Este trabalho foi apoiado por uma Aliança Nacional para a Pesquisa sobre Esquizofrenia e Depressão (NARSAD) Young Investigator Award e National Institutes of Health conceder R01NS073635 a AV.

1. Preparação de reagentes e soluções <ol><li> Revestimento de 35 mm de prato de cultura: O colagénio tipo 1 solução é fornecida como uma solução aquosa de ácido acético 20 mM (~ 100 mg de proteína / tubo de ensaio). Dilui-se um volume adequado de solução de colagénio a uma concentração de trabalho de 0,01% a partir da cultura de tecido estéril, de grau água. Pratos de revestimento com 1 ml de solução de colagénio durante 3-4 horas à temperatura ambiente (TA) ou a 37 ° C, ou durante a noite a 2-8 ° C. Retire o excesso de solução do prato revestido e deixe-a secar durante a noite. Lave o prato com água grau cultura de tecido ou PBS antes de adicionar mídia. Placas revestidas podem ser armazenadas a 4 ° C durante um mês. </li><li> Prepare Eagle Modificado por Dulbecco completo (DMEM). Suplementar o meio DMEM (500 ml) com 10% FBS e solução de antibiótico-antimicótico (concentração 1x; 1 ml/100 ml). DMEM completo pode ser armazenado a 4 ° C durante dois meses. </li><li> Prepara-se uma alíquota (50 ml) de meio DMEM com DNase I (0,001 mg / ml), (referido como DMEM-DNase I). </li><li> Preparar outra alíquota (10 ml) de meio DMEM com colagenase e dispase (1 mg / mL) (referido como DMEM-collagenase/dispase). </li><li> Prepare celular endotelial Meio de Cultura (ECCM, 500 ml), suplementado com EGF (5 ug), ECGS (100 mg) e 5 ul de solução de antibiótico-antimicótico. ECCM pode ser armazenado a 4 ° C durante dois meses. </li><li> Pré-aquecer todos os meios antes de usar. </li><li> Preparar tampão de purificação: PBS, pH 7,2, com 0,5% de BSA e EDTA a 2 mM. Manter tampão frio (2-8 ° C). </li></ol> 2. Remoção de Embriões e Dissecção da Telencéfalo Todos os experimentos com animais de laboratório são aprovados pelos cuidados com os animais e uso de comitês de McLean Hospital e em conformidade com as diretrizes do NIH para o cuidado e uso de animais de laboratório. <ol><li> Use ratinhos CD1 grávidas cronometrados (dia embrionário 15). O dia de tampão vaginal descoberta é considerado embrionário dia 0 (E0). Barragens CD1 geralmente produzem grandes ninhadas, então 10-12 embriãos são esperados a partir de uma barragem grávida. </li><li> Para histerotomia, anestesiar ratas grávidas com uma injecção intraperitoneal de cetamina (100 mg / kg) / xilazina (5 mg / kg). No caso dos ratos, aproximadamente 20-30 g, entregar 0,3 ml de uma solução anestésica de 10 ml / kg de cetamina / xilazina e assegurar que a dor e desconforto é mínima durante as injecções intraperitoneais de anestésico. Use restrição de mão para as injeções. </li><li> Verifique anestesia profunda por toe pitada. Retirar embriões de ratinhos anestesiados profundamente. Decapitar cada embrião imediatamente após a remoção da mãe. Coloque as cabeças embrionárias em placas de Petri estéreis em PBS gelado. </li><li> Siga histerectomia por eutanásia imediata com overdose de anestésico (130 mg / kg de pentobarbital, ip), um método de eutanásia, o que é consistente com as diretrizes AVMA para a eutanásia de animais: 2013 Edition. </li><li> Sob um microscópio estéreo, retirar o cérebro da cabeça com uma tesoura MicroTip finas e uma pinça fina. Nota: A dissecar alta qualidadeião é essencial para remover com sucesso o pial membrana do cérebro embrionário. Isto é conseguido através da prática. Uma pinça fina e tesoura MicroTip também são ferramentas fundamentais para a obtenção de uma boa dissecção. </li><li> Use a pinça para obter um controle firme sobre o cérebro e as tesouras MicroTip para descascar a membrana pial. Retire o mesencéfalo e metencéfalo e isolar o telencéfalo. Transferir telencéfalo pial-livre de todos os embriões para a 35 milímetros prato de cultura com 2 ml de PBS em gelo. </li></ol> 3. Isolamento celular <ol><li> Picar o telencéfalo em fragmentos de 1-2 mm com uma lâmina de bisturi e recolher o tecido em um tubo Falcon de 15 ml contendo 2 ml de DMEM completo. </li><li> Dissociar tecido suave mas completamente com uma pipeta de 1 ml até aglomerados desaparecer e uma suspensão leitosa é alcançado. </li><li> Centrífuga células dissociadas em 800-1.000 xg durante 5 min à temperatura ambiente. </li><li> Remova cuidadosamente o sobrenadante e ressuspender o pellet em