Publications by authors named "Ruurdtje Hoekstra"

43 Publications

Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells.

Toxicol Appl Pharmacol 2020 07 16;399:115055. Epub 2020 May 16.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands. Electronic address:

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.
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http://dx.doi.org/10.1016/j.taap.2020.115055DOI Listing
July 2020

Genome-wide expression profiling reveals increased stability and mitochondrial energy metabolism of the human liver cell line HepaRG-CAR.

Cytotechnology 2020 Jun 4;72(3):377-395. Epub 2020 Mar 4.

Tytgat Institute for Liver and Intestinal Research, AG&M, Amsterdam UMC, University of Amsterdam, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.

Human liver cell line HepaRG is a well-known source of human hepatocyte-like cells which, however, displays limited biotransformation and a tendency to transform after 20 passages. The new HepaRG-CAR cell line overexpressing constitutive androstane receptor (CAR, NR1I3), a regulator of detoxification and energy metabolism outperforms the parental HepaRG cell line in various liver functions. To further characterize this cell line and assess its stability we compared HepaRG-CAR with HepaRG cells at different passages for their expression profile, ammonia and lactate metabolism, bile acid and reactive oxygen species (ROS) production. Transcriptomic profiling of HepaRG-CAR vs. HepaRG early-passage revealed downregulation of hypoxia, glycolysis and proliferation and upregulation of oxidative phosphorylation genesets. In addition CAR overexpression downregulated the mTORC1 signaling pathway, which, as mediator of proliferation and metabolic reprogramming, may play an important role in the establishment of the HepaRG-CAR phenotype. The ammonia and lactate metabolism and bile acid production of HepaRG-CAR cells was stable for 10 additional passages compared to HepaRG cells. Interestingly, bile acid production was 4.5-fold higher in HepaRG-CAR vs. HepaRG cells, whereas lactate and ROS production were 2.7- and 2.0-fold lower, respectively. Principal component analysis showed clustering of HepaRG-CAR (early- and late-passage) and HepaRG early-passage and not with HepaRG late-passage indicating that passaging exerted larger effect on the transcriptional profile of HepaRG than HepaRG-CAR cells. In conclusion, overexpression of CAR in HepaRG cells improves their bile acid production, mitochondrial energy metabolism, and stability, with the latter possibly due to reduced ROS production, resulting in an optimized source of human hepatocytes.
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http://dx.doi.org/10.1007/s10616-020-00384-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225227PMC
June 2020

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

Hepatology 2020 07 8;72(1):257-270. Epub 2020 Apr 8.

Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

Background And Aims: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited.

Approach And Results: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes.

Conclusions: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.
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http://dx.doi.org/10.1002/hep.31037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496924PMC
July 2020

End-stage liver failure: filling the treatment gap at the intensive care unit.

J Artif Organs 2020 Jun 18;23(2):113-123. Epub 2019 Sep 18.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Academic Medical Center, Meibergdreef 69-71, S1-176, 1105 BK, Amsterdam, The Netherlands.

End-stage liver failure is a condition of collapsing liver function with mortality rates up to 80. Liver transplantation is the only lifesaving therapy. There is an unmet need for therapy to extend the waiting time for liver transplantation or regeneration of the native liver. Here we review the state-of-the-art of non-cell based and cell-based artificial liver support systems, cell transplantation and plasma exchange, with the first therapy relying on detoxification, while the others aim to correct also other failing liver functions and/or modulate the immune response. Meta-analyses on the effect of non-cell based systems show contradictory outcomes for different types of albumin purification devices. For bioartificial livers proof of concept has been shown in animals with liver failure. However, large clinical trials with two different systems did not show a survival benefit. Two clinical trials with plasma exchange and one with transplantation of mesenchymal stem cells showed positive outcomes on survival. Detoxification therapies lack adequacy for most patients. Correction of additional liver functions, and also modulation of the immune system hold promise for future therapy of liver failure.
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http://dx.doi.org/10.1007/s10047-019-01133-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228976PMC
June 2020

Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions.

