<p class="jove_content">This work was supported by funding from the Overstreet Foundation.</p>


<p class="jove_title">1.  Basic Set Up Before Proceeding to Dissection:</p>
<ol>
<li>Appropriate volume of complete NSC medium is prepared by mixing NeuroCult NSC Basal Medium and NeuroCult NSC Proliferation Supplements at a 9:1 ratio, respectively.</li>
<li>The medium is warmed up in a 37&deg;C water bath.</li>
<li>Cold HEPES-buffered minimum essential medium (HEM) with high concentration of antibiotics (10%) is prepared for dissection and washing purpose. Alternatively, NSC basal medium with antibiotics supplementation may also be used fo...

<ol>
	<li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> <em style='font-style: italic'>Science</em>. <b>255</b>, 1707-1710 (1992).</li><li>Morshead, C. M. <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+cells+in+the+adult+mammalian+forebrain:+a+relatively+quiescent+subpopulation+of+subependymal+cells.">Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.</a> <em style='font-style: italic'>Neuron</em>. <b>13</b>, 1071-1082 (1994).</li><li>Craig, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+growth+factor+expansion+of+endogenous+subependymal+neural+precursor+cell+populations+in+the+adult+mouse+brain.">In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.</a> <em style='font-style: italic'>J Neurosci</em>. <b>16</b>, 2649-2658 (1996).</li><li>Golmohammadi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+the+frequency+and+distribution+of+stem+and+progenitor+cells+in+the+adult+mouse+brain.">Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.</a> <em style='font-style: italic'>Stem Cells</em>. <b>26</b>, 979-987 (2008).</li><li>Azari, H., Rahman, M., Sharififar, S., BA, R. e. y. n. o. l. d. s. <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+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> <em style='font-style: italic'>J Vis Exp</em>. , (2010).</li><li>Reynolds, B. A., Rietze, R. L. <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+cells+and+neurospheres--re-evaluating+the+relationship.">Neural stem cells and neurospheres--re-evaluating the relationship.</a> <em style='font-style: italic'>Nat Methods</em>. <b>2</b>, 333-336 (2005).</li><li>Louis, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+neural+stem+and+progenitor+cells+in+the+neural+colony-forming+cell+assay.">Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.</a> <em style='font-style: italic'>Stem Cells</em>. <b>26</b>, 988-996 (2008).</li></ol>

<p class="jove_content">The neurosphere assay is the method of choice for the isolation and expansion of neural stem cells <sup>1-5</sup> because of its simplicity and reproducibility. This assay is an invaluable tool for large-scale generation of undifferentiated CNS precursor cells, which could be used for both <em>in vitro</em> and <em>in vivo</em> studies. It should be emphasized that neurospheres could be generated from both the bona fide neural stem cells and more restricted progenitors. Therefore, calculating the neurosphere forming freq...

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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>NeuroCult NSC Basal Medium</td>
<td>Medium</td>
<td>Stem Cell Technologies</td>
<td>05700</td>
<td>&nbsp;</td>
<tr>
<td>NeuroCult NSC Proliferation Supplements</td>
<td>Medium supplement</td>
<td>Stem Cell Technologies</td>
<td>05701</td>
<td>&nbsp;</td>
<tr>
<td>%0.05 trypsin-EDTA</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25300-062</td>
<td>&nbsp;</td>
<tr>
<td>Soybean trypsin inhibitor </td>
<td>Reagent</td>
<td>Sigma</td>
<td>T6522</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep </td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>*MEM</td>
<td>Reagent</td>
<td>Gibco</td>
<td>41500-018</td>
<td>HEM component</td>
</tr>
<tr>
<td>*HEPES</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4034</td>
<td>HEM component</td>
</tr>
<tr>
<td>*Distilled water</td>
<td>Reagent </td>
<td>Gibco</td>
<td>15230-147</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer </td>
<td>Sieve</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>T25 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>136196</td>
<td>&nbsp;</td>
<tr>
<td>T80 flask</td>
<td>Culture ware</td>
<td>Nalge Nunc international</td>
<td>178905</td>
<td>&nbsp;</td>
<tr>
<td>15 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>50 ml tubes</td>
<td>Culture ware</td>
<td>BD Falcon</td>
<td>352070</td>
<td>&nbsp;</td>
<tr>
<td>Fine curved forceps</td>
<td>Surgical tools</td>
<td>Fine Science Tools</td>
<td>11251&#x2010;35</td>
<td>&nbsp;</td>
<tr>
<td>Small fine forceps</td>
<td>Surgical tools</td>
<td>Fine Science Tools</td>
<td>11272&#x2010;30</td>
<td>&nbsp;</td>
<tr>
<td>Small forceps</td>
<td>Surgical tools</td>
<td>Fine Science Tools</td>
<td>11050&#x2010;10</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Surgical tools</td>
<td>World Percision Instruments, Inc.</td>
<td>500216</td>
<td>&nbsp;</td>
<tr>
<td>EGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>2028-EG</td>
<td>&nbsp;</td>
<tr>
<td>b-FGF</td>
<td>Growth factor</td>
<td>R&amp;D</td>
<td>3139-FB</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>Growth factor</td>
<td>Sigma</td>
<td>H4784</td>
<td>Reconstituted in PBS</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			<p class="jove_content">*To make HEM, mix 1&#215;10L packet of MEM and160ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4&deg;C.</p>		</div>
		

establishing embryonic mouse neural stem cell culture using the neurosphere assay

<p class="jove_content">In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These <em>in vitro</em> expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.</p>

Watch this Scientific Journal Video about Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay at JoVE.com

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

<p class="jove_content">This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.</p>

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

establishing-embryonic-mouse-neural-stem-cell-culture-using

Research

JoVE Journal

Neuroscience

24.5K visualizações.  Shiraz University of Medical Sciences, Shiraz, Iran. Este protocolo de vídeo demonstra a aplicação do ensaio neurosphere para o isolamento e expansão de células-tronco neurais do eminências ganglionares do dia 14 de embriões de rato-cérebro.