Improved reproducibility in preparing precision-cut liver tissue slices

Zimmermann, Martina; Lampe, Johanna W.; Lange, Sebastian; Smirnow, Irina; Königsrainer, Alfred; Hann-von-Weyhern, Claus; Fend, Falko; Gregor, Michael; Bitzer, Michael; Lauer, Ulrich M.

doi:10.1007/s10616-009-9246-4

Cited by 40 publications

(39 citation statements)

References 12 publications

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“…Most research groups that successfully prepare and use precision-cut liver slices use the Krumdieck slicer, Vitron slicer or Leica VT1200 S microtome (Krumdieck et al 1980; de Graaf et al 2010; Parrish et al 1995; Zimmermann et al 2009). Recently, the EMS-4000 tissue slicer was introduced, which is mainly used to prepare lung slices (Khan et al 2010a); however, others have performed pilot experiments with rat liver and they have shown that viable PCLS can be prepared (M.H.…”

Section: Liver In Vitro Models In Pharmacology Toxicology and Basic mentioning

confidence: 99%

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

Hewitt²,

et al. 2013

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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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

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Section: Liver In Vitro Models In Pharmacology Toxicology and Basic mentioning

confidence: 99%

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

Hewitt²,

et al. 2013

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“…An alternative to a Weck microtome is a vibrating microtome, which can reproducibly generate thinner slices from firm but not soft tissues (cf 10 ), but is much slower. Very thin slices (100–200 μm) may show a proportionately larger wounding response and are more fragile.…”

Section: Notesmentioning

confidence: 99%

Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices

Fan¹,

Lane²,

Higashi³

2016

BIO-PROTOCOL

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An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg 1 , by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis. In our approach, we maintain freshly resected tissue slices (both cancerous and non-cancerous from the same organ of the same subject) in cell culture media, and treat with appropriate stable isotope-enriched nutrients, e.g. 13 C 6 -glucose or 13 C 5 , 15 N 2 -glutamine. These slices are viable for at least 24 h, and make it possible to eliminate systemic influence on the target tissue metabolism while maintaining the original 3D cellular architecture. It is therefore an excellent pre-clinical platform for assessing the effect of therapeutic agents on target tissue metabolism and their therapeutic efficacy on individual patients 2,3 .

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“…This ranged from the use of advanced instrumentation to the adaptation of adequate culture conditions based on extensive experimental trials conducted before undertaking this study. Briefly, compared with previous instruments available, the instrumentation (Vibratome VT1200; Leica) allows for rapid cutting of each tumor tissue slice in 20-40 s with the significant advantage of controlling the thickness of the slice at a micrometer level (17); it also minimizes the vertical deflection of the blade, and therefore, it preserves the integrity of delicate specimens and ensures a higher number of viable cells on the section surface. In addition, with this technique, no agar or agarose embedding is necessary to successfully cut uneven specimens.…”

Section: Technical Improvements Aimed At Achieving Consistent and Relmentioning

confidence: 99%

Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors

Vaira

Fedele

Pyne

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

242

261

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Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial-stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global geneexpression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy.drug response | ex vivo | human tumor tissue culture | PI3K

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Improved reproducibility in preparing precision-cut liver tissue slices

Cited by 40 publications

References 12 publications

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

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

Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices

Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors

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