ACLS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Z.
Right arrow Articles by Li, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Z.
Right arrow Articles by Li, G.
Annals of Clinical & Laboratory Science 35:7-14 (2005)
© 2005 Association of Clinical Scientists

Configuration of a New Bioartificial Liver Support System and In Vitro Evaluation of its Functions

Zhong Chen1,2, Yitao Ding3 and Genxi Li1
1 National Key Lab of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing,2 Department of General Surgery, Affiliated Hospital, Nantong University, Nantong,3 Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing University, Nanjing, Jiangsu Province, People’s Republic of China

Address correspondence to Zhong Chen, Ph.D., National Key Lab of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Hankou Road, 210093, Nanjing People’s Republic of China; tel 86 513 5052351; fax 86 513 5106369; e-mail zchen9999{at}sina.com.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The aim of this study was to configure a new bioartificial liver (BAL) support system and evaluate its functions in vitro. Chinese experimental miniature pig hepatocytes were isolated by an in situ recirculating collagenase perfusion method and were cultured in serum-free medium with restriction attachment and spinner technique to form hepatocyte spheroid suspensions containing 1.0 x 1010 hepatocytes. The BAL support system was configured by inoculating the hepatocyte spheroids into the cell circuit of a hollow fiber bioreactor (BIOLIV A3A, Cell Biotech Limited, HK, China). The number and viability of hepatocytes, the levels of alanine aminotransferase (ALT), total bilirubin (TBi), and albumin (ALB) in the circulating hepatocyte suspension and RPMI-1640 medium, and lidocaine metabolism were determined during 6 hr of circulation in the BAL devices. Independent experiments were performed 5 times. There were no significant changes in the number and viability of the hepatocytes during the circulation period. The BAL support system demonstrated substantial albumin synthesis and lidocaine metabolism. The results indicate that the new BAL support system has the ability to perform liver functions and could be used to treat liver failure or provide temporary liver support in patients who are candidates for liver transplantation.

(received 16 August 2004; accepted 23 October 2004)

Keywords: bioartificial liver, porcine hepatocyte culture, liver transplantation, lidocaine metabolism


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Liver failure is associated with high mortality. Currently, orthotopic liver transplantation (OLT) is the most effective way to improve survival in patients with acute liver failure [1]. Because of the shortage of liver donors, long-term immuno-rejection, and the high cost of transplantation, artificial liver support systems need to be developed. Artificial liver has the potential for providing temporary support for patients with acute hepatic failure and for patients awaiting OLT[2,3]. Bioartificial liver (BAL) devices containing primary hepatocytes have been studied in animal models for several years and some systems are now undergoing clinical trials [1,4–5]. The major problem is how best to configure a BAL support system. To attain enhanced efficacy of liver support, several BAL configurations have been proposed. The long-term maintenance of hepatocyte viability and function is crucial for any BAL support system. Some systems use hepatocyte suspensions, while others use cultured hepatocytes attached to mircrocarrier beads or multicellular spheroid aggregates [6–10]. In regard to bioreactor design, various bioreactor configurations have been proposed that employ glass plates, hollow fiber membranes, encapsulation in biological matrices, and 3-dimensional carrier materials [11–15].

In this study, porcine hepatocytes were isolated by an in situ recirculating method with collagenase perfusion and spinner culture in order to prepare hepatocyte spheroids. A new BAL support system was configured by introducing the hepatocyte spheroids into cell circuit of a BIOLIV A3A hollow fiber bioreactor [16] and the functions of the BAL support system were evaluated in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals.  Chinese experimental miniature pigs (n = 5, body weight 2.5 to 4.0 kg, male and female) were provided by Beijing Agricultural University and were housed in our research center for 2 wk prior to the experiment. The research protocol complied with Chinese guidelines for humane care of experimental animals. The pigs were allowed free access to water and were fasted for 12 hr before the experiment. One pig was used in each experiment.

