Limits...
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.

Show MeSH

Related in: MedlinePlus

Primary mouse hepatocytes and liver endothelial cells: Phase II biotransformation in HepaChip® and standard 2D cell cultures. Sulfotransferase (a) and UDP glucuronosyltransferase (b) activities were measured using 7-hydroxycoumarin as a substrate (b). Data represent mean values ± SD of four mice
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3753504&req=5

Fig22: Primary mouse hepatocytes and liver endothelial cells: Phase II biotransformation in HepaChip® and standard 2D cell cultures. Sulfotransferase (a) and UDP glucuronosyltransferase (b) activities were measured using 7-hydroxycoumarin as a substrate (b). Data represent mean values ± SD of four mice

Mentions: The HepaChip® in vitro microfluidic system has been tested for activities of several phase I and phase II metabolizing enzymes (Figs. 21, 22) and compared to standard 96-well plate co-cultures of hepatocytes and endothelial cells. Freshly isolated mouse hepatocytes and cryopreserved liver endothelial cells have been applied for short-term incubations up to 3 h each day with a substance cocktail containing 10 μM 7-hydroxycoumarin, 20 μM testosterone and 20 μM phenacetin in Williams E medium supplemented with 0.1 μM dexamethasone. Incubations of hepatocytes without test substances were also included. Data on testosterone hydroxylation and phenacetin deacetylation to acetaminophen by CYP3A4 and enzymes CYP1A2, respectively, are summarized in Fig. 21; data on 7-hydroxycoumarin-sulfate formation by sulfotransferase and 7-hydroxycoumarin-glucuronide formation by UGTs are summarized in Fig. 22. From these data, it is evident that activities in HepaChip® cultures are at each time point significantly higher than in standard cultures. These results fit well with the increased urea and albumin secretion rate (Fig. 23) which is obvious higher in HepaChip® compared to standard 96-well plate cultures.Fig. 21


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)

Primary mouse hepatocytes and liver endothelial cells: Phase II biotransformation in HepaChip® and standard 2D cell cultures. Sulfotransferase (a) and UDP glucuronosyltransferase (b) activities were measured using 7-hydroxycoumarin as a substrate (b). Data represent mean values ± SD of four mice
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig22: Primary mouse hepatocytes and liver endothelial cells: Phase II biotransformation in HepaChip® and standard 2D cell cultures. Sulfotransferase (a) and UDP glucuronosyltransferase (b) activities were measured using 7-hydroxycoumarin as a substrate (b). Data represent mean values ± SD of four mice
Mentions: The HepaChip® in vitro microfluidic system has been tested for activities of several phase I and phase II metabolizing enzymes (Figs. 21, 22) and compared to standard 96-well plate co-cultures of hepatocytes and endothelial cells. Freshly isolated mouse hepatocytes and cryopreserved liver endothelial cells have been applied for short-term incubations up to 3 h each day with a substance cocktail containing 10 μM 7-hydroxycoumarin, 20 μM testosterone and 20 μM phenacetin in Williams E medium supplemented with 0.1 μM dexamethasone. Incubations of hepatocytes without test substances were also included. Data on testosterone hydroxylation and phenacetin deacetylation to acetaminophen by CYP3A4 and enzymes CYP1A2, respectively, are summarized in Fig. 21; data on 7-hydroxycoumarin-sulfate formation by sulfotransferase and 7-hydroxycoumarin-glucuronide formation by UGTs are summarized in Fig. 22. From these data, it is evident that activities in HepaChip® cultures are at each time point significantly higher than in standard cultures. These results fit well with the increased urea and albumin secretion rate (Fig. 23) which is obvious higher in HepaChip® compared to standard 96-well plate cultures.Fig. 21

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