<ol>
	<li>Heslop, J. 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=Mechanistic+evaluation+of+primary+human+hepatocyte+culture+using+global+proteomic+analysis+reveals+a+selective+dedifferentiation+profile.">Mechanistic evaluation of primary human hepatocyte culture using global proteomic analysis reveals a selective dedifferentiation profile.</a> Arch Toxicol. 91 (1), 439-452 (2017).</li><li>Ietto, 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=Multicellular+liver+organoids:+Generation+and+importance+of+diverse+specialized+cellular+components.">Multicellular liver organoids: Generation and importance of diverse specialized cellular components.</a> Cells. 12 (10), 1429 (2023).</li><li>Dunn, J. C., Tompkins, R. G., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocytes+in+collagen+sandwich:+evidence+for+transcriptional+and+translational+regulation.">Hepatocytes in collagen sandwich: evidence for transcriptional and translational regulation.</a> J Cell Biol. 116 (4), 1043-1053 (1992).</li><li>Keemink, J., Oorts, M., Annaert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Hepatocytes+in+sandwich+culture.">Primary Hepatocytes in sandwich culture.</a> Methods in Mol Biol (Clifton, N.J). 1250, 175-188 (2015).</li><li>Kaur, I., 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=Primary+hepatocyte+isolation+and+cultures:+Technical+aspects,+challenges+and+advancements.">Primary hepatocyte isolation and cultures: Technical aspects, challenges and advancements.</a> Bioengineering (Basel, Switzerland). 10 (2), 131 (2023).</li><li>Thompson, W. L., Takebe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+liver+model+systems+in+a+dish.">Human liver model systems in a dish.</a> Dev Growth Differ. 63 (1), 47-58 (2021).</li><li>Takebe, T., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+by+design.">Organoids by design.</a> Science (New York, N.Y.). 364 (6444), 956-959 (2019).</li><li>Nantasanti, S., de Bruin, A., Rothuizen, J., Penning, L. C., Schotanus, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+organoids+are+a+powerful+tool+for+the+study+of+liver+disease+and+personalized+treatment+design+in+humans+and+animals.">Concise review: organoids are a powerful tool for the study of liver disease and personalized treatment design in humans and animals.</a> Stem Cells Transl Med. 5 (3), 325-330 (2016).</li><li>Raju, 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=In+vitro+pluripotent+stem+cell+differentiation+to+hepatocyte+ceases+further+maturation+at+an+equivalent+stage+of+e15+in+mouse+embryonic+liver+development.">In vitro pluripotent stem cell differentiation to hepatocyte ceases further maturation at an equivalent stage of e15 in mouse embryonic liver development.</a> Stem Cells Dev. 27 (13), 910-921 (2018).</li><li>Peng, W. 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=Inflammatory+cytokine+TNFalpha+promotes+the+long-term+expansion+of+primary+hepatocytes+in+3D+culture.">Inflammatory cytokine TNFalpha promotes the long-term expansion of primary hepatocytes in 3D culture.</a> Cell. 175 (6), 1607-1619.e15 (2018).</li><li>Hu, 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=Long-term+expansion+of+functional+mouse+and+human+hepatocytes+as+3d+organoids.">Long-term expansion of functional mouse and human hepatocytes as 3d organoids.</a> Cell. 175 (6), 1591-1606.e19 (2018).</li><li>Michalopoulos, G. K., Bowen, W. C., Mul&#232;, K., Lopez-Talavera, J. C., Mars, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocytes+undergo+phenotypic+transformation+to+biliary+epithelium+in+organoid+cultures.">Hepatocytes undergo phenotypic transformation to biliary epithelium in organoid cultures.</a> Hepatology (Baltimore, Md). 36 (2), 278-283 (2002).</li><li>Schaub, J. 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=De+novo+formation+of+the+biliary+system+by+TGF&#946;-mediated+hepatocyte+transdifferentiation.">De novo formation of the biliary system by TGF&#946;-mediated hepatocyte transdifferentiation.</a> Nature. 557 (7704), 247-251 (2018).</li><li>Lancaster, M. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+in+a+dish:+modeling+development+and+disease+using+organoid+technologies.">Organogenesis in a dish: modeling development and disease using organoid technologies.</a> Science (New York, N.Y.). 345 (6194), 1247125 (2014).</li><li>Huch, M., Koo, B. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+mouse+and+human+development+using+organoid+cultures.">Modeling mouse and human development using organoid cultures.</a> Development (Cambridge, England). 142 (18), 3113-3125 (2015).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods in Cell Biol. 13, 29-83 (1976).</li><li>Ng, I. 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=Isolation+of+primary+rat+hepatocytes+with+multiparameter+perfusion+control.">Isolation of primary rat hepatocytes with multiparameter perfusion control.</a> J Vis Exp. (170), e62289 (2021).</li><li>Shen, L., Hillebrand, A., Wang, D. Q. -. H., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+hepatic+cells.">Isolation and primary culture of rat hepatic cells.</a> J Vis Exp. (64), e3917 (2012).</li><li>Cabral, 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=Purification+of+hepatocytes+and+sinusoidal+endothelial+cells+from+mouse+liver+perfusion.">Purification of hepatocytes and sinusoidal endothelial cells from mouse liver perfusion.</a> J Vis Exp. (132), e56993 (2018).</li><li>Charni-Natan, M., Goldstein, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+primary+mouse+hepatocyte+isolation.">Protocol for primary mouse hepatocyte isolation.</a> STAR protoc. 1 (2), 100086 (2020).</li><li>Smith, A. J., Clutton, R. E., Lilley, E., Hansen, K. E. A., Brattelid, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PREPARE:+Guidelines+for+planning+animal+research+and+testing.">PREPARE: Guidelines for planning animal research and testing.</a> Lab Anim. 52 (2), 135-141 (2018).</li><li>Kluiver, T. A., Kraaier, L. J., Peng, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+murine+primary+hepatocyte+organoids.">Long-term expansion of murine primary hepatocyte organoids.</a> Methods in Molecular Biol (Clifton, N.J). 2544, 1-13 (2022).</li><li>Shulman, M., Nahmias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+and+coculture+of+primary+rat+and+human+hepatocytes.">Long-term culture and coculture of primary rat and human hepatocytes.</a> Methods in Molecular Biol (Clifton, N.J). 945, 287-302 (2013).</li><li>Knobeloch, D., 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=Human+hepatocytes:+isolation,+culture,+and+quality+procedures.">Human hepatocytes: isolation, culture, and quality procedures.</a> Methods in Molecular Biol (Clifton, N.J). 806, 99-120 (2012).</li><li>Queral, A. E., DeAngelo, A. B., Garrett, C. T. <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+different+collagenases+on+the+isolation+of+viable+hepatocytes+from+rat+liver.">Effect of different collagenases on the isolation of viable hepatocytes from rat liver.</a> Anal Biochem. 138 (1), 235-237 (1984).</li></ol>

