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

Os autores agradecem a Dra. Katharina Schallmoser para fornecer pHPL e Margaretha Frühwirth e Daniela Thaler para assistência técnica excelente.

A.) ECFC ISOLATON DIA 1: <ol><li> Antes de começar a preparar com a experiência da célula endotelial Crescimento meio de cultura Médio-2 (EGM-2). Suplemento 500ml endotelial Basal Medium-2 com heparina (1ml de 1000 U / ml), solução de 100x 5ml de penicilina / estreptomicina (todos Sigma, St. Louis, MO; <a href="http://www.sigmaaldrich.com/">www.sigmaaldrich.com</a> ), L-glutamina (concentração final de 2 mM ), e 50 ml de lisado de plaquetas agrupadas humano (pHPL). Em seguida, adicion...

<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>

Este isolamento romance e método de expansão é um processo simples e eficiente que é mais eficiente e menos morosa, evitando qualquer separação de células (sem gradiente de densidade necessário) e menos caro do que as técnicas convencionais. Por meio de contato pHPL aos antígenos bovina e xenoimmunization subseqüentes putativa é excluído. Durante o período de cultivo pequenos coágulos podem se formar, causada pela pHPL. Baseado em nossa experiência esses coágulos não afetam a formação de colônias e ...

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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

Este trabalho apresenta uma estratégia de recuperação da novela para colônia de células progenitoras endoteliais formando (ECFCs) de heparinizado mas de outra maneira não-manipulado sangue periférico humano adulto dentro de uma média de 12 dias. Após a expansão em grande escala&gt; 1x10 8 ECFCs pode ser obtido para mais testes. Vantajosamente usando pHPL o contato de células humanas com antígenos soro bovino pode ser excluída. Por citometria de fluxo e imunohistoquímica das células isoladas podem ser caracterizadas como ECFC e sua funcionalidade vitro para formar estruturas vasculares, como pode ser testado em um ensaio de Matrigel. Além disso essas células podem ser subcutânea em um modelo de mouse para forma funcional, vasos perfundidos in vivo. Após a expansão a longo prazo e proliferação de criopreservação, função e estabilidade genômica parecem ser preservada 3,4. Este isolamento de proteína animal-livre e método de expansão é de fácil aplicação para gerar uma grande quantidade de ECFCs.

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

Endotelial colônia formando células progenitoras (ECFCs) são uma ferramenta promissora para o estudo da homeostase vascular e reparação.<sup&gt; 1,2</sup&gt; Este artigo introduz um novo método de soro animal-livre para isolamento e expansão de ECFC heparinizado de sangue humano adulto periférica com pool de lisado de plaquetas humanas (pHPL) diminuindo o risco de anti-bovino imunização.

Isolamento e expansão em grande escala de Adulto Humanos endotelial Colony Células Progenitoras Forming

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

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