Name: murine precision-cut liver slices as an ex vivo model of liver biology
Uploaded: 2020-03-14T13:00:00
Description: Watch this Scientific Journal Video about Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology at JoVE.com

这项工作得到了澳大利亚国家卫生和医学研究委员会（NHMRC）的研究资助（赠款号） 的支持。APP1048740 和 APP1142394 到 G.A.R. ;APP1160323 到 J.E.E.T.， J.K.O.， G.A.R.）.A. Ramm 基金由澳大利亚 NHMRC 的高级研究研究奖学金提供支持（赠款号APP1061332）。曼努埃尔·费尔南德斯-罗霍得到西班牙马德里的TALENTO方案（T1-BIO-1854）的支持。

所有动物实验均根据澳大利亚关于为科学目的护理和使用动物的法典进行，经该研究所动物伦理委员会批准。雄性C57BL/6小鼠（15-20周大）来自澳大利亚西澳大利亚州动物资源中心。
注：所有与样品接触的解决方案、介质、仪器、硬件和管材都必须用70%乙醇溶液消毒或彻底消毒，并使用无菌技术进行处理，以尽量减少培养污染的风险。
1. 振动器的设置
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<ol>
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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>

该议定书演示了鼠PCLS分离和组织培养的应用，这些程序旨在评估生存能力和效用，以及利用生化测定、组织学和qPCR检查肝病理学外源性中介的影响。PCLS组织培养在啮齿动物和人类的实验效用已在各种应用中得到证明，包括微RNA15/RNA9/蛋白质表达16、代谢17、病毒感染动力学10、18、18感染信号<sup class=...

大多数慢性肝病（即病毒性肝炎、非酒精性性性性性性肝炎、胆汁性肝病和肝癌）的发病机制涉及多种不同肝细胞类型之间的复杂相互作用，这些细胞类型驱动炎症、纤维生成和癌症发展11、2。2为了了解这些慢性肝基疾病背后的分子机制，必须研究多种肝细胞类型之间的相互作用。虽然多个肝细胞系（以及最近，器官）可以在体外培养，但这些模型不能准确模拟肝微环境3的复杂结构、功能和细胞多样性。此外，培养的肝细胞（特别是转化的细胞系）可能偏离其原始来源生物学。动物模型被实验性地用于研究多种肝细胞类型之间的相互作用。然而，由于肝外器官（例如，在测试潜在治疗药物时），实验操作的范围可能会显著缩小。
在组织培养中使用精密切肝片（PCLS）是首次用于药物代谢和毒性研究的实验技术，它涉及切割可行的超薄（约100~250 μm厚）肝片。这允许直接实验操作肝组织前活体4。该技术填补了体内动物研究与体外细胞培养方法之间的实验差距，克服了两种方法的许多缺点（即，对整个动物可进行的实验范围的实际限制，以及利用体外细胞培养方法丧失结构/功能和细胞多样性）。
此外，与整个动物研究相比，PCLS大大提高了实验能力。由于一只小鼠可以产生超过48个肝切片，这也方便了使用来自同一肝脏的控制和治疗组。此外，该技术将肝脏组织与其他器官系统物理分离;因此，在测试外源性刺激的效果时，它消除了整个动物中可能发生的潜在非目标效应。
在此协议中，PCLS 使用带有横向振动刀片的振动器生成。其他研究已经成功地使用了克鲁姆迪克组织切片机，如奥林加和舒潘5所述。在振动器中，刀片的横向振动可防止剪切应力引起的超薄组织的撕裂，因为刀片被推入组织。振动片和克鲁姆迪克组织切片器都能有效工作，无需对肝脏组织进行结构嵌入，从而简化切片程序。该技术还可用于从患病肝脏创建PCLS，包括那些从小鼠模型纤维化/肝硬化6和肝硬化7。
除了展示PCLS的制备和组织培养所需的技术外，本报告还通过测量腺苷三磷酸（ATP）水平并检查组织组织组织学来评估坏死和纤维化，来检查这些前活体组织的生存能力。作为一项具有代表性的实验程序，PCLS以三种不同的胆汁酸（糖胆酸、黄骨酸和胆酸）的病理生理浓度进行治疗，以模拟胆静肝损伤。在胆汁性肝损伤的情况下，特别是胆碱酸在囊性纤维化相关肝病8的儿童血清和胆汁中都明显增加。
肝祖细胞也接受了在体外用陶罗基酸治疗，以模拟患者观察到的高灰胆酸水平，这种治疗导致肝祖细胞对胆小（胆碱）表型9的增殖和分化增加。随后，PCLS在体内处理，其陶罗乔酸水平升高，并观察到胆碱基标记的增加。这支持体外观察，在小儿囊性纤维化相关肝病9中，陶罗基酸驱动胆汁增殖和/或分化。

