This work was supported by research grants from the National Health and Medical Research Council (NHMRC) of Australia (Grant No. APP1048740 and APP1142394 to G.A.R.; APP1160323 to J.E.E.T., J.K.O., G.A.R.). Grant A. Ramm is supported by a Senior Research Fellowship from the NHMRC of Australia (Grant No. APP1061332). Manuel Fernandez-Rojo was supported by the TALENTO program of Madrid, Spain (T1-BIO-1854).

All animal experiments were performed in accordance with the Australian code for the care and use of animals for scientific purposes at QIMR Berghofer Medical Research Institute with approval from the institute animal ethics committee. Male C57BL/6 mice (15&#8722;20 weeks old) were obtained from the Animal Resources Centre, WA, Australia.
NOTE: All solutions, media, instruments, hardware, and tubes that contact the samples must be sterilized or thoroughly disinfected with a 70% ethanol solutio...

<ol>
	<li>Sircana, A., Paschetta, E., Saba, F., Molinaro, F., Musso, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Insight+into+the+Role+of+Fibrosis+in+Nonalcoholic+Steatohepatitis-Related+Hepatocellular+Carcinoma.">Recent Insight into the Role of Fibrosis in Nonalcoholic Steatohepatitis-Related Hepatocellular Carcinoma.</a> International Journal of Molecular Sciences. 20 (7), 1745 (2019).</li><li>Kohn-Gaone, J., Gogoi-Tiwari, J., Ramm, G. A., Olynyk, J. K., Tirnitz-Parker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+liver+progenitor+cells+during+liver+regeneration,+fibrogenesis,+and+carcinogenesis.">The role of liver progenitor cells during liver regeneration, fibrogenesis, and carcinogenesis.</a> American Journal of Physiology-Gastrointestinal Liver Physiology. 310 (3), 143-154 (2016).</li><li>Ouchi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Steatohepatitis+in+Humans+with+Pluripotent+Stem+Cell-Derived+Organoids.">Modeling Steatohepatitis in Humans with Pluripotent Stem Cell-Derived Organoids.</a> Cell Metabolism. 30 (2), 374-384 (2019).</li><li>Vickers, A. E., Fisher, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+slices+for+the+evaluation+of+human+drug+toxicity.">Organ slices for the evaluation of human drug toxicity.</a> Chemico-Biological Interactions. 150 (1), 87-96 (2004).</li><li>Olinga, P., Schuppan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+liver+slices:+a+tool+to+model+the+liver+ex+vivo.">Precision-cut liver slices: a tool to model the liver ex vivo.</a> Journal of Hepatology. 58 (6), 1252-1253 (2013).</li><li>Paish, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Bioreactor+Technology+for+Modeling+Fibrosis+in+Human+and+Rodent+Precision-Cut+Liver+Slices.">A Bioreactor Technology for Modeling Fibrosis in Human and Rodent Precision-Cut Liver Slices.</a> Hepatology. 70 (4), 1377-1391 (2019).</li><li>Prins, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pathophysiological+Model+of+Non-Alcoholic+Fatty+Liver+Disease+Using+Precision-Cut+Liver+Slices.">A Pathophysiological Model of Non-Alcoholic Fatty Liver Disease Using Precision-Cut Liver Slices.</a> Nutrients. 11 (3), 507 (2019).</li><li>Ramm, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrogenesis+in+pediatric+cholestatic+liver+disease:+role+of+taurocholate+and+hepatocyte-derived+monocyte+chemotaxis+protein-1+in+hepatic+stellate+cell+recruitment.">Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.</a> Hepatology. 49 (2), 533-544 (2009).</li><li>Pozniak, K. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taurocholate+Induces+Biliary+Differentiation+of+Liver+Progenitor+Cells+Causing+Hepatic+Stellate+Cell+Chemotaxis+in+the+Ductular+Reaction:+Role+in+Pediatric+Cystic+Fibrosis+Liver+Disease.">Taurocholate Induces Biliary Differentiation of Liver Progenitor Cells Causing Hepatic Stellate Cell Chemotaxis in the Ductular Reaction: Role in Pediatric Cystic Fibrosis Liver Disease.</a> The American Journal of Pathology. 187 (12), 2744-2757 (2017).</li><li>Clouzeau-Girard, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+bile+acids+on+biliary+epithelial+cell+proliferation+and+portal+fibroblast+activation+using+rat+liver+slices.">Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices.</a> Lab Investigation. 86 (3), 275-285 (2006).</li><li>Szalowska, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oxygen+concentration+and+selected+protocol+factors+on+viability+and+gene+expression+of+mouse+liver+slices.">Effect of oxygen concentration and selected protocol factors on viability and gene expression of mouse liver slices.</a> Toxicology in Vitro. 27 (5), 1513-1524 (2013).</li><li>Koch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+precision-cut+liver+slices+(PCLS):+a+new+tool+for+studying+tumor+microenvironments+and+cell+signaling+ex+vivo.">Murine precision-cut liver slices (PCLS): a new tool for studying tumor microenvironments and cell signaling ex vivo.</a> Cell Communication and Signaling. 12, 73 (2014).</li><li>Granitzny, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+high+quality+rat+precision+cut+liver+slices+during+culture+to+study+hepatotoxic+responses:+Acetaminophen+as+a+model+compound.">Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound.</a> Toxicology in Vitro. 42, 200-213 (2017).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+human+liver+slice+cultures+as+an+immunological+platform.">Precision-cut human liver slice cultures as an immunological platform.