Esta pesquisa é apoiada pela concessão de CARREIRA NSF, a Fundação Hartwell, e da American Heart Association (# 0830384N).

1. Preparação <ol><li> Autoclave em um instrumento coberto bandeja os seguintes itens: <ol><li> Pinça serrilhada - Para manuseio do tecido folheto </li><li> Tesoura de tecido (8 cm) - Para corte de tecido e folheto cúspides </li><li> Cotonetes de algodão - Para isolar a camada endotelial do folheto ou cúspide </li></ol></li><li> Fazer a solução de colagenase estéril <ol><li> Adicionar 4,0 gramas de pó DMEM para 250 mL de água de 18 mohms. </li><li> Adicionar 1,11 gramas de bicarbonato de sódio. </li><li> Add (600 U / mL) 180.000 unidades de colagenase. </li><li> Adicionar 1% (3 mL) Penicilina / Estreptomicina. </li><li> Ajustar o pH da solução para 7,2. </li><li> Levar a solução até 270 mL. </li><li> Esterilizar a solução, passando por um filtro de 0,2 mm. </li><li> Adicionar 10% (30ml) de soro fetal bovino estéril. </li></ol></li><li> Faça médio de células endoteliais estéril <ol><li> Adicionar 6,7 gramas de pó DMEM para 400 mL de água de 18 mohms. </li><li> Adicionar 1,85 gramas de bicarbonato de sódio. </li><li> Adicionar (50 U / mL) 25.000 unidades de heparina. </li><li> Adicionar 1% (5 mL) Penicilina / Estreptomicina. </li><li> Ajustar o pH da solução para 7,2. </li><li> Levar a solução até 450 mL. </li><li> Esterilizar a solução, passando por um filtro de 0,2 mm. </li><li> Adicionar 10% (50 ml) de soro fetal bovino estéril. </li></ol></li><li> Faça médio de células estéreis intersticial <ol><li> Adicionar 13,37 gramas de pó DMEM para 800 mL de água de 18 mohms. </li><li> Adicionar 3,7 gramas de bicarbonato de sódio. </li><li> Adicionar 1% (10 ml) Penicilina / Estreptomicina. </li><li> Ajustar o pH da solução para 7,2. </li><li> Levar a solução até 900 mL. </li><li> Esterilizar a solução, passando por um filtro de 0,2 mm. </li><li> Adicionar 10% (100 mL) de soro bovino estéril. </li></ol></li></ol> 2. Isolamento dos folhetos da válvula cardíaca <ol><li> A raiz da aorta especiais de consumo imediato e de forma asséptica do coração após o sacrifício. </li><li> Lave bem a raiz da aorta de sangue com o frio DPBS estéril. É imperativo para remover todos os componentes do sangue o mais rapidamente possível para limitar o VEC morte e contaminação bacteriana. Antibióticos e antimicóticos não são aconselhados, nesta fase, uma vez que são potencialmente prejudiciais para o VEC. </li><li> Isolar folhetos da válvula (3 por válvula) diretamente a partir da raiz e coloque em um tubo cônico 15 mL estéril preenchida com 12 mL DPBS frio. Agite várias vezes para remover os resíduos e reabastecer com DPBS fresco. </li><li> Transporte para o laboratório em gelo. </li><li> À chegada ao laboratório, colocar o recipiente com o tecido sob o capô estéril. </li></ol> 3. Isolamento da camada endotelial <ol><li> Encha um prato estéril 35mm com 3 mL de solução de colagenase frio por válvula (3 folhetos). </li><li> Coloque todos os três folhetos do tubo de 15 mL para o prato cheio com a solução de colagenase. </li><li> Incubar o tecido por 5-10 minutos a 37 ° C. </li><li> Remover suavemente a camada endotelial girando um algodão estéril e seco sobre a superfície do folheto. O sentido de rotação ea quantidade de cisalhamento aplicada é crítica para a pureza de sua amostra. A rotação da haste deve ficar em uma direção oposta ao movimento linear de sua mão criando uma tesoura controlada. Este corte é o que levanta a células endoteliais do tecido. A quantidade de força aplicada deve ser suficiente para sentir a resistência do tecido, mas, não penetram na membrana basal. </li><li> Ocasionalmente, dab o swab dentro da solução de colagenase para desalojar as células das fibras ponta. Depois de esfregar está completa, a textura da camada endotelial deve se sentir um pouco mais suave do que antes. </li><li> Recolher a suspensão de células / colagenase e transferir para um tubo estéril nova 15mL. </li><li> Centrifugar os tubos a 1000 rpm por 5 minutos para sedimentar as células isoladas e aspire o sobrenadante. Se isolar células intersticiais, bem como, realizar esse protocolo, enquanto essas células estão sendo centrifugado. </li><li> Adicionar 3 ml de meio de suínos endoteliais para os 15 tubos