DMEM-DNase I. Gently dissociar a cells novamente utilizando uma pipeta ml e centrifugar a 800-1.000 xg durante 5 min à temperatura ambiente. </li><li> Ressuspender o sedimento em DMEM completo quente. Células filtrar através de uma malha de nylon estéril 70 mM. Coletar as células filtradas e manter em gelo. </li><li> Recolher as porções de células retidas na malha e digerir ainda mais com DMEM-collagenase/dispase (2 ml) durante 1-2 minutos à temperatura ambiente. Lavar as células 3x em DMEM-ADNase I através de centrifugação a 800-1.000 xg durante 5 min à temperatura ambiente. Agrupar as células com as células filtrados recolhidos no passo 3.5. Lave as células uma vez com DMEM totais completa. Prossiga para determinar o número de células e etapas de rotulagem magnéticos. Nota: É importante para se obter uma suspensão de célula única antes da marcação magnética. </li></ol> 4. Rotulagem Magnetic <ol><li> Este protocolo mostra a rotulagem magnética de 10 7 células. Para o número de células inferior a 10 7, use o mesmo volume de reagente e para o número de células superior a 10 7, escala de todos os volumes de reagentes e volumes totaisem conformidade (por exemplo, para 2 x 10 7 células totais, utilizar duas vezes o volume de todos os reagentes indicados). Manter todos os reagentes e as células em gelo. </li><li> Determinar o número de células com um hemocitômetro. </li><li> Suspensão de células de centrifugar a 300 xg durante 10 min à temperatura ambiente. Aspirar o sobrenadante completamente. </li><li> Adicionar 90 μl/10 7 células de tampão de purificação para a pelete de células seguido de 10 ul de MicroBeads CD31. Os Microbeads fornecidos pelo fabricante são conjugados a anticorpo monoclonal anti-CD31 mouse. </li><li> Misture bem e incubar por 15 min na geladeira. Nota: As temperaturas mais elevadas e / ou aumentando o tempo de incubação, durante a etiquetagem magnético pode resultar numa ligação não específica. </li><li> Lavar as células por adição de 1-2 ml/10 7 células de tampão de purificação e centrifugar a 300 xg durante 10 min. Remover as células do sobrenadante e ressuspender em 500 ul de tampão de purificação. Vá para a separação magnética ou etapa de purificação. </li></ol> 5. Purification <ol><li> Coloque coluna MS no campo magnético de MACS Separador. </li><li> Lavar coluna com 500 mL de tampão de purificação. </li><li> Aplicar a suspensão de células para dentro da coluna. Carregar número adequado de células para a coluna MS como o número de células mais elevada pode obstruir a coluna. Cuidado: Cada coluna MS pode acomodar um número máximo de 2 x 10 7 células, para um número de células superior, utilizar mais colunas. </li><li> Coletar e descartar flow-through contendo células não marcadas. </li><li> Lavar coluna com 500 ul de tampão de purificação de três vezes. Durante as etapas de lavagem, certifique-se de que o reservatório da coluna está vazia antes de adicionar alíquotas tampão de purificação posteriores. </li><li> Remover coluna do separador MACS e coloque-o em um tubo Falcon de 15 ml. </li><li> Um êmbolo é fornecido juntamente com a coluna MS. Pipeta 1 ml de tampão de purificação na coluna de MS e imediatamente lavar as células magneticamente rotulados com firmeza empurrando o êmbolo para a coluna. </li><li> Placa celsl em uma placa de cultura de 35 milímetros pré-revestidos com colagénio do tipo 1 e crescer células em ECCM numa incubadora a 37 ° C com 95% de O2 e 5% de CO 2. </li><li> Troca da mídia a cada quatro dias. Use PVECs para experiências ou subcultura após 10-12 dias, quando o prato é confluente. </li></ol> 6. Subcultura de PV ECS <ol><li> Retire o ECCM e lavar a monocamada de PVECS com 1-2 ml de PBS estéril. </li><li> Adicionar cuidadosamente 1,5 ml de 0,25% de solução de tripsina-EDTA para cobrir a monocamada de células. </li><li> Devolver o prato para a incubadora. Dentro de 5-7 minutos, as células irão separar da superfície do prato. </li><li> Após as células terem destacado, imediatamente adicionar um volume igual de inibidor de tripsina e tritura-se várias vezes. </li><li> Transferência das células para um tubo estéril de 15 ml Falcon. Centrifuga-se o tubo a 500 xg durante 5 min. </li><li> Remover o sobrenadante e ressuspender o sedimento em 1 ml de pré-aquecido ECCM. </li><li> Semente das células para a expansão da cultura, imenvelhecimento ou outros ensaios. </li></ol>

