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Ruurdtje Hoekstra,
Geert A A Nibourg,
Tessa V van der Hoeven,
Gabrielle Plomer,
Jurgen Seppen,
Mariette T Ackermans,
Sandrine Camus,
Wim Kulik,
Thomas M van Gulik,
Ronald P Oude Elferink,
Robert A F M Chamuleau
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ABSTRACT: The human liver cell line HepaRG has been recognized as a promising source for in vitro testing of metabolism and toxicity of compounds. However, currently the hepatic differentiation of these cells relies on exposure to dimethylsulfoxide (DMSO), which, as a side-effect, has a damaging effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a 3D bioreactor that has previously been shown to upregulate various liver functions of cultured cells. We therefore cultured HepaRG cells in the AMC-BAL without DMSO and characterized the drug metabolism. Within 14 days of culture, the HepaRG-AMC-BALs contained highly polarized viable liver-like tissue with heterogeneous expression of cytochrome P450 (CYP) 3A4. We found a substantial metabolism of the tested substrates, ranging from 26% (UDP-glucuronosyltransferase 1A1), 47% (CYP3A4) to 240% (CYP2C9) of primary human hepatocytes. The CYP3A4 activity could be induced 2-fold by rifampicin, while CYP2C9 activity remained equally high. The HepaRG-AMC-BAL produced bile acids at 43% the rate of primary human hepatocytes and demonstrated hydroxylation, conjugation, and transport of bile salts. Concluding, culturing HepaRG cells in the AMC-BAL yields substantial phase 1 and phase 2 drug metabolism, while maintaining high viability, rendering DMSO addition superfluous for the promotion of drug metabolism. Therefore, AMC-BAL culturing makes the HepaRG cells more suitable for testing metabolism and toxicity of drugs.
Drug metabolism and disposition: the biological fate of chemicals 12/2012; · 3.74 Impact Factor
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ABSTRACT: BACKGROUND & AIMS: The AMC-bioartificial liver loaded with the human hepatoma cell line HepaRG as biocomponent (HepaRG-AMC-BAL) has recently proven efficacious in rats with acute liver failure (ALF). However, its efficacy may be affected by cytotoxic components of ALF plasma during treatment. In this study, we investigated the effects of ALF-plasma on the HepaRG-AMC-BAL. METHODS: HepaRG-AMC-BALs were connected to the blood circulation of rats with total liver ischaemia, either during the first 5 h after induction of ischaemia (mild ALF group), or during the following 10 h (severe ALF group). After disconnection, the BALs were assessed for cell leakage, gene transcript levels, ammonia elimination, urea production, cytochrome P450 3A4 activity, apolipoprotein A 1 production, glucose and amino acid metabolism. RESULTS: Cell leakage increased 2.5-fold in the severe ALF group, but remained limited in all groups. Hepatic gene transcript levels decreased (max 40-fold) or remained stable. In contrast, hepatic functions increased slightly or remained stable. Particularly, urea production increased 1.5-fold, with a concurrent increase in arginase 2 transcription and arginine consumption, with a trend towards reduced conversion of ammonia into urea. The amino acid consumption increased, however, the net glucose consumption remained stable. CONCLUSIONS: The HepaRG-AMC-BAL retains functionality after both mild and severe exposure to ALF plasma, but urea production may be increasingly derived from arginase 2 activity instead of urea cycle activity. Nevertheless, the increase in cell leakage and decrease in various hepatic transcript levels suggest that a decrease in hepatic functionality may follow upon extended exposure to ALF plasma.