mL, centrifugar uma segunda vez, e mídia aspirar. Esta segunda centrifugação ajuda a filtrar alguns dos materiais indesejáveis, tais como fibras de ponta. </li><li> Re-suspender as células endoteliais centrifugadas em 5 mL de meio de suínos endoteliais e placa de as células em um pré-revestido frasco de T-25 com colágeno (use um frasco por tubo de centrifugação). </li><li> Permitir que as células crescer pelo menos 2-3 dias antes de mudar o meio endoteliais. Isto ajuda a recuperar as células e dividir uma vez que o processo de isolamento é bastante dura eo rendimento das células pode ser baixo. É fundamental para as células passagem perto confluência vez que a inibição de contato pode levar à transformação celular. </li></ol> 4. Preparação de 60 Dish Cultura mm para isolamento específico Side <ol><li> A linha de 60 mm de vidro placas de Petri com papel de alumínio (2 pratos de vidro por leaflet). A folha de alumínio ajuda a remover a parafina, de modo que a placa de Petri de vidro pode ser reutilizado para outros isolamentos. </li><li> Lugar contas de parafina dentro dos pratos, cerca de metade cheio, e cobertura para autoclave. </li><li> Uma vez que a autoclave é completo e de parafina é derretida, mova o ensemble prato para uma superfície fria plana. Como a parafina esfria, ele vai endurecer e criar uma camada que irá apoiar punções de agulha. </li><li> Após 30 minutos, o prato esterilizado pode então ser usado como uma câmara de isolamento para imobilizar o tecido folheto. </li></ol> 5. Isolamento de camada específica Side endotelial <ol><li> Remove os folhetos dos tubos 15mL e coloque no prato de cultura preparado 60mm para isolamento específico lado. </li><li> Manipular o folheto de modo que o lado ventricularis é a face para baixo sobre a superfície de parafina deixando o lado fibrosa expostos. Pin das bordas do folheto para expor a camada endotelial. </li><li> Coloque algumas gotas de colagenase frio em cada superfície (para cima-facing) e incubar endoteliais do tecido por 5-10 minutos a 37 ° C. </li><li> Como antes, remover suavemente a camada endotelial girando um algodão estéril e seco sobre a superfície do folheto. O sentido de rotação ea quantidade de cisalhamento aplicada é crítica para a pureza de sua amostra. A rotação da haste deve ficar em uma direção oposta ao movimento linear de sua mão criando uma tesoura controlada. Este corte é o que levanta a células endoteliais do tecido e nada mais. A quantidade de força aplicada deve ser suficiente para sentir a resistência do tecido, mas, não penetram no tecido. </li><li> Ocasionalmente, dab o swab dentro da solução de colagenase para desalojar as células das fibras ponta. Depois de esfregar está completa, a textura da camada endotelial deve se sentir um pouco mais suave do que antes. </li><li> Recolher a suspensão de células / colagenase e transferir para um tubo estéril nova 15mL. Indicam especificidade lado na etiqueta (folhetos da válvula mesmo pode ser agrupados). </li><li> Transferir os folhetos para um prato nova cultura para que o lado ventricularis está agora exposta e repita os passos (5,3-5,6). </li><li> Uma vez que todos suspensão celular / colagenase é coletado, centrifugar os tubos a 1000 rpm por 5 minutos para sedimentar as células isoladas e aspire o sobrenadante. Se isolar células intersticiais, bem como, realizar esse protocolo, enquanto essas células estão sendo centrifugado. </li><li> Adicionar 3 ml de meio de suínos endoteliais para os tubos, centrífuga uma segunda vez, e aspire mídia. Esta segunda centrifugação ajuda a filtrar alguns dos materiais indesejáveis, tais como fibras de ponta. </li><li> Re-suspender as células endoteliais centrifugadas em 5 mL de meio de suínos endoteliais e placa de as células em um pré-revestido frasco de T-25 com colagem (use um frasco por tubo de centrifugação). </li><li> Permitir que as células crescer pelo menos 2-3 dias antes de mudar o meio endoteliais. Isto ajuda a recuperar as células e dividir uma vez que o processo de isolamento é bastante dura. Se você observar o rendimento ser muito baixa, considerar a mudança para um pequeno frasco ou placa bem desde a adesão célula-célula promove o crescimento celular. Lembre-se de passagem perto confluência vez que a inibição