<ol>
	<li>Shen, Q., S, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+stimulate+self-renewal+and+expand+neurogenesis+of+neural+stem+cells.">Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.</a> Science. 304, 1338-1340 (2004).</li><li>Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+three-dimensional+system+to+study+interactions+between+endothelial+cells+and+neural+cells+of+the+developing+central+nervous+system.">A novel three-dimensional system to study interactions between endothelial cells and neural cells of the developing central nervous system.</a> BMC Neurosci. 8, 3 (2007).</li><li>Rauch, M. F., Michaud, M., Xu, H., Madri, J. A., Lavik, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+primary+neural+progenitor+and+endothelial+cells+in+a+macroporous+gel+promotes+stable+vascular+networks+in+vivo.">Co-culture of primary neural progenitor and endothelial cells in a macroporous gel promotes stable vascular networks in vivo.</a> 19, 1469-1485 (2008).</li><li>Vasudevan, A., Long, J. E., Crandall, J. E., Rubenstein, J. L., Bhide, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartment-specific+transcription+factors+orchestrate+angiogenesis+gradients+in+the+embryonic+brain.">Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain.</a> Nat. Neurosci. 11, 429-439 (2008).</li><li>Vasudevan, A., Bhide, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+embryonic+CNS:+a+new+twist+on+an+old+tale.">Angiogenesis in the embryonic CNS: a new twist on an old tale.</a> Cell Adh. Migr. 2, 167-169 (2008).</li><li>Won, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+vascular+networks+synchronize+GABA+neuron+migration+in+the+embryonic+forebrain.">Autonomous vascular networks synchronize GABA neuron migration in the embryonic forebrain.</a> Nat. Commun. 4, 2149-2162 (2013).</li><li>Dong, Q. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+strategy+for+isolation+of+endothelial+cells+from+murine+tissues.+Characterization+of+two+endothelial+cell+lines+from+the+murine+lung+and+subcutaneous+sponge+implants.">A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants.</a> Arterioscler. Thromb. Vasc. Biol. 17, 1599-1604 (1997).</li><li>Erblich, B., Zhu, L., Etgen, A. M., Dobrenis, K., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+colony+stimulation+factor-1+receptor+results+in+loss+of+microglia,+disrupted+brain+development+and+olfactory+deficits.">Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.</a> PLoS One. 6, (2011).</li><li>Wichterle, H., Garcia-Verdugo, J. M., Herrera, D. G., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Young+neurons+from+medial+ganglionic+eminence+disperse+in+adult+and+embryonic+brain.">Young neurons from medial ganglionic eminence disperse in adult and embryonic brain.</a> 2, 461-466 (1999).</li><li>Snapyan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculature+guides+migrating+neuronal+precursors+in+the+adult+mammalian+forebrain+via+brain-derived+neurotrophic+factor+signaling.">Vasculature guides migrating neuronal precursors in the adult mammalian forebrain via brain-derived neurotrophic factor signaling.</a> J. Neurosci. 29, 4172-4188 (2009).</li><li>Whitman, M. C., Fan, W., Rela, L., Rodriguez-Gil, D. J., Greer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+form+a+migratory+scaffold+in+the+rostral+migratory+stream.">Blood vessels form a migratory scaffold in the rostral migratory stream.</a> J. Comp. Neurol. 516, 94-104 (2009).</li></ol>

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PVEC do são mais fisiologicamente relevante do que as células endoteliais adultas do cérebro e células endoteliais provenientes de outras fontes de tecidos para estudos sobre interações neurovasculares e também têm potencial terapêutico. Para a preparação PVEC, é princípio fundamental com dissecção de trabalhar rápido para conseguir uma boa viabilidade desde as células mortas pode ligar de forma inespecífica a MicroBeads CD31. Além disso, se a suspensão de uma única célula não é conseguido antes ...

A angiogênese, neurogênese e migração neuronal são eventos críticos no sistema nervoso central (SNC) de desenvolvimento, reparo e regeneração. Vários estudos elegantes mostraram que células endoteliais estimular a proliferação neuronal e vice-versa através da liberação de fatores solúveis e pelo contato direto. Descobrimos que é curioso que na maioria dos estudos 1-3, enquanto progenitoras neuronais / células estaminais neurais são isoladas a partir do cérebro embrionário, que são co-cultivados com células endoteliais do cérebro adulto, outras fontes de tecido adulto, ou com linhas de células endoteliais. Isto pode em parte ser devido aos problemas técnicos associados com o isolamento e cultivo de populações puras de células endoteliais do cérebro embrionário. No entanto, a angiogénese, a neurogénese e migração neuronal são eventos que ocorrem em simultâneo, em ordens de magnitude mais robusto no cérebro embrionário do que no cérebro adulto normal. A rede vascular periventricular do embryonic prosencéfalo (telencéfalo) origina a partir de um navio localizado no primórdio gânglios basais e desenvolve-se na forma de um gradiente de ordenada ventral dorsal telencéfalo por dia embrionário 11 (E11), 4,5. Este plexo de vasos da rede vascular periventricular são distintos dos vasos pial com base em origens, localização anatômica, os padrões de crescimento e regulação do desenvolvimento 4,5. A direção de propagação do gradiente angiogênese periventricular corresponde ao gradiente neurogenético transversal telencefálico. Dentro do telencéfalo, o gradiente de angiogénese periventricular e o gradiente de neurónios GABA migram tangencialmente sobrepõe espacialmente assim 6. No que diz respeito ao tempo, a inclinação da angiogénese é, antes da inclinação e do neurónio GABA gradiente neurogenético por cerca de um dia. Assim, as células endoteliais periventriculares são espacialmente e temporalmente bem posicionada para fornecer pistas importantes para apoiar a neurogênese e telencefálico4,6 migração neuronal. Portanto, o uso de células endoteliais periventriculares embrionárias em experimentos de co-cultura com células progenitoras e / ou neurônios neuronais forneceria um modelo mais favorável para o estudo de interações neurovasculares e desenvolvimento de novos caminhos para o tratamento de doenças neurodegenerativas ou da lesão cerebral isquêmica / traumático. Ressaltamos a importância de remover a membrana pial, não só para limitar a contaminação de células epiteliais, mas também para separar as células endoteliais pial que são molecularmente e funcionalmente distinto de células endoteliais da rede vascular periventricular 4,6 (PVECs denominado para distinguir de pial ECs) . Aqui, descrevemos o método que usam rotineiramente em nosso laboratório para obter um rendimento rico e puro de PVECs. Estas células endoteliais são preparados a partir de prosencéfalo embrionário isolado a partir de um único rato cronometrado-grávida. Eles podem ser expandidas, subcultivadas e congelado para baixo com êxito para utilização futura.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DNase I</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:147px;">D-4527</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagen, Type 1 solution from rat tail </td>
			<td>Sigma</td>
			<td>C3867</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS</td>
			<td>Quality Biologicals</td>
			<td style="width:147px;">114057-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>M4055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td style="width:183px;">Sigma</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS column</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD31 microbeads</td>
			<td>Miltenyi Biotech</td>
			<td>130-097-418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS separator</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS multi stand</td>