Liver international: official journal of the International Association for the Study of the Liver 12/2012; · 3.82 Impact Factor
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ABSTRACT: Purpose: We recently demonstrated the high liver functionality of the human liver cell line HepaRG, including ammonia eliminating capacity, making it a valuable biocomponent of a bioartificial liver (BAL) to support patients with acute liver failure (ALF). This cell line further gains detoxification properties when cultured with dimethyl sulfoxide (DMSO). In this paper we describe whether its functionality is compromised by the toxic effects of ALF plasma, as has been shown for primary hepatocytes. Methods: We exposed -DMSO and +DMSO HepaRG cultures during 16 hours to healthy plasma and ALF-rat plasma. The cultures were analyzed for lipid accumulation, cell leakage, apolipoprotein A-1 production, nitrogen metabolism and transcript levels of hepatic genes. Results: The -DMSO cultures showed increased cell leakage after healthy and ALF plasma exposure in contrast to +DMSO cultures, but otherwise the -DMSO and +DMSO cultures were equally affected by exposure to the plasmas. Exposure to both plasmas caused lipid accumulation and decreased transcript levels of various hepatic genes. ALF plasma decreased urea cycle activity, but increased urea production from arginine by upregulated arginase 2. However, total ammonia elimination was not affected by exposure to either plasma, indicating its predominant elimination by fixation into amino acids. In addition, apolipoprotein A-1 production remained constant. Conclusions: HepaRG cells are negatively affected by rat plasma, even of healthy origin. However, their ammonia eliminating capacity is relatively resistant, underlining their suitability for BAL application. DMSO pre-treatment may increase their viability in plasma.
The International journal of artificial organs 09/2012; · 1.86 Impact Factor
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ABSTRACT: Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver-failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC-BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β-hydroxylation of testosterone showed 2-20-fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG-BAL); and (3) in the current set-up the mass balance of substrate, metabolite, or cell damage markers between in-and out-flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG-BAL). Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering 06/2012; · 3.95 Impact Factor
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ABSTRACT: INTRODUCTION: Bioartificial livers (BALs) are urgently needed to bridge severe liver failure patients to liver transplantation or liver regeneration. When based on primary hepatocytes, their efficacy has been shown in animal experiments and their safety was confirmed in clinical trials. However, a proliferative human cell source with therapeutic functionality is needed to secure availability and move BAL application forward. AREAS COVERED: This review compares the performance of BALs based on proliferative human biocomponents and primary hepatocytes. This review evaluates relevant studies identified by searching the MEDLINE database until July 2011 and some of our own unpublished data. EXPERT OPINION: All the discussed hepatocyte-like biocomponents show deficiencies in their hepatic functionality compared with primary hepatocytes, particularly functions occurring late in liver development. Nonetheless, the HepaRG, HepG2-GS-CYP3A4, and mesenchymal stem cells show efficacy in a statistically well-powered animal model of acute liver failure, when applied in a BAL device. Various methods to gain higher functionality of BALs, including genetic modification, the usage of combinatory cell sources, and improvement of culture methods, have scarcely been applied, but may further pave the path for BAL application. Clinical implementation of a BAL based on a human proliferative biocomponent is still several years away.
Expert opinion on biological therapy 05/2012; 12(7):905-21. · 3.22 Impact Factor
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ABSTRACT: Liver function after hepatic ischemia-reperfusion (I/R) injury and partial liver resection (PHx) is influenced by the extent of PHx, hepatocellular damage, and liver regeneration. This study investigates the effect of minor PHx with increasing degrees of I/R-induced damage on postoperative liver function parameters and compares the indocyanine green (ICG) clearance test with (99m)Tc-mebrofenin hepatobiliary scintigraphy (HBS) for quantitative measurement of hepatic function in a standardized rat model.
Rats were subjected to 70% partial liver I/R combined with resection of the nonischemic lobes. Various degrees of hepatic damage were induced by 0, 15, 30, 45, and 60 min ischemia. Prothrombin time and bilirubin were used as indirect parameters of liver function. (99m)Tc-mebrofenin HBS and ICG clearance were used as dynamic quantitative liver function tests.