de contato pode levar à transformação celular. </li></ol> 6. Isolamento de células intersticiais <ol><li> Encha um tubo de centrifugação estéril 15 mL com 10 mL de solução de colagenase por válvula (3 folhetos). </li><li> Depois de esfregar os folhetos de células endoteliais, imediatamente colocá-los no tubo apropriado 15mL com a solução de colagenase. </li><li> Incubar por aproximadamente 12 a 18 horas (agitar suavemente se desejar). </li><li> Misture delicadamente o tecido degradadas com uma pipeta sorológica, até suspensão de células / colagenase fica homogeneizado. Esta homogeneização ajuda a quebrar o tecido e liberar as células intersticiais. </li><li> Centrifugar o tecido digerido por 5 minutos a 1000 rpm e aspire o sobrenadante. </li><li> Adicionar 5 mL de médio suína intersticial para os tubos de 15mL, centrífuga uma segunda vez, e aspire sobrenadante. </li><li> Re-suspender as células endoteliais centrifugadas em 5 mL de meio de suínos intersticial e placa de as células em um T-75 frasco (use um frasco por tubo de centrifugação). </li><li> Permitir que as células crescer pelo menos 1-2 dias antes de mudar o meio de célula intersticial. Haverá restos de tecido muito mais do que com as células endoteliais, mas, que é esperado. As células também deve crescer a confluência mais rápido que as células endoteliais por causa do rendimento das células e sua natureza. </li></ol> 7. Imagens representante <img src="/files/ftp_upload/2158/2158fig1.jpg" alt="Figura 1" /> Figura 1. A morfologia das células isoladas em 2-3 dias após o isolamento. (A) VEC apresentam uma morfologia típica endoteliais e formar grupos para promover o crescimento. (B) VIC morfologia é semelhante a miofibroblastos que geralmente são fusiformes e espalhe uniformemente em todo o frasco. <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/2158/2158fig2.jpg" alt="Figura 2" /> Figura 2. A morfologia das células isoladas na confluência. (A) VEC apresentam uma morfologia típica endoteliais que geralmente são de paralelepípedos e contactar o crescimento inibido. (B) VIC morfologia é semelhante a miofibroblastos que geralmente são fusiformes e não contato com crescimento inibido. <img src="/files/ftp_upload/2158/2158fig3.jpg" alt="Figura 3" /> Figura 3. As características da função de células isoladas 6. (A) VEC estão associados com altos níveis de captação de LDL acetilada (vermelho). (B) VIC estão associados com baixos níveis de captação de LDL acetilada. <img src="/files/ftp_upload/2158/2158fig4.jpg" alt="Figura 4" /> Figura 4. Marcadores celulares de células isoladas 6. (A) fenótipo VEC é contribuído para coloração positiva para o Fator de Von Willebrand (verde), azul (núcleos). (B) é o fenótipo VIC contribuiu para coloração negativa de Von Willebrand. <img src="/files/ftp_upload/2158/2158fig5.jpg" alt="Figura 5" /> Figura 5. Marcadores celulares de células isoladas. (A) fenótipo VEC é contribuído para coloração negativa para alfa-SMA (verde), azul (núcleos). (B) é o fenótipo VIC contribuiu para coloração positiva para a SMA alpha. <table border="1"><tbody><tr><td> Dissociação Agent </td><td> Dissociação Técnica </td><td> Coleta de célula </td><td> Quantidade de células </td><td> Célula Pureza </td><td> Contaminação </td></tr><tr><td> EDTA (3mm) sem CaCl 2 </td><td> 5, 20, 60 min. antes disso CaCl 2 </td><td> 20, 60, 120 min. antes da coleta </td><td> - </td><td> + / - </td><td> + + + </td></tr><tr><td> EDTA (6mm) sem CaCl 2 </td><td> 5, 20, 60 min. antes disso CaCl 2 </td><td> 20, 60, 120 min. antes da coleta </td><td> + / - </td><td> + </td><td> + + + </td></tr><tr><td> Tripsina-EDTA (0,5 g / L) </td><td> 5, 10, 15 min. antes de desactivação </td><td> Médias coletadas imediatamente </td><td> + </td><td> + </td><td> + + </td></tr><tr><td> Colagenase II (300 U / mL) </td><td> 5, 10, 15 min. antes de desactivação </td><td> Médias coletadas imediatamente </td><td> -, +, + + </td><td> -, + +, + </td><td> + </td></tr><tr><td> Colagenase II (600 U / mL) </td><td> 5, 10, 15 min. antes de desactivação </td><td> Médias coletadas imediatamente </td><td> +, + +, + + + </td><td> + +, + +, - </td><td> - </td></tr></tbody></table> Tabela 1. Resultados preliminares de valvular protocolos de isolamento das células endoteliais.