Hepatocyte isolation and culture.  Collagenase, RPMI-1640 medium, hepatocyte growth factor, nerve growth factor, epidermal growth factor, and N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) were from Gibco BRL Life Technologies, Grand Island, NY, USA. Insulin, glucagon, transferrin, linoleic acid, glutamine, bovine serum albumin, Na2SeO3, CuSO4·5H2O, ZnSO4·7H2O, poly (2-hydroxyethyl methacrylate) (poly-HEMA), penicillin, and streptomycin were from Sigma-Aldrich Chemie Gmbh, Steinheim, Germany. Porcine hepatocytes were isolated by an in situ recirculating collagenase perfusion method as previously described [17–19]. The pigs were initially anesthetized with ketamine (50 mg/kg). The abdomen was entered through a median incision. After iv administration of heparin (125 U/kg), the portal vein and infrahepatic vena cava were ligated, respectively, and their proximal portions were cannulated with silicone tubing. The liver was perfused in situ at 37°C for 15 min with 1.5–2.0 L of Ca2+,Mg2+-free Hanks solution. The perfusion was followed by recirculation of 250 ml Hanks solution supplemented with 0.05% (w/v) collagenase at 37°C until the liver surface became grained and the tissue became disrupted. The liver was resected and divided into several parts. The tissue was washed and weighed, after which it was put into a sterile bottle and digested with 0.05% (w/v) collagenase at 37°C for 15 min. Following the enzymatic digestion, the hepatocyte suspension was passed through a 120 µm stainless steel sieve. It was washed with Hanks solution immediately and hepatocytes were isolated by centrifugation (1000 rpm, 5 min) 3 times at 4°C. Cell viability was assayed by the trypan blue exclusion test.

Hepatocytes were inoculated at a density of 5 x107/ml in serum-free RPMI-1640 medium supplemented with 200 µg/L hydrocortisone, 1 mg/L hepatocyte growth factor, 10 µg/L nerve growth factor, 20 µg/L epidermal growth factor, 100 µg/L insulin, 4 µg/L glucagon, 6.25 mg/L transferrin, 10 mg/L linoleic acid, 2 mmol/L glutamine, 0.5 g/L bovine serum albumin, 3 nmol/L Na2SeO3, 0.1 µmol/L CuSO4·5H2O, 50 pmol/L ZnSO4·7H2O, 15 mmol/L N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 200 mg/L cefoperazone sodium, 100,000 U/L penicillin, and 100 mg/L streptomycin. To inhibit cell attachment and to induce the formation of cell spheroids in suspension, 250 ml bottles were coated with poly-HEMA. About 100 ml of hepatocyte suspension was placed in each bottle. The bottles were placed in an incubator (5% CO2, 37°C) for 20 hr and slowly rotated (12 revolutions/hr). The hepatocytes were counted and their viability was determined by the trypan blue exclusion test. The morphology of the hepatocytes was inspected with an inverse microscope.

Configuration of the BAL support system.  We developed a BAL support system that contained a hollow fiber cartridge (BIOLIV A3A, Cell Biotech Ltd, Hong Kong, China) with a nominal molecular weight cut-off of 70 kDa, a pore size of 200 nm, and a surface area 0.06 m2. The BAL support system is diagrammed in Fig. 1Go. The total cell circuit volume (including the bioreactor) was 250 ml. Hepatocyte spheroids in serum-free RPMI-1640 medium containing 1.0 x 1010 primary porcine hepatocytes were infused into the outer space of of the hollow fibers and the medium was circulated at 20 ml/min with continuous O2 input (2 L/min). The bioreactor was kept at 37.5°C in an incubator. The RPMI-1640 medium flowed through the lumen of the hollow fibers at a rate of 30 ml/min for 6 hr.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. Schematic diagram of the BAL support system. The BAL system has two circuits. Pump 1 circulates an oxygenated suspension of porcine hepatocyte spheroids in the outer space of the hollow fiber bioreactor (BIOLIV A3A). Pump 2 circulates RPMI-1640 medium in the inner space (lumen) of the hollow fiber bioreactor. The entire BAL support system is housed in an incubator at 37°C.

 
Cell number and viability assays.  The number and % viability of hepatocytes in the cell circuit pre-circulation and at 2, 4, and 6 hr of circulation were assayed by cell counting and trypan blue exclusion.

Biochemical analyses.  Independent experiments were performed 5 times with the BAL devices. Samples of the hepatocyte suspension and RMPI-1640 medium were obtained at 2 hr intervals during 6 hr of circulation. Changes of alanine aminotransferase (ALT), total bilirubin (TBi), and albumin (ALB) levels in the circulating hepatocytes and the medium were determined with an automatic biochemical analyzer (MEGA, Toshiba, Japan).

Lidocaine metabolism test.  The experimental and control groups (no cells) were set up. Lidocaine (40 mg/L) was added to RPMI-1640 medium when the circulation began. Samples (0.5 ml) of RPMI-1640 medium was obtained after 15 min, 1, 2, 4, and 6 hr of circulation. Monoethylgycinexylidide (MEGX) was determined by a fluoresence polarization immunoassay (TDX analyzer, Abbott Laboratories, North Chicago, IL, USA).