After 24 h reperfusion hepatocellular damage increased with prolonged ischemia times. Hepatocellular damage and liver regeneration were closely interrelated. Moderate I/R-induced damage enhanced regeneration, while extensive damage debilitates the regenerative capacity. PHx alone resulted in no significant decrease in liver uptake function measured by HBS or ICG. Increasing severity of hepatic I/R injury had a differential effect on ICG clearance and (99m)Tc-mebrofenin uptake and excretion.
The specific impact of 30% PHx combined with progressive ischemia times is different for each liver function test. Albeit (99m)Tc-mebrofenin HBS and the ICG clearance test provide complementary quantitative information to biochemical parameters, they only quantify one or two components of liver function. ICG and (99m)Tc-mebrofenin uptake profiles differed significantly, suggesting that the specific hepatic transporters may be distinct.
Journal of Surgical Research 01/2012; 172(1):85-94. · 2.25 Impact Factor
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ABSTRACT: A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into hepatocytes and bile duct cells. Metabolism and synthesis of HepaRG monolayer cultures is relatively high and their drug metabolism can be enhanced upon treatment with 2% dimethyl sulfoxide (DMSO). However, their potential for bioartificial liver application has not been assessed so far. Therefore, HepaRG cells were cultured in the Academic Medical Center bioartificial liver (AMC-BAL) with and without DMSO and assessed for their hepatic functionality in vitro and in a rat model of acute liver failure. HepaRG-AMC-BALs cultured without DMSO eliminated ammonia and lactate, and produced apolipoprotein A-1 at rates comparable to freshly isolated hepatocytes. Cytochrome P450 3A4 transcript levels and activity were high with 88% and 37%, respectively, of the level of hepatocytes. DMSO treatment of HepaRG-AMC-BALs reduced the cell population and the abovementioned functions drastically. Therefore, solely HepaRG-AMC-BALs cultured without DMSO were tested for efficacy in rats with acute liver failure (n = 6). HepaRG-AMC-BAL treatment increased survival time of acute liver failure rats ∼50% compared to acellular-BAL treatment. Moreover, HepaRG-AMC-BAL treatment decreased the progression of hepatic encephalopathy, kidney failure, and ammonia accumulation. These results demonstrate that the HepaRG-AMC-BAL is a promising bioartificial liver for clinical application.
PLoS ONE 01/2012; 7(6):e38778. · 4.09 Impact Factor
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ABSTRACT: For bioartificial liver application, cells should meet the following minimal requirements: ammonia elimination, drug metabolism and blood protein synthesis. Here we explore the suitability of HepaRG cells, a human cell line reported to differentiate into hepatocyte clusters and surrounding biliary epithelial-like cells at high density and after exposure to dimethyl sulfoxide (DMSO). The effect of carbamoyl-glutamate (CG), an activator of urea cycle enzyme carbamoylphosphate synthetase (CPS) was studied additionally. The effects of DMSO and/or CG were assessed in presence of (15)NH(4)Cl on HepaRG cells in monolayer. We tested hepatocyte-specific functions at transcript and biochemical level, cell damage parameters and performed immunostainings. Ureagenesis, ammonia/galactose elimination and albumin, glutamine synthetase and CPS transcript levels were higher in -DMSO than +DMSO cultures, probably due to a higher cell content and/or cluster-neighbouring regions contributing to their functionality. DMSO treatment increased cytochrome P450 (CYP) transcript levels and CYP3A4 activity, but also cell damage and repressed hepatic functionality in cluster-neighbouring regions. The levels of ammonia elimination, apolipoprotein A-1 production, and transcription of CYP3A4, CYP2B6 and albumin reached those of primary hepatocytes in either the + or -DMSO cultures. Preconditioning with CG increased conversion of (15)NH(4)Cl into (15)N-urea 4-fold only in -DMSO cultures. Hence, HepaRG cells show high metabolic and synthetic functionality in the absence of DMSO, however, their drug metabolism is only high in the presence of DMSO. An unparalleled broad hepatic functionality, suitable for bioartificial liver application, can be accomplished by combining CG treated -DMSO cultures with +DMSO cultures.
