Name: establishing primary adult fibroblast cultures from rodents
Uploaded: 2010-10-05T17:00:00
Description: Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

<p class="jove_content">We thank Dr. Steven Austad for providing us with the first version of this protocol.  This work was supported by grants from the NIH and the Ellison Medical Foundation to V.G. and A.S.</p>

<p class="jove_title">1. Before Starting</p>
<ol>
  <li> Sterilize scissors and forceps with 70% ethanol.</li>
  <li> Place small magnetic stirrer inside a 30 mL beaker, cover with two layers of foil, and autoclave.</li>
  <li> Prepare a 28 Wunsch units/mL stock solution of Liberase Blendzyme 3 in sterile water. Make 0.5 mL aliquots and freeze at -20&deg;C. Thaw a new aliquot before every use. The solution may appear cloudy after thawing. Vortex the solution until it becomes clear.</li>
  <li> Warm up the cell culture media.</li>
</ol>
<...

<ol>
	<li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+.">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> <em style='font-style: italic'>Cell</em>. <b>7</b>, 813-823 (2008).</li><li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts.">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> <em style='font-style: italic'>Nat Cell Biol</em>. <b>5</b>, 741-747 (2003).</li></ol>

<p class="jove_content">Normal primary fibroblasts provide an excellent alternative to established cancer cell lines in biological research. An important advantage of fibroblasts is that they do not carry mutations in oncogenes and tumor suppressors and maintain intact cell cycle checkpoints. This makes normal fibroblasts a preferred system for the studies of cell cycle regulation, DNA repair, and apoptosis. The protocol described here provides a simple recipe for isolation and maintenance of primary fibroblasts. This protocol was used to succe...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM/F12 media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FBS), Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>01437-036</td>
<td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15420-096</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td><strong>Liberase TM Research Grade</strong></td>
<td>&nbsp;</td>
<td>Roche</td>
<td><strong>05401127001</strong></td>
<td>Replacement enzyme.</td>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A note from the authors:
Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.</td>
<tr>
<td>EMEM media</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>30-203</td>
<td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td>
</tr>
<tr>
<td>Feathered #21 disposable, sterile scalpel</td>
<td>&nbsp;</td>
<td>Multiple suppliers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Three Gas Control incubator</td>
<td>&nbsp;</td>
<td>Forma or Heraeus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

establishing primary adult fibroblast cultures from rodents

<p class="jove_content">The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes.  Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization.  Hence, new cultures need to be established regularly.  The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge.  Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients.  Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained.  Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs.  We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.</p>

Watch this Scientific Journal Video about Establishing Primary Adult Fibroblast Cultures From Rodents at JoVE.com

Establishing Primary Adult Fibroblast Cultures From Rodents

<p class="jove_content">This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.</p>

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

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