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1.
Toxicol Appl Pharmacol ; 399: 115055, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32428594

RESUMO

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.


Assuntos
Amiodarona/farmacologia , Biotransformação/fisiologia , Hepatócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Receptor Constitutivo de Androstano , Metabolismo Energético/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado Artificial , Mitocôndrias/metabolismo
2.
Cytotechnology ; 72(3): 377-395, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32130581

RESUMO

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.

3.
J Artif Organs ; 23(2): 113-123, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31535298

RESUMO

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.


Assuntos
Doença Hepática Terminal/terapia , Fígado Artificial , Troca Plasmática , Plasmaferese , Animais , Transplante de Células , Humanos , Unidades de Terapia Intensiva
4.
Mitochondrion ; 47: 298-308, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30802674

RESUMO

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.


Assuntos
Carbamoil-Fosfato Sintase (Amônia)/biossíntese , Técnicas de Cultura de Células , Regulação Enzimológica da Expressão Gênica , Mitocôndrias Hepáticas/enzimologia , Regulação para Cima , Ureia/metabolismo , Amônia/metabolismo , Arginase/biossíntese , Arginase/genética , Arginina/genética , Arginina/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/genética , Linhagem Celular , Humanos , Mitocôndrias Hepáticas/genética , Ornitina Carbamoiltransferase/biossíntese , Ornitina Carbamoiltransferase/genética
5.
Cell Physiol Biochem ; 48(5): 2189-2204, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30110678

RESUMO

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.


Assuntos
Técnicas de Cultura de Células/métodos , Falência Hepática Aguda/patologia , Fígado Artificial , Plasma/química , Animais , Arginase/genética , Arginase/metabolismo , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Falência Hepática Aguda/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Plasma/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Suínos , Regulação para Cima/efeitos dos fármacos
6.
PLoS One ; 13(4): e0193664, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29672606

RESUMO

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.


Assuntos
Técnicas de Cultura de Células/métodos , Metabolismo Energético/fisiologia , Hepatócitos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Linhagem Celular , Citocromo P-450 CYP3A/metabolismo , Hepatócitos/citologia , Humanos
7.
J Cell Commun Signal ; 12(3): 575-588, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29399736

RESUMO

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%O2), normoxia (21%O2) or hyperoxia (40%O2). 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.

8.
Mitochondrion ; 39: 30-42, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28844938

RESUMO

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%O2), 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% O2, 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.


Assuntos
Hepatócitos/fisiologia , Fígado Artificial , Técnicas de Cultura de Órgãos/métodos , Biogênese de Organelas , Linhagem Celular , Meios de Cultura/química , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Oxigênio/metabolismo
9.
Biofabrication ; 9(3): 035001, 2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-28664876

RESUMO

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.


Assuntos
Fígado Artificial , Células-Tronco/citologia , Engenharia Tecidual/métodos , Meios de Transporte , Animais , Carcinogênese/patologia , Técnicas de Cultura de Células , Proliferação de Células , Criopreservação , Células HeLa , Humanos , Camundongos Nus , Camundongos SCID , Temperatura , Fatores de Tempo
10.
Drug Metab Dispos ; 45(1): 56-67, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27780834

RESUMO

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.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/química , Dimetil Sulfóxido/farmacologia , Fígado/metabolismo , Preparações Farmacêuticas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Receptor Constitutivo de Androstano , Avaliação Pré-Clínica de Medicamentos , Vetores Genéticos , Humanos , Lentivirus/genética , Desintoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , Modelos Biológicos
11.
Int J Biol Sci ; 12(8): 964-78, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27489500

RESUMO

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.


Assuntos
Fígado Artificial , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Biosens Bioelectron ; 53: 129-34, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24135544

RESUMO

The aim of this study was to assess the classification accuracy of an e-Nose in detecting acute liver failure (ALF) in rats. Exhaled breath from 14 rats was repeatedly sampled by e-Nose (8 sensors) and an additional external CO2 sensor at three stages: healthy period; portacaval shunt; and during the development of ALF due to surgically induced complete liver ischemia. We performed principal component analysis (PCA) on the (grouped) sensor data in each stage and the classification accuracy of the first two principal components was assessed by the leave-one-out approach. In addition we performed gas chromatography-mass spectrometry (GC-MS) analysis of the exhaled breath from three rats. The first and second principal components from the PCA analysis of e-Nose data accounted for more than 95% variance in the data. Measurements in the ALF stage were contrasted with the measurements in the control stage. Leave-one-out validation showed classification accuracy of 96%. This accuracy was reached after 3h of ALF development, and was reached already after 2h when data of an external CO2 sensor were also included. GC-MS identified 2-butanol, 2-butanone, 2-pentanone and 1-propanol to be possibly elevated in the ALF stage. This is the first study to demonstrate that ALF in rats can be detected by e-Nose data analysis of the exhaled breath. Confirmation of these results in humans will be an important step forward in the non-invasive diagnosis of ALF.


