这项工作是由多学科临床研究中心（IZKF）蒙斯特（SEED 03/12，SB），由德意志研究联合会（DFG）的支持下，细胞功能于运动卓越的集群（EXC 1003 - CIM），明斯特大学（SB和SGM），并由否则克朗 - 费森尤斯基金会（2012_A88 SB和SGM）。我们感谢海克百隆以出色的插图。

根据动物保护的德国法律，并进行了所有的动物实验经当地主管部门（“雷克灵豪森，德国LandesamtfürNatur，UMWELT UND Verbraucherschutz NRW; Fachbereich 87，Tiergesundheit，Tierschutz”）。 1.一般评论的小鼠实验<ol><li>执行使用小鼠按照各自的机构动物护理和使用委员会的指导方针的所有实验。 </li><li>保持小鼠的无病原体的条件下，使他们能够获得食物和水随意。 </li><li>使用年龄和性别匹配的小鼠实验组，因为生物特性可以与年龄和性别而有所不同。 </li><li>只要有可能尽量减少，改进或替代动物实验。 </li></ol> 2.准备Percoll梯度的<ol><li>混合19毫升的1×PBS中，用1ml 10×PBS中，加入1ml的FCS和10毫升的Percoll。 </li><li>执行STE激怒过滤在玻璃管中（过滤器孔的直径：0.2微米）。之后存储所述溶液在4℃下。 </li></ol> 3.制备内皮细胞培养基和细胞培养板的涂层<ol><li>内皮细胞培养基： <ol><li> 500毫升介质的制备：（约20％）混合400毫升的DMEM培养基中（约80％），用100ml的PDS 0.25 ml的bFGF（20微克/毫升;约0.05％），0.5 ml肝素（100μl/孔毫升，约0.1％）和0.5ml pyrumycin（4毫克/毫升;约0.1％）。添加pyrumycin仅在第2天培养物中使用的细胞培养基。 </li><li>执行在玻璃管中的无菌过滤（过滤器孔的直径：0.2微米）。之后存储所述溶液在4℃下。 </li></ol></li><li>涂层的细胞培养板的： <ol><li> 1毫升涂料溶液，制备500μl的DDH 2 O的，400μl的IV型胶原蛋白（0.4毫克/毫升）和100μl的纤连蛋白（0.1毫克/毫升）的溶液中。 </li><li> EAC覆盖的整个表面ħ良好的细胞培养板用涂布液的。存储该板在4℃下过夜。 </li></ol></li></ol> 4.隔离小鼠脑微血管内皮细胞（MBMEC） <ol><li>牺牲10成年小鼠（8 - 12周开始，C57BL / 6）颈椎脱臼。此后隔离鼠标大脑和存储于5ml的PBS（警告：！工作不育）。 </li><li>除去脑梗，小脑和丘脑用钳子。通过仔细滚动的大脑在无菌吸墨纸分离脑膜。收集所产生的脑膜无前脑。 </li><li>大脑转移到50ml Falcon管中填充有13.5毫升DMEM中。 </li><li>剁碎的组织，先用25毫升移液管，然后用10毫升吸管，直到介质变为乳白色。 </li><li>然后消化的组织具有0.6 ml胶原酶CLS2（10毫克/毫升的DMEM）和0.2毫升DNA酶（1mg / ml的PBS中）在Dulbecco改良的Eagle培养基（DMEM）中进行1小时的混合物中，在37℃在定轨摇R成为180转。 </li><li>加入10 mL的DMEM加入到组织悬浮液和离心将细胞在1000×g离心10分钟，在4℃下。 </li><li>弃去上清液（警告：！小球容易失去） </li><li>以除去髓磷脂，悬浮用25毫升吸管将沉淀在25毫升BSA的DMEM中（20％重量/体积）（约25倍），和离心机在1000×g离心20分钟，在4℃下。 </li><li>弃去上层髓鞘层（乳白色）与碎玻璃巴斯德吸管直径较大，然后弃去BSA层具有正常巴斯德吸管。 </li><li>重悬沉淀9毫升的DMEM和加入1 ml的胶原酶/分散酶（最终浓度为1毫克/毫升）和0.1ml DNA酶。消化以180rpm的溶液1小时，在37℃下于轨道摇床（警告：不使用移液管重悬 - 细胞丢失）。 </li><li>在消化期间，离心的Percoll溶液来设置使用超速离心机的密度梯度，在3000×g离心1小时，在4℃下，用加速度，无线thout刹车。 </li><li>加入10 mL的DMEM至消化的细胞悬液。离心该悬浮液在1000×g离心在4℃下10分钟。然后弃去上清液和悬浮颗粒在2毫升的DMEM。 </li><li>小心添加重悬的细胞在Percoll梯度离心的顶部，在700×g离心10分钟，在4℃下无加速和制动。注：细胞是作为云中的相间可见。 </li><li>拿约。 12毫升相间与细胞用长无菌针头，并将其放置在一个新50毫升隼用5ml DMEM中。 </li><li>再次离心细胞在1,000×g离心在4℃下10分钟。随后重悬在内皮细胞培养基中的沉淀物（用约0.2毫升培养基的每1cm细胞培养板中的2面）。 </li><li>除去从涂覆的细胞培养板涂布液（参见步骤3.2）和冲洗每孔用无菌PBS中连续两次洗涤步骤。 </li><li>保持在内皮细​​胞培养在37℃，细胞培养基和5％的CO 2在无菌恒温箱。每两至三天更换培养基。分裂的细胞在90％汇合。 </li></ol>

