3-D Coculture of Hepatic Sinusoidal Cells with Primary Hepatocytes—Design of an Organotypical Model

Bader, Augustinus; Knop, Erich; Kern, Armin; Böker, K. H. W.; Frühauf, Nils R.; Crome, Olaf; Esselmann, Hermann; Pape, C.; Kempka, G.; Sewing, Karl–Friedrich

doi:10.1006/excr.1996.0222

Cited by 111 publications

(63 citation statements)

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“…Other models have been developed, such as the spheroid or sandwich methods, but they have been poorly described with regard to their potential to maintain drug metabolism capacities (Bader et al, 1994(Bader et al, , 1996Koebe et al, 1994;Niwa et al, 1996). In addition, no regulation studies have been reported as yet using these model systems.…”

Section: Discussionmentioning

confidence: 99%

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Lerche

Fautrel

Shaw³

et al. 1997

European Journal of Biochemistry

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In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CUP), including CYPlAU2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase a (GSTa), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin 0-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities.Two general patterns of accumulation of liver-specific mRNAs were observed. CYPl A1 /2, CYP2B1/2, CYP3All2, GSTa, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYPl A1/2 (13.6-fold) and GSTu (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (1 1 .Zfold), GSTu (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells.Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B 1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments.Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.

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Section: Discussionmentioning

confidence: 99%

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Lerche

Fautrel

Shaw³

et al. 1997

European Journal of Biochemistry

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“…fibroblasts, endothelial cells) (Langenbach et al 1979;Talamini et al 1998). However, the co-culture of mature hepatocytes with non-parenchymal (stromal) cells from the liver itself has been shown to be more effective in modulating cell differentiation (Bader et al 1996;Bhatia et al 1999;Ries et al 2000). Various two and three dimensional co-culture models have demonstrated their ability to mimic the liver microenvironment, suggesting that each hepatic non-parenchymal cell population (sinusoidal endothelium, Kupffer cells and hepatic stellate cells (HSCs)) may contribute to the overall beneficial effect on hepatocytes (Morin and Normand 1986;Okamoto et al 1998;Riccalton-Banks et al 2003; Thomas et al 2005;Zinchenko et al 2006a).…”

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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“…[29][30][31][32][33][34][35][36][37][38] Several strategies have previously been proposed to culture LSECs while retaining their phenotype. [37][38][39][40][41][42][43] LSECs, cultured in media supplemented with lipids and oleic acid, in particular, was shown to maintain their metabolic and endocytotic activity for up to 5 days by influencing Akt/PKB and ERK signaling pathways. 39 Another study with culturing LSECs and hepatocytes in a three-dimensional (3D) microreactor shows retention of LSEC phenotype expression for 13 days, primarily through modulation of vascular endothelial growth factor in the culture media.…”

mentioning

confidence: 99%

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

Bale

Golberg

Jindal

et al. 2015

Tissue Engineering Part C: Methods

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Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of nonparenchymal cells (NPCs), which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell interactions and cumulative drug response. Hepatocytes and liver sinusoidal endothelial cells (LSECs) account for *80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multicellular physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel (1) in a sandwich and (2) as an intervening extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 (SE-1) antibody demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin production of the cocultures was 10-15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel-based hepatocyte-LSEC cocultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel-based hepatocyte-LSEC coculture models are promising for in vitro toxicity testing, and liver model development studies.

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3-D Coculture of Hepatic Sinusoidal Cells with Primary Hepatocytes—Design of an Organotypical Model

Cited by 111 publications

References 0 publications

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

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

Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells

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