Assuntos
Técnicas Biossensoriais/métodos , Dióxido de Carbono/isolamento & purificação , Nariz Eletrônico , Falência Hepática Aguda/diagnóstico , Animais , Testes Respiratórios/métodos , Butanóis/isolamento & purificação , Butanonas/isolamento & purificação , Expiração/fisiologia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Falência Hepática Aguda/fisiopatologia , Pentanonas/isolamento & purificação , Ratos
13.
Int J Biochem Cell Biol ; 45(8): 1860-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23770120

RESUMO

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.


Assuntos
Reatores Biológicos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Fígado/fisiologia , Aminoácidos/metabolismo , Aspartato Aminotransferases/metabolismo , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , L-Lactato Desidrogenase/metabolismo , Neoplasias Hepáticas/genética , Fígado Artificial , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas
15.
Liver Int ; 33(4): 516-24, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23387413

RESUMO

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.


Assuntos
Falência Hepática Aguda/terapia , Fígado Artificial , Fígado/metabolismo , Aminoácidos/metabolismo , Amônia/metabolismo , Animais , Apolipoproteína A-I/metabolismo , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Fígado/patologia , Falência Hepática Aguda/sangue , Falência Hepática Aguda/genética , Falência Hepática Aguda/patologia , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Fatores de Tempo , Transcrição Gênica , Ureia/metabolismo
16.
Drug Metab Dispos ; 41(3): 562-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23238784

RESUMO

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.


Assuntos
Ácidos e Sais Biliares/metabolismo , Dimetil Sulfóxido/farmacologia , Hepatócitos/metabolismo , Fígado Artificial , Hidrocarboneto de Aril Hidroxilases/metabolismo , Bilirrubina/metabolismo , Reatores Biológicos , Biotransformação , Técnicas de Cultura de Células , Linhagem Celular , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A/metabolismo , Dimetil Sulfóxido/toxicidade , Glucuronosiltransferase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Hidroxilação , Isoenzimas , Cinética , Desintoxicação Metabólica Fase I , Desintoxicação Metabólica Fase II , Rifampina/farmacologia , Especificidade por Substrato , Testosterona , Tolbutamida/metabolismo
17.
Liver Int ; 33(1): 40-52, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22429562

RESUMO

BACKGROUND: An early and proper diagnosis of acute on chronic liver failure (ACLF), together with the identification of indicators associated with disease severity is critical for outcome prediction and therapy. OBJECTIVE: To systematically identify and summarize prognostic indicators for patients with ACLF and to evaluate the predictive value of these indicators. METHODS: Embase and Ovid-Medline were searched for English-language articles. The search criteria focused on identifying clinical trials and observational studies reporting on indicators used for prediction of mortality in patients with ACLF. RESULTS: Of 2382 studies identified, 19 were included for detailed analysis. Thirteen different definitions of ACLF were found. The main differences were related to acute deterioration in liver function, coagulopathy and hyperbilirubinaemia/jaundice. Seventy three prognostic indicators and their association with mortality were extracted and categorized into seven categories: general markers (n = 13), viral markers (n = 6), bio-markers (n = 22), hemodynamics (n = 1), morphology/histology (n = 17), scoring systems (n = 10) and treatments (n = 4). CONCLUSIONS: The ambiguity and variability in the definition of ACLF and in its predictive indicators hampers comparability among studies. There is a need for a single uniform definition of ACLF. Also absence of a gold standard is an obstacle to render one indicator superior to another. The age, hepatic encephalopathy, model for end-stage liver disease score, total bilirubin and International normalized ratio (prothrombin time) appeared to be promising candidates for evaluation in future studies. The result of this review may be useful as a starting point in developing a standard list of indicators for clinical outcome that concur with the clinicians' subjective views on prognosis in ACLF.


Assuntos
Doença Hepática Terminal/diagnóstico , Doença Hepática Terminal/mortalidade , Indicadores Básicos de Saúde , Falência Hepática Aguda/diagnóstico , Falência Hepática Aguda/mortalidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Criança , Progressão da Doença , Diagnóstico Precoce , Doença Hepática Terminal/classificação , Feminino , Humanos , Falência Hepática Aguda/classificação , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Medição de Risco , Fatores de Risco , Índice de Gravidade de Doença , Terminologia como Assunto , Adulto Jovem
18.
PLoS One ; 7(12): e50952, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23272081