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The authors declare that they have no competing financial interests.

血-脑屏障功能障碍的标志的各种有害的CNS疾病的，血脑屏障的例如在多发性硬化的击穿广泛地促进CNS的浸润通过自身反应性免疫细胞和使少突胶质细胞的相遇和破坏。在缺血性卒中血脑屏障的破坏和随后形成的脑水肿是影响二次梗塞生长和患者6的整体存活的关键因素。此外，通过血脑屏障的偏远地区的变化局灶性脑缺血性损伤能够诱导广泛的功能改变的大脑。但是，基本的分子机制知之甚少5。与此相反，高密度和外排转运体的多样性功能BMECs降低的有益药物的浓度，例如化学治疗剂对脑恶性肿瘤或抗生素的感染性疾病。因此，血脑酒吧更深入的了解垒的生理和病理生理条件下的功能，需要开发药物制剂，其能够在偏爱的方向21来专门调节屏障功能。可靠的 BBB 体外模型是不可缺少的工具来研究在BBB的监管机制。 许多永生化的脑内皮细胞系已经建立，并用作体外血脑屏障模型22。这些细胞系提供了主要BMECs一定的优势，因为他们成长得更快，并保持一定的BBB特点在几个段落。然而，内皮细胞系不能完全替代作为重要性质改变，与实验结果的体内情况的可转移性相互混合的原代细胞。例如，小鼠脑内皮细胞株bEnd.5表示不连续分布的紧密连接蛋白的仅低水平导致高paracellular渗透率23。 BEnd.3，另一个常用的小鼠脑内皮细胞系，表明致密，连续分布的紧密连接，高跨内皮电阻，几个外排转运和低旁细胞通透性。然而，bEND.3细胞层相比主BMECs缺乏对于某些跨细胞和细胞旁基板24的透过一个真实歧视。 在原代细胞培养物的制剂中，污染的细胞可以显著与实验结果的有效性产生干扰。在所描述的协议，pyrumicin用作选择剂为内皮细胞，以达到高纯度。然而，细胞培养物的常规质量控制是不可避免的需要，以保证可靠的实验。有几种方法用于质量控制，除了内皮细胞的典型形态特性（ 见图1A），所述transendothelia升阻力可能被测量或内皮细胞标记物的表达或如CD31（参见图1B）血管性血友病因子（vWF）可能被评估。 脑内皮细胞从一个鼠脑分离的数目是相对低的，并建立适当的细胞培养物进行多次实验，一些小鼠的脑中必须被汇集。在我们的经验中，至少10只需要被用于一个实验这可能是一个限制因素，根据各动物设施的资源。为了避免偏差或混杂因素，只小鼠具有相同的遗传和环境背景应该被汇集到一起。与此相反，牛和猪的脑内皮细胞制剂提供了大量在一个脑可用脑内皮细胞。然而，除了简单的滋生和住房，可转基因小鼠的广泛和不断增加的剧目呈现的主要鼠BMECs作为一种理想模型来研究血 - 脑屏障的功能。 关于BBB和分子机制的职能几个主要结果都来自于2D组织培养系统的研究。最近，3D培养系统已被开发25，其中内皮细胞置于一胶原蛋白基质，使他们以形成腔和管状网络内。这些新的细胞培养系统允许更精确再现血管过程中的完整的生物体内 。的NVU在三维细胞培养系统的进一步的细胞，例如周细胞和星形胶质细胞的参与可能发生革命性的体外的血-脑屏障功能研究;第一个模型最近已开发出26。 有故障排除一些建议。首先无菌加工的是对内皮细胞培养物的质量是必不可少的。其次，pyrumycin应该只用于细胞添加在第2天培养物中使用的培养基，再应用程序可能会导致增加的MBMEC细胞死亡，低质量的屏障功能。第三，根据制造商的说明在此协议应用的酶，应使用和储存;否则他们的正常功能可能会受到影响。此外，细胞培养板的涂层可能会修正，如果内皮细胞不重视。纤连蛋白和胶原蛋白的浓度可能会改变或其他基质蛋白可能被添加到涂料溶液中。最后，年龄，性别，对动物实验室内的年份和环境条件的季节是可能影响的MBMEC隔离和实验可比较的质量的重要因素。它应当确保条件可比独立实验之间。 所描述的协议应被认为是一个基本的神经免疫方法以分离鼠BMECs。将分离的细胞可能为Used的为各种各样的应用程序，以进一步了解血脑屏障功能，如迁移测定，基因和蛋白质表达研究，电生理评估或渗透性实验。如上所述，BMECs的三维细胞培养系统可能提供新的机会来研究涉及血脑屏障在NVU的上下文中的调节的复杂机制。因此，初级内皮细胞的分离将仍然是一个非常宝贵的技术，BBB研究，在未来的领域。

