Name: isolation and large scale expansion of adult human endothelial colony forming progenitor cells
Uploaded: 2009-10-28T07:00:00
Description: Watch this Scientific Journal Video about Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells at JoVE.com

De auteurs danken dr. Katharina Schallmoser voor het verstrekken van pHPL en Margaretha Frühwirth en Daniela Thaler voor een uitstekende technische ondersteuning.

A.) ECFC ISOLATON DAG 1: <ol><li> Voordat u begint met het experiment voor te bereiden de celkweekmedium Endothelial Growth Medium-2 (EGM-2). Supplement 500ml Endotheliale basismedium-2 met heparine (1 ml van 1000 U / ml), 5 ml 100x oplossing van penicilline / streptomycine (alle Sigma, St. Louis, MO, <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L-glutamine (2 mM eindconcentratie ), en 50 ml gepoolde humane bloedplaatjes lysaat (pHPL). Voeg vervolgens 0,2 ml hydrocortiso...

<ol>
	<li>Solovey, A., Lin, Y., Browne, P., Choong, S., Wayner, E., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+activated+endothelial+cells+in+sickle+cell+anemia.">Circulating activated endothelial cells in sickle cell anemia.</a> N Engl J Med. 337, 1584-1590 (1997).</li><li>Yoder, M. C., Mead, L. E., Prater, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Schallmoser, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+large-scale+expansion+of+functional+mesenchymal+stem+cells+from+unmanipulated+bone+marrow+without+animal+serum.">Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.</a> Tissue Eng Part C Methods. 14, 185-196 (2008).</li><li>Reinisch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+large-scale+expanded+endothelial+colony-forming+cells+function+in+vitro+and+in+vivo.">Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo.</a> Blood. , (2009).</li><li>Ricardo, R., Phelan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+and+Determining+the+Viability+of+Cultured+Cells.">Counting and Determining the Viability of Cultured Cells.</a> J Vis Exp. , (2008).</li></ol>

Deze nieuwe isolatie en uitbreiding methode is een eenvoudig en efficiënt proces dat is efficiënter en minder tijdrovend door het vermijden van elke cel scheiding (geen dichtheidsgradiënt nodig) en minder duur dan conventionele technieken. Door gebruik te maken pHPL contact met runderen antigenen en vermeende daaropvolgende xenoimmunization is uitgesloten. Gedurende de periode van cultuur kleine stolsels kunnen vormen, veroorzaakt door de pHPL. Gebaseerd op onze ervaring van deze stolsels geen effect kolonie vorming ...

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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>Endothelial basal Medium-2</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>500ml</td>
</tr>
<tr>
<td>EGM-2 SingleQuots</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>EGF, hFGF-B, VEGF, IGF-1, Hydrocortisone, ascorbic acid</td>
</tr>
<tr>
<td>preservative-free Heparin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>&nbsp;</td>
<td>10U/ml per EGM-2</td>
</tr>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>2mM per EGM-2</td>
</tr>
<tr>
<td>Penicillin and Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>&nbsp;</td>
<td>100U/mL Penicillin and 100&mu;g/mL Streptomycin per EGM-2</td>
</tr>
<tr>
<td>Trypsin/1mM EDTA </td>
<td>&nbsp;</td>
<td>Gibco Cell Culture, Invitrogen </td>
<td>&nbsp;</td>
<td>7ml for T75 flask 70ml for CF-4</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Four-layered cell factory (CF-4) </td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Funnel</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>T75cm2 culture flask (with vented cap)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 mL vacutainer tubes (preservative-free sodium heparin) </td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>&nbsp;</td>
<td>preloaded with 108 IU/6ml of preservative-free sodium heparin</td>
</tr>
<tr>
<td>50ml Falcon tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>5ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>25ml Stripetts</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Squeezer</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0,2 pore size sterile filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>2x 500ml per EGM-2</td>
</tr>		</tbody>
		</table>
		</div>

isolation and large scale expansion of adult human endothelial colony forming progenitor cells

Deze paper introduceert een nieuwe recovery strategie voor de endotheliale kolonievormende progenitor cellen (ECFCs) van heparine, maar anders ongemanipuleerde volwassen menselijk perifeer bloed binnen een gemiddelde van 12 dagen. Na de grootschalige expansie&gt; 1x10 8 ECFCs kan worden verkregen voor verdere tests. Bij voorkeur door gebruik te maken pHPL het contact van menselijke cellen met bovine serum antigenen kunnen worden uitgesloten. Door flow cytometrie en immunohistochemie de geïsoleerde cellen kan worden gekarakteriseerd als ECFC en hun in vitro functionaliteit om vasculaire vorm als structuren kunnen worden getest in een Matrigel assay. Verder deze cellen kunnen subcutaan worden geïnjecteerd in een muismodel voor functionele, perfusie schepen in vivo te vormen. Na een lange termijn uitbreiding en cryopreservatie proliferatie, functie en genomische stabiliteit lijken te worden behouden. 3,4 Dit dier-eiwit vrij isolatie en uitbreiding methode is gemakkelijk toepasbaar op een grote hoeveelheid ECFCs te genereren.

Watch this Scientific Journal Video about Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells at JoVE.com

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

Endotheliale kolonievormende progenitor cellen (ECFCs) zijn een veelbelovend instrument om vasculaire homeostase en reparatie te bestuderen.<sup&gt; 1,2</sup&gt; Dit artikel introduceert een nieuwe dier-serum vrije methode voor de isolatie en de uitbreiding van ECFC van gehepariniseerd volwassen humaan perifeer bloed met gepoolde humane bloedplaatjes lysaat (pHPL) het verminderen van het risico van anti-bovine immunisatie.

Isolatie en grootschalige expansie van volwassen humane endotheelcellen kolonievormende Progenitor Cellen

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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