Statistical analysis.  Results were expressed as means ± SD; statistical differences were evaluated by ANOVA and paired-sample t tests. The threshold for statistical significance was p <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Number and viability of hepatocytes.  The yield of isolated hepatocytes from a single porcine liver averaged 1.52 x 1010 ± 0.25 x1010 (range = 1.0 x 1010 to 2.5 x 1010). At the start of the experiments, hepatocyte viability averaged 97%. Hepatocyte spheroids formed after incubation for 20 hr in the serum-free medium with restricted attachment and spinner culture. The average viability of hepatocytes was maintained at 95% throughout the experiments. As shown in Figs. 2Go and 3Go, there were no significant changes in the number or viability of porcine hepatocytes during the 6 hr of circulation.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Total numbers of porcine hepatocytes (x 1010) in the cell circuit of the BAL support system, based on cell counts at the start of circulation and 2, 4, and 6 hr thereafter. The circles with error bars show the means ± SD of results from 5 independent experiments.

 


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3. Viability of porcine hepatocytes (%) in the cell circuit of the BAL support system, based on trypan blue exclusion tests at the start of circulation and 2, 4, and 6 hr thereafter. The circles with error bars show the means ± SD of results from 5 independent experiments.

 
ALT, TBi, and ALB levels in hepatocyte suspensions and RPMI-1640 medium during circulation.  ALT, TBi, ALB levels in hepatocyte suspensions and RPMI-1640 medium during circulation experiments are shown in Figs. 4GoGo to 6Go. In the hepatocyte suspensions, there were no significant changes of ALT activity during 6 hr of circulation, whereas the TBi and ALB levels increased gradually (p <0.05). In the RPMI-1640 medium, the pre-circulation levels of ALT, TBi and ALB were lower than in hepatocyte suspensions (p <0.05). During 6 hr of circulation, there was no significant change of ALT activity, whereas the TBi and ALB levels gradually increased (p <0.05). At 2, 4, and 6 hr of circulation, there were no significant differences of TBi levels in RPMI-1640 medium vs the hepatocyte suspensions, but the ALB concentration was lower in the RPMI-1640 medium than in hepatocyte suspensions at all times (p <0.05).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4. Time-course of alanine aminotransferase activity (ALT, U/L) in samples from the two circuits of the BAL support system at the start of circulation and 2, 4, and 6 hr thereafter, based on 5 independent experiments. The triangles with error bars show the ALT activity (mean ± SD) in samples of the circulating suspension of hepatocyte spheroids; the circles with error bars show the ALT activity in the samples of RPMI-1640 medium. *p <0.05 for ALT activity in the hepatocyte suspensions vs the corresponding RPMI-1640 samples.

 


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. Time-course of total bilirubin concentration (TBi, mmol/L) in the two circuits of the BAL support system at the start of circulation and 2, 4, and 6 hr thereafter, based on 5 independent experiments. The triangles with error bars show the TBi levels (mean ± SD) in the circulating suspension of hepatocyte spheroids; the circles with error bars show the TBi levels in the samples of RPMI-1640 medium. *p <0.05 for TBi concentration in hepatocyte suspensions vs the corresponding RPMI-1640 samples.

 


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6. Time-course of albumin concentration (ALB, g/L) in the two circuits of the BAL support system at the start of circulation and 2, 4, and 6 hr thereafter, based on 5 independent experiments. The triangles with error bars show the ALB levels (mean ± SD) in the circulating suspension of hepatocyte spheroids; the circles with error bars show the ALB levels in the samples of RPMI-1640 medium. *p <0.05 for ALB concentration in hepatocyte suspensions vs the corresponding RPMI-1640 samples.

 
Lidocaine metabolism test.  In the samples of hepatocyte suspensions, MEXG levels averaged 32.3 ± 3.7, 45.7 ± 3.9, 39.2 ± 5.3, 33.2 ± 4.2, and 21.3 ± 3.8 µg/L after circulation for 15 min, 1, 2, 4, and 6 hr, respectively. The MEXG level peaked at 1 hr and then declined gradually. MEXG could not be detected in the corresponding samples of RMPI-1640 medium at any time.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Treatment of acute hepatic failure is a formidable clinical challenge. In China, an area with high incidence of viral hepatitis, the morbidity and mortality from acute and chronic hepatic failure are very high. At present, OLT is the most effective therapy, but the patients’ serious condition, the scarcity of donor organs, and high costs limit the general availabilty of OLT. An artificial liver support system could serve as a bridging technique until a donor organ becomes available [2,3]. Furthermore, it might allow some patients with acute hepatic failure to recover their hepatic function.

