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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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Self-assembly of sinusoid-like structures in vitro by co-culturing hepatocytes and endothelial cells. Endothelial cells were seeded on Matrigel. The endothelial cells rapidly formed tubes. Subsequently, freshly isolated hepatocytes were randomly seeded in the culture. The hepatocytes directionally migrated toward the artificial vasculature and adhered to it. The image shows hepatocytes decorating an endothelial cell tube at day four in co-culture. Double-immunofluorescence staining for endothelial cells (CD31, red) and hepatocytes (CK-18, green). Scale bar 200 µm (from Nahmias et al. 2006b)
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Fig26: Self-assembly of sinusoid-like structures in vitro by co-culturing hepatocytes and endothelial cells. Endothelial cells were seeded on Matrigel. The endothelial cells rapidly formed tubes. Subsequently, freshly isolated hepatocytes were randomly seeded in the culture. The hepatocytes directionally migrated toward the artificial vasculature and adhered to it. The image shows hepatocytes decorating an endothelial cell tube at day four in co-culture. Double-immunofluorescence staining for endothelial cells (CD31, red) and hepatocytes (CK-18, green). Scale bar 200 µm (from Nahmias et al. 2006b)

Mentions: Perhaps the most intriguing approach to liver bioengineering exploits the propensity of cells to histotypic self-organization. It is well known that a mixture of different types of isolated cells has an intrinsic ability to self-organize. Cells of the same type tend to coalesce in a mixture following a typical length and time-scale. This ability relies mainly on a differential cell–cell adhesion. This principle can be exploited in liver bioengineering. Nahmoas et al. (2006b) have generated vascular structures formed by seeding endothelial cells on basement membrane ECM. Hepatocytes seeded in contact with the bioengineered vasculature migrate and adhere to the vessel. Structures resembling the liver sinusoids, with hepatocyte plates, bile canaliculi and a vascular lumen are formed by the self-organization of the hepatocytes and the endothelial cells (Fig. 26). These bioengineered sinusoids maintain CYP expression and activity, as well as an expression and secretion rate of albumin stable for over 2 months. A further approach to the controlled self-assembly of functional liver constructs is the formation of spheroids, described in detail in section “Hepatospheres.” The maximum diameter that can be reached by liver cell spheroids is limited by the diffusion of oxygen and nutrients. A necrotic core develops in a spheroid as its diameter exceeds 200–300 μm. Several approaches have been tested in order to overcome the size limitation of the spheroids and obtain larger bioengineered constructs. Inamori et al. (2009) have obtained a large vascularized liver construct by self-assembly of hepatocyte spheroids coated with endothelial cells. Spheroids with a diameter of 100–150 μm were obtained from rat hepatocytes. The spheroids were first coated with collagen I by immersion in a diluted solution. The collagen-coated hepatocyte spheroids were then co-cultured with endothelial cells (HUVEC). Due to the collagen barrier, the HUVEC covered the spheroid surface without invading it. Subsequently, the HUVEC-coated spheroids were packed in a plastic hollow fiber and cultured further. After nine days of culture, the spheroids were fused with each other and the endothelial cells have formed a dense vascular network (Fig. S3; see 10.1007/s00204-013-1078-5). This is an interesting approach to bioengineer the liver parenchyma by employing a scaffold-free self-assembly strategy. A similar approach was pursued (Kelm et al. 2006) to engineer vascularized cartilage by culturing spheroids in large custom-shaped agarose molds.Fig. 26


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)

Self-assembly of sinusoid-like structures in vitro by co-culturing hepatocytes and endothelial cells. Endothelial cells were seeded on Matrigel. The endothelial cells rapidly formed tubes. Subsequently, freshly isolated hepatocytes were randomly seeded in the culture. The hepatocytes directionally migrated toward the artificial vasculature and adhered to it. The image shows hepatocytes decorating an endothelial cell tube at day four in co-culture. Double-immunofluorescence staining for endothelial cells (CD31, red) and hepatocytes (CK-18, green). Scale bar 200 µm (from Nahmias et al. 2006b)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig26: Self-assembly of sinusoid-like structures in vitro by co-culturing hepatocytes and endothelial cells. Endothelial cells were seeded on Matrigel. The endothelial cells rapidly formed tubes. Subsequently, freshly isolated hepatocytes were randomly seeded in the culture. The hepatocytes directionally migrated toward the artificial vasculature and adhered to it. The image shows hepatocytes decorating an endothelial cell tube at day four in co-culture. Double-immunofluorescence staining for endothelial cells (CD31, red) and hepatocytes (CK-18, green). Scale bar 200 µm (from Nahmias et al. 2006b)
Mentions: Perhaps the most intriguing approach to liver bioengineering exploits the propensity of cells to histotypic self-organization. It is well known that a mixture of different types of isolated cells has an intrinsic ability to self-organize. Cells of the same type tend to coalesce in a mixture following a typical length and time-scale. This ability relies mainly on a differential cell–cell adhesion. This principle can be exploited in liver bioengineering. Nahmoas et al. (2006b) have generated vascular structures formed by seeding endothelial cells on basement membrane ECM. Hepatocytes seeded in contact with the bioengineered vasculature migrate and adhere to the vessel. Structures resembling the liver sinusoids, with hepatocyte plates, bile canaliculi and a vascular lumen are formed by the self-organization of the hepatocytes and the endothelial cells (Fig. 26). These bioengineered sinusoids maintain CYP expression and activity, as well as an expression and secretion rate of albumin stable for over 2 months. A further approach to the controlled self-assembly of functional liver constructs is the formation of spheroids, described in detail in section “Hepatospheres.” The maximum diameter that can be reached by liver cell spheroids is limited by the diffusion of oxygen and nutrients. A necrotic core develops in a spheroid as its diameter exceeds 200–300 μm. Several approaches have been tested in order to overcome the size limitation of the spheroids and obtain larger bioengineered constructs. Inamori et al. (2009) have obtained a large vascularized liver construct by self-assembly of hepatocyte spheroids coated with endothelial cells. Spheroids with a diameter of 100–150 μm were obtained from rat hepatocytes. The spheroids were first coated with collagen I by immersion in a diluted solution. The collagen-coated hepatocyte spheroids were then co-cultured with endothelial cells (HUVEC). Due to the collagen barrier, the HUVEC covered the spheroid surface without invading it. Subsequently, the HUVEC-coated spheroids were packed in a plastic hollow fiber and cultured further. After nine days of culture, the spheroids were fused with each other and the endothelial cells have formed a dense vascular network (Fig. S3; see 10.1007/s00204-013-1078-5). This is an interesting approach to bioengineer the liver parenchyma by employing a scaffold-free self-assembly strategy. A similar approach was pursued (Kelm et al. 2006) to engineer vascularized cartilage by culturing spheroids in large custom-shaped agarose molds.Fig. 26

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