Expression of Liver Functions in Imrnortalised Rot Hepotocyte Cell Lines

MacDonald, Caroline; Vass, Maureen; Willett, Brian J.; Scott, A.; Grant, Helen

doi:10.1177/096032719401300613

Cited by 26 publications

(10 citation statements)

References 27 publications

(4 reference statements)

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“…Investigation of testosterone hydroxylations, EROD activity, and GST expression are accepted measures of hepatocyte differentiation 17, 20–22, 31, 32. Testosterone metabolism was measured in cells cultured on the membranes for 48 h, and again these were compared with the profile of metabolism generated by cells cultured on collagen‐coated tissue culture dishes (Table II).…”

Section: Resultsmentioning

confidence: 99%

“…A second aspect of cell function used for measuring cell differentiation is the expression of glutathione S ‐transferases (GSTs), a major de‐toxification pathway in liver tissue. Expression of GST isoenzymes changes as liver cells de‐differentiate and age in culture 20–22. With time in culture, expression of alpha‐GST declines, and that of pi‐GST increases.…”

Section: Introductionmentioning

confidence: 99%

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The viability and function of primary rat hepatocytes cultured on polymeric membranes developed for hybrid artificial liver devices

Grant

Morgan

Henderson

et al. 2005

J Biomedical Materials Res

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Bioartificial liver devices require membranes to support the function and viability of hepatocytes because they are anchorage-dependent cells. This study investigated the ability of several polymeric membranes to support the functions of primary hepatocyte cultures. Tailor-made membranes were sought by synthesizing acrylonitrile copolymers with different comonomers resulting in ionic, hydrophilic, or reactive functional groups on the polymer surface. Hepatocyte morphology and viability were assessed by confocal microscopy, and function by the content and activities of cytochrome P450, and the expression of glutathione S-transferases. Hydrophilic membranes (polyacrylonitrile and acrylonitrile copolymerized with 2-acrylamino-2-methyl-propane sulfonic acid) were more biocompatible than hydrophobic membranes such as polysulfone. The chemistry of the hydrophilic group was important; amine groups had a deleterious effect on maintenance of the primary hepatocytes. The biocompatibility of hydrophobic membranes was improved by collagen coating. Improving the chemistry of membranes for artificial liver devices will enhance the phenotypic stability of the cells, enabling us to prolong treatment times for patients.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The viability and function of primary rat hepatocytes cultured on polymeric membranes developed for hybrid artificial liver devices

Grant

Morgan

Henderson

et al. 2005

J Biomedical Materials Res

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“…[94] -necrotic cells/ scar tissue at edges of the slice -bile cannot be collected and analyzed -presence of necrotic cells might affect active transport of drug through the outer cells [204][205][206][207] Freshly isolated hepatocytes in suspension -reasonably high throughput -most drug metabolizing enzymes well-preserved at in vivo levels -easy to use -zone specific metabolism and toxicity may be studied depending upon the method of isolation -lack of cell polarity limits use for drug transporter studies -lacks functional bile canaliculi -limited survival (2-4 hrs.) [94] -lack of cell-cell and cell-marix contacts -viability of isolated human hepatocytes may be variable [208][209][210][211] Primary Hepatocyte Cultures -throughput is a function of technology used to preserve the tissue function -relatively easy to use -differentiated function maintained in many short term and some long term cultures -potential for use in chronic toxicity studies and drug-drug interaction studies -loss in drug metabolizing enzyme activities in long term culture -may or may not have functional bile canaliculi -no single system has yet been able to preserve all the different liver specific functions in vitro -culture may need special supplements in media [4,11,15,16,23,24,34,35,91,93,144,212,213] Cell Lines -unlimited availability -some liver specific functions have been shown to be maintained -lacks in vivo phenotype -only a small set of hepatic functions expressed at levels different from in vivo liver [214][215][216][217][218] Intracellular fraction: microsomes -high throughput system -maintain expression of Phase I enzymes -can be used in evaluating intrinsic clearance, covalent binding and drug inhibition studies -can be recovered from frozen tissues -lacks Phase II and other cytosolic enzymes -can be used only for limited studies -inadequate representation of the diversity of hepatic functions [32,…”

Section: Introductionmentioning

confidence: 99%

A Microscale In Vitro Physiological Model of the Liver: Predictive Screens for Drug Metabolism and Enzyme Induction

Sivaraman

Leach

Townsend

et al. 2005

CDM

285

230

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In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

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“…In vitro techniques most commonly used to study the expression of hepatic metabolizing enzymes and cytotoxicity include precision-cut liver slices (7,15), primary cultures of hepatocytes (13,22,45,57), and immortalized cell lines (11,38). However, without exception, application of these in vitro systems is limited.…”

Section: Introductionmentioning

confidence: 99%

Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes

Dávila

Morris

1999

In Vitro Cell.Dev.Biol.-Animal

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The induction of cytochrome P450 (CYP450) and Phase II conjugating enzymes by prototypical hepatic enzyme inducers was studied in adult male rat hepatocytes. Hepatocytes were suspended and cultured in diluted Matrigel in a basal serum-free Dulbecco's modified Eagle medium and exposed to the prototypical liver enzyme inducers, 3-methylcholanthrene, phenobarbital, hydrocortisone, and clofibrate for 48 h. Total RNA and microsomes were isolated and prepared, respectively, at 72 h. The expression of CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A1, CYP3A2, CYP4A1, fatty acyl-CoA oxidase, uridine diphosphate-glucuronosyltransferase, glutathione-S-transferase, and sulfotransferase was determined at the mRNA level with reverse transcriptase polymerase chain reaction (RT-PCR). The expression of CYP1A1, CYP2B1, CYP2C11, CYP2E1, and CYP4A1 was also measured at the apoprotein level by Western immunoblotting. Using these culture and expression analysis techniques, we have found that the expression of these metabolic enzymes can be maintained in culture for up to 7 d at the mRNA and apoprotein levels. In addition, hepatocytes were found to respond to chemical enzyme inducers with marked increases in enzyme expression at either the mRNA or protein level and in a concentration-related fashion. Cells were responsive to enzyme induction as early as 24 h after initial plating. The results obtained from this investigation indicate that the presence of diluted Matrigel (at a concentration of 0.35 mg/ml), the use of low concentrations of insulin (1 microM), hydrocortisone (0.1 microM), and serum-free culture medium can maintain the differentiated phenotype and responsiveness of cultured hepatocytes to chemical-induced metabolic enzyme expression. Under the conditions used in this study, enzyme induction in adult male rat hepatocytes shows close agreement with enzyme induction observed in the livers of rats exposed to these or similar prototypical enzyme inducers. Rat hepatocytes cultured in the presence of diluted Matrigel coupled with enzyme mRNA expression analysis with RT-PCR are proven to be a valuable and important in vitro toxicological approach to assess the chemical-induced changes in expression of liver CYP450 and Phase II conjugating enzymes.

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Expression of Liver Functions in Imrnortalised Rot Hepotocyte Cell Lines

Cited by 26 publications

References 27 publications

The viability and function of primary rat hepatocytes cultured on polymeric membranes developed for hybrid artificial liver devices

The viability and function of primary rat hepatocytes cultured on polymeric membranes developed for hybrid artificial liver devices

A Microscale In Vitro Physiological Model of the Liver: Predictive Screens for Drug Metabolism and Enzyme Induction

Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes

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