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	<tbody>
		<tr>
			<td>A83-01- ALK5 Inhibitor IV</td>
			<td>Twin Helix</td>
			<td>&#160;T3031</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher Scientific</td>
			<td>0080085SA</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX Connect Real-Time PCR Detection System&#160;</td>
			<td>&#160;Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Twin Helix</td>
			<td>T2310</td>
			<td></td>
		</tr>
		<tr>
			<td>Click EdU Alexa 488 imaging kit</td>
			<td>Thermofisher Scientific</td>
			<td>C10499</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Type I, solution from rat tail</td>
			<td>Merck</td>
			<td>C3867-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone&#160;</td>
			<td>Merck</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Merck</td>
			<td>&#160;E9644</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Euroclone</td>
			<td>ECS0180L</td>
			<td></td>
		</tr>
		<tr>
			<td>GELTREX LDEV FREE RGF BME</td>
			<td>Thermofisher Scientific</td>
			<td>A1413202</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium 25000 IU/5 ml</td>
			<td>B. Braun Melsungen AG</td>
			<td>B01AB01</td>
			<td></td>
		</tr>
		<tr>
			<td>HGF</td>
			<td>Peprotech</td>
			<td>&#160;100-39H&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium solution 100x&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Digest Medium&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>&#160;17703-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Liver Perfusion Medium</td>
			<td>Thermofisher Scientific</td>
			<td>17701038</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Thermofisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Merck</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Merck</td>
			<td>&#160;N-0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Merck</td>
			<td>M7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>Aurogene</td>
			<td>ant-nr-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer 1X</td>
			<td>PanReac AppliChem</td>
			<td>A0964,9050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution 100x</td>
			<td>Euroclone</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Santa Cruz</td>
			<td>sc-296039A</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec&#8482;&#160;</td>
			<td>MS-4/12 Reglo Digital Pump</td>
			<td></td>
		</tr>
		<tr>
			<td>TNFa</td>
			<td>Peprotech</td>
			<td>&#160;300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>TRI Reagent</td>
			<td>Merck</td>
			<td>T9424</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing&#160;</td>
			<td>Ismatec&#8482;&#160;</td>
			<td>&#160;ID.2,79mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Williams&#39; E Medium, no glutamine</td>
			<td>Thermofisher Scientific</td>
			<td>31415029</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Twin Helix</td>
			<td>T1725</td>
			<td></td>
		</tr>
	</tbody>
</table>

rat liver perfusion and primary hepatocytes isolation: an old procedure crucial for cutting-edge 3d organoids culture

Perfusion der Rattenleber und Isolierung primärer Hepatozyten: ein altes Verfahren, das für die hochmoderne 3D-Organoidkultur entscheidend ist

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due ...

rat-liver-perfusion-primary-hepatocytes-isolation-an-old-procedure

Research

JoVE Journal

Biology

Rat Liver Perfusion and Primary Hepatocytes Isolation: An Old Procedure Crucial for Cutting-Edge 3D Organoids Culture

423 Views.  Italian Liver Foundation. Introduction.Primary hepatocytes are an important tool for in vitro liver related studies. However, the expansion and maintenance of these cells have been historically challenging as they lose morphology and functionality after a few days in culture. 3D organoids are a cutting-edge tool able to recapitulate tissues in a dish for a long-term culture, overcoming the problem of the inability to expand to the primary hepatocytes in vitro.The overall goal of the present work is to describe ...