Name: seven steps to stellate cells
Uploaded: 2011-05-10T13:00:00
Description: Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

This work was funded by the Smith Family Award for Excellence in Biomedical Research and NIH RO1 AI083426-01 (F.W.). Patrick Maschmeyer and Melanie Flach are supported by PhD fellowships from the Boehringer Ingelheim Foundation.

C57BL/6 mice should be used at ~20 weeks of age or older. The use of male mice weighing 25-30g is recommended. The yield of stellate cells can be increased by feeding mice a vitamin A-enriched diet for 2 months prior to stellate cell isolation. Approximately 2x105 hepatic stellate cells can be purified from the liver of one C57BL/6 mouse, whereas the yield of stellate cells from Balb/c mice is considerably higher. The following protocol is adjusted to 5 mice. Animal care and experimentation were performed in a...

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	<li>Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

Hepatic stellate cells regulate essential physiological and pathophysiological processes in the liver. Moreover, stellate cells possess antigen presenting properties, rendering them an important component of the hepatic immune response. Although hepatic stellate cells comprise 10-15% of the total cell number in the liver, isolation of these cells is challenging due to their localization in the perisinusoidal space of Disse.

 Here we present a straightforward method to isolate hepatic ...

<table border="1">
<tbody>
 <tr>
 <th>Name Of Reagent</th>
 <th>Company</th>
 <th>Catalog No.</th>
 </tr>
 <tr>
 <td>70&mu;m Cell Strainer</td>
 <td>BD</td>
 <td>352350</td>
 </tr>
 <tr>
 <td>CaCL2 x 2H2O</td>
 <td>Sigma-Aldrich</td>
 <td>C3306</td>
 </tr>
 <tr>
 <td>Collagenase P</td>
 <td>Roche</td>
 <td>11249002001</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11960</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td>10104159001</td>
 </tr>
 <tr>
 <td>DPBS</td>
 <td>Cellgro</td>
 <td>21-031-cv</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Fluka</td>
 <td>3777</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Gibco</td>
 <td>16000</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>Sigma-Aldrich</td>
 <td>G7528</td>
 </tr>
 <tr>
 <td>Gradient Centrifugation Tubes</td>
 <td>Greiner Bio-One</td>
 <td>163160</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Gibco</td>
 <td>15630</td>
 </tr>
 <tr>
 <td>KCL</td>
 <td>Sigma-Aldrich</td>
 <td>P9541</td>
 </tr>
 <tr>
 <td>KH2PO4</td>
 <td>Sigma-Aldrich</td>
 <td>P9791</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 </tr>
 <tr>
 <td>MgCl2 x 6H2O</td>
 <td>Fluka</td>
 <td>63068</td>
 </tr>
 <tr>
 <td>MgSO4 x 7H2O</td>
 <td>Sigma-Aldrich</td>
 <td>M2773</td>
 </tr>
 <tr>
 <td>Na2HPO4 x 2H2O</td>
 <td>Fluka</td>
 <td>71643</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma-Aldrich</td>
 <td>S3014</td>
 </tr>
 <tr>
 <td>NaH2PO4 x H2O</td>
 <td>Fluka</td>
 <td>71507</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Fluka</td>
 <td>71628</td>
 </tr>
 <tr>
 <td>Nycodenz</td>
 <td>Accudenz AG</td>
 <td>AN7050/BLK</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Gibco</td>
 <td>15140</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Cole Parmer</td>
 <td>HV-7523-70</td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>Sigma-Aldrich</td>
 <td>P4633</td>
 </tr>
 <tr>
 <td>Pronase E</td>
 <td>Calbiochem</td>
 <td>7433-2</td>
 </tr>
 <tr>
 <td>Silicone Tube</td>
 <td>Masterflex</td>
 <td>HV-96440-14</td>
 </tr>
 <tr>
 <td>Sodium Pyruvate</td>
 <td>Gibco</td>
 <td>11360</td>
 </tr>
 <tr>
 <td>Tissue culture flask (25cm2)</td>
 <td>BD</td>
 <td>353108</td>
 </tr>
 <tr>
 <td>Winged Infusion Set</td>
 <td>Terumo</td>
 <td>1SV27EL</td>
 </tr>
 </tbody>
</table>

seven steps to stellate cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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