</a> Journal of Immunological Methods. 455, 71-79 (2018).</li><li>Zarybnicky, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-Individual+Variability+in+Acute+Toxicity+of+R-Pulegone+and+R-Menthofuran+in+Human+Liver+Slices+and+Their+Influence+on+miRNA+Expression+Changes+in+Comparison+to+Acetaminophen.">Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen.</a> International Journal of Molecular Sciences. 19 (6), 1805 (2018).</li><li>van de Bovenkamp, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+liver+slices+as+a+new+model+to+study+toxicity-induced+hepatic+stellate+cell+activation+in+a+physiologic+milieu.">Precision-cut liver slices as a new model to study toxicity-induced hepatic stellate cell activation in a physiologic milieu.</a> Toxicology Sciences. 85 (1), 632-638 (2005).</li><li>Buettner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+analysis+of+hepatic+glucose+output+and+insulin+action+using+a+liver+slice+culture+system.">Efficient analysis of hepatic glucose output and insulin action using a liver slice culture system.</a> Hormone and Metabolic Research. 37 (3), 127-132 (2005).</li><li>Lagaye, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-hepatitis+C+virus+potency+of+a+new+autophagy+inhibitor+using+human+liver+slices+model.">Anti-hepatitis C virus potency of a new autophagy inhibitor using human liver slices model.</a> World Journal of Hepatology. 8 (21), 902-914 (2016).</li><li>Gobert, G. N., Nawaratna, S. K., Harvie, M., Ramm, G. A., McManus, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+model+for+studying+hepatic+schistosomiasis+and+the+effect+of+released+protein+from+dying+eggs.">An ex vivo model for studying hepatic schistosomiasis and the effect of released protein from dying eggs.</a> PLoS Neglected Tropical Diseases. 9 (5), 0003760 (2015).</li><li>Jaiswal, S. K., Gupta, V. K., Ansari, M. D., Siddiqi, N. J., Sharma, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+C+acts+as+a+hepatoprotectant+in+carbofuran+treated+rat+liver+slices+in+vitro.">Vitamin C acts as a hepatoprotectant in carbofuran treated rat liver slices in vitro.</a> Toxicology Reports. 4, 265-273 (2017).</li><li>Plazar, J., Hreljac, I., Pirih, P., Filipic, M., Groothuis, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+xenobiotic-induced+DNA+damage+by+the+comet+assay+applied+to+human+and+rat+precision-cut+liver+slices.">Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices.</a> Toxicology in Vitro. 21 (6), 1134-1142 (2007).</li><li>van de Bovenkamp, M., Groothuis, G. M., Meijer, D. K., Olinga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+fibrotic+rat+liver+slices+as+a+new+model+to+test+the+effects+of+anti-fibrotic+drugs+in+vitro.">Precision-cut fibrotic rat liver slices as a new model to test the effects of anti-fibrotic drugs in vitro.</a> Journal of Hepatology. 45 (5), 696-703 (2006).</li><li>Guyot, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrogenic+cell+phenotype+modifications+during+remodelling+of+normal+and+pathological+human+liver+in+cultured+slices.">Fibrogenic cell phenotype modifications during remodelling of normal and pathological human liver in cultured slices.</a> Liver International. 30 (10), 1529-1540 (2010).</li><li>Bigaeva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+organ-specific+features+of+fibrogenesis+using+murine+precision-cut+tissue+slices.">Exploring organ-specific features of fibrogenesis using murine precision-cut tissue slices.</a> Biochim Biophys Acta - Molecular Basis Disease. 1866 (1), 165582 (2020).</li><li>Kiziltas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+in+pathophysiology+of+liver+diseases.">Toll-like receptors in pathophysiology of liver diseases.</a> World Journal of Hepatology. 8 (32), 1354-1369 (2016).</li><li>Mencin, A., Kluwe, J., Schwabe, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+as+targets+in+chronic+liver+diseases.">Toll-like receptors as targets in chronic liver diseases.</a> Gut. 58 (5), 704-720 (2009).</li><li>Finot, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Stimulation+with+the+Tumor+Necrosis+Factor+alpha+and+the+Epidermal+Growth+Factor+Promotes+the+Proliferation+of+Hepatocytes+in+Rat+Liver+Cultured+Slices.">Combined Stimulation with the Tumor Necrosis Factor alpha and the Epidermal Growth Factor Promotes the Proliferation of Hepatocytes in Rat Liver Cultured Slices.</a> International Journal of Hepatology. 2012, 785786 (2012).</li><li>Marshall, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+between+hepatocyte+G1+arrest,+impaired+hepatic+regeneration,+and+fibrosis+in+chronic+hepatitis+C+virus+infection.">Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection.</a> Gastroenterology. 128 (1), 33-42 (2005).</li><li>Alpini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+acid+feeding+increased+proliferative+activity+and+apical+bile+acid+transporter+expression+in+both+small+and+large+rat+cholangiocytes.">Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes.</a> Hepatology. 34 (5), 868-876 (2001).</li><li>Studer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugated+bile+acids+activate+the+sphingosine-1-phosphate+receptor+2+in+primary+rodent+hepatocytes.">Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes.</a> Hepatology. 55 (1), 267-276 (2012).</li></ol>