The international journal of biochemistry & cell biology 06/2011; 43(10):1483-9. · 4.89 Impact Factor
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ABSTRACT: To bridge patients with acute liver failure to transplantation or liver regeneration, a bioartificial liver (BAL) is urgently needed. A BAL consists of an extracorporeal bioreactor loaded with a bioactive mass that would preferably be of human origin and display high hepatic functionality, including detoxification. The human hepatoma cell line HepG2 exhibits many hepatic functions, but its detoxification function is low. In this study, we investigated whether stable overexpression of pregnane X receptor (PXR), a master regulator of diverse detoxification functions in the liver [eg, cytochrome P450 3A (CYP3A) activity], would increase the potential of HepG2 for BAL application. Stable overexpression was achieved by lentiviral expression of the human PXR gene, which yielded cell line cBAL119. In monolayer cultures of cBAL119 cells, PXR transcript levels increased 29-fold versus HepG2 cells. Upon activation of PXR by rifampicin, the messenger RNA levels of CYP3A4, CYP3A5, and CYP3A7 increased 49- to 213-fold versus HepG2 cells. According to reporter gene assays with different inducers, the highest increase in CYP3A4 promoter activity (131-fold) was observed upon induction with rifampicin. Inside BALs, the proliferation rates, as measured by the DNA content, were comparable between the 2 cell lines. The rate of testosterone 6beta-hydroxylation, a measure of CYP3A function inside BALs, increased 4-fold in cBAL119 BALs versus HepG2 BALs. Other functions, such as apolipoprotein A1 synthesis, urea synthesis, glucose consumption, and lactate production, remained unchanged or increased. Thus, stable PXR overexpression markedly increases the potential of HepG2 for BAL application.
Liver Transplantation 09/2010; 16(9):1075-85. · 3.39 Impact Factor
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Tanja Deurholt,
Niek P van Til,
Aniska A Chhatta,
Lysbeth ten Bloemendaal,
Ruth Schwartlander,
Catherine Payne,
John N Plevris,
Igor M Sauer,
Robert Afm Chamuleau,
Ronald Pj Oude Elferink,
Jurgen Seppen, Ruurdtje Hoekstra
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ABSTRACT: A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications.
Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion.
This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.
BMC Biotechnology 01/2009; 9:89. · 2.35 Impact Factor
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Tanja Deurholt,
van Til Niek,
Aniska Chhatta,
ten Bloemendaal Lysbeth,
Ruth Schwartlander,
Catherine Payne,
John Plevris,
Igor Sauer,
Robert Chamuleau,
Ronald Elferink,
Jurgen Seppen, Ruurdtje Hoekstra
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ABSTRACT: Abstract
Background
A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications.
Results
Cell clones derived from human fetal liver cells were immortalized by over-expression of telomerase reverse transcriptase. The resulting cell line, cBAL111, displayed hepatic functionality similar to the parental cells prior to immortalization, and did not grow in soft agar. Cell line cBAL111 expressed markers of immature hepatocytes, like glutathione S transferase and cytokeratin 19, as well as progenitor cell marker CD146 and was negative for lidocaine elimination. On the other hand, the cBAL111 cells produced urea, albumin and cytokeratin 18 and eliminated galactose. In contrast to hepatic cell lines NKNT-3 and HepG2, all hepatic functions were expressed in cBAL111, although there was considerable variation in their levels compared with primary mature hepatocytes. When transplanted in the spleen of immunodeficient mice, cBAL111 engrafted into the liver and partly differentiated into hepatocytes showing expression of human albumin and carbamoylphosphate synthetase without signs of cell fusion.
Conclusion
This novel liver cell line has the potential to differentiate into mature hepatocytes to be used for in vitro hepatocyte applications.