<html>脑微血管内皮细胞（脑微血管内皮细胞）形式的单层，它们是高度专业化的血 - 脑屏障（BBB）的一个组成部分。 BMECs由连到基膜连接蛋白互连。与周皮细胞，平滑肌细胞，星形细胞端脚和循环血细胞，他们建立了所谓的神经血管单元（NVU）1。对于血液和中央神经系统（CNS）的之间的不可渗透屏障之前的概念中，NVU是一个动态的，高度特异性的，规范的接口，其控制流体，分子和细胞脑血管和中枢神经系统2之间的过渡。该NVU的功能障碍或失调可发起和/或向多种中枢神经系统的神经血管，感染，炎症性或退化性疾病，如缺血性中风，HIV脑病，多发性硬化症，阿尔茨海默氏症或帕金森氏症3-6。 _content“&gt;的脑微血管内皮细胞单层紧密由一个连接复合体密封的密封（TJ）构成，并且粘着结（AJ）7，高电阻和血脑屏障的低旁细胞渗透性主要基于TJ蛋白8的紧密连接是由密蛋白和occludin的家族的跨膜蛋白，其通过接头分子连接到内皮细胞的细胞骨架，形成的配合物，如在zonulaoccludens（ZO）的蛋白质ZO1-3 4。粘着结主要是组装的完整的膜蛋白血管内皮（VE）-cadherin，这是与通过连接素8的骨架。 血脑屏障的紧密密封防止血液和脑脊液的底物和细胞的自由交流。此规则的例外是亲脂性的，小分子的分子量&lt;400道尔顿，其能够穿过BBB通过脂质介导的扩散9。的通路较大和/或亲水性分子，如葡萄糖，氨基酸，肽，蛋白质和许多药物被限制在高度控制的跨细胞运输系统10，它可以分为五大类：载体介导的转运，离子运输，主动外排转运，受体介导的运输和胞膜窖介导的转运4。这些转运系统有助于维持中枢神经系统，这是需要一个准确的信号的产生，传递和整合的动态平衡。此外，BMECs都能够通过各种ectoenzymes的表达主动地控制不同的分子的过渡。这些酶被定位于细胞表面，并修改特定的内源性和外源性底物阻碍或允许所述BBB 11的过渡。 而中枢神经系统被认为是一种免疫特权器官经过较长时间，最近的研究结果表明相当活跃，并严格监管的免疫监测网系统eillance中枢神经系统。在BMECs正审慎参与免疫细胞轮回的调节。通过在其表面上选择素的淋巴细胞中的表达被选择性诱导的松散附着于内皮。与特定的受体上的白细胞遇到趋化因子的分泌导致的表达或白细胞整联的构象变化，例如LFA-1（淋巴细胞功能相关抗原-1）和VLA-4（极晚期抗原-4）。的整介导通过结合它们的内皮counterreceptors， 例如 VCAM-I（血管细胞黏附分子），ICAM-I（细胞间粘附分子），使得轮回成之间或穿过血脑屏障12-14 BMECs脑实质牢固的粘合。这些和其他的发现强调了内皮细胞自身的调节免疫细胞迁移的活性的作用。 此外，BMECs作为NVU的部分都参与了脑血流里的调节nked局部神经元代谢需要。当星形细胞刺激内皮细胞产生的血管活性物质，如一氧化氮导致血管平滑肌细胞15的松弛。 血管生成和神经发生的显影以及在成人脑中显示平行构图和显影，共享调控1,4的许多特性。内皮细胞是至关重要参与这些过程16，17。 综上所述，BMECs提供必要的功能，以保证中枢神经系统的正常发育和功能。血脑屏障功能障碍与许多严重的神经系统疾病。然而，只有很少的目标已经确定在脑血管系统的界面为特定和有效的处理18。简化的体外模型已被用于理解所涉及的功能和复杂的生理系统的调节对罗机制NG时间。此稿件和鼠BMECs的体外实验描述的那样，考虑到各种特定的基因敲除小鼠，菌株的分离，可能会提供进一步的了解血脑屏障功能和调节生理和病理条件下开辟新的治疗途径下。