Preliminary clinical trials indicate that BAL is a promising therapy for acute hepatic failure and a bridge to liver transplantation [1]. An ideal BAL system would provide all of the hepatic functions. BAL systems differ from non-biologic artificial liver devices in the synthesis of essential metabolites and the selective removal of toxic substances, which are carried out by the cultured hepatocytes. The choice of hepatocyte components, the methods of isolation and culture, and the number of hepatocytes are key points in the configuration of BAL [1].

In theory, human hepatocytes are the optimal choice, but the scarcity of donor livers restricts their use in BAL. Since porcine hepatocytes are similar to human hepatocytes in regard to morphology and biological functions, they can serve as a heterogeneous resource for BAL [17,18]. In recent years, a BAL device based on porcine hepatocytes was admitted into clinical trials by the Food and Drug Administration in the USA.

Configuration of BAL needs a large number of hepatocytes with high viability. In this study, porcine hepatocytes were isolated in situ by recirculating collagenase perfusion [19]. The method consumed less collagenase and yielded better cell viability (ie, 97%) than other methods. The number of hepatocytes is also an important parameter of BAL, since 10–30% of hepatocytes of the normal human liver are needed to maintain normal hepatic functions [20]. Matsmura et al [21] proposed that a BAL requires 150 to 450 g of liver. In clinical studies of BAL, the number of cultured hepatocytes ranges from 109 to 1010 [7]. The BAL device that we configured in the present study contained at least 1.0 x 1010 hepatocytes. Large numbers of hepatocytes must be cultured at high density because of the limitation of BAL volume.

It has been reported that cell-cell interaction has an important role in maintaining the viability and functions of hepatocytes [22]. In the present study, hepatocytes were incubated in serum-free medium and poly-HEMA-coated bottles by a continuous rotational method in order to restrict their attachment to the wall and promote the formation of hepatocyte spheroids. This method not only facilitates cell-cell interaction and maintains cell functions, but it also solves the requirement of high density culture in BAL [22]. It reduces the possibility of immune reactions by using serum-free medium with hormones and various growth factors [7]. The present study demonstrated the formation of hepatocyte spheroids by spinner culture with 97% viability, compared to the 75 to 85% viability of hepatocytes that has previously been obtained by a common culture technique [16].

The design of the bioreactor is another key point in BAL. The ideal bioreactor should provide a good environment for hepatocyte growth and metabolism as well as for the efficient exchange of substances. At present, the most common device is a hollow fiber bioreactor with many small hollow fibers made from semipermeable membranes. The device has two independent compartments that are separated by the hollow fiber semipermeable membranes. The intratubular space is used to perfuse blood, while the extratubular space is used to culture hepatocytes. The patient’s blood or plasma flows through the bioreactor, exchanging substances with hepatocytes through the semipermeable membranes. The membranes also provide immuno-isolation. For optimal substance exchange, hollow fiber chambers with high molecular weight cut-off (MWCO) membranes are best [23]. But for optimal immuno-isolation, hollow fiber chambers with low MWCO membranes are best. In view of the molecular weight of albumin (68 kDa),a hollow fiber membrane with 200 nm pore size and a MWCO of 70 kDa was chosen for the present study. The membrane allowed passage of some relatively small molecules, such as albumin, but restricted the passage of lymphocytes and higher molecular weight proteins.

The faults of hollow fiber bioreactors include maldistribution of hepatocytes, poor adherence of hepatocytes and the formation of large aggregates of hepatocytes during circulation. Such problems affect the functions of the hepatocytes, block the membrane pores, and impair substance exchange and oxygen transport. Hay et al [24] reported that the oxygen supply was inadequate in a polysolfone hollow fiber bioreactor (Hepatix Co., La Jolla, CA, USA). Therefore, in our new BAL system, we devised a cell circuit with continuous O2 input. Although number of hepatocytes in a bioreactor was at least 1010 and the density of hepatocytes attained 5 x 107/ml, the hepatocytes received sufficient nutrients and oxygen supply. As a result, the hepatocyte viability remained high throughout the 6 hr of circulation.