The protocol demonstrates the application of murine PCLS isolation and tissue culture, and the procedures are designed to assess both viability and utility as well as examine impacts of exogenous mediators of liver pathobiology using biochemical assays, histology, and qPCR. The experimental utility of PCLS tissue culture in rodents and humans has been demonstrated in a wide range of applications, including experimental investigations in microRNA15/RNA9/protein expression<su...

The pathogenesis of most chronic liver diseases (i.e., viral hepatitis, nonalcoholic steatohepatitis, cholestatic liver injury and liver cancer) involves complex interactions between multiple different liver cell types that drive inflammation, fibrogenesis, and cancer development1,2. To understand the molecular mechanisms underlying these chronic liver-based diseases, the interactions between multiple liver cell types must be investigated. While multiple hepatic cell lines (and more recently, organoids) can be cultured in vitro, these models do not accurately emulate the complex structure, function, and cellular diversity of the hepatic microenvironment3. Furthermore, cultured liver cells (in particular, transformed cell lines) may deviate from their original source biology. Animal models are used experimentally to investigate the interactions between multiple liver cell types. However, they may become significantly reduced in scope for experimental manipulation, due to significant off-target effects in extrahepatic organs (e.g., when testing potential therapeutics).
The use of precision-cut liver slices (PCLS) in tissue culture is an experimental technique first used in drug metabolism and toxicity studies, and it involves the cutting of viable, ultrathin (around 100&#8722;250 &#181;m thick) liver slices. This permits the direct experimental manipulation of liver tissue ex vivo4. The technique bridges an experimental gap between in vivo animal studies and in vitro cell culture methods, overcoming many drawbacks of both methods (i.e., practical limits on the range of experiments that can be performed in whole animals as well as loss of structure/function and cellular diversity with in vitro cell culture methods).
Furthermore, PCLS vastly increases experimental capacity compared to whole animal studies. As one mouse can produce more than 48 liver slices, this also facilitates the use of both control and treatment groups from the same liver. In addition, the technique physically separates the liver tissue from other organ systems; therefore, it removes potential off-target effects that can occur in whole animals when testing the effects of exogenous stimuli.
In this protocol, PCLS are generated using a vibratome with a laterally vibrating blade. Other studies have successfully used a Krumdieck tissue slicer, as described in Olinga and Schuppan5. In the vibratome, lateral vibration of the blade prevents tearing of the ultrathin tissue caused by shear stress, as the blade is pushed into the tissue. Both the vibratome and Krumdieck tissue slicer work effectively without structural embedding of liver tissue, which streamlines the slicing procedure. This technique can also be used to create PCLS from diseased livers, including those from mouse models of fibrosis/cirrhosis6 and hepatic steatosis7.
In addition to demonstrating the techniques required for preparation and tissue culture of PCLS, this report also examines the viability of these ex vivo tissues by measuring adenosine triphosphate (ATP) levels and examining tissue histology to assess necrosis and fibrosis. As a representative experimental procedure, PCLS are treated with pathophysiological concentrations of three different bile acids (glycocholic, taurocholic, and cholic acids) to simulate cholestatic liver injury. In the context of cholestatic liver injury, taurocholic acid in particular has been shown to be significantly increased in both the serum and bile of children with cystic fibrosis-associated liver disease8.
Liver progenitor cells have also been treated in vitro with taurocholic acid to simulate the elevated taurocholic acid levels observed in patients, and this treatment caused increased proliferation and differentiation of liver progenitor cells towards a biliary (cholangiocyte) phenotype9. Subsequently, PCLS were treated ex vivo with elevated levels of taurocholic acid, and increased cholangiocyte markers were observed. This supports the in vitro observation that taurocholic acid drives biliary proliferation and/or differentiation in the context of pediatric cystic fibrosis-associated liver disease9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm Petri Dish</td>