RESUMO

INTRODUCTION: Acute liver failure is a rare disease with high mortality and liver transplantation is the only life saving therapy. Accurate prognosis of ALF is crucial for proper intervention. AIM: To identify and characterize newly developed prognostic models of mortality for ALF patients, assess study quality, identify important variables and provide recommendations for the development of improved models in the future. METHODS: The online databases MEDLINE® (1950-2012) and EMBASE® (1980-2012) were searched for English-language articles that reported original data from clinical trials or observational studies on prognostic models in ALF patients. Studies were included if they developed a new model or modified existing prognostic models. The studies were evaluated based on an existing framework for scoring the methodological and reporting quality of prognostic models. RESULTS: Twenty studies were included, of which 18 reported on newly developed models, 1 on modification of the Kings College Criteria (KCC) and 1 on the Model for End-Stage Liver Disease (MELD). Ten studies compared the newly developed models to previously existing models (e.g. KCC); they all reported that the new models were superior. In the 12-point methodological quality score, only one study scored full points. On the 38-point reporting score, no study scored full points. There was a general lack of reporting on missing values. In addition, none of the studies used performance measures for calibration and accuracy (e.g. Hosmer-Lemeshow statistics, Brier score), and only 5 studies used the AUC as a measure of discrimination. CONCLUSIONS: There are many studies on prognostic models for ALF but they show methodological and reporting limitations. Future studies could be improved by better reporting and handling of missing data, the inclusion of model calibration aspects, use of absolute risk measures, explicit considerations for variable selection, the use of a more extensive set of reference models and more thorough validation.


Assuntos
Gastroenterologia/métodos , Falência Hepática Aguda/diagnóstico , Falência Hepática Aguda/mortalidade , Algoritmos , Área Sob a Curva , Calibragem , Feminino , Gastroenterologia/normas , Humanos , Falência Hepática Aguda/terapia , Masculino , Modelos Estatísticos , Valor Preditivo dos Testes , Probabilidade , Prognóstico , Reprodutibilidade dos Testes , Risco , Índice de Gravidade de Doença , Resultado do Tratamento
19.
Int J Artif Organs ; 35(11): 1006-14, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23065880

RESUMO

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.


Assuntos
Hepatócitos/fisiologia , Falência Hepática Aguda/sangue , Falência Hepática Aguda/patologia , Plasma/fisiologia , Animais , Arginase/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Hepatócitos/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/fisiologia , Fígado Artificial , Ratos
20.
Crit Care ; 16(5): R200, 2012 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-23078781

RESUMO

INTRODUCTION: The Risk, Injury, Failure, Loss, and End-Stage Renal Disease (RIFLE) is a consensus-based classification system for diagnosing acute kidney insufficiency (AKI), based on serum creatinine (SCr) and urine output criteria (RIFLESCr+UO). The urine output criteria, however, are frequently discarded and many studies in the literature applied only the SCr criteria (RIFLESCr). We diagnosed AKI using both RIFLE methods and compared the effects on time to AKI diagnosis, AKI incidence and AKI severity. METHODS: This was a prospective observational cohort study during four months in adult critically ill patients admitted to the ICU for at least 48 hours. During the first week patients were scored daily for AKI according to RIFLESCr+UO and RIFLESCr. We assessed urine output hourly and fluid balance daily. The baseline SCr was estimated if a recent pre-ICU admission SCr was unknown. Based on the two RIFLE methods for each patient we determined time to AKI diagnosis (AKI-0) and maximum RIFLE grade. RESULTS: We studied 260 patients. A pre-ICU admission SCr was available in 101 (39%) patients. The two RIFLE methods resulted in statistically significantly different outcomes for incidence of AKI, diagnosis of AKI for individual patients, distribution of AKI-0 and distribution of the maximum RIFLE grade. Discarding the RIFLE urine criteria for AKI diagnosis significantly underestimated the presence and grade of AKI on admission and during the first ICU week (P < 0,001) and significantly delayed the diagnosis of AKI (P < 0.001). Based on RIFLESCr 45 patients had no AKI on admission but subsequently developed AKI. In 24 of these patients (53%) AKI would have been diagnosed at least one day earlier if the RIFLE urine criteria had been applied. Mortality rate in the AKI population was 38% based on RIFLESCr and 24% based on RIFLESCr+UO (P = 0.02). CONCLUSIONS: The use of RIFLE without the urine criteria significantly underscores the incidence and grade of AKI, significantly delays the diagnosis of AKI and is associated with higher mortality.


Assuntos
Injúria Renal Aguda/diagnóstico , Creatinina/sangue , Urina , Injúria Renal Aguda/sangue , Injúria Renal Aguda/mortalidade , Injúria Renal Aguda/urina , Estado Terminal , Feminino , Mortalidade Hospitalar , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Índice de Gravidade de Doença , Micção
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