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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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Related in: MedlinePlus

Setup for isolated rat liver perfusion in the open, non-recirculating, constant flow system. a The liver is posted on a balance pan, which allows for continuous monitoring of liver mass. Perivascular nerve stimulation is achieved by means of a platinum electrode placed around the portal vein. b Organ photometry/fluorimetry allows to monitor redox transitions in the NAD(P)H/NAD(P)+ system using nicotinamide nucleotide-specific wavelength pair 350–377 nm
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Fig7: Setup for isolated rat liver perfusion in the open, non-recirculating, constant flow system. a The liver is posted on a balance pan, which allows for continuous monitoring of liver mass. Perivascular nerve stimulation is achieved by means of a platinum electrode placed around the portal vein. b Organ photometry/fluorimetry allows to monitor redox transitions in the NAD(P)H/NAD(P)+ system using nicotinamide nucleotide-specific wavelength pair 350–377 nm

Mentions: An example of a perfusion setup is schematically depicted in Fig. 7. The perfusion fluid is delivered from a reservoir by a roller pump at a constant speed (“constant flow system”) to a silicon tube oxygenator, where the perfusion fluid is warmed up to 37 °C and equilibrated with an appropriate gas mixture, for example O2/CO2 (95/5; v/v), when the perfusion fluid is Krebs–Henseleit buffer. Alternatively, a “constant pressure system” can be used, which keeps the perfusion pressure constant, whereas the perfusion flow varies with alterations of perfusion resistance. Appropriate additions of metabolites, hormones or drugs to the influent perfusate can be performed by precise micropumps for defined time periods. The perfusate enters the liver via the cannulated portal vein and is drained into effluent by a cannula fixed in the superior caval vein. The hepatic artery usually is tied off; however, perfusion can also be performed bivascularly, i.e. via the portal vein and the hepatic artery (Sahin and Rowland 1998; Gardemann et al. 1991). Under these conditions, the portal versus hepatic arterial flow ratio should be about 4:1 (Gardemann et al. 1991). When required, the bile duct is cannulated and bile samples can be taken for analysis, as is the case with the effluent perfusate samples. Besides the portal pressure, the oxygen concentration and pH in effluent perfusate are monitored continuously, as well as the effluent Ca2+ and K+ concentrations using ion-sensitive electrodes, which are placed in the effluent perfusate. One lobe of the liver may be placed in the light beam of a dual wavelength organ photometer (Sies et al. 1974). A platinum electrode may be placed around the portal vein and hepatic artery, and perivascular hepatic nerves can be stimulated electrically with 20 V, a frequency of 20 Hz and an impulse duration of 2 ms (Hartmann et al. 1982; Häussinger et al. 1987). Placing the liver on a balance pan not only allows for monitoring liver mass, whose baseline constancy is one surrogate marker for excellent perfusion quality, but also allows the measurement of substrate- and hormone-induced changes in the hepatocellular water content in the absence of alterations of perfusion pressure and flow (vom Dahl et al. 1991). Finally, a liver lobe can be placed into a light beam with specific wavelengths for organ photometry and fluorescence recordings (Sies 1978; Sies et al. 1974).Fig. 7


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)

Setup for isolated rat liver perfusion in the open, non-recirculating, constant flow system. a The liver is posted on a balance pan, which allows for continuous monitoring of liver mass. Perivascular nerve stimulation is achieved by means of a platinum electrode placed around the portal vein. b Organ photometry/fluorimetry allows to monitor redox transitions in the NAD(P)H/NAD(P)+ system using nicotinamide nucleotide-specific wavelength pair 350–377 nm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: Setup for isolated rat liver perfusion in the open, non-recirculating, constant flow system. a The liver is posted on a balance pan, which allows for continuous monitoring of liver mass. Perivascular nerve stimulation is achieved by means of a platinum electrode placed around the portal vein. b Organ photometry/fluorimetry allows to monitor redox transitions in the NAD(P)H/NAD(P)+ system using nicotinamide nucleotide-specific wavelength pair 350–377 nm
Mentions: An example of a perfusion setup is schematically depicted in Fig. 7. The perfusion fluid is delivered from a reservoir by a roller pump at a constant speed (“constant flow system”) to a silicon tube oxygenator, where the perfusion fluid is warmed up to 37 °C and equilibrated with an appropriate gas mixture, for example O2/CO2 (95/5; v/v), when the perfusion fluid is Krebs–Henseleit buffer. Alternatively, a “constant pressure system” can be used, which keeps the perfusion pressure constant, whereas the perfusion flow varies with alterations of perfusion resistance. Appropriate additions of metabolites, hormones or drugs to the influent perfusate can be performed by precise micropumps for defined time periods. The perfusate enters the liver via the cannulated portal vein and is drained into effluent by a cannula fixed in the superior caval vein. The hepatic artery usually is tied off; however, perfusion can also be performed bivascularly, i.e. via the portal vein and the hepatic artery (Sahin and Rowland 1998; Gardemann et al. 1991). Under these conditions, the portal versus hepatic arterial flow ratio should be about 4:1 (Gardemann et al. 1991). When required, the bile duct is cannulated and bile samples can be taken for analysis, as is the case with the effluent perfusate samples. Besides the portal pressure, the oxygen concentration and pH in effluent perfusate are monitored continuously, as well as the effluent Ca2+ and K+ concentrations using ion-sensitive electrodes, which are placed in the effluent perfusate. One lobe of the liver may be placed in the light beam of a dual wavelength organ photometer (Sies et al. 1974). A platinum electrode may be placed around the portal vein and hepatic artery, and perivascular hepatic nerves can be stimulated electrically with 20 V, a frequency of 20 Hz and an impulse duration of 2 ms (Hartmann et al. 1982; Häussinger et al. 1987). Placing the liver on a balance pan not only allows for monitoring liver mass, whose baseline constancy is one surrogate marker for excellent perfusion quality, but also allows the measurement of substrate- and hormone-induced changes in the hepatocellular water content in the absence of alterations of perfusion pressure and flow (vom Dahl et al. 1991). Finally, a liver lobe can be placed into a light beam with specific wavelengths for organ photometry and fluorescence recordings (Sies 1978; Sies et al. 1974).Fig. 7

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