BMC Biotechnology. 01/2009;
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ABSTRACT: Mechanisms underlying hepatic zonation are not completely elucidated. In vitro test systems may provide new insights into current hypotheses. In this study, zonally expressed proteins, i.e. glutamine synthetase (GS; pericentral) and carbamoylphosphate synthetase (CPS; periportal), were tested for their expression patterns in the bioartificial liver of the Academic Medical Center (AMC-BAL).
Distribution and organization of porcine hepatocytes inside the AMC-BAL as well as GS and CPS expression were analyzed (immuno-)histochemically in time. Ten zonally expressed proteins were analyzed by RT-PCR on cell isolate and bioreactor samples. General metabolic and hepatocyte-specific functions were determined as well.
Viable hepatocyte layers of approximately 150 microm were observed around gas capillaries, whereas inside the matrix, single cells or small aggregates were present. GS protein and mRNA levels were upregulated in time. GS protein was preferentially expressed in hepatocytes adjacent to oxygen-supplying capillaries and in previously CPS-positive hepatocytes. No shift towards a periportal or pericentral phenotype was observed from RT-PCR analysis.
Induction of GS expression inside the AMC-BAL is not dependent of (low) oxygen tensions and hepatic nuclear factor 4alpha transcript levels. GS expression might be related to (1) low substrate levels and/or autocrine soluble factors, or (2) to cytoskeleton interactions, putatively associated with the beta-catenin signaling pathway.
Cells Tissues Organs 04/2008; 188(3):259-69. · 2.20 Impact Factor
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ABSTRACT: Long-term culturing of primary porcine hepatocytes (PPH) inside the Academic Medical Center (AMC)-bioartificial liver is characterized by increased anaerobic glycolysis. Recommendations to increase oxygen availability were proposed in a previous numerical study and were experimentally evaluated in this study. Original bioreactors as well as new configuration bioreactors with 2.2-fold thinner nonwoven matrix and 2-fold more capillaries were loaded with PPHs and oxygenated with different gas oxygen pressures resulting in medium pO(2) (pO(2-med)) of either 135-150 mm Hg or 235-250 mm Hg. After 6 days culturing, new configuration bioreactors with pO(2-med )of 250 mm Hg showed significantly reduced anaerobic glycolysis, 60% higher liver-specific functions, and increased transcript levels of five liver-specific genes compared to the standard bioreactor cultures. Changed bioreactor configuration and increasing pO(2-med) contributed equally to these improvements. Histological examination demonstrated small differences in cell organization. In conclusion, higher metabolic stability and liver-specific functionality was achieved by enhanced oxygen availability based on a prior modeling concept.
Artificial Organs 03/2008; 32(2):116-26. · 2.00 Impact Factor
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ABSTRACT: Clinical use of bioartificial livers (BAL) relies heavily on the development of human liver cell lines. The aim of this study was to assess the potential of the recently developed human fetal liver cell line cBAL111 for application in the AMC-BAL.
Laboratory-scale AMC-BAL bioreactors were loaded with 20 or 200 million cBAL111 cells and were cultured for 3 days. Parameters for hepatocyte-specific function and general metabolism were determined daily using tests with culture medium or 100% human serum. The bioreactors were also analyzed for mRNA levels of liver-specific genes and histology.
cBAL111 eliminated ammonia at a rate up to 49% of that in primary porcine hepatocytes (PPH), despite a low (1.1%) urea production. Transcript levels of glutamine synthetase (GS) were 570% of that in human liver, whereas genes of the urea cycle showed low expression. GS expression was confirmed immunohistochemically, and glutamine was produced by the cells. cBAL111 eliminated galactose (90.1% of PPH) and lidocaine (0.1% of PPH) and produced albumin (6% of PPH). Human serum did not increase function of cBAL111.
cBAL111 showed liver-specific functionality when cultured inside the AMC-BAL and eliminated ammonia mainly by the activity of GS, and not through the urea cycle.
