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>准备完整的Dulbecco改良的Eagle培养基（DMEM）。补充的DMEM培养基（500ml）中，用10％FBS和抗生素 - 抗真菌剂溶液（1倍浓度; 1 ml/100毫升）。完全DMEM可以储存在4℃下2个月。 </li><li>制备的等分试样（50ml）中的DMEM与DNA酶I（0.001毫克/毫升），（简称为DMEM-DNA酶I）。 </li><li>制备的DMEM另一等份（10毫升）用胶原酶和分散酶（1毫克/毫升）（简称DMEM-collagenase/dispase）。 </li><li>制备内皮细胞培养基（ECCM，500毫升）中，补充有EGF（5微克），ECGS（100mg）和5微升的抗生素 - 抗真菌剂溶液。 ECCM可以储存在4℃下2个月。 </li><li>使用前Prewarm所有媒体。 </li><li>制备纯化缓冲液：PBS，pH 7.2的含有0.5％BSA和2mM EDTA中。保留缓冲区冷（2-8℃）。 </li></ol> 2。去除胚胎端脑和夹层使用实验动物的所有实验都是由麦克林医院的动物护理和使用委员会批准，并符合实验动物的护理和使用美国国立卫生研究院的指导方针。 <ol><li>使用定时怀孕CD1小鼠（胚胎15天）。阴道塞发现之日被认为是0天胚胎（E0）。 CD1水坝通常会产生较大的窝，使胚胎10-12S是从怀孕坝预期。 </li><li>对于子宫切开术，麻醉孕鼠腹腔注射氯胺酮（100毫克/千克）/甲苯噻嗪（5毫克/千克）。对于小鼠约20-30克，交付0.3 10毫升/千克氯胺酮/甲苯​​噻嗪麻醉的溶液，并确保疼痛，并在麻醉剂的腹膜内注射窘迫是最小的。用手克制为注射剂。 </li><li>由脚趾捏检查深度麻醉。从深度麻醉小鼠的胚胎取出。取出后立即从母亲斩首每个胚胎。将胚胎头在无菌培养皿中冰冷的PBS。 </li><li> 2013版：通过直接安乐死与麻醉剂过量（130毫克/千克戊巴比妥，IP），安乐死，这是与动物的安乐死AVMA准则相一致的方法，按照子宫切除术。 </li><li>在立体显微镜，从头部取出大脑细微尖剪刀和细镊子。注：一个高品质的解剖离子是必不可少的成功从胚胎脑取出软脑膜膜。这是通过实践来实现。细镊子和剪刀微尖也很关键工具，获得良好的解剖。 </li><li>使用镊子，以获得有关大脑和微尖剪刀牢牢把握剥开软脑膜膜。取出脑和后脑和隔离端脑。所有的胚胎软脑膜转移无端脑为35 mm培养皿用2毫升的PBS在冰。 </li></ol> 3。细胞分离<ol><li>百果端脑到1-2毫米的碎片用手术刀刀片和在含2 ml完全DMEM的15毫升猎鹰管收集组织。 </li><li>轻轻但彻底分离组织与1毫升吸管，直到团块消失，乳白色混悬液的实现。 </li><li>离心分离细胞在800-1000×g离心5分钟，在室温。 </li><li>小心地取下的DMEM-DNA酶一，上清，重悬沉淀轻轻分离的CELLS再次用1ml移液器和离心机在800-1000×g离心5分钟，在室温。 </li><li>重悬沉淀在温暖的完全DMEM。通过无菌70μm的尼龙网过滤细胞。过滤收集细胞，并保持在冰上。 </li><li>收集的细胞的部分保留在网和在RT下1-2分钟，使用DMEM-collagenase/dispase（2毫升）进一步消化。在DMEM-DNA酶I通过离心800-1000×g离心5分钟，在室温洗涤细胞3倍。汇集这些细胞收集在步骤3.5中的过滤单元。用完全DMEM洗一次细胞总数。继续进行以确定细胞数量和磁性标签的步骤。注：获得磁标记之前的单细胞悬浮液是重要的。 </li></ol> 4。磁性标签<ol><li>这个协议显示10 7个细胞的磁性标记。为细胞数低于10 7，使用相同的试剂量和细胞数大于10 7，扩展所有试剂体积和总体积相应地（ 例如 2×10 7细胞总数，使用所有显示的试剂量两次）。保持在冰中的所有试剂和细胞。 </li><li>确定与血球细胞数。 </li><li>离心细胞悬浮液在300×g下在室温下10分钟。吸上清完全。 </li><li>加90μl/107细胞的纯化缓冲液将细胞沉淀，接着用10微升CD31微珠的。由制造商提供的微珠偶联单克隆抗小鼠CD31抗体。 </li><li>拌匀，静置在冰箱15分钟。注意：较高的温度和/或磁标记中较长的孵育时间可能会导致非特异性结合。 </li><li>通过在300×g离心10分钟，加入1-2 ml/10 7细胞的纯化缓冲液和离心洗细胞。于500μl纯化缓冲取出上清，重悬细胞。进行到磁性分离或纯化步骤。 </li></ol> 5。浦rification <ol><li>放置MS柱中的MACS分离器的磁场。 </li><li>冲洗柱，用500微升的纯化缓冲液中。 </li><li>应用细胞悬液上柱。装入电池的适当数量的MS柱为较高的细胞数可能会阻塞列。注意：每个MS柱可容纳2×10 7个细胞的最大数目，以更高的细胞数，使用更多的列。 </li><li>收集并丢弃流过含有未标记细胞。 </li><li>用500μL的纯化缓冲液3次洗柱。在洗涤步骤，确保该列水库是将后续的纯化缓冲液分装前空。 </li><li>从MACS分离器除去柱，并将其放置在15毫升猎鹰管。 </li><li>柱塞沿与MS列提供。移液管加入1ml净化缓冲到MS柱，并立即冲洗掉磁标记的细胞紧紧推柱塞压入列。 </li><li> CEL板LS在35毫米培养皿中预先涂有胶原蛋白1型和在培养箱设定为37℃，用95％O 2和5％CO 2培养细胞中ECCM。 </li><li>改变介质每四天。使用PVECs用于实验或传代培养后10-12天，当菜是融合。 </li></ol> 6。光伏精英的亚文化<ol><li>取出ECCM和冲洗PVECS的单层用1-2毫升无菌PBS。 </li><li>轻轻地加入1.5毫升0.25％胰蛋白酶-EDTA溶液以覆盖细胞单层。 </li><li>返回菜的孵化器。在5-7分钟，将细胞分离从盘的表面上。 </li><li>后的细胞脱离，马上添加胰蛋白酶抑制剂的等体积的和磨碎几次。 </li><li>细胞转移到无菌的15毫升猎鹰管。离心管中，在500×g离心5分钟。 </li><li>去除上清，重悬沉淀于1毫升预热抗干扰的。 </li><li>种子细胞扩大培养，IM老化或其他实验。 </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>

没有利益冲突的声明。

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</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

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 ...

isolation-culture-endothelial-cells-from-embryonic

Research

JoVE Journal

Neuroscience

15.3K 次观看.  Harvard Medical School. 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。 

视频: 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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 ...

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.