Serum-Free, Long-Term Cultures of Human Hepatocytes: Maintenance of Cell Morphology, Transcription Factors, and Liver-Specific Functions

Runge, Dieter; Jäger, Dana; Lubecki, Kimberly A.; Stolz, Donna B.; Karathanasis, Sotirios K.; Kietzmann, Thomas; Strom, Stephen C.; Jungermann, Kurt; Michalopoulos, George K.

doi:10.1006/bbrc.2000.2215

Cited by 94 publications

(69 citation statements)

References 40 publications

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“…Rat hepatocytes were isolated from male Fisher 344 rats (Harlan, Indianapolis, IN) using the modified two-step collagenase perfusion technique. 19,20 Freshly isolated hepatocytes of Ͼ90% viability, as assessed by trypan blue exclusion, were added to plating medium (minimal essential medium containing 50 g/ml bovine insulin and 0.1% gentamicin). Hepatocytes were plated on rat-tail collagen Icoated cultureware at a density of 3 to 4 ϫ 10 6 cells/ 100-mm plastic dish or 1 to 2 ϫ 10 5 cells/22-mm collagen-coated glass coverslip (BD Biocoat, Bedford, MA), incubated at 37°C (5% CO 2 ), and checked for adherence of monolayers after 2 to 4 hours.…”

Section: Hepatocyte Cell Culturementioning

confidence: 99%

Peroxisomal Localization of Hypoxia-Inducible Factors and Hypoxia-Inducible Factor Regulatory Hydroxylases in Primary Rat Hepatocytes Exposed to Hypoxia-Reoxygenation

Khan

Michalopoulos

Stolz

2006

The American Journal of Pathology

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Many signals involved in pathophysiology are controlled by hypoxia-inducible factors (HIFs), transcription factors that induce expression of hypoxiaresponsive genes. HIFs are post-translationally regulated by a family of O 2 -dependent HIF hydroxylases: four prolyl 4-hydroxylases and an asparaginyl hydroxylase. Most of these enzymes are abundant in resting liver, which is itself unique because of its physiological O 2 gradient, and they can exist in both nuclear and cytoplasmic pools. In this study, we analyzed the cellular localization of endogenous HIFs and their regulatory hydroxylases in primary rat hepatocytes cultured under hypoxia-reoxygenation conditions. In hepatocytes, hypoxia targeted HIF-1␣ to the peroxisome, rather than the nucleus, where it co-localized with von Hippel-Lindau tumor suppressor protein and the HIF hydroxylases. Confocal immunofluorescence microscopy demonstrated that the HIF hydroxylases translocated from the nucleus to the cytoplasm in response to hypoxia, with increased accumulation in peroxisomes on reoxygenation. These results were confirmed via immunotransmission electron microscopy and Western blotting. Surprisingly, in resting liver tissue, perivenous localization of the HIF hydroxylases was observed, consistent with areas of low pO 2 . In conclusion, these studies establish the peroxisome as a highly relevant site of subcellular localization and function for the endogenous HIF pathway in

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Section: Hepatocyte Cell Culturementioning

confidence: 99%

Peroxisomal Localization of Hypoxia-Inducible Factors and Hypoxia-Inducible Factor Regulatory Hydroxylases in Primary Rat Hepatocytes Exposed to Hypoxia-Reoxygenation

Khan

Michalopoulos

Stolz

2006

The American Journal of Pathology

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“…When in cell culture, primary hepatocytes are known to lose their differentiated phenotype and vitality rapidly on isolation from their native microenvironment (Arterburn et al 1995;Runge et al 2000). Therefore, many investigators have been developing sophisticated cell culture systems aiming to maintain the vitality and specific metabolic functions (Elaut et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

Krause

Saghatolislam

Koenig

et al. 2009

In Vitro Cell.Dev.Biol.-Animal

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Primary hepatocytes lose their differentiated functions rapidly when in culture. Our aim was to maintain the differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes were placed into collagen-coated culture dishes in the presence of HSCs at different ages of pre-culture, with or without direct cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place of vital cells. Changes in morphology and enhancement of phosphoenolpyruvate carboxykinase (PCK) activity by glucagon were used to determine the differentiated status of hepatocytes in 2d-short-term culture. HSCs proved able to maintain the differentiated function of hepatocytes in coculture either by direct cell contacts or via factors derived from HSC-conditioned medium. In comparison, however, without cellular contact to hepatocytes five to ten times as many HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent, resting HSCs (precultured for 1-2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of hepatocytes with a lipid extract or a particulate fraction from HSCs clearly displayed a very strong beneficial effect on enzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids.

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“…According to a theoretical calculation, an adequate mass of up to at least 1-2 · 10 10 viable hepatocytes is necessary to maintain normal human liver function and is needed in a liver support system to treat patients with liver failure (Morsiani et al 2002a;van de Kerkhove et al 2005b). The methods used for isolation of primary human hepatocytes are less than optimal because of the limited number of donor organs (Dou et al 1992;Runge et al 2000). Therefore, other cell sources, including animal (xenogeneic) hepatocytes, hepatoblastoma cell lines, and immortalized hepatocytes have been used for their application in bioartificial liver support systems (Tsiaoussis et al 2001).…”

Section: Cell Sourcementioning

confidence: 99%

Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems

Diekmann¹,

Bader

Schmitmeier

2006

Cytotechnology

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The liver is the most important organ for the biotransformation of xenobiotics, and the failure to treat acute or acute-on-chronic liver failure causes high mortality rates in affected patients. Due to the lack of donor livers and the limited possibility of the clinical management there has been growing interest in the development of extracorporeal liver support systems as a bridge to liver transplantation or to support recovery during hepatic failure. Earlier attempts to provide liver support comprised non-biological therapies based on the use of conventional detoxification procedures, such as filtration and dialysis. These techniques, however, failed to meet the expected efficacy in terms of the overall survival rate due to the inadequate support of several essential liver-specific functions. For this reason, several bioartificial liver support systems using isolated viable hepatocytes have been constructed to improve the outcome of treatment for patients with fulminant liver failure by delivering essential hepatic functions. However, controlled trials (phase I/II) with these systems have shown no significant survival benefits despite the systems' contribution to improvements in clinical and biochemical parameters. For the development of improved liver support systems, critical issues, such as the cell source and culture conditions for the longterm maintenance of liver-specific functions in vitro, are reviewed in this article. We also discuss aspects concerning the performance, biotolerance and logistics of the selected bioartificial liver support systems that have been or are currently being preclinically and clinically evaluated.

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Serum-Free, Long-Term Cultures of Human Hepatocytes: Maintenance of Cell Morphology, Transcription Factors, and Liver-Specific Functions

Cited by 94 publications

References 40 publications

Peroxisomal Localization of Hypoxia-Inducible Factors and Hypoxia-Inducible Factor Regulatory Hydroxylases in Primary Rat Hepatocytes Exposed to Hypoxia-Reoxygenation

Peroxisomal Localization of Hypoxia-Inducible Factors and Hypoxia-Inducible Factor Regulatory Hydroxylases in Primary Rat Hepatocytes Exposed to Hypoxia-Reoxygenation

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems

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