Journal of Hepatology 03/2008; 48(2):266-75. · 9.26 Impact Factor
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ABSTRACT: Long-term culturing of primary porcine hepatocytes (PPH) inside the Academic Medical Center (AMC)-bioartificial liver is characterized by increased anaerobic glycolysis. Recommendations to increase oxygen availability were proposed in a previous numerical study and were experimentally evaluated in this study. Original bioreactors as well as new configuration bioreactors with 2.2-fold thinner nonwoven matrix and 2-fold more capillaries were loaded with PPHs and oxygenated with different gas oxygen pressures resulting in medium pO2 (pO2-med) of either 135-150 mm Hg or 235-250 mm Hg. After 6 days culturing, new configuration bioreactors with pO2-med of 250 mm Hg showed significantly reduced anaerobic glycolysis, 60% higher liver-specific functions, and increased transcript levels of five liver-specific genes compared to the standard bioreactor cultures. Changed bioreactor configuration and increasing pO2-med contributed equally to these improvements. Histological examination demonstrated small differences in cell organization. In conclusion, higher metabolic stability and liver-specific functionality was achieved by enhanced oxygen availability based on a prior modeling concept.
Artificial Organs 01/2008; 32(2):116 - 126. · 2.00 Impact Factor
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ABSTRACT: A comprehensive understanding of the mechanisms that underlie hepatic differentiation inside a bioartificial liver (BAL) device is obtained when functional, histological, and gene expression analyses can be combined. We therefore developed a novel cell-sampling technique that enabled us to analyze adherent hepatocytes inside a BAL device during a 5-day culture period, without the necessity of terminating the culture. Biochemical data showed that hepatocyte-specific functions were relatively stable, despite an increase in glycolytic activity. Quantitative reverse transcriptase polymerase chain reaction analysis of hepatic genes cytochrome p450 3A29, albumin, glutamine synthetase, alpha-1 antitrypsin, and carbamoyl-phosphate synthetase, but also de-differentiation marker pi-class glutathione S transferase showed stable messenger ribonucleic acid (mRNA) levels from day 1 to 5. In contrast, mRNA levels of alpha-fetoprotein, pro- and anti-apoptotic genes Bax-alpha and Bcl-X(L), metabolic genes lactate dehydrogenase and uncoupling protein 2, and cytoskeleton genes alpha- and beta-tubulin and beta-actin increased in 5 days. Histological analysis revealed viable tissue-like structures with adaptation to the in vitro environment. We conclude that hepatocytes show a tendency for de-differentiation shortly after seeding but thereafter remain acceptably differentiated during 5 days of culture. Furthermore, partly impaired mitochondrial function is suggestive for local hypoxic regions and may trigger the observed metabolic changes. Anti-apoptotic activity seems to balance pro-apoptotic activity. This new cell-sampling technique facilitates the analysis of dynamic processes of hepatocyte culture inside a BAL.
Tissue Engineering 07/2007; 13(6):1235-46. · 4.02 Impact Factor
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ABSTRACT: The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)-BAL during a 7-day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2-fold more ammonia and produced 3-fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5-fold higher than MPHs and 6.6-fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue-like structures inside the AMC-BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading.
