Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies

Coundouris, John; Grant, M.H.; Engeset, J; Petrie, J C; Hawksworth, Gabrielle

doi:10.3109/00498259309059449

Cited by 57 publications

(25 citation statements)

References 22 publications

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“…In this respect, cryopreservation is especially important for hepatocytes from species such as humans, where tissue supply is limited. There are several reports of the cryopreservation of human hepatocytes (Chesne et al, 1993;Coundouris et al, 1993). Furthermore, a few groups have reported the evaluation of metabolic pathways, enzyme induction, and inhibition using cryopreserved human hepatocytes applied to drug metabolism research (Ruegg et al, 1997;Li et al, 1999;Hewitt et al, 2001).…”

Section: Fig 3 Correlation Of CL Intin Vitro Between Freshly Isolamentioning

confidence: 99%

“…Cryopreservation would greatly enhance the utility of human hepatocytes because cryopreserved hepatocytes could be used at any time for experiments. Successful cryopreservation of human hepatocytes has been reported by several researchers (Chesne et al, 1993;Coundouris et al, 1993). However, there have been few reports on the prediction of hepatic clearance using human cryopreserved hepatocytes.…”

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confidence: 99%

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Utility of Hepatocytes in Predicting Drug Metabolism: Comparison of Hepatic Intrinsic Clearance in Rats and Humans in Vivo and in Vitro

Naritomi¹,

Terashita²,

Kagayama³

et al. 2003

Drug Metab Dispos

185

146

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:We investigated hepatic in vitro intrinsic clearance (CL int,in vitro ) in freshly isolated or cryopreserved hepatocytes and compared with CL int,in vivo by using nine model compounds, FK1052, FK480, diazepam, diltiazem, troglitazone, quinotolast, FK079, zidovudine, and acetaminophen, in rats and humans. The compounds showed a broad range of in vivo hepatic extraction ratios (rat, 0.05-0.93; humans, 0.03-0.76) and were metabolized by hepatic P450, UDPglucuronosyltransferase, sulfotransferase, and/or esterase. CL int,in vitro was determined from substrate disappearance rate at 1 M in hepatocytes. CL int,in vivo was calculated from in vivo pharmacokinetic data using two frequently used mathematical models (the well stirred and dispersion models). When estimating rat CL int,in vitro in freshly isolated hepatocytes, the rat scaling factor values (CL int,in vivo /CL int,in vitro ) showed marked difference among the model compounds (0.2-73.1-fold). The rat CL int,in vitro values in freshly isolated hepatocytes were in good agreement with these in cryopreserved hepatocytes. Human CL int,in vitro were determined by use of cryopreserved hepatocytes. When human CL int,in vitro was regarded as the predicted CL int,in vivo , the observed and predicted CL int,in vivo for FK1052, FK480, troglitazone, and FK079 differed markedly (12.4-199.0-fold). In contrast, using human CL int,in vitro corrected with the rat scaling factors yielded better predictions of CL int,in vivo that were mostly within 5-fold of the actual values. These results make the evaluation using hepatocytes more useful and provide a basis for predicting hepatic clearance using hepatocytes.Recently, pharmacokinetic investigation has played an increasingly important role in drug discovery. In particular, it is very important to predict human hepatic metabolic clearance because most drugs are eliminated from the body predominantly by hepatic metabolism. For predicting hepatic clearance, theoretical aspects of in vitro/in vivo scaling based on a physiological model and clearance concepts have been developed (Roberts and Rowland, 1986). Application of this method has been successful in predicting in vivo hepatic clearance in rats for many drugs metabolized by P450 1 from in vitro metabolism data using rat liver microsomes and isolated hepatocytes (Sugiyama et al., 1988;Houston and Carlile, 1997). Because human liver samples have become more readily available, it would also be very useful to predict in vivo outcomes from in vitro data in humans. However, there has been relatively limited application of this approach (Hoener, 1994), and there have been some failed attempts at the prediction of human hepatic clearance. For example, Iwatsubo et al. (1997) and Houston and Carlile (1997) reported that CL int,in vitro generally exhibited a positive correlation with CL int,in vivo , but in some cases animal or human clearance values were not well predicted from in vitro studies. To improve the predict...

