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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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Induction of lipid accumulation in hepatocytes in vitro. Oil red O staining of primary human hepatocytes (PHHs) incubated with b: palmitate (0.2 mM) or (a) FFA-free BSA, which served as a control
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Fig40: Induction of lipid accumulation in hepatocytes in vitro. Oil red O staining of primary human hepatocytes (PHHs) incubated with b: palmitate (0.2 mM) or (a) FFA-free BSA, which served as a control

Mentions: In an in vitro model, cellular lipid accumulation was induced in primary human hepatocytes by adding free fatty acids (FFAs) to the culture medium (Wobser et al. 2009). FFAs appear to be the major mediators of excessive hepatic lipid accumulation. The rate of hepatic FFA uptake is not regulated, and therefore, is proportional to plasma FFA concentrations (Teli et al. 1995). In humans with NAFLD, circulating FFAs are commonly elevated, and their plasma levels correlate with disease severity (Nehra et al. 2001). In their model, Wobser et al. (2009) used palmitate (C16:0), the most prevalent long-chain saturated fatty acid found in the circulation where it is bound to albumin in physiological ratio of 2:1. In states of insulin resistance and obesity, serum fatty acid levels are commonly elevated, yielding ratios as high as 7.5:1 (Kleinfeld et al. 1996). Thus, human hepatocytes were incubated with palmitate complexed to bovine serum albumin in a molar ratio of 6.7:1, thereby mimicking hyperlipidemic conditions. Exposure to palmitate induced an accumulation of cytosolic lipid droplets in human hepatocytes, as detected by confocal microscopy after staining with Oil Red O (Fig. 40). Colorimetric analysis revealed a dose-dependent increase in intracellular triglyceride concentration in hepatocytes exposed to 0.1–0.4 mM palmitate (Fig. 41). Measurement of the (residual) palmitate concentration in the supernatant revealed a complete uptake of palmitate by hepatocytes incubated for 24 h with palmitate at a concentration of 0.1 or 0.2 mM, and after 24-h incubation with 0.4 mM palmitate, approximately 85 % of the palmitate was taken up by the hepatocytes. Importantly, the viability of human hepatocytes was not affected by the stimulation with palmitate in a concentration of up to 0.4 mM. This in vitro model of hepatocellular lipid accumulation has been applied already in several subsequent studies to assess the effect of hepatic steatosis on the expression of defined genes and signal transduction pathways (Wanninger et al. 2011; Schnabl et al. 2011; Kirovski et al. 2010). Notably, changes observed in human hepatocytes in response to in vitro-induced steatosis were in line with elevated expression levels in human NAFLD tissue compared to normal hepatic tissue (Wanninger et al. 2011; Schnabl et al. 2011; Kirovski et al. 2010). Furthermore, palmitate dose dependently induced ROS formation in human hepatocytes in this model (Schnabl et al. 2011). Oxidative stress has been suggested to be major consequence of cellular lipid overload, and to contribute significantly to inflammatory liver damage and fibrogenesis in NASH, when the capacity of hepatocytes to safely store excess FFAs in form of triglyceride in lipid droplets is depleted (Rombouts and Marra 2010). Together, these findings indicate that palmitate-induced lipid accumulation in human hepatocytes in vitro is a suitable model to resemble the in vivo situation in human NAFLD.Fig. 41


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)

Induction of lipid accumulation in hepatocytes in vitro. Oil red O staining of primary human hepatocytes (PHHs) incubated with b: palmitate (0.2 mM) or (a) FFA-free BSA, which served as a control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig40: Induction of lipid accumulation in hepatocytes in vitro. Oil red O staining of primary human hepatocytes (PHHs) incubated with b: palmitate (0.2 mM) or (a) FFA-free BSA, which served as a control
Mentions: In an in vitro model, cellular lipid accumulation was induced in primary human hepatocytes by adding free fatty acids (FFAs) to the culture medium (Wobser et al. 2009). FFAs appear to be the major mediators of excessive hepatic lipid accumulation. The rate of hepatic FFA uptake is not regulated, and therefore, is proportional to plasma FFA concentrations (Teli et al. 1995). In humans with NAFLD, circulating FFAs are commonly elevated, and their plasma levels correlate with disease severity (Nehra et al. 2001). In their model, Wobser et al. (2009) used palmitate (C16:0), the most prevalent long-chain saturated fatty acid found in the circulation where it is bound to albumin in physiological ratio of 2:1. In states of insulin resistance and obesity, serum fatty acid levels are commonly elevated, yielding ratios as high as 7.5:1 (Kleinfeld et al. 1996). Thus, human hepatocytes were incubated with palmitate complexed to bovine serum albumin in a molar ratio of 6.7:1, thereby mimicking hyperlipidemic conditions. Exposure to palmitate induced an accumulation of cytosolic lipid droplets in human hepatocytes, as detected by confocal microscopy after staining with Oil Red O (Fig. 40). Colorimetric analysis revealed a dose-dependent increase in intracellular triglyceride concentration in hepatocytes exposed to 0.1–0.4 mM palmitate (Fig. 41). Measurement of the (residual) palmitate concentration in the supernatant revealed a complete uptake of palmitate by hepatocytes incubated for 24 h with palmitate at a concentration of 0.1 or 0.2 mM, and after 24-h incubation with 0.4 mM palmitate, approximately 85 % of the palmitate was taken up by the hepatocytes. Importantly, the viability of human hepatocytes was not affected by the stimulation with palmitate in a concentration of up to 0.4 mM. This in vitro model of hepatocellular lipid accumulation has been applied already in several subsequent studies to assess the effect of hepatic steatosis on the expression of defined genes and signal transduction pathways (Wanninger et al. 2011; Schnabl et al. 2011; Kirovski et al. 2010). Notably, changes observed in human hepatocytes in response to in vitro-induced steatosis were in line with elevated expression levels in human NAFLD tissue compared to normal hepatic tissue (Wanninger et al. 2011; Schnabl et al. 2011; Kirovski et al. 2010). Furthermore, palmitate dose dependently induced ROS formation in human hepatocytes in this model (Schnabl et al. 2011). Oxidative stress has been suggested to be major consequence of cellular lipid overload, and to contribute significantly to inflammatory liver damage and fibrogenesis in NASH, when the capacity of hepatocytes to safely store excess FFAs in form of triglyceride in lipid droplets is depleted (Rombouts and Marra 2010). Together, these findings indicate that palmitate-induced lipid accumulation in human hepatocytes in vitro is a suitable model to resemble the in vivo situation in human NAFLD.Fig. 41

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