Our results showed that the TBi and ALB levels in the hepatocyte suspensions and in the RPMI-1640 medium increased during 6 hr of circulation. There were no significant differences of TBi levels in RPMI-1640 medium vs hepatocyte suspensions after 2 hr of circulation. The ALB level in the RPMI-1640 samples was lower than in the hepatocyte suspensions at all periods of circulation. The ALB levels in RPMI-1640 medium and hepatocyte suspension averaged 2.8 g/L and 3.8 g/L, respectively, after 6 hr of circulation. During the 6 hr of circulation, there were no significant changes of ALT levels in RPMI-1640 medium or hepatocyte suspensions. The level of ALT was lower in RPMI-1640 medium than in hepatocyte suspensions at all times. Bilirubin and albumin could readily cross the semi-permeable membrane because of their low molecular weights. The results indicate that albumin synthesized by hepatocytes in our BAL support system can cross into into the circulating stream in the intratubular space of the hollow fibers.

The biotransformation functions of the hepatic cytochrome P450 (CYP) system may determine whether patients recover from liver failure. Therefore it is important to evaluate the biotransformation functions of BAL [25]. Lidocaine is quickly metabolized mainly in liver. About 90% of lidocaine is transformed into monoethylglycinexylidide (MEGX) and glycinexylidide (GX) by CYP-mediated, hepatocyte-dependent dealklation reactions. The blood MEGX level of healthy persons increases rapidly during the period from 0 to 15 min after iv injection of lidocaine. The MEGX level reaches a peak at 15 min and stays elevated from 15 to 60 min [26]. In contrast, in patients with hepatic impairment, blood MEGX level increases slowly and reaches a peak at about 4 hr after iv injection of lidocaine [27]. The lidocaine metabolism test has been used to estimate the biotransformation functions of BAL [25]. Iwata et al [25] reported that a hollow-fiber BAL device maintained lidocaine metabolism during a 10-day observation period. In the present study, the MEGX level in the new BAL device averaged 32.3 ± 3.7 µg/L after 15 min of circulation and reached its peak (45.7 ± 3.9 µg/L) after 1 hr of circulation. Thereafter, the MEGX level declined gradually. These findings indicate that the BAL displays the biotransformation function of the hepatocyte cytochrome P450 system and could partially replace the hepatic functions of detoxification and metabolism. Such measurements of lidocaine metabolism in a BAL support system have not previously been reported.

In conclusion, we developed a new hollow fiber BAL support system with two circuits. Porcine hepatocyte spheroids are infused and oxygen flows into the cell circuit. In vitro studies showed that the BAL device has an ability to support liver functions and appears to have potential advantages for clinical use in patients with liver failure.