Mitochondrion 2019 07 22;47:298-308. Epub 2019 Feb 22.

Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands; Amsterdam UMC, University of Amsterdam, Surgical Laboratory, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Electronic address:

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.
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http://dx.doi.org/10.1016/j.mito.2019.02.005DOI Listing
July 2019

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity.

Cell Physiol Biochem 2018 15;48(5):2189-2204. Epub 2018 Aug 15.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (AMC), Amsterdam, the Netherlands.

Background/aims: For applicability of cell-based therapies aimed at the treatment of liver failure, such as bioartificial livers (BALs) and hepatocyte transplantation, it is essential that the applied hepatocytes tolerate exposure to the patient plasma. However, plasma from both healthy donors and acute liver failure (ALF) patients is detrimental to hepatocytes and hepatic cell lines, such as HepaRG. We aimed to elucidate the underlying mechanisms of plasma-induced toxicity against HepaRG cells in order to ultimately develop methods to reduce this toxicity and render HepaRG-BAL treatment more effective.

Methods: Differentiated HepaRG cells cultured in monolayers and laboratory-scale BALs were exposed to culture medium, healthy human plasma, healthy porcine plasma and ALF porcine plasma. Healthy human plasma was fractionated based on size- and polarity, albumin depleted and heat treated to characterize the toxic fraction. The cells were assessed for viability by total protein content and trypan blue staining. Their hepatic differentiation was assessed on transcript level through qRT-PCR and microarray analysis, and on functional level for Cytochrome P450 3A4 activity and ammonia elimination. Mitochondrial damage was assessed by JC-1 staining and mitochondrial gene transcription.

Results: Sixteen hours of healthy human plasma exposure did not affect viability, however, hepatic gene-transcript levels decreased dramatically and dose-dependently within four hours of exposure. These changes were associated with early NF-kB signaling and a shift from mitochondrial energy metabolism towards glycolysis. Healthy human plasma-toxicity was associated with the dose-dependent presence of heat-resistant, albumin-bound and (partly) hydrophobic toxic compound(s). HepaRG cells cultured in BALs were partially protected from plasma-toxicity, which was mainly attributable to medium perfusion and/or 3D configuration applied during BAL culturing. The detrimental human plasma effects were reversible in BAL-cultured cells. Porcine ALF-plasma elicited mitotoxicity additional to the basal detrimental effect of porcine healthy plasma, which were only partially reversible.

Conclusion: A specific fraction of human plasma reduces hepatic differentiation of HepaRG cultures, in association with early NF-κB activation. In addition, ALF-plasma elicits mitotoxic effects. These findings allow for a targeted approach in preventing plasma-induced cell damage.
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http://dx.doi.org/10.1159/000492560DOI Listing
September 2018

A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines.

PLoS One 2018 19;13(4):e0193664. Epub 2018 Apr 19.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.

Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the in-vitro maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193664PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908182PMC
July 2018

Improved oxygenation dramatically alters metabolism and gene expression in cultured primary mouse hepatocytes.

Hepatol Commun 2018 03 9;2(3):299-312. Epub 2018 Feb 9.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center University of Amsterdam Amsterdam the Netherlands.

Primary hepatocyte culture is an important system for the study of liver functions. , hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in static cultures is much less than that by blood circulation . Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia-inducible factors (HIF)-1/2α western blotting, and real-time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF-2α expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3-phosphate dehydrogenase [], glucose transporter 1 [], pyruvate dehydrogenase kinase 1 [], and lactate dehydrogenase A []) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (), apolipoprotein B (), and acetyl-coenzyme A carboxylase 1 (), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4α () and farnesoid X receptor () were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF-2 signaling was involved in hypoxia-induced down-regulation of . : Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an model. ( 2018;2:299-312).
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http://dx.doi.org/10.1002/hep4.1140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831026PMC
March 2018

Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines.

J Cell Commun Signal 2018 Sep 4;12(3):575-588. Epub 2018 Feb 4.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (AMC), Meibergdreef 69-71, 1105BK, Amsterdam, The Netherlands.

The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O), normoxia (21%O) or hyperoxia (40%O). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1α.
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http://dx.doi.org/10.1007/s12079-018-0456-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039343PMC
September 2018

AMC-Bio-Artificial Liver culturing enhances mitochondrial biogenesis in human liver cell lines: The role of oxygen, medium perfusion and 3D configuration.

Mitochondrion 2018 03 24;39:30-42. Epub 2017 Aug 24.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (AMC), University of Amsterdam, Postbus 22660, 1100 DD Amsterdam, The Netherlands; Surgical Laboratory, Academic Medical Center (AMC), University of Amsterdam, Postbus 22660, 1100 DD Amsterdam, The Netherlands. Electronic address:

Background: Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we investigated the background of the differences between BAL-cultures and monolayers.

Methods: We performed whole-genome microarray analysis on HepaRG monolayer and BAL-cultures. Next, mitochondrial biogenesis was studied in monolayer and BAL-cultures of HepaRG and C3A. The driving forces for mitochondrial biogenesis by BAL-culturing were investigated in representative culture models differing in oxygenation level, medium flow or 2D vs 3D configuration.

Results: Gene-sets related to mitochondrial energy metabolism were most prominently up-regulated in HepaRG-BAL vs monolayer cultures. This was confirmed by a 2.4-fold higher mitochondrial abundance with increased expression of mitochondrial OxPhos complexes. Moreover, the transcript levels of mitochondria-encoded genes were up to 3.6-fold induced and mitochondrial membrane potential activity was 8.3-fold increased in BAL vs monolayers. Culturing with 40% O, dynamic medium flow and/or in 3D increased the mitochondrial abundance and expression of mitochondrial complexes vs standard monolayer culturing. The stimulatory effect of the BAL culture on mitochondrial biogenesis was confirmed in C3A cells in which mitochondrial abundance increased 2.2-fold with induction of mitochondria-encoded genes.

Conclusions And General Significance: The increased functionality of liver cell lines upon AMC-BAL culturing is associated with increased mitochondrial biogenesis. High oxygenation, medium perfusion and 3D configuration contribute to the up-regulation of the mitochondrial biogenesis.
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http://dx.doi.org/10.1016/j.mito.2017.08.011DOI Listing
March 2018

Scaling-up of a HepaRG progenitor cell based bioartificial liver: optimization for clinical application and transport.

Biofabrication 2017 Jun 30;9(3):035001. Epub 2017 Jun 30.

Surgical laboratory, Academic Medical Center, University of Amsterdam, The Netherlands. Tytgat Institute for Liver and Intestinal Research, Academic Medical Centre, University of Amsterdam, The Netherlands.

A new generation of bioartificial livers, based on differentiated proliferative hepatocyte sources, has been developed. Several practicable and regulatory demands have to be addressed before these can be clinically evaluated. We identified three main hurdles: (1) expansion and preservation of the biocomponent, (2) development of scaled-up culture conditions and (3) transport of the device to the bedside. In this study we address these three issues for the HepaRG-progenitor cell line-loaded AMC-Bioartificial Liver. (1) HepaRG cells were expanded in large quantities and then cryopreserved or loaded directly into bioreactors. After 3 weeks of culture, key hepatic functions (ammonia/lactate elimination, apolipoprotein A1 synthesis and cytochrome P450 3A4 activity) did not differ significantly between the two groups. (2) Bioartificial livers were scaled up from 9 ml to 540 ml priming volume, with preservation of normalized hepatic functionality. Quantification of amino acid consumption revealed rapid depletion of several amino acids. (3) Whole-device cryopreservation and cooled preservation induced significant loss of hepatic functionality, whereas simulated transport from culture-facility to the bedside in a clinical-grade transport unit with controlled temperature maintenance, medium perfusion and gas supply did not affect functionality. In addition, we assessed tumorigenicity of HepaRG cells in immune-incompetent mice and found no tumor formation of HepaRG cells (n = 12), while HeLa cells induced formation of carcinomas in eight out of 12 mice in 140 days.
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http://dx.doi.org/10.1088/1758-5090/aa7657DOI Listing
June 2017

Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells.

Drug Metab Dispos 2017 01 25;45(1):56-67. Epub 2016 Oct 25.

Department of Experimental Surgery (V.A.M., R.A.F.M.C., R.H.), and the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (V.A.M., D.R.W., R.P.J.O.E., R.A.F.M.C., R.H.), Amsterdam, the Netherlands; and Biopredic International, Saint-Grégoire, France (V.S.)

Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression.
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http://dx.doi.org/10.1124/dmd.116.072603DOI Listing
January 2017

Selecting Cells for Bioartificial Liver Devices and the Importance of a 3D Culture Environment: A Functional Comparison between the HepaRG and C3A Cell Lines.

Int J Biol Sci 2016 24;12(8):964-78. Epub 2016 Jun 24.

1. Surgical laboratory, Academic Medical Center, University of Amsterdam, the Netherlands.; 2. Tytgat Institute for Liver and Intestinal Research, Academic Medical Centre, University of Amsterdam, the Netherlands.

Recently, the first clinical trials on Bioartificial Livers (BALs) loaded with a proliferative human hepatocyte cell source have started. There are two cell lines that are currently in an advanced state of BAL development; HepaRG and HepG2/C3A. In this study we aimed to compare both cell lines on applicability in BALs and to identify possible strategies for further improvement. We tested both cell lines in monolayer- and BAL cultures on growth characteristics, hepatic differentiation, nitrogen-, carbohydrate-, amino acid- and xenobiotic metabolism. Interestingly, both cell lines adapted the hepatocyte phenotype more closely when cultured in BALs; e.g. monolayer cultures produced lactate, while BAL cultures showed diminished lactate production (C3A) or conversion to elimination (HepaRG), and urea cycle activity increased upon BAL culturing in both cell lines. HepaRG-BALs outperformed C3A-BALs on xenobiotic metabolism, ammonia elimination and lactate elimination, while protein synthesis was comparable. In BAL cultures of both cell lines ammonia elimination correlated positively with glutamine production and glutamate consumption, suggesting ammonia elimination was mainly driven by the balance between glutaminase and glutamine synthetase activity. Both cell lines lacked significant urea cycle activity and both required multiple culture weeks before reaching optimal differentiation in BALs. In conclusion, culturing in BALs enhanced hepatic functionality of both cell lines and from these, the HepaRG cells are the most promising proliferative cell source for BAL application.
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http://dx.doi.org/10.7150/ijbs.15165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971735PMC
November 2017

Long-term culture of genome-stable bipotent stem cells from adult human liver.

Cell 2015 Jan 18;160(1-2):299-312. Epub 2014 Dec 18.

Hubrecht Institute-KNAW, University Medical Centre Utrecht, CancerGenomics.nl, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands. Electronic address:

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.
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http://dx.doi.org/10.1016/j.cell.2014.11.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313365PMC
January 2015

Bioartificial livers in vitro and in vivo: tailoring biocomponents to the expanding variety of applications.

Expert Opin Biol Ther 2014 Dec 4;14(12):1745-60. Epub 2014 Nov 4.

Surgical Laboratory, and Tytgat Institute for Liver and Intestinal Research, Academic Medical Center , Meibergdreef 9, 1105 AZ Amsterdam , The Netherlands.

Introduction: Bioartificial livers (BALs) were originally developed to treat patients suffering from severe liver failure and relied on primary hepatocytes or on hepatoblastoma-derived cell lines. Currently, new in vitro BAL applications are emerging, including drug toxicity testing, disease modeling and basic clinical research, and in recent years, advances in the field of stem cell biology have resulted in potential alternative cell sources.

Areas Covered: This review identifies the demands of clinical and in vitro BAL applications to their biocomponent and summarizes the functionality and developmental state of BAL technology and cell types currently available. Relevant studies identified by searching the MEDLINE database until April 2014 were reviewed, supplemented with some of our own unpublished data.

Expert Opinion: BALs have the potential to meet demands currently left unmet in both clinical and in vitro applications. All the reviewed biocomponents show limitations towards one or more BAL applications. However, the generation of stem cell-derived hepatocyte-like cells is progressing rapidly, so the criteria for patient-specific drug toxicity screening and disease modeling are probably met in the near future. HepaRG cells are the most promising biocomponent for clinical BAL application, based on their proliferative and differentiation capacity.
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http://dx.doi.org/10.1517/14712598.2014.950651DOI Listing
December 2014

Human fetal liver cells for regulated ex vivo erythropoietin gene therapy.

Mol Ther Methods Clin Dev 2014 12;1:14003. Epub 2014 Mar 12.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam , Amsterdam, The Netherlands.

Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.
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http://dx.doi.org/10.1038/mtm.2014.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362349PMC
May 2015

Increased hepatic functionality of the human hepatoma cell line HepaRG cultured in the AMC bioreactor.

Int J Biochem Cell Biol 2013 Aug 11;45(8):1860-8. Epub 2013 Jun 11.

Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

The clinical application of a bioartificial liver (BAL) depends on the availability of a human cell source with high hepatic functionality, such as the human hepatoma cell line HepaRG. This cell line has demonstrated high hepatic functionality, but the effect of BAL culture on its functionality in time is not known. Therefore, we studied the characteristics of the HepaRG-AMC-BAL over time, and compared the functionality of the HepaRG-AMC-BAL with monolayer cultures of HepaRG cells, normalized for protein (bioactive mass) and DNA (cell number). Histological analysis of 14-day-old BALs demonstrated functional heterogeneity similar to that of monolayer cultures. Hepatic functionality of the HepaRG-AMC-BALs increased during 2-3 weeks of culture. The majority of the measured protein-normalized hepatic functions were already higher in day 14 BAL cultures compared to monolayer cultures, including ammonia elimination (3.2-fold), urea production (1.5-fold), conversion of (15)N-ammonia into (15)N-urea (1.4-fold), and cytochrome P450 3A4 activity (7.9-fold). Lactate production in monolayer cultures switched into lactate consumption in the BAL cultures, a hallmark of primary hepatocytes. When normalized for DNA, only cytochrome P450 3A4 activity was 2.5-fold higher in the BAL cultures compared to monolayer cultures and lactate production switched to consumption, whereas urea production and (15)N-urea production were 1.5- to 2-fold lower. The different outcomes for protein and DNA normalized functions probably relate to a smaller cell volume of HepaRG cells when cultured in the AMC-BAL. Cell damage was 4-fold lower in day 14 BAL cultures compared to monolayer cultures. Transcript levels of cytochrome P450 1A2, 2B6, 3A4 and 3A7 genes and of regulatory genes hepatic nuclear factor 4α and pregnane X receptor increased in time in BAL cultures and reached higher levels than in monolayer cultures. Lastly, metabolism of amino acids, particularly the alanine consumption and ornithine production of HepaRG-AMC-BALs more resembled that of primary hepatocytes than monolayer HepaRG cultures. We conclude therefore that BAL culture of HepaRG cells increases its hepatic functionality, particularly when normalized for biomass, both over time, and compared to monolayer, and this is associated with a reduction in cell damage, upregulation of both regulatory and structural hepatic genes, and changes in amino-acid metabolism. These results underline the potential of HepaRG cells for BAL application.
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http://dx.doi.org/10.1016/j.biocel.2013.05.038DOI Listing
August 2013

Effects of acute-liver-failure-plasma exposure on hepatic functionality of HepaRG-AMC-bioartificial liver.

Liver Int 2013 Apr 7;33(4):516-24. Epub 2013 Feb 7.

Dept. of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

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.
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http://dx.doi.org/10.1111/liv.12090DOI Listing
April 2013

Phase 1 and phase 2 drug metabolism and bile acid production of HepaRG cells in a bioartificial liver in absence of dimethyl sulfoxide.

Drug Metab Dispos 2013 Mar 13;41(3):562-7. Epub 2012 Dec 13.

Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands.

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 cytotoxic effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a three-dimensional 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 CYP3A4. 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, whereas CYP2C9 activity remained equally high. The HepaRG-AMC-BAL secreted 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.
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http://dx.doi.org/10.1124/dmd.112.049098DOI Listing
March 2013

The effect of rat acute-liver-failure plasma on HepaRG cells.

Int J Artif Organs 2012 Nov;35(11):1006-14

Surgical Laboratory, Academic Medical Center, Amsterdam, the Netherlands.

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.
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http://dx.doi.org/10.5301/ijao.5000121DOI Listing
November 2012

Perfusion flow rate substantially contributes to the performance of the HepaRG-AMC-bioartificial liver.

Biotechnol Bioeng 2012 Dec 4;109(12):3182-8. Epub 2012 Jul 4.

Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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).
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http://dx.doi.org/10.1002/bit.24586DOI Listing
December 2012

Liver progenitor cell line HepaRG differentiated in a bioartificial liver effectively supplies liver support to rats with acute liver failure.

PLoS One 2012 18;7(6):e38778. Epub 2012 Jun 18.

Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038778PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3377721PMC
December 2012

Proliferative human cell sources applied as biocomponent in bioartificial livers: a review.

Expert Opin Biol Ther 2012 Jul 31;12(7):905-21. Epub 2012 May 31.

University of Amsterdam, Academic Medical Center, Department of Experimental Surgery (Surgical Laboratory; IWO 1A.1-117), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1517/14712598.2012.685714DOI Listing
July 2012

The HepaRG cell line is suitable for bioartificial liver application.

Int J Biochem Cell Biol 2011 Oct 24;43(10):1483-9. Epub 2011 Jun 24.

Surgical Laboratory, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.biocel.2011.06.011DOI Listing
October 2011

Quantitative assessment of liver function after ischemia-reperfusion injury and partial hepatectomy in rats.

J Surg Res 2012 Jan 21;172(1):85-94. Epub 2010 Jul 21.

Department of Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1016/j.jss.2010.06.038DOI Listing
January 2012

Stable overexpression of pregnane X receptor in HepG2 cells increases its potential for bioartificial liver application.

Liver Transpl 2010 Sep;16(9):1075-85

Department of Experimental Surgery and University of Amsterdam, Amsterdam, the Netherlands.

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.
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http://dx.doi.org/10.1002/lt.22110DOI Listing
September 2010

Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes.

BMC Biotechnol 2009 Oct 21;9:89. Epub 2009 Oct 21.

AMC Liver Center, Meibergdreef 69-71, 1105 BK Amsterdam, the Netherlands.

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.
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http://dx.doi.org/10.1186/1472-6750-9-89DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770505PMC
October 2009

Expression of glutamine synthetase and carbamoylphosphate synthetase i in a bioartificial liver: markers for the development of zonation in vitro.

Cells Tissues Organs 2008 20;188(3):259-69. Epub 2008 Mar 20.

Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Background: 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).

Methods: 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.

Results: 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.

Conclusion: 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.
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http://dx.doi.org/10.1159/000121609DOI Listing
January 2009

Evaluation of a new immortalized human fetal liver cell line (cBAL111) for application in bioartificial liver.

J Hepatol 2008 Feb 17;48(2):266-75. Epub 2007 Dec 17.

Department of Surgery (Surgical Laboratory; IWO-1-172), Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

Background/aims: 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1016/j.jhep.2007.09.018DOI Listing
February 2008

Enhanced oxygen availability improves liver-specific functions of the AMC bioartificial liver.

Artif Organs 2008 Feb 14;32(2):116-26. Epub 2007 Nov 14.

Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1111/j.1525-1594.2007.00500.xDOI Listing
February 2008