Name: passaging human neural stem cells
Uploaded: 2007-08-22T00:00:00
Description: Watch this Scientific Journal Video about Passaging Human Neural Stem Cells at JoVE.com

<p class="jove_content">The authors gratefully acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children's Hospital, Orange County Research Institute for providing hNSPCs and initial instruction in their culturing.</p>

<p class="jove_content"><em>Note</em>: For routine culturing of our hNSPCs, we change 50-100% of the media every other day and usually passage them 1:2 once a week. The culture media contains 20% BIT-9500, 1X antibiotic/antimycotic, and growth factors (EGF, FGF, and PDGF each at 40 ng/ml) in DMEM:F12 base media.</p>
<p class="jove_step">Preparing the Coated Flask and Dissociating the Cells</p>
<ol>
<li>To prepare new flasks for the passaged cells, coat T25 flasks with 10 &micro;g/ml human fibronectin in EMEM for 4 hours, or overnight, in a 37&d...

<ol>
	<li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> <em style='font-style: italic'>J. Neurosci. Res</em>. <b>83</b>, 845-856 (2006).</li><li>Nethercott, H. N., Maxwell, H., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+culture.">Neural stem cell culture.</a> <em style='font-style: italic'>Human Stem Cell Manual: A Laboratory Guide</em>. , (2007).</li><li>Palmer, T. D., Schwartz, P. H., Taupin, P., Kaspar, B., Stein, S. A., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture.+Progenitor+cells+from+human+brain+after+death.">Cell culture. Progenitor cells from human brain after death.</a> <em style='font-style: italic'>Nature</em>. <b>411</b>, 42-43 (2001).</li><li>Schwartz, P. H., Bryant, P. J., Fuja, T. J., Su, H., O'Dowd, D. K., Klassen, H. <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+characterization+of+neural+progenitor+cells+from+post-mortem+human+cortex.">Isolation and characterization of neural progenitor cells from post-mortem human cortex.</a> <em style='font-style: italic'>J Neurosci Res</em>. <b>74</b>, 838-851 (2003).</li></ol>

<p class="jove_content">We have found that this protocol provides reliable cultures of hNSPCs.  One critical factor for our cells is that they need to be kept as fairly dense cultures and cannot be passaged to the point that the cells are sparse.  In our hands, sparse cultures grow very slowly or completely cease dividing.  For this reason, we usually split our cultures 1:2 or 1:3 when a culture is extremely dense.  Coating the surface of a culture dish with fibronectin is important since it promotes good cell attachment and migration, but, unl...

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		<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>BIT-9500 (BSA, Insulin, Transferrin)</td>
<td>Reagent</td>
<td>Stem Cell Technologies</td>
<td>09500</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic-Antimycotic</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>DMEM:F12 (1X)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>11330-032</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10437-028</td>
<td>&nbsp;</td>
<tr>
<td>FGF, human basic recombinant</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>100-18B</td>
<td>&nbsp;</td>
<tr>
<td>PDGF-AB</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>100-00AB</td>
<td>&nbsp;</td>
<tr>
<td>EGF, human recombinant</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>CB40052</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Cell Culture Flask, 25 cm2</td>
<td>Tool</td>
<td>Corning</td>
<td>10-126-28</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Fibronectin, human, natural</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>CB40008A</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>Human Neural Stem/Precursor Cells</td>
<td>Cells</td>
<td>National Human Neural Stem Cell Resource http://www.nhnscr.org/default.htm </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cell Dissociation Buffer</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>13150-016</td>
<td>&nbsp;</td>		</tbody>
		</table>
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passaging human neural stem cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

Watch this Scientific Journal Video about Passaging Human Neural Stem Cells at JoVE.com

Passaging Human Neural Stem Cells

<p class="jove_content">The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.</p>

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

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