetal hepatocyte line, cBAL111, is 
facing the same dilemma (Poyck et al., 2008; Deurholt et 
al., 2009). Even the stem cell derived hepatocytes, which 
are now considered promising sources for liver replace-
ment therapy, still have functional deficiencies which are 
difficult to overcome (Dalgetty et al., 2009; Kung and 
Forbes, 2009). No wonder some researchers hold the 
view that no cell source so far is suitable for BAL support 
(Pless, 2010; Chamuleau et al., 2005). Probably, clinical 
trials of BALs can be carried out only on the condition that 
great progress is made in the promotion of hepatocyte 
differentiation in vitro. Maybe future studies should focus 
on such issues as differentiation promoting factor, matrix 
and co-culture, so that an in vivo-like environment can be 
provided (Chamuleau et al., 2005).  

In conclusion, HepLi-4 cells in the choanoid fluidized 
bed bioreactor based BAL system showed some 
beneficial metabolic effects on FHF pigs, but still cannot 
be called a suitable cell source for BAL. Future research 
should put more insights into interpreting the conditions 
for hepatocyte differentiation. 
 

 

ACKNOWLEDGEMENTS 

 

We wish to thank our work partners in Zhejiang Academy 



 
 
 

 

of Traditional Chinese Medicine (Hangzhou, China) for 
providing the experimental space. This work was sup-
ported by grants from the High Technology Research and 
Development Program of China (2011AA020104), the 
National Natural Science Foundation of China 
(30630023) and Science Fund for Creative Research 
Groups of the National Natural Science Foundation of 
China (81121002). 
 

 
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