			<td>Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer 70 &#181;m</td>
			<td style="width:183px;">BD Bioscience</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic and antimycotic solution</td>
			<td>Sigma</td>
			<td>A5955</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase/Dispase</td>
			<td>Roche</td>
			<td>10269638001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM </td>
			<td style="width:183px;">Lonza</td>
			<td>12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm culture dish</td>
			<td style="width:183px;">BD Bioscience</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml falcon tube </td>
			<td style="width:183px;">BD Bioscience</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml falcon tube</td>
			<td style="width:183px;">BD Bioscience</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">ECCM kit</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">355054</td>
			<td>Kit Includes Endothelial cell growth supplement, EGF and Soybean Trypsin Inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endothelial Cell Growth Supplement (ECGS)</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">354006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RBECGM</td>
			<td style="width:183px;">Cell applications </td>
			<td style="width:147px;">R819-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM F12 </td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">10565-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glutamax</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">305050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">tissue culture grade water</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">15230162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">0.25% Trypsin</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">15050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Soyabean trypsin inhibitor</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">5425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Matrigel</td>
			<td style="width:183px;">BD Bioscience</td>
			<td style="width:147px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qtracker 655 Cell Labeling Kit</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">Q25021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">CellLight Plasma Membrane-RFP, BacMam 2.0</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:147px;">C10608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Biotinylated Isolectin B4 antibody</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:147px;">L2140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Anti-Von Willebrand factor</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:147px;">F3520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Anti-CD31/PECAM-1</td>
			<td style="width:183px;">BD Pharmingen</td>
			<td style="width:147px;">550274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Vectashield Hardset Mounting media with DAPI</td>
			<td style="width:183px;">Vector Laboratories</td>
			<td style="width:147px;">H-1500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Ketamine</td>
			<td style="width:183px;">Butler Schein Animal Health Supply</td>
			<td style="width:147px;">44028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Xylazine</td>
			<td style="width:183px;">Lloyd Laboratories</td>
			<td style="width:147px;">1009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stereomicroscope</td>
			<td style="width:183px;">Motic</td>
			<td style="width:147px;">SMZ-168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">hemocytometer</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:147px;">267110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">inverted microscope</td>
			<td style="width:183px;">Olympus CK-40</td>
			<td style="width:147px;">CK-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flouroscent microscope</td>
			<td style="width:183px;">Olympus FSX-100</td>
			<td style="width:147px;">FSX-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fine forceps</td>
			<td style="width:183px;">Roboz surgical instrument</td>
			<td>7 inox</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Fine microtip scissors</td>
			<td style="width:183px;">Roboz surgical instrument</td>
			<td>RS5611</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of endothelial cells from the embryonic forebrain