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Section: Fig 3 Correlation Of CL Intin Vitro Between Freshly Isolamentioning

confidence: 99%

mentioning

confidence: 99%

Utility of Hepatocytes in Predicting Drug Metabolism: Comparison of Hepatic Intrinsic Clearance in Rats and Humans in Vivo and in Vitro

Naritomi¹,

Terashita²,

Kagayama³

et al. 2003

Drug Metab Dispos

185

146

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show abstract

“…3 Furthermore, because of interracial or sex-related differences and differences in storage periods (cell viability), the drug-metabolizing activity varies greatly among cells, causing problems in reproducibility. 4,5 For these reasons, much has been expected of liver cell lines that have uniform and stable properties, can be used for long-term experiments, are highly differentiated, and retain human liver functions. …”

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confidence: 99%

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver

Iwahori

2003

Hepatology

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CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver. The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) line FLC-5. A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells. The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture. Western blot analysis also demonstrated an increase in expression of the CYP3A protein. When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6␤-hydroxy testosterone as a metabolite was formed. D rug-drug interactions can be categorized as mediated by metabolic inhibition and enzyme induction. Numerous studies have been conducted on metabolic inhibition. It is now possible, to some extent, to predict drug-drug interactions in vivo based on the results in vitro. Enzyme induction has been studied using experimental animals, but, because of the existence of interspecies differences, it is often difficult to extrapolate the results of animal studies directly to humans. 1 To resolve these problems, enzyme-induction experiments have been performed using human primary cell culture systems, and experimental models established using these cell cultures have been recognized to be useful in the evaluation of enzyme induction. 2 However, longterm primary culture of human hepatocytes can reduce the functions of liver enzymes, as reflected, for example, by a decrease in the level of cytochrome P450 (CYP), an enzyme mainly catalyzing phase-I reactions of drug metabolism, 4 days after isolation, making it difficult to use these cells for prolonged periods of time. 3 Furthermore, because of interracial or sex-related differences and differences in storage periods (cell viability), the drug-metabolizing activity varies greatly among cells, causing problems in reproducibility. 4,5 For these reasons, much has been expected of liver cell lines that have uniform and stable properties, can be used for long-term experiments, are highly differentiated, and retain human liver functions.

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“…On the other hand, glutathione S-transferase and glutathione reductase activities of cryopreserved human hepatocytes were reduced to less than 40% of the enzyme activity of fresh hepatocytes (Coundouris et al, 1993). Drug transporter activities were also well preserved in cryopreserved rat hepatocytes (Houle et al, 2003).…”

Section: Cytochrome P450 In Cryopreserved Hepatocytesmentioning

confidence: 73%

Metabolic Activity of Cytochrome P450 Isoforms in Hepatocytes Cryopreserved with Wheat Protein Extract

Grondin

Hamel²,

Sarhan³

et al. 2008

Drug Metab Dispos

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ABSTRACT:The drug discovery and development process requires adequate safety testing for drug toxicity before new drugs can be administered to patients. Hepatocytes are used in vitro to screen compounds for hepatotoxicity, induction of drug-metabolizing enzymes such as cytochrome P450 (P450) isoforms, drug-drug interactions, and establish human relevance for metabolism. Cryopreservation makes it possible to preserve a large quantity of functional hepatocytes. Techniques for cryopreservation of hepatocytes are mainly based on dimethyl sulfoxide (DMSO). However, analyses of metabolic capacities of cryopreserved hepatocytes are often limited by loss of functional integrity of hepatocytes after thawing. Therefore, it is necessary to improve techniques of cryopreservation. We have developed a new cryopreservation technology for mammalian cells based on a wheat protein extract (WPE).We determined whether the WPE can better preserve activities of major P450 isoforms both in suspension and monolayer cultures of hepatocytes. This was achieved by comparing basal and inducible or metabolic activities of isoforms CYP1A1, CYP1A2, CYP2C6, CYP2D2, and CYP3A in rat hepatocytes that were cryopreserved with WPE, relative to fresh cells and those cryopreserved with DMSO. We conclusively show that rat hepatocytes cryopreserved with WPE retain their metabolic competency and their ability to respond to classical P450 inducers when compared with freshly isolated hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for rat hepatocytes. They are an efficient, nontoxic, economic natural product and universal cryoprotectant that is superior to DMSO, which has limitations because of cellular toxicity.

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Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies

Cited by 57 publications

References 22 publications

Utility of Hepatocytes in Predicting Drug Metabolism: Comparison of Hepatic Intrinsic Clearance in Rats and Humans in Vivo and in Vitro

Utility of Hepatocytes in Predicting Drug Metabolism: Comparison of Hepatic Intrinsic Clearance in Rats and Humans in Vivo and in Vitro

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver

Metabolic Activity of Cytochrome P450 Isoforms in Hepatocytes Cryopreserved with Wheat Protein Extract

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