			<td>GREINER</td>
			<td>664160</td>
			<td>Sterile Dish</td>
		</tr>
		<tr>
			<td>12 Well Tissue Culture Plate Flat Bottom</td>
			<td>Greiner Bio-one</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol Solution (made with AR Grade)</td>
			<td>Chem-Supply Pty Ltd</td>
			<td>EA043-20L-P</td>
			<td>Disinfection solution</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Chem-Supply Pty Ltd</td>
			<td>AA008-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholic acid</td>
			<td>Sigma-Aldrich</td>
			<td>C1129-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate Super Glue</td>
			<td>Parfix, DuluxGroup (Australia)</td>
			<td></td>
			<td>Other brands should work</td>
		</tr>
		<tr>
			<td>Disposable Single Edge Safety Razor Blades</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Board</td>
			<td>Made in-house</td>
			<td></td>
			<td>Sterile material over polystyrene</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GE Healthcare Australia Pty Ltd</td>
			<td>SH30084.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps sharp point 130 mm long</td>
			<td>ThermoFisher Scientific</td>
			<td>MET2115-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma Steri-Cycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>371</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycocholic acid hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G2878-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>ISOLATE II RNA Mini Kit</td>
			<td>Bioline (Aust) Pty Ltd</td>
			<td>BIO-52073</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 50 ml</td>
			<td>Provet</td>
			<td>KETAI1</td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Henseleit Buffer with Added Glucose 2000 mg/L</td>
			<td>Sigma-Aldrich</td>
			<td>K3753</td>
			<td>Can also be made in house</td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td>Hepa air filtration</td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liq 100 ml</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Picro Sirius Red</td>
			<td>ABCAM Australia Pty Ltd</td>
			<td>ab246832</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips Abt 1000 &#181;l Filter Interpath</td>
			<td>Interpath</td>
			<td>24800</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips Abt 10 &#181;l Filter Interpath</td>
			<td>Interpath</td>
			<td>24300</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips Abt 200 &#181;l Filter Interpath</td>
			<td>Interpath</td>
			<td>24700</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips Abt 20 &#181;l Filter Interpath</td>
			<td>Interpath</td>
			<td>24500</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys Homogeniser</td>
			<td>Bertin Instruments</td>
			<td>P000669-PR240-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Protractor</td>
			<td>Generic</td>
			<td></td>
			<td>To measure blade angle</td>
		</tr>
		<tr>
			<td>Quantstudio 5 QPCR Fixed 384 Block</td>
			<td>Applied Biosystems/ ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade Holder</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SensiFAST cDNA Synthesis Kit</td>
			<td>Bioline (Aust) PTY LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small Paintbrush with Plastic Handle</td>
			<td>Mixed</td>
			<td></td>
			<td>Plastic handle resists ethanol</td>
		</tr>
		<tr>
			<td>Square-Head Foreceps</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50 ml Plastic Tubes</td>
			<td>Corning Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Clamps</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Forceps</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Pins</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Taurochoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T-4009-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome SYS-NVSLM1 Motorized Vibroslice</td>
			<td>World Precision Instruments</td>
			<td>SYS-NVSLM1</td>
			<td>With thermoelectric cooling</td>
		</tr>
		<tr>
			<td>Williams Medium E</td>
			<td>Life Technologies Australia Pty Ltd</td>
			<td>12551032</td>
			<td>2.0 g/l glucose</td>
		</tr>
		<tr>
			<td>Xylazine 100 mg/mL 50 mL</td>
			<td>Provet</td>
			<td>XYLAZ4</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine precision-cut liver slices as an ex vivo model of liver biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