Células endoteliais cerebrais embrionárias podem servir como uma ferramenta importante no estudo da angiogênese eo desenvolvimento neurovascular e interações. As duas redes vasculares do cérebro anterior embrionário, pial e periventricular, são espacialmente distinta e têm diferentes origens e padrões de crescimento. As células endoteliais das redes vasculares pial e periventriculares têm perfis de expressão de genes únicos e funções. Aqui apresenta-se um protocolo de passo-a-passo para o isolamento, a cultura, e a verificação de populações puras de células endoteliais a partir da rede vascular periventricular (PVECs) do prosencéfalo embrionário (telencéfalo). Nesta abordagem, telencéfalo desprovido de membrana pial obtido a partir do dia embrionário 15 ratinhos é picada, digeridos com colagenase / dispase e disperso mecanicamente numa suspensão de células individuais. PVECs são purificados a partir de suspensão de células utilizando selecção positiva com anticorpo conjugado com MicroBeads anti-CD-31/PECAM-1 usando um forte magnéticométodo de separação. Células purificadas são cultivadas em colágeno 1 revestidas placas de cultura em meio de cultura de células endoteliais, até que se tornar confluentes e mais subcultivadas. PVECs obtidos com esta exposição de paralelepípedos protocolo e fenótipos fusiformes, visualizadas por microscopia de luz de contraste de fase e microscopia de fluorescência. Pureza de culturas PVEC foi estabelecida com marcadores de células endoteliais. Em nossas mãos, este método confiável e consistente produz populações puras de PVECs. Este protocolo irá beneficiar estudos para obtenção de conhecimentos mecanicistas em prosencéfalo angiogênese, compreender as interacções PVEC e transversais conversações com os tipos de células neuronais e tem um enorme potencial para a angiogênese terapêutica.

A caracterização fenotípica dos PVECs do dia 1-12 é mostrado por microscopia óptica de contraste de fase (figura 2). As células ligadas ao prato no dia 1 mostra a morfologia característica de divisão celular (Figura 2A). Entre 5-8 dias, PVECs transição de pedra de calçada ao fuso morfologia em forma típica de células endoteliais e mais parecido com o seu estado in vivo (Figuras 2B e 2C). Por volta do dia 12, a cultura atinge PVEC c...

Watch this Scientific Journal Video about Isolation and Culture of Endothelial Cells from the Embryonic Forebrain at JoVE.com

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Este vídeo demonstra uma estratégia fácil e confiável para a preparação de culturas puras de células endoteliais do cérebro anterior embrionário dentro de 10-12 dias e será útil para a pesquisa focada em muitos aspectos da angiogênese cerebral.

Isolamento e cultura de células endoteliais da Embryonic posencéfalo

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

isolation-culture-endothelial-cells-from-embryonic

Research

JoVE Journal

Neuroscience

15.3K visualizações.  Harvard Medical School.  Este vídeo demonstra uma estratégia fácil e confiável para a preparação de culturas puras de células endoteliais do cérebro anterior embrionário dentro de 10-12 dias e será útil para a pesquisa focada em muitos aspectos da angiogênese cerebral. 

Vídeo: Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Neurociência

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectina baseado em isolamento e cultura de motoneurônios embrionárias de rato

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e Cultura de neurônios do hipocampo de ratos pré-natal

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolamento e cultivo de células neurais embrionárias de ratos: um protocolo rápido

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolamento e cultivo de células da crista neural do tubo neural embrionário murino

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

Isolamento da Primária Murino Cérebro microvascular células endoteliais

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolamento, cultura e Long-Term Manutenção da Primária mesencefálico Dopaminergic neurônios dos cérebros de roedores Embryonic

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.