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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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Relation of predicted CLint, in vitro and CLint, in vivo for hepatocytes (a) and microsomes (b). Dashed lines represent unity, fitted power functions and (a) upper and lower limits of bias correction for hepatocytes. From: Hallifax et al. 2010
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Fig35: Relation of predicted CLint, in vitro and CLint, in vivo for hepatocytes (a) and microsomes (b). Dashed lines represent unity, fitted power functions and (a) upper and lower limits of bias correction for hepatocytes. From: Hallifax et al. 2010

Mentions: Commercially available cryopreserved human hepatocytes, used in suspension, are the most established hepatic cell system for prediction of human clearance, are generally considered to be superior to alternative primary hepatocyte culture systems and have substantially replaced the previous microsome standard model (Blanchard et al. 2005; Griffin and Houston 2004; Soars et al. 2007b). At the same time, it has been recognized that predictions from primary hepatocytes are frequently inaccurate. A recent assessment of published predictions of CL found that human cryopreserved hepatocytes, on average, underpredicted in vivo CLint by about fourfold, whereas human liver microsomes underpredicted by about fivefold (Hallifax and Houston 2010), consolidating previous reports (Naritomi et al. 2003; Bachmann et al. 2003; Riley et al. 2005; Brown et al. 2007; Stringer et al. 2008). Some tendency toward underprediction using microsomes is anticipated given their incomplete set of CL pathways (including phase II metabolism and uptake transport), although this extent of average underprediction suggests additional methodological shortcomings (Lam and Benet 2004; Parker and Houston 2008). However, while microsomal CLint was approximately proportional to in vivo CLint (throughout a wide range), CLint in hepatocytes tended to diverge from in vivo with increasing CLint from relatively unbiased predictions for low CL drugs to poorer predictions (relative to microsomes) for high CL drugs (Hallifax et al. 2010) (Fig. 35). This inconsistency between the systems implies that the causes of underprediction have a substantial in vitro component as opposed to possible extrapolation methodological bias, as discussed above.Fig. 36


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)

Relation of predicted CLint, in vitro and CLint, in vivo for hepatocytes (a) and microsomes (b). Dashed lines represent unity, fitted power functions and (a) upper and lower limits of bias correction for hepatocytes. From: Hallifax et al. 2010
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig35: Relation of predicted CLint, in vitro and CLint, in vivo for hepatocytes (a) and microsomes (b). Dashed lines represent unity, fitted power functions and (a) upper and lower limits of bias correction for hepatocytes. From: Hallifax et al. 2010
Mentions: Commercially available cryopreserved human hepatocytes, used in suspension, are the most established hepatic cell system for prediction of human clearance, are generally considered to be superior to alternative primary hepatocyte culture systems and have substantially replaced the previous microsome standard model (Blanchard et al. 2005; Griffin and Houston 2004; Soars et al. 2007b). At the same time, it has been recognized that predictions from primary hepatocytes are frequently inaccurate. A recent assessment of published predictions of CL found that human cryopreserved hepatocytes, on average, underpredicted in vivo CLint by about fourfold, whereas human liver microsomes underpredicted by about fivefold (Hallifax and Houston 2010), consolidating previous reports (Naritomi et al. 2003; Bachmann et al. 2003; Riley et al. 2005; Brown et al. 2007; Stringer et al. 2008). Some tendency toward underprediction using microsomes is anticipated given their incomplete set of CL pathways (including phase II metabolism and uptake transport), although this extent of average underprediction suggests additional methodological shortcomings (Lam and Benet 2004; Parker and Houston 2008). However, while microsomal CLint was approximately proportional to in vivo CLint (throughout a wide range), CLint in hepatocytes tended to diverge from in vivo with increasing CLint from relatively unbiased predictions for low CL drugs to poorer predictions (relative to microsomes) for high CL drugs (Hallifax et al. 2010) (Fig. 35). This inconsistency between the systems implies that the causes of underprediction have a substantial in vitro component as opposed to possible extrapolation methodological bias, as discussed above.Fig. 36

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