Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor

Bartolo, Loredana De; Morelli, Sabrina; Giorno, Lidietta; Rende, Maria; Memoli, Bruno; Procino, Alfredo; Andreucci, Vittorio E.; Bader, Augustinus; Drioli, Enrico

doi:10.1016/j.biomaterials.2006.05.015

Cited by 69 publications

(55 citation statements)

References 29 publications

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“…Freshly isolated human hepatocytes represent a good in vitro liver model because they are able to perform the full range of in vivo hepatic drug biotransformation pathways and retain many of the uptake and efflux functions of liver cells (De Bartolo et al, 2006). However, the use of fresh human hepatocytes has several drawbacks, such as scarce and unpredictable availability, interdonor variability, and significant variation in cell functions, especially cytochrome P450 (P450) activities (Luo et al, 2002;Ohtsuki et al, 2012;Rogue et al, 2012;Schaefer et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

In Vitro Evaluation of Major In Vivo Drug Metabolic Pathways Using Primary Human Hepatocytes and HepaRG Cells in Suspension and a Dynamic Three-Dimensional Bioreactor System

Darnell

Ulvestad

Ellis

et al. 2012

J Pharmacol Exp Ther

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Major human specific metabolites, not detected during in vivo and in vitro preclinical studies, may cause unexpected drug interactions and toxicity in human and delays in clinical programs. Thus, reliable preclinical tools for the detection of major human metabolites are of high importance. The aim of this study was to compare major drug metabolic pathways in HepaRG cells, a human hepatoma cell line, to fresh human hepatocytes, cryopreserved human hepatocytes, and human in vivo data. Furthermore, the maintenance of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) activities in a dynamic three-dimensional (3D) bioreactor were evaluated over time by using HepaRG cells and human hepatocytes.14 Cdiclofenac and a candidate from AstraZeneca's drug development program, 14 C-AZD6610, which are metabolized by P450 and UGT in vivo, were used as model substrates. The proportion of relevant biotransformation pathways of the investigated drug was clearly different in the various cell systems. The hydroxylation route was favored in primary human hepatocytes, whereas the glucuronidation route was favored in HepaRG cells. The human in vivo metabolite profile of AZD6610 was best represented by human hepatocytes, whereas all major diclofenac metabolites were detected in HepaRG cells. Moreover, the metabolite profiles in cryopreserved and fresh human hepatocytes were essentially the same. The liver bioreactor using both fresh human hepatocytes and HepaRG cells retained biotransformation capacity over 1 week. Thus, the incubation time can be increased from a few hours in suspension to several days in 3D cultures, which opens up for detection of metabolites from slowly metabolized drugs.

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Section: Introductionmentioning

confidence: 99%

In Vitro Evaluation of Major In Vivo Drug Metabolic Pathways Using Primary Human Hepatocytes and HepaRG Cells in Suspension and a Dynamic Three-Dimensional Bioreactor System

Darnell

Ulvestad

Ellis

et al. 2012

J Pharmacol Exp Ther

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“…Freshly isolated human hepatocytes represent a good model of the liver because they are able to perform the full range of known in vivo drug biotransformation pathways and retain many of the uptake and efflux functions of the liver cells (De Bartolo et al, 2006). However, the use of primary human hepatocytes has several drawbacks such as scarce and unpredictable availability, huge variation in cell functions, especially cytochrome P450 (P450) activities, as well as a variable response to P450 inducers (Luo et al, 2002;Madan et al, 2003;Abadie-Viollon et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Cytochrome P450-Dependent Metabolism in HepaRG Cells Cultured in a Dynamic Three-Dimensional Bioreactor