To determine the cell viability of PCLS over time, tissue ATP levels were measured. ATP levels are typically proportional to viability. PCLS (around 15 mm2 in area) were cultured in normal William&#39;s E medium with 10% FBS, then at specific timepoints, liver slices were removed from tissue culture and homogenized with both ATP and protein (for normalization) concentrations (<a href="https://www.jove.com/pdf-materials/60992?refresh=1" target="_blank">Table of Materials</a>) being measured (<s...

Watch this Scientific Journal Video about Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology at JoVE.com

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

This video demonstrates the production and application of precision-cut liver slices as an experimental ex vivo model of liver tissue biology. Precision-cut liver slices occupy an experimental niche that exists between in vivo animal studies and in vitro cell culture methods. This technique has several key benefits over in vivo animal studies, including the ability to use control and treatment samples from the same liver, the isolation of liver tissue to remove off-target effects from other organ systems, and the ability to use reagents that might have a negative effect if applied to a whole living mouse, for example, toxic pathway inhibitors.The technique involves using a laterally vibrating blade to cut ultra-thin, liver slices of viable liver tissue followed by laboratory tissue culture. The vibrations of the blade prevents tearing caused by shear stress. In this example, we'll show the techniques for preparations of viable ex vivo liver slices, and demonstrate the utility of liver slice culture in example experiments where this ex vivo tissue is treated with bile acids to stimulate cholestatic liver injury to assess the mechanisms of hepatic fibrogenesis.Insert the blade into the cutting arm. Disinfect the blade and the cutting arm with a 70%ethanol solution. Always be careful to avoid contact with the blade.Disinfect the buffer tray with a 70%ethanol solution, and wipe with a sterile tissue. Insert the buffer tray into the vibratome and tighten the mounting screw. Set the cutting arm to the maximum available height.Check the blade angle. On our vibratome, we use a 10-degree angle from horizontal, with the blade sloping down towards the sample. Make sure the blade angle is tightly fixed.Disinfect the specimen holder, again, with a 70%ethanol solution. Connect the cooling water for the Peltier thermoelectric cooler, located under the buffer tray. Some vibratome models use an ice bath for cooling instead.Fix the water-out tube to a drain, and turn on the water. Set the cooler to four degrees Celsius. Add ice-cold Krebs-Henseleit buffer to the buffer tray.All surgical instruments and materials should be sterile. Mice should be deeply anesthetized or euthanized. Skin surfaces on the mice were disinfected by wetting with 70%ethanol solution.Make a midline incision into the skin from the base of the abdominal cavity to the diaphragm. Using clean forceps and scissors, open the abdominal cavity. Remove the liver quickly, without damaging the lobes.Push the liver downwards, and cut the connective tissue at the top of the liver. Push the liver upwards. Hold the center vascular area of the liver with the forceps and pull upward.Cut connective tissue blood vessels. Take care not to damage the liver lobes, and also, take care not to cut into the gastrointestinal tract. Place the removed liver into a sterile dish of ice-cold Krebs-Henseleit buffer.Separate the liver into individual lobes. Select one liver lobe and place flat side down on a new sterile dish. Keep other lobes on ice in Krebs-Henseleit buffer.Trim the edges of the liver lobes. Trimming is important, particularly at the edge that will contact the cutting blade first, as having a tissue edge relatively perpendicular to the cutting blade will prevent tissue compression that can occur at shallow angles. Cutting the tissue around the other three edges removes much of the fibrous capsules at the tissue edge, and typically allows easier removal of the tissue slices.Place a thin layer of cyanoacrylate glue on the front edge of the specimen holder, slightly larger than the trimmed liver lobes. Remove any residue of buffer from the tissue using sterile absorbent material. Place liver lobe on cyanoacrylate glue, with the largest edge facing towards the front.Allow to cure in air for one to two minutes. Place the tissue attached to the specimen holder into the vibratome. Set the vibratome speed.Lower the cutting blade until it's located just above the liver lobe. Run the vibrating cutting arm over the sample. Vibration of the blade on this machine is activated by the foot pedal.Return the blade backwards, and