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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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a Confocal image of whole-mount staining of primary rat liver microtissue in co-culture with non-parenchymal cells. The endothelial marker ICAM-1 is visualized by red, bile canalicular marker DPPIV is shown by green, and nuclei are by blue fluorescence. The picture was kindly provided by Dr. Seddik Hammad, IfaDo, Dortmund. b Paraffin section and immunostaining of primary rat liver microtissue in co-culture with NPCs. Kupffer macrophage marker CD68 is visualized by green color, whereas nuclei appear in gray
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Fig18: a Confocal image of whole-mount staining of primary rat liver microtissue in co-culture with non-parenchymal cells. The endothelial marker ICAM-1 is visualized by red, bile canalicular marker DPPIV is shown by green, and nuclei are by blue fluorescence. The picture was kindly provided by Dr. Seddik Hammad, IfaDo, Dortmund. b Paraffin section and immunostaining of primary rat liver microtissue in co-culture with NPCs. Kupffer macrophage marker CD68 is visualized by green color, whereas nuclei appear in gray

Mentions: In order to enhance hepatocyte-specific functions of hepatospheres (see section “Hepatospheres”), co-culture approaches were performed with different cell types. It was shown that co-culture of rat hepatocytes with mouse NIH3T3 (Seo et al. 2006b; Chia et al. 2005) had beneficial effects on albumin secretion, ammonia elimination rate and CYP1A1 activity. Co-culture of rat hepatocytes with the rat hepatic stellate cell line HSC-T6 resulted in heterospheroids with elevated albumin and CYP2B1/2 expression. The HSC-T6 cells invaded into the hepatocyte spheroids before retracting to their original location at the periphery of the MT, leaving sinusoid-like spaces (Abu-Absi et al. 2002). Similarly, coating of rat hepatocyte spheroids with human umbilical vein endothelial cells (HUVECs) resulted in invasion of HUVECs into the hepatocyte spheroid and establishment of a dense vascular network (Inamori et al. 2009). The previously discussed approaches use cell lines mixed with primary cells but to fully exploit the potential of co-culture as a means of achieving a more organotypic model, primary hepatocytes would ideally to be co-cultured with primary cells from the same organ. This can be achieved by isolation of primary hepatocytes and enrichment of NPCs, such as endothelial cells, Kupffer and stellate cells. This cell mixture can be co-cultured with hepatocytes in hanging drops, which results in tissue re-formation within 2–3 days. Interestingly, the endothelial cells re-organize themselves to completely surround the hepatocyte core, as visualized by whole-mount confocal microscopy in Fig. 18a. The endothelial cells were stained with ICAM-1 marker, whereas hepatocyte polarity and bile canaliculi were visualized with DPPIV marker. The self-sorting of cells can be explained by the differential expression of cadherins on the two cell types. According to the differential cell adhesion hypothesis, the cell type with the higher expression of cadherins builds the core of a tissue, whereas the cell type with the lower expression of cadherins surrounds the core (Foty and Steinberg 2005). Moreover, the Kupffer cells, which are present in the NPC fraction, distribute themselves within the hepatosphere (Fig. 18b). Kupffer cells are very motile within the tissue in vivo as well as in the hepatosphere, since they do not form direct cell–cell contacts to hepatocytes. Importantly, Kupffer cells can be stimulated with inflammatory stimuli, such as lipopolysaccharide. In response to this stimulus, the macrophages secrete cytokines, which trigger local inflammation (unpublished results). Thus, it is possible to study inflammation-mediated toxicity.Fig. 18


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)

a Confocal image of whole-mount staining of primary rat liver microtissue in co-culture with non-parenchymal cells. The endothelial marker ICAM-1 is visualized by red, bile canalicular marker DPPIV is shown by green, and nuclei are by blue fluorescence. The picture was kindly provided by Dr. Seddik Hammad, IfaDo, Dortmund. b Paraffin section and immunostaining of primary rat liver microtissue in co-culture with NPCs. Kupffer macrophage marker CD68 is visualized by green color, whereas nuclei appear in gray
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Related In: Results  -  Collection

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Fig18: a Confocal image of whole-mount staining of primary rat liver microtissue in co-culture with non-parenchymal cells. The endothelial marker ICAM-1 is visualized by red, bile canalicular marker DPPIV is shown by green, and nuclei are by blue fluorescence. The picture was kindly provided by Dr. Seddik Hammad, IfaDo, Dortmund. b Paraffin section and immunostaining of primary rat liver microtissue in co-culture with NPCs. Kupffer macrophage marker CD68 is visualized by green color, whereas nuclei appear in gray
Mentions: In order to enhance hepatocyte-specific functions of hepatospheres (see section “Hepatospheres”), co-culture approaches were performed with different cell types. It was shown that co-culture of rat hepatocytes with mouse NIH3T3 (Seo et al. 2006b; Chia et al. 2005) had beneficial effects on albumin secretion, ammonia elimination rate and CYP1A1 activity. Co-culture of rat hepatocytes with the rat hepatic stellate cell line HSC-T6 resulted in heterospheroids with elevated albumin and CYP2B1/2 expression. The HSC-T6 cells invaded into the hepatocyte spheroids before retracting to their original location at the periphery of the MT, leaving sinusoid-like spaces (Abu-Absi et al. 2002). Similarly, coating of rat hepatocyte spheroids with human umbilical vein endothelial cells (HUVECs) resulted in invasion of HUVECs into the hepatocyte spheroid and establishment of a dense vascular network (Inamori et al. 2009). The previously discussed approaches use cell lines mixed with primary cells but to fully exploit the potential of co-culture as a means of achieving a more organotypic model, primary hepatocytes would ideally to be co-cultured with primary cells from the same organ. This can be achieved by isolation of primary hepatocytes and enrichment of NPCs, such as endothelial cells, Kupffer and stellate cells. This cell mixture can be co-cultured with hepatocytes in hanging drops, which results in tissue re-formation within 2–3 days. Interestingly, the endothelial cells re-organize themselves to completely surround the hepatocyte core, as visualized by whole-mount confocal microscopy in Fig. 18a. The endothelial cells were stained with ICAM-1 marker, whereas hepatocyte polarity and bile canaliculi were visualized with DPPIV marker. The self-sorting of cells can be explained by the differential expression of cadherins on the two cell types. According to the differential cell adhesion hypothesis, the cell type with the higher expression of cadherins builds the core of a tissue, whereas the cell type with the lower expression of cadherins surrounds the core (Foty and Steinberg 2005). Moreover, the Kupffer cells, which are present in the NPC fraction, distribute themselves within the hepatosphere (Fig. 18b). Kupffer cells are very motile within the tissue in vivo as well as in the hepatosphere, since they do not form direct cell–cell contacts to hepatocytes. Importantly, Kupffer cells can be stimulated with inflammatory stimuli, such as lipopolysaccharide. In response to this stimulus, the macrophages secrete cytokines, which trigger local inflammation (unpublished results). Thus, it is possible to study inflammation-mediated toxicity.Fig. 18

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