<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

了解肝损伤、肝纤维化和肝硬化的机制，这些机制是慢性肝病（即病毒性肝炎、非酒精性脂肪性肝病、代谢性肝病和肝癌）的基础，需要实验性操作动物模型和体外细胞培养物。这两种技术都有局限性，例如对大量动物进行体内操作的要求。然而，体外细胞培养物不再现多细胞肝环境的结构和功能。使用精密切肝片是一种技术，其中保持一致的可行的小鼠肝脏切片在实验室组织培养进行实验操作。该技术占据了动物研究和体外细胞培养方法之间的实验空白。提出的协议描述了一种从小鼠身上分离和培养精确切肝片的简单而可靠的方法。作为该技术的应用，前活体肝切片用胆汁酸进行治疗，以模拟胆静肝损伤，并最终评估肝纤维成因的机制。

为了确定PCLS随时间的细胞可行性，测量了组织ATP水平。ATP 水平通常与可行性成正比。PCLS（面积约15 mm2区域）在正常威廉的E介质中培养，具有10%的FBS，然后在特定的时间点，肝切片从组织培养中去除，并同质化，测量ATP和蛋白质（用于正常化）浓度（<a href="https://www.jove.com/pdf-materials/60992?refresh=1" target="_blank">材料表</a>）。Figure 1A对于这样的...