Darnell¹,

Schreiter²,

Zeilinger³

et al. 2011

Drug Metab Dispos

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ABSTRACT:Reliable and stable in vitro cellular systems maintaining specific liver functions important for drug metabolism and disposition are urgently needed in preclinical drug discovery and development research. The cell line HepaRG exhibits promising properties such as expression and function of drug-metabolizing enzymes and transporter proteins, which resemble those found in freshly isolated human hepatocytes. In this study, HepaRG cells were cultured up to 68 days in a three-dimensional multicompartment capillary membrane bioreactor, which enables high-density cell culture under dynamic conditions. The activity of drug-metabolizing cytochrome P450 (P450) enzymes was investigated by a cocktail of substrates for CYP1A1/2 (phenacetin), CYP2C9 (diclofenac), CYP2B6 (bupropion), and CYP3A4 (midazolam). The model P450 substrates, which were introduced to the bioreactor system mimicking in vivo bolus doses, showed stable metabolism over the entire experimental period of several weeks with the exception of bupropion hydroxylase, which increased over time. Ketoconazole treatment decreased the CYP3A4 activity by 69%, and rifampicin induced the CYP3A4-and CYP2B6-dependent activity 6-fold, which predicts well the magnitude of changes observed in vivo. Moreover, polarity of transporter expression and formation of tissue-like structures including bile canaliculi were demonstrated by immune histochemistry. The long-lasting bioreactor system using HepaRG cells thus provides a promising and stable liver-like in vitro model for continuous investigations of the hepatic kinetics of drugs and of drug-drug interactions, which well predict the situation in vivo in humans.

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“…[11][12][13][14][15][16][17][18] For example, Suzuki et al used a polytetrafluorethylene (PTFE) membrane to support collagen sandwich-cultured hepatocytes. 19 Tilles et al 14 and de Bartolo et al [20][21][22] have also demonstrated the benefits of using oxygen (O 2 )-permeable membranes to improve O 2 delivery to the cellular spaces in flat-plate configurations. In general, tissue function is modulated by the communication of cells with extracellular matrices, soluble factors, and other cells.…”

mentioning

confidence: 99%

The Effectiveness of a Novel Cartridge-Based Bioreactor Design in Supporting Liver Cells

Niu

Hammond

Coger

2009

Tissue Engineering Part A

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There are a number of applications-ranging from temporary strategies for organ failure to pharmaceutical testing-that rely on effective bioreactor designs. The significance of these devices is that they provide an environment for maintaining cells in a way that allows them to perform key cellular and tissue functions. In the current study, a novel cartridge-based bioreactor was developed and evaluated. Its unique features include its capacity for cell support and the adaptable design of its cellular space. Specifically, it is able to accommodate functional and reasonably sized tissue (>2.0Â10 8 cells), and can be easily modified to support a range of anchorage-dependent cells. To evaluate its efficacy, it was applied to liver support in the current study. This involved evaluating the performance of rat primary hepatocytes within the unique cartridges in culture-sans bioreactor-and after being loaded within the novel bioreactor. Compared to collagen sandwich culture functional controls, hepatocytes within the unique cartridge design demonstrated significantly higher albumin production and urea secretion rates when cultured under dynamic flow conditions-reaching peak values of 170 AE 22 mg=10 6 cells=day and 195 AE 18 mg=10 6 cells=day, respectively. The bioreactor's effectiveness in supporting live and functioning primary hepatocytes is also presented. Cell viability at the end of 15 days of culture in the new bioreactor was 84 AE 18%, suggesting that the new design is effective in maintaining primary hepatocytes for at least 2 weeks in culture. Liver-specific functions of urea secretion, albumin synthesis, and cytochrome P450 activity were also assessed. The results indicate that hepatocytes are able to achieve good functional performance when cultured within the novel bioreactor. This is especially true in the case of cytochrome P450 activity, where by day 15 of culture, hepatocytes within the bioreactor reached values that were 56.6% higher than achieved by the collagen sandwich functional control cultures. The success of the novel cartridge-based bioreactor in supporting hepatocytes with good viability and functional performance suggests that it is an effective design for supporting anchorage-dependent cells.

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Long-term maintenance of human hepatocytes in oxygen-permeable membrane bioreactor

Cited by 69 publications

References 29 publications

In Vitro Evaluation of Major In Vivo Drug Metabolic Pathways Using Primary Human Hepatocytes and HepaRG Cells in Suspension and a Dynamic Three-Dimensional Bioreactor System

In Vitro Evaluation of Major In Vivo Drug Metabolic Pathways Using Primary Human Hepatocytes and HepaRG Cells in Suspension and a Dynamic Three-Dimensional Bioreactor System

Cytochrome P450-Dependent Metabolism in HepaRG Cells Cultured in a Dynamic Three-Dimensional Bioreactor

The Effectiveness of a Novel Cartridge-Based Bioreactor Design in Supporting Liver Cells

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