<ol>
	<li>Thompson, R. P., Fitzharris, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+the+truncus+arteriosus+of+the+chick+embryo+heart:+the+formation+and+migration+of+mesenchymal+tissue.">Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.</a> Am J Anat. 154, 545-556 (1979).</li><li>Johnson, C. M., Fass, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+cardiac+valvular+endothelial+cells+in+culture:+A+relative+deficiency+of+fibronectin+synthesis+in+vitro.">Porcine cardiac valvular endothelial cells in culture: A relative deficiency of fibronectin synthesis in vitro.</a> Lab Invest. 49 (5), 589-598 (1983).</li><li>Manduteanu, I., Popov, D., Radu, A., Simionescu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calf+cardiac+valvular+endothelial+cells+in+culture:+production+of+glycosaminoglycans,+prostacyclin+and+fibronectin.">Calf cardiac valvular endothelial cells in culture: production of glycosaminoglycans, prostacyclin and fibronectin.</a> J Mol Cell Cardiol. 20 (2), 103-118 (1988).</li><li>Cheunyg, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+isolating+and+purifying+porcine+aortic+valve+endothelial+cells.">Techniques for isolating and purifying porcine aortic valve endothelial cells.</a> JHVD. 17 (6), 674-681 (2008).</li><li>Paranya, G., Vineberg, S., Dvorin, E., Kaushal, S., Roth, S. J., Rabkin, E., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> Am J Pathol. 159 (4), 1335-1343 (2001).</li><li>Butcher, J. T., Penrod, A., Garcia, A. J. M., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+morphology+and+focal+adhesion+development+of+valvular+endothelial+cells+in+static+and+fluid+flow+environments.">Unique morphology and focal adhesion development of valvular endothelial cells in static and fluid flow environments.</a> Arterioscler Thromb Vasc Bio. 24 (1), 1429-1434 (2004).</li><li>Simmons, C. A., Grant, G. R., Manduchi, E., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+heterogeneity+of+endothelial+phenotypes+correlates+with+side-specific+vulnerability+to+calcification+in+normal+porcine+aortic+valves.">Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.</a> Circ Res. 96, 792-799 (2005).</li><li>Butcher, J. T., Tressel, S., Johnson, T., Turner, D., Sorescu, G., Jo, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Profiles+of+Valvular+and+Vascular+Endothelial+Cells+Reveal+Phenotypic+Differences:+Influence+of+Shear+Stress.">Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress.</a> Arterioscler Thromb Vasc Biol. 26, 69-69 (2006).</li><li>Parachuri, S., Yang, J. H., Aikawa, E., Melero-Martin, J. M., Khan, Z. A., Loukogeorgakis, S., Schoen, F. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Pulmonary+Valve+Progenitor+Cells+Exhibit+Endothelial/Mesenchymal+Plasticity+in+Response+to+Vascular+Endothelial+Growth+Factor-A+and+Transforming+Growth+Factor-&#946;2.">Human Pulmonary Valve Progenitor Cells Exhibit Endothelial/Mesenchymal Plasticity in Response to Vascular Endothelial Growth Factor-A and Transforming Growth Factor-&#946;2.</a> Circ Res. 99 (8), 861-869 (2006).</li><li>Shi, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+circulating+bone+marrow-derived+endothelial+cells.">Evidence for circulating bone marrow-derived endothelial cells.</a> Blood. 92, 362-367 (1998).</li><li>Rehman, J., Li, J., Orschell, C. M., March, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+endothelial+progenitor+cells+are+derived+from+monocyte/macrophages+and+secrete+angiogenic+growth+factors.">Peripheral blood endothelial progenitor cells are derived from monocyte/macrophages and secrete angiogenic growth factors.</a> Circulation. 107, 1164-1169 (2003).</li></ol>