   Acknowledgments
 
This study was supported by a grant from the Health Department of Jiangsu Province, People’s Republic of China.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Watanabe FD, Mullon CJ, Hewitt WR, Arkadopoulos N, Kahaku E, Eguchi S, Khalili T, Arnaout W, Shackleton CR, Rozga J, Soloman B, Demetriou AA. Clinical experience with a bioartificial liver in the treatment of liver failure: a phase I clinical trial. Ann Surg 1997;225: 484–494.[Medline]
  2. Arkadoupoulos N, Rozga J, Demetriou AA. Liver assist system: state of art. Int J Artif Organs 1998;21:781–787.[Medline]
  3. Friedman AL. Why bioartificial liver support remains the Holy Grail. ASAIO J 1998;44:241–243.[Medline]
  4. Cuervas-Mons V, Colas A, Rivera JA, Prados E. In vivo efficacy of a bioartificial liver in improving spontaneous recovery from fulminant hepatic failure: a controlled study in pigs. Transplantation 2000;69:337–344.[Medline]
  5. Sussman NL, Chong MG, Koussayer T, He DE, Shang TA, Whisennand HH, Kelly JH. Reversal of fulminant hepatic failure using an extracorporeal liver assist device. Hepatology 1992;16:60–65.[Medline]
  6. Chen SC, Mullon C, Kahaku E,Watenabe F, Hewitt W, Eguchi S, Middleton Y, Arkadopoulos N, Rozga J, Solomon B, Demetriou AA. Treatment of severe liver failure with a bioartificial liver. Ann NY Acad Sci 1997; 831:350–360.[Medline]
  7. Patzer JF, Lopez RC, Zhu Y, Wang ZF, Mazariegos GV, Fung JJ. Bioartificial liver assist devices in support of patients with liver failure. HBPD INT 2002;1:18–25.
  8. Gerlach JC. Development of a hybrid liver support system: a review. Int J Artif Organs 1996;19:645–654.[Medline]
  9. McLaughlin BE, Tosone CM, Custer LM, Mullon C. Overview of extracorporeal liver support systems and clinical results. Ann NY Acad Sci 1999;875:310–325.[Medline]
  10. Pahernik SA, Thasler WE, Doser M, Gomez-Lechon MJ, Castell MJ, Planck H, Koebe HG. High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane based biomatrices. Cell Tissues Organs 2001; 168:170–177.
  11. Miyoshi H, Yanagi K, Fukuda H, Ohshima N. Long-term performance of albumin secretion of hepatocytes cultured in a packed-bed reactor utilizing porous resin. Artif Organs 1996;20:803–807.[Medline]
  12. Arnaout WS, Moscioni AD, Barbour RL, Demetriou AA. Development of bioartificial liver: bilirubin conjugation in Gunn rats. J Surg Res 1990;48:379–382.[Medline]
  13. Fremond B, Malandain C, Guyomard C, Chesne C, GuillouzoA, 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.[Medline]
  14. Gerlach JC, Encke J, Hole O, Muller C, Ryan CJ, Neuhaus P. Bioreactor for a larger scale hepatocyte in-vitro perfusion. Transplantation 1994;58:984–988.[Medline]
  15. Nyberg SL, Shirabe K, Peshwa MV, Sielaff TD, Crotty PL, Mann HJ, Remmel RP, Payne WB, Hu WS, Ferra FB. Extracorporeal application of a gel-entrapment, bioartificial liver: demonstration of drug metabolism and other biochemical functions. Cell Transplant 1993;2:441–452.[Medline]
  16. Xue YL, Zhao SF, Zhang ZY, Wang YF, Li XJ, Huang XQ, Luo Y, Huang YC, Liu CG. Effects of a bioartificial liver support system on acetaminophen-induced acute liver failure canines. World J Gastroenterol 1999;5:308–311.[Medline]
  17. Chen Z, Ding Y, Zhang H. Cryopreservation of suckling pig hepatocytes. Ann Clin Lab Sci 2001;31:391–398.[Abstract/Free Full Text]
  18. Chen Z, Ding Y, Zhang H. Morphology, viability and functions of suckling pig hepatocytes cultured in serum-free medium at high density. Dig Surg 2002;19:184–193.[Medline]
  19. Chen Z, Ding Y. In situ recirculating collagenase perfusion method for isolation of pig hepatocytes. Cell Biol 2003; 25:124–127.
  20. Nagasue N, Yukaya H, Ogawa Y, Kohno H, Nakamura T. Human liver regeneration after major hepatic resection: a study of normal liver and livers with chronic hepatitis and cirrhosis. Ann Surg 1987;206:30–39.[Medline]
  21. Matsmura KN, Guevara GR, Huston H. Hybrid bio-artificial liver in hepatic failure: preliminary clinical report. Surgery 1987;101:99–103.[Medline]
  22. Lazar A, Peshwa MV, Wu FJ, Chi CM, Cerra FB, Hu WS. Formation of porcine hepatocyte spheroids for use in a bioartificial liver. Cell Transplant 1995;4:259–268.[Medline]
  23. Matsushita T, Amiot B, Hardin J, Platt JL, Nyberg SL. Membrane pore size impacts performance of a xenogeneic bioartificial liver. Transplantation 2003;76,1299–1305.[Medline]
  24. Hay PD, Veitch AR, Smith MD, Cousins RB, Gaylor JD. Oxygen transfer in a diffusion-limited hollow fiber bioreactor liver. Artif Organs, 2000;24:278–288.[Medline]
  25. Iwata H, Sajiki T, Maeda H, Park YG, Zhu B, Satoh S, Uesugi T, Ikai I, Yamaoka Y, Ikada Y. In vitro evaluation of metabolic functions of a bioartificial liver. ASAIO J, 1999;45:299–306.[Medline]
  26. Thomson AH, Elliot HL, Kelman AW, Meredith PA, Whiting B. The pharmacokinetics and pharmaco-dynamics of lignocaine and MEGX in healthy subjects. J Pharmacokinet Biopharm 1987;15:101–115.[Medline]
  27. Conti F, Dousset B, Cherruau B, Guérin C, Soubrane O, Houssin D, Calmus Y. Use of lidocaine metabolism to test liver function during the long-term follow-up of liver transplant recipients. Clin Transplant 2004;18:235–241.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Z.
Right arrow Articles by Li, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Z.
Right arrow Articles by Li, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS