Name: isolation and culture of endothelial cells from the embryonic forebrain
Uploaded: 2014-01-23T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Endothelial Cells from the Embryonic Forebrain at JoVE.com

这项工作是由国家联盟研究精神分裂症与抑郁症（NARSAD）青年研究者奖和国家卫生研究所授予R01NS073635到AV支持。

1。试剂和溶液的制备<ol><li> 35毫米培养皿中涂：胶原蛋白1型溶液是作为在20mM醋酸（〜100 mg蛋白质/瓶）的水溶液。稀释的胶原溶液适当体积的0.01％用无菌组织培养级水的工作浓度。外套菜用1毫升3-4小时胶原溶液在室温（RT）或37℃，或隔夜在2-8°C。从涂层菜除去多余的溶液，并允许它干通宵。添加介质冲洗前用组织培养级水或PBS的菜。包被的培养皿可以被存储在4℃下一个月。 </li><li>准备完整?...

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	<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>

PVEC的比成年人的大脑内皮细胞和内皮细胞的其他组织来源的研究侧重于神经血管互动更加生理有关，也有治疗潜力。对于PVEC的准备，关键是开始与清扫工作速度快，实现了良好的生存能力，因为死细胞可以非特异性结合CD31微珠。此外，如果单细胞悬浮液之前不与磁性标签的步骤实现的，这将导致故障，因为细胞团块会堵塞柱子。这种方法的一个优点是，没有必要对孵育，用胰蛋白酶对从磁珠分...

血管生成，神经发生和神经细胞迁移是在中枢神经系统（CNS）的开发，修复和再生的关键事件。几个雅致的研究表明，内皮细胞通过可溶性因子释放和通过直接接触刺激神经细胞的增殖，反之亦然。我们发现它奇怪的是，在这些研究大部分1-3中，虽然神经祖细胞/神经干细胞是从胚胎中分离脑，它们被共培养从成年脑，其它成体组织来源的，或与内皮细胞系内皮细胞。这可能部分是由于从胚胎脑分离和培养内皮细胞纯人口相关的技术困难。但是，血管生成，神经发生和神经元迁移是发生在幅度更健壮的胚胎大脑的命令比在正常成人大脑并发事件。该embryoni的脑室周围血管网Ç前脑（端脑）来源于位于基底节原基内的容器中，并在从腹侧的有序梯度的形式开发由胚胎11天（E11）4,5至背端脑。脑室周围血管网的血管丛这是基于出身，解剖部位，生长模式和发育调控从4,5软脑膜血管显着。脑室周围血管生成渐变的传播方向的端脑的横向神经源性梯度相匹配。在端脑，脑室周围血管梯度和GABA神经元迁移的梯度沿切线方向重叠空间，以及6。关于时机，血管生成梯度提前神经源性梯度和GABA神经元梯度由大约一天。因此，脑室周围内皮细胞在空间上和时间上很好地提供关键线索，以支持端脑和神经神经细胞迁移4,6。因此，利用胚胎脑室周围内皮细胞共培养的实验与神经元祖细胞和/或神经元会为研究神经血管的相互作用和开发新的途径治疗神经退行性疾病或缺血/创伤性脑损伤的一个更有利的模式。 我们强调除去脑膜膜的重要性，不仅限制上皮细胞污染，而且分开的软脑膜血管内皮细胞，其分子和从脑室周围血管网4,6（称为PVECs从软脑膜内皮区分）的内皮细胞的功能不同的。在这里，我们描述了我们经常使用在我们的实验室获得了丰富而纯净的PVECs产量的方法。这些内皮细胞是从胚胎前脑从单一的定时怀孕的小鼠中分离制得。它们可以被扩大，传代培养，并冷冻下成功地供将来使用。

<table border="0" cellpadding="0" cellspacing="0" width="768">
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		<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

胚胎脑内皮细胞可以作为血管生成和血管神经发育和相互作用的研究中的重要工具。胚胎前脑，脑膜及脑室周围两个血管网络，在空间上是独特的，有着不同的起源和增长模式。从软脑膜和脑室周围血管网内皮细胞具有独特的基因表达和功能。在这里，我们提出了一个一步一步的协议进行分离，培养，和验证从胚胎前脑（端脑）的脑室周围血管网（PVECs）内皮细胞的纯种群。在这种方法中，端脑缺乏从胚胎第15天的小鼠获得软脑膜膜被剁碎，用胶原酶消化/分​​散酶，和分散机械成单细胞悬液。 PVECs从细胞悬液，用积极的选择与anti-CD-31/PECAM-1抗体结合到微珠使用强磁性纯化分离方法。纯化的细胞在胶原1涂布的培养皿在内皮细胞培养基中培养，直到它们变得汇合，并进一步传代培养。 PVECs本协议展览鹅卵石和梭形表型获得，如通过可视化相衬光学显微镜和荧光显微镜。建立与血管内皮细胞标记PVEC文化的纯洁性。在我们的手中，这种方法可靠，一致得到PVECs纯人群。该协议将有利于研究旨在获得机械见解脑血管，了解PVEC的相互作用，并与神经细胞类型的交叉谈判，并拥有巨大的潜力，治疗性血管生成。

从1-12天PVECs的表型特征示出由相位对比光学显微镜（ 图2）。连接到第1天秀形态的细胞分裂（ 图2A）的特性菜的细胞。间5-8天，从鹅卵石PVECs过渡到梭形形态典型为内皮细胞和更类似于其在体内的状态（ 图2B和2C）。通过12天的PVEC文化达到充分融合（ 图2D）。 PVECs可以很容易地传代培养，扩大殖民地（ 图2E-G）。建?...

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

该视频演示了一个简单而可靠的战略准备内皮细胞从内10-12天的胚胎前脑的纯培养物，并将成为研究主要集中在脑血管的许多方面非常有用。

从胚胎前脑内皮细胞的分离和培养

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

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Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

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

科研

神经科学

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

从人类毛囊神经嵴干细胞的分离和培养的

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

基于凝集素的分离和培养的小鼠胚胎运动神经元

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

从产前小鼠海马神经元的分离和培养

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

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

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

从小鼠胚胎神经管的神经嵴细胞的分离和培养

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

原代鼠脑微血管内皮细胞的分离

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

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

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

分离和成人神经干细胞的培养从鼠标Subcallosal区

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