Não há conflitos de interesse declarados.

Uma compreensão da biologia valvular tem sido prejudicada por dificuldades técnicas de isolamento e as populações de cultura pura de células endoteliais valvular. Técnicas de isolamento típico envolvem digestão enzimática da matriz subjacente basal ou dissociação química de ligações adesivas endotelial 2,3. Experimentos de isolamento preliminares foram avaliados qualitativamente por diferentes agentes dissociação e períodos de incubação. Os resultados destes experimentos mostraram que EDTA ...

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isolation of valvular endothelial cells

Válvulas do coração é o único responsável por manter o fluxo sanguíneo unidirecional através do sistema cardiovascular. Esses finos, tecidos fibrosos são submetidos a tensões mecânicas significativas como abrir e fechar vários bilhões de vezes ao longo de uma vida. A resistência incrível destes tecidos é devido ao valvular residente endotelial (VEC) e células intersticiais (VIC) que constantemente reparação e remodelação em resposta a sinais locais mecânico e biológico. Apenas recentemente nós começamos a compreender os comportamentos ímpares dessas células, para o qual a experimentação in vitro tem desempenhado um papel fundamental. Particularmente desafiante é o isolamento e cultura de VEC. Cuidado especial deve ser usado a partir do momento em que o tecido é removido do host através de revestimento final. Aqui apresentamos os protocolos para o isolamento direto, o isolamento lado específico, cultura e verificação das populações puras de VEC. Usamos digestão enzimática seguido por uma técnica suave swab raspagem para retirar células da superfície somente. Estas células são então coletadas em um tubo e centrifugado em um pellet. O pellet é ressuspenso em seguida, e banhado em frascos de cultura pré-revestida com colágeno da matriz I. Fenótipo VEC é confirmado pelo contato inibiu o crescimento ea expressão de marcadores endoteliais específicos, tais como PECAM1 (CD31), fator de von Willebrand (vWF), e expressão negativa de alfa-actina de músculo liso (α-SMA). As características funcionais do VEC estão associados com altos níveis de LDL acetilada. Ao contrário de células endoteliais vasculares, VEC tem a capacidade única de transformar em mesênquima, que normalmente ocorre durante a formação embrionária válvula 1. Isso também pode ocorrer durante significativamente confluentes pós prolongada na cultura in vitro, assim que o cuidado deve ser feito para passagem em ou perto de confluência. Após o isolamento VEC, populações puras de VIC podem ser facilmente adquiridos.

Watch this Scientific Journal Video about Isolation of Valvular Endothelial Cells at JoVE.com

Isolation of Valvular Endothelial Cells

Nós fornecemos um método para isolar e populações de cultura pura de células endoteliais válvula cardíaca (VEC). VEC pode ser isolado de ambos os lados da cúspide ou folheto e imediatamente a seguir, a célula intersticial subjacente isolamento (VIC) é simples.

Isolamento de Valvular Células Endoteliais

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Research

JoVE Journal

Biology

20.2K visualizações.  Cornell University. Nós fornecemos um método para isolar e populações de cultura pura de células endoteliais válvula cardíaca (VEC). VEC pode ser isolado de ambos os lados da cúspide ou folheto e imediatamente a seguir, a célula intersticial subjacente isolamento (VIC) é simples.

Vídeo: Isolation of Valvular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Isolamento de células endoteliais da veia umbilical humana (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biologia

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

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

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 ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolamento e Cultura da Avian embrionárias Células Progenitoras Valvular

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Isolamento de Células Embrionárias ventriculares endoteliais

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

O isolamento de células endoteliais de Murino Válvula

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Isolamento de células de camundongo Coronária endoteliais

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Isolamento e cultura de células endoteliais aórticas primárias de suínos em miniatura

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Isolamento e Caracterização de Células Endoteliais Hemorlicais do Fígado Primário do Rato

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Methods Article

Isolamento de Valvular Células Endoteliais