Liver Transplantation 04/2007; 13(4):589-98. · 3.39 Impact Factor
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ABSTRACT: The selection of a cell type for bioartificial liver (BAL) systems for the treatment of patients with acute liver failure is in part determined by issues concerning patient safety and cell availability. Consequently, mature porcine hepatocytes (MPHs) have been widely applied in BAL systems. The success of clinical BAL application systems is, however, largely dependent on the functionality and stability of hepatocytes. Therefore, we compared herein the general metabolic and functional activities of MPHs with mature human hepatocytes (MHHs) in the Academic Medical Center (AMC)-BAL during a 7-day culture period. We also tested fetal human hepatocytes (FHHs), since their proliferation capacity is higher than MHHs and their function is increased compared to human liver cell lines. The results showed large differences between the 3 cell types. MHHs eliminated 2-fold more ammonia and produced 3-fold more urea than MPHs, whereas FHHs produced ammonia. Lidocaine elimination of FHHs was 3.5-fold higher than MPHs and 6.6-fold higher than of MHHs. Albumin production was not different between the 3 cell types. MPHs and FHHs became increasingly glycolytic, whereas MHHs remained metabolically stable during the whole culture period. MHHs and MPHs formed tissue-like structures inside the AMC-BAL. In conclusion, we propose that FHHs can be considered as a suitable cell type for pharmacological studies inside a bioreactor. However, we conclude that MHHs are the preferred cell source for loading a BAL device for clinical use, because of their high ammonia eliminating capacity and metabolic stability. MPHs should be considered as the best alternative cell source for BAL application, although their phenotypic instability urges application within 1 or 2 days after loading. Liver Transpl 13:589–598, 2007. © 2007 AASLD.
Liver Transplantation 03/2007; 13(4):589 - 598. · 3.39 Impact Factor
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ABSTRACT: The majority of cholangiocarcinoma patients present with advanced incurable disease. Therefore development of new therapeutic modalities including adenoviral gene therapy is of paramount importance. We set out to identify tumour specific promoters which have low activity in human liver cells and retain their specificity in an adenoviral vector.
mRNA levels of cyclo-oxygenase-2, cytokeratin-19, mucin-1, midkine and telomerase reverse transcriptase (TERT) were determined in human liver, cholangiocarcinoma (resection specimens and cell lines), primary human hepatocytes, cholangiocytes and endothelial cells by Reverse Transcriptase-quantitative PCR. The activity of candidate promoters in adenoviral vectors was then determined in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes.
mRNA levels of all tested tumour markers were significantly higher in cholangiocarcinoma than in normal liver. Based on low expression in hepatocytes, either in combination with low expression in primary cholangiocytes or endothelial cells, the cytokeratin-19, mucin-1 and TERT promoters were selected for further analyses. In an adenoviral vector, the activity of the TERT or cytokeratin-19 promoters were low in normal human hepatocytes and cholangiocytes, and high in cholangiocarcinoma cell lines.
The TERT and Cytokeratin-19 promoters are highly expressed in cholangiocarcinoma and seem suitable to restrict adenoviral gene therapy to these intra-hepatic tumours.
Journal of Hepatology 02/2006; 44(1):126-33. · 9.26 Impact Factor
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ABSTRACT: Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we evaluated the hepatic function of HFLCs under proliferative conditions, to determine whether HFLCs can replace mature hepatocytes for in vitro applications. HFLCs were isolated from fetal livers of 16 weeks gestation. Hepatic functions of HFLCs were determined in primary culture and after expansion in vitro. Clonal derivatives were selected and tested for hepatic functionality. Results were compared to primary mature human hepatocytes in vitro. No differences were observed between primary HFLCs and mature human hepatocytes in albumin production and mRNA levels of various liver-specific genes. Ureagenesis was 4.4-fold lower and ammonia elimination was absent in HFLCs. Expanding HFLCs decreased hepatic functions and increased cell stretching. In contrast, clonal derivatives had stable functionality and morphology and responded to differentiation stimuli. Although their hepatic functions were higher than in passaged HFLCs, functionality was at least 20 times lower compared to mature human hepatocytes. HFLCs cannot replace mature human hepatocytes in in vitro applications requiring extensive in vitro expansion, because this is associated with decreased hepatic functionality. Selecting functional subpopulations can, at least partly, prevent this. In addition, defining conditions that support hepatic differentiation is necessary to obtain HFLC cultures suitable for in vitro hepatic applications.
Cell Transplantation 02/2006; 15(8-9):811-22. · 5.13 Impact Factor