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

该协议为从小鼠中生产可行的精确切肝片提供了一种简单可靠的方法。前活体组织样本可在实验室组织培养条件下维持多日，为检查肝病理生物学提供了灵活的模型。

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

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

本视频演示了精密切肝片作为肝组织生物学实验外活体模型的制作和应用。精密切肝片占据着体内动物研究和体外细胞培养方法之间的实验利基。这项技术比体内动物研究具有几个关键优势，包括使用同一肝脏的控制和治疗样本的能力，分离肝脏组织以去除其他器官系统的离目标效应的能力，以及使用试剂的能力，如果应用于整个活的小鼠，例如有毒通路抑制剂，可能会产生负面影响。该技术涉及使用横向振动刀片切割超薄的活肝组织肝片，然后实验室组织培养。刀片的振动可防止剪切应力导致撕裂。在此示例中，我们将展示可行的外活肝切片的制剂技术，并演示肝片培养在示例实验中的效用，其中这种外活体组织用胆汁酸进行治疗，以刺激胆汁性肝损伤，以评估肝纤维化机制。将刀片插入切割臂。使用 70%乙醇溶液对刀片和切割臂进行消毒。始终小心避免与刀片接触。用 70% 乙醇溶液对缓冲盘进行消毒，然后用无菌组织擦拭。将缓冲托盘插入振动器并拧紧安装螺钉。将切割臂设置为最大可用高度。检查刀片角度。在我们的振动器上，我们使用从水平到 10 度角，刀片向下倾斜到样品上。确保刀片角度已紧固定。再次使用 70% 乙醇溶液对试样支架进行消毒。连接位于缓冲盘下的 Peltier 热电冷却器的冷却水。一些振动器型号使用冰浴进行冷却。将出水管固定到排水沟上，然后打开水。将冷却器设置为 4 摄氏度。向缓冲盘添加冰冷的克雷布斯-亨塞莱特缓冲器。所有手术器械和材料都应无菌。老鼠应该被深深地麻醉或安乐死。用70%乙醇溶液对小鼠的皮肤表面进行润湿。将中线切口切入从腹腔底部到隔膜的皮肤。使用干净的钳子和剪刀，打开腹腔。快速切除肝脏，不损害叶。向下推肝脏，切割肝脏顶部的结缔组织。向上推肝脏。用钳子握住肝脏的中心血管区域，向上拉。切割结缔组织血管。注意不要损害肝叶，也注意不要切入胃肠道。将切除的肝脏放入冰冷的克雷布斯-亨塞莱特缓冲液的无菌盘中。将肝脏分离成单独的叶。选择一个肝叶，并放在一个新的无菌菜平侧。在克雷布斯-亨塞莱特缓冲区中将其他叶放在冰上。修剪肝叶的边缘。修剪很重要，特别是在首先接触切削刀片的边缘，因为组织边缘相对垂直于切削刀片，将防止在浅角下可能发生组织压缩。切割其他三个边缘周围的组织可去除组织边缘的纤维胶囊，并且通常允许更容易去除组织切片。将薄层氰丙烯酸酯放在试样支架的前边缘，比修剪过的肝叶稍大。使用无菌吸水材料清除组织中任何缓冲液残留物。将肝叶放在氰丙烯酸酯胶水上，最大边缘朝向前面。允许在空气中固化一到两分钟。将附着在试样支架上的组织放入振动器中。设置振动器速度。降低切割刀片，直到它位于肝叶的上方。将振动切割臂运行在样品上。此机器上的铲刀振动由脚踏板激活。向后返回刀片，将刀片高度降低 250 微米。重复此过程，直到去除组织顶层。丢弃第一片或两片，因为这些将包含Glisson的胶囊，因此，不会包含太多的功能性肝组织。要切割肝片，通过转动手柄，慢慢将振动刀片推进到组织中。在切割过程中，使用小画笔轻轻引导组织。使用画笔拾取切割组织部分，然后将组织部分放入含有 Krebs-Henseleit 缓冲液的无菌管中进行储存。不使用时，请将根放在冰上。有些时候，组织在切割过程中确实会撕裂，但在大多数情况下，组织仍然可用。切组织切片，直到靠近氰丙烯酸酯胶水。不要切入胶水。重复与其他肝叶，这是坐在冰上，直到获得所需的组织切片的数量。要清洁试样支架，请使用刀片刮掉胶组织混合物，或者使用丙酮或二甲基硫化物等溶剂软化混合物。在组织培养罩中，移液器将一个含有10%胎儿牛血清和抗菌素的Williams'E培养素mL放入12井板中。轻轻旋转混合物，倒入无菌盘中，取出组织切片。将组织切成大致均匀的大小。在此示例中，我们针对表面面积约为 50 毫米方的组织切片。注意检查组织切片的一致性。较暗的切片表明组织厚度增加，应丢弃。较轻的切片表明存在固化的氰丙烯酸酯胶水，应丢弃。将组织切片转移到含有一毫升介质的 12 井板中。在正常组织培养条件下孵育肝片。如果可能，使用方法来提高组织切片的氧气供应。在后天，使用手动移液器更换介质，以防止吸气导致组织损失。精密切肝片现已可供实验使用。对于我们的应用，我们已经用胆汁酸治疗了两天肝片，并在裂解缓冲液中均质化，用于信使RNA提取和qPCR分析。为了检查精密切肝切片在一段时间内的可行性，我们使用ATP水平作为细胞活力的代理。ATP水平被规范化为蛋白质，并在隔离后多个时间点测量。即时和一小时的样本中，ATP水平有所下降，但恢复时间为三小时。此后，ATP 水平维持了五天，尽管随着时间的推移呈下降趋势。H&E 染色用于检查在培养中保存长达五天的精密切肝片。组织形态是暗示一些坏死发生在第二天，严重的坏死发生在第五天。由于第二天至第五天发生坏死，我们建议将这项技术的实验用途限制在三天左右。然而，通过使用更好的氧气输送技术来增强组织切片。精密切肝片似乎也具有增厚的胶原纤维相对于时间在培养。这是自发纤维化过程的暗示。在示例实验中，用三种单独的胆汁酸处理两天，以模拟肝胆汁。研究三个胆黄细胞特异性基因的mRNA表达，这两种结合胆汁酸，甘油酸和龙酸，显著增加了细胞蛋白-19和康奈辛-43的表达。这与胆血管细胞的发展是一致的。看完此视频后，您应该对如何创建精确切割的肝切片以及如何进行基本组织培养有一个很好的理解。精密切肝片可用于非常广泛的应用，包括RNA表达、蛋白质表达、表观遗传学、DNA结合、代谢、感染机制、癌症入侵、药理学、细胞生物学和分泌研究的实验研究，举几例。

Research

JoVE Journal

Medicine

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

一个人力<em&gt;前体内</em&gt;动脉粥样硬化斑块模型研究病变的生物学

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

科研

医学

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

使用穆林精密切肺片对肺动脉内下氧肺血管收缩进行视频形态分析

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Subnormothermic的技术<em&gt;前体内</em&gt;肝脏灌注的存储，评估和修复边际移植肝

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

该<em&gt;卵内</em&gt;鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

大动物模型肝组织血浆凝固的评价

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

体外常温肝灌注的小动物模型

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction

氯乙烯和高脂肪饮食作为环境与肥胖相互作用的典范

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

精密切肺切片作为 离体 肺血管结构和收缩力研究的有效工具

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...