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Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gómez-Lechón MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG - Arch. Toxicol. (2013)

Bottom Line: When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes.One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation.Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139, Dortmund, Germany.

ABSTRACT
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

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Retrograde/antegrade liver perfusion for studies on hepatocyte heterogeneity. The technique was first used in studies on hepatic ammonia metabolism (Häussinger 1983). Urea cycle enzymes are located in periportal hepatocytes, whereas glutamine synthetase is located in a small perivenous hepatocyte population surrounding the terminal hepatic venule. Under conditions of limited ammonia supply, the metabolic fate of ammonia is dependent on the direction of liver perfusion
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Fig10: Retrograde/antegrade liver perfusion for studies on hepatocyte heterogeneity. The technique was first used in studies on hepatic ammonia metabolism (Häussinger 1983). Urea cycle enzymes are located in periportal hepatocytes, whereas glutamine synthetase is located in a small perivenous hepatocyte population surrounding the terminal hepatic venule. Under conditions of limited ammonia supply, the metabolic fate of ammonia is dependent on the direction of liver perfusion

Mentions: Many metabolic steps are heterogeneously distributed along the liver acinus. Metabolic interactions between different subacinar hepatocyte populations have been established in ammonium, glutamine and eicosanoid metabolism (Häussinger 1983; Groothuis et al. 1982; Häussinger and Stehle 1988). This functional hepatocyte heterogeneity can be studied in isolated perfused rat liver, in which the structural organization is preserved, by the retrograde–antegrade perfusion technique (Häussinger 1983). When two metabolic pathways are differently localized in the acinus and share a common substrate, under conditions of a rate-limiting substrate supply, one of these pathways will be favored depending upon the direction of perfusion. As for controls, the metabolic flux differences between retro- and antegrade perfusion must be abolished either when excess substrate is added or when one of these competing pathways is inhibited. Such competing pathways are urea and glutamine synthesis from ammonium ions (Fig. 10), and by means of this technique, the periportal localization of urea synthesis and glutaminase and the perivenous localization of glutamine synthetase and the existence of the intercellular glutamine cycle in the structurally and metabolically intact liver were demonstrated (Häussinger 1983). The antegrade/retrograde perfusion technique was also successfully employed with regard to intra-acinar eicosanoid metabolism (Häussinger and Stehle 1988). Here it was shown that prostanoids can be formed inside the liver acinus, but do not leave the liver due to their removal by perivenous hepatocytes (so-called perivenous scavenger cells) (Häussinger and Stehle 1988; Morimoto et al. 1993). Oxygen microelectrodes and microlightguides (Ji et al. 1980) have been developed for the registration of oxygen tension and fluorescence at different sublobular regions; however, these techniques are restricted to signals obtained from the hepatocytes located at the surface of a lobe. Perfusion experiments after destruction of the perivenous compartment of the liver acinus without impairment of the periportal zone have also been used for studies on hepatocyte heterogeneity (Häussinger and Gerok 1984).Fig. 10


Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, Bode JG, Bolleyn J, Borner C, Böttger J, Braeuning A, Budinsky RA, Burkhardt B, Cameron NR, Camussi G, Cho CS, Choi YJ, Craig Rowlands J, Dahmen U, Damm G, Dirsch O, Donato MT, Dong J, Dooley S, Drasdo D, Eakins R, Ferreira KS, Fonsato V, Fraczek J, Gebhardt R, Gibson A, Glanemann M, Goldring CE, Gómez-Lechón MJ, Groothuis GM, Gustavsson L, Guyot C, Hallifax D, Hammad S, Hayward A, Häussinger D, Hellerbrand C, Hewitt P, Hoehme S, Holzhütter HG, Houston JB, Hrach J, Ito K, Jaeschke H, Keitel V, Kelm JM, Kevin Park B, Kordes C, Kullak-Ublick GA, LeCluyse EL, Lu P, Luebke-Wheeler J, Lutz A, Maltman DJ, Matz-Soja M, McMullen P, Merfort I, Messner S, Meyer C, Mwinyi J, Naisbitt DJ, Nussler AK, Olinga P, Pampaloni F, Pi J, Pluta L, Przyborski SA, Ramachandran A, Rogiers V, Rowe C, Schelcher C, Schmich K, Schwarz M, Singh B, Stelzer EH, Stieger B, Stöber R, Sugiyama Y, Tetta C, Thasler WE, Vanhaecke T, Vinken M, Weiss TS, Widera A, Woods CG, Xu JJ, Yarborough KM, Hengstler JG - Arch. Toxicol. (2013)

Retrograde/antegrade liver perfusion for studies on hepatocyte heterogeneity. The technique was first used in studies on hepatic ammonia metabolism (Häussinger 1983). Urea cycle enzymes are located in periportal hepatocytes, whereas glutamine synthetase is located in a small perivenous hepatocyte population surrounding the terminal hepatic venule. Under conditions of limited ammonia supply, the metabolic fate of ammonia is dependent on the direction of liver perfusion
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3753504&req=5

Fig10: Retrograde/antegrade liver perfusion for studies on hepatocyte heterogeneity. The technique was first used in studies on hepatic ammonia metabolism (Häussinger 1983). Urea cycle enzymes are located in periportal hepatocytes, whereas glutamine synthetase is located in a small perivenous hepatocyte population surrounding the terminal hepatic venule. Under conditions of limited ammonia supply, the metabolic fate of ammonia is dependent on the direction of liver perfusion
Mentions: Many metabolic steps are heterogeneously distributed along the liver acinus. Metabolic interactions between different subacinar hepatocyte populations have been established in ammonium, glutamine and eicosanoid metabolism (Häussinger 1983; Groothuis et al. 1982; Häussinger and Stehle 1988). This functional hepatocyte heterogeneity can be studied in isolated perfused rat liver, in which the structural organization is preserved, by the retrograde–antegrade perfusion technique (Häussinger 1983). When two metabolic pathways are differently localized in the acinus and share a common substrate, under conditions of a rate-limiting substrate supply, one of these pathways will be favored depending upon the direction of perfusion. As for controls, the metabolic flux differences between retro- and antegrade perfusion must be abolished either when excess substrate is added or when one of these competing pathways is inhibited. Such competing pathways are urea and glutamine synthesis from ammonium ions (Fig. 10), and by means of this technique, the periportal localization of urea synthesis and glutaminase and the perivenous localization of glutamine synthetase and the existence of the intercellular glutamine cycle in the structurally and metabolically intact liver were demonstrated (Häussinger 1983). The antegrade/retrograde perfusion technique was also successfully employed with regard to intra-acinar eicosanoid metabolism (Häussinger and Stehle 1988). Here it was shown that prostanoids can be formed inside the liver acinus, but do not leave the liver due to their removal by perivenous hepatocytes (so-called perivenous scavenger cells) (Häussinger and Stehle 1988; Morimoto et al. 1993). Oxygen microelectrodes and microlightguides (Ji et al. 1980) have been developed for the registration of oxygen tension and fluorescence at different sublobular regions; however, these techniques are restricted to signals obtained from the hepatocytes located at the surface of a lobe. Perfusion experiments after destruction of the perivenous compartment of the liver acinus without impairment of the periportal zone have also been used for studies on hepatocyte heterogeneity (Häussinger and Gerok 1984).Fig. 10

Bottom Line: When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes.One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation.Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Research Centre for Working Environment and Human Factors (IFADO), 44139, Dortmund, Germany.

ABSTRACT
This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Show MeSH
Related in: MedlinePlus