lower the blade height by 250 micrometers. Repeat this process until the top layer of tissue is removed. Discard the first one or two slices, as these will contain Glisson's capsule, and therefore, won't contain much functional liver tissue.To cut liver slices, slowly advance the vibrating blade into the tissue by turning the handle. Use a small paintbrush to gently guide the tissue during the cutting process. Use the paintbrush to pick up the cut tissue section, and then place the tissue section into a sterile tube containing Krebs-Henseleit buffer for storage.When not in use, keep this tube on ice. Some of the time, the tissue does tear during the cutting process, but for most purposes, the tissue is still usable. Cut the tissue slices until near the cyanoacrylate glue.Don't cut into the glue. Repeat with the other liver lobes, which are sitting on ice, until the required number of tissue slices is obtained. To clean the specimen holder, either use a blade to scrape the glue-tissue mixture off, or the mixture can be softened using solvents such as acetone or dimethyl sulfoxide.In a tissue culture hood, pipette one mL of Williams'E medium containing 10%fetal bovine serum and antimicrobials into a 12-well plate. Remove the tissue slices by gently swirling the mixture and tip into a sterile dish. Cut the tissue into a roughly uniform size.In this example, we aimed for tissue slices with a surface area around 50 millimeters square. Take care to examine the tissue slices for consistency. Darker slices suggest the tissue thickness is increased, and should be discarded.Lighter slices suggest the presence of cured cyanoacrylate glue, and should be discarded. Transfer the tissue slices to the 12-well plate containing one milliliter of media. Incubate the liver slices under normal tissue culture conditions.If possible, use methods to enhance oxygen availability to the tissue slices. On the day after, change the media using a manual pipette to prevent loss of tissue by suction. The precision-cut liver slices are now ready for experimental use.For our application, we have treated the liver slices with bile acids for two days, and homogenized in lysis buffer for messenger RNA extraction and qPCR analysis. To examine the viability of precision-cut liver slices over time, we used ATP levels as a proxy for cell viability. ATP levels were normalized to protein, and were measured at multiple time points post-isolation.ATP levels were decreased in the immediate and one-hour samples, however this recovered by three hours. Following this, the ATP levels were maintained for five days, although they trended downwards over time. H&E staining was used to examine precision-cut liver slices that were maintained in culture for up to five days.Tissue morphology was suggestive of some necrosis occurring at day two, with severe necrosis happening by day five. Because of the necrosis happening between days two and five, we'd recommend the experimental utility of this technique is limited to around three days. However, this could be enhanced by using better oxygen delivery techniques to the tissue slices.Precision-cut liver slices also appear to have thickening of collagen fibers relative to time in culture. This is suggestive of spontaneous fibrogenic processes occurring. In the example experiment, the precision-cut liver slices were treated with three separate bile acids for two days, to mimic hepatic cholestasis.Looking at the mRNA expression of three cholangiocyte-specific genes, the two conjugated bile acids, glycocholic and taurocholic acids, significantly increased the expression of both cytokeratin-19 and connexin-43. This is consistent with cholangiocyte development. After watching this video, you should have a good understanding of how to create precision-cut liver slices, and how to perform basic tissue culture.Precision-cut liver slices can be used for a very wide range of applications, including experimental investigations in RNA expression, protein expression, epigenetics, DNA binding, metabolism, infection mechanisms, cancer invasion, pharmacology, cell biology, and secretion studies, just to name a few.

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JoVE Journal

Medicine

15.0K visualizações.  QIMR Berghofer Medical Research Institute. Este vídeo demonstra a produção e a aplicação de fatias de fígado cortadas com precisão como um modelo experimental ex vivo de biologia do tecido hepático. As fatias hepáticas cortadas com precisão ocupam um nicho experimental que existe entre estudos in vivo em animais e métodos de cultura celular in vitro. Esta técnica tem vários benefícios fundamentais sobre estudos in vivo em animais, incluindo a capacidade de usar amostras de controle e tratamento do mesmo fígado, o isolam...