Name: isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs
Uploaded: 2010-03-22T17:30:00
Description: Watch this Scientific Journal Video about Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs at JoVE.com

The work was supported in part by National Institute of Health (R01GM079688, R21RR024439, and R21GM075838), National Science Foundation (CBET 0941055), and the MSU Foundation.

1. Isolation of Rat MSCs
Mesenchymal stem cells were isolated from 6-8 week old Sprague-Dawley female rat as previously described 1, 2. Isolated MSCs can adhere to plastic surface and easily expand during in vitro culture.
<ol>
<li> The animal was put into an anaesthesia chamber and anaesthetized for around five minutes. During the anaesthesia, observe the rate of blinking, breathing and motor activity. Remove the animal from the chamber immediately after it stops motor activit...

<ol>
	<li>de Hemptinne, I., Vermeiren, C., Maloteaux, J. M., Hermans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+glial+glutamate+transporters+in+adult+mesenchymal+stem+cells.">Induction of glial glutamate transporters in adult mesenchymal stem cells.</a> J Neurochem. 91, 155-166 (2004).</li><li>Porter, R. M., Huckle, W. R., Goldstein, A. S. <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+dexamethasone+withdrawal+on+osteoblastic+differentiation+of+bone+marrow+stromal+cells.">Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells.</a> J Cell Biochem. 90, 13-22 (2003).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <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:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25, 2739-2749 (2007).</li><li>Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells:+from+basic+biology+to+clinical+applications.">Human mesenchymal stem cells: from basic biology to clinical applications.</a> Gene Ther. 15, 109-116 (2008).</li><li>Erica, L., Herzog, L. C., Diane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krause+Plasticity+of+marrow-derived+stem+cells.">Krause Plasticity of marrow-derived stem cells.</a> Blood. 102, 3483-3493 (2003).</li><li>Colter, D. C., Sekiya, I., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+subpopulation+of+rapidly+self-renewing+and+multipotential+adult+stem+cells+in+colonies+of+human+marrow+stromal+cells.">Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells.</a> Proc Natl Acad Sci U S A. 98, 7841-7845 (2001).</li></ol>

This protocol describes how to isolate and enrich MSCs. A method to separate the single-colony derived cells is also incorporated. There are several steps that are important for a successful isolation, enrichment and colony separation. While doing cell isolation from rat, it is recommended to filter through a cell strainer or a sterile nylon mesh of similar size to get rid of the blood clots and bone debris. After plating the cells overnight, many dead cells will be floating in the medium and the dead cells are removed b...

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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>1X D-PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14040-133</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>BD Imagnet</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552311</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG2a</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555572</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG1</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555747</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD54</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL054B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD90</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL005B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD45</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL001B</td>
<td>&nbsp;</td>
<tr>
<td>BD Imag buffer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552362</td>
<td>&nbsp;</td>
<tr>
<td>Round-bottom tube</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin particles</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>557812</td>
<td>&nbsp;</td>
<tr>
<td>SA-PE</td>
<td>&nbsp;</td>
<td>R&amp;D systems</td>
<td>F0040</td>
<td>&nbsp;</td>
<tr>
<td>Cloning cylinder</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2059</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Watch this Scientific Journal Video about Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs at JoVE.com

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

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