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">bFGF</td>
			<td style="width:133px;">Peprotech</td>
			<td style="width:123px;">100-18B</td>
			<td style="width:331px;">Basic fibroblast growth factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">BSA</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">A9418</td>
			<td>Bovine serum albumine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Collagenase CLS2</td>
			<td style="width:133px;">Worthington</td>
			<td style="width:123px;">LS004176</td>
			<td>High relative level of protease activity</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Collagenase-dispase</td>
			<td style="width:133px;">Roche</td>
			<td align="right" style="width:123px;">10269638001</td>
			<td style="width:331px;">Collagenase from V. alginolyticus, Dispase from B. polymyxa</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Collagen IV</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">C0543</td>
			<td style="width:331px;">From Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMEM</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">D5030</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DNAse</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">D4513</td>
			<td>Deoxyribonuclease I from bovine pancreas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCS</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">F6178</td>
			<td>Fetal calf serum (also named fetal bovine serum)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Heparin</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">H3393</td>
			<td>Anticoagulant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">PDS</td>
			<td style="width:133px;">First Link UK Ltd.</td>
			<td style="width:123px;">60-00-850</td>
			<td>Plasma-derived serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Percoll</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">P1644</td>
			<td>Warm to room temperature before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pyrumycin</td>
			<td style="width:133px;">Sigma Aldrich</td>
			<td style="width:123px;">P8833</td>
			<td>Should only be used in first 2 d of culture</td>
		</tr>
	</tbody>
</table>

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.

立即分离后，鼠BMECs和污染细胞形成密集体漂浮在细胞培养基中（ 图1A中 ，第0天）。与pyrumycin治疗导致选择鼠BMECs的，因为它们表达高水平的外排转运体，如P-糖蛋白导致毒素的相对电阻。污染的细胞是更容易pyrumycin介导的细胞毒性19。期间pyrumicin选择的过程中，污染的细胞进入细胞凋亡或坏死性细胞死亡，而MBMECs开始附着到IV型胶原蛋白/纤连蛋白包被的细胞培养板（ 图1A中 ，第1天与第2天）。直到第5天，在MBMECs增殖到约的密度。 80 - 90％。它们的特征是典型的纺锤形外形。在汇合时，它们形成紧凑，纵向排列与非重叠的接触抑制性细胞（ 图1A，第5天）的单层。通过pyrumicin选择，最高可达99％MBMEC的高纯度达到这表现在血管内皮细胞标志物CD31（PECAM1; 图1B）。该MBMECs形成紧紧紧密连接蛋白相互连接密封单层。后7 - 8天的培养后，内皮细胞层建立稳定值的电阻（20之间达到的典型值- 30Ω×cm之间2）和电容（ 图1C），此时点的功能测定法如小区迁移实验3，20,21和渗透试验， 如用伊文思蓝或右旋糖酐，应执行。 <img alt="图1" src="/files/ftp_upload/52204/52204fig1highres.jpg" width="500" /> 图1.形态和鼠BMECs的功能特性。 （A）代表的时间由透射光显微观察培养的BMECs课程SCOPY超过5天。比例尺适用于所有的图像。在第5天的CD31（绿色）和DAPI（蓝色）（B）的免疫荧光染色的内皮细胞标记物CD31，（C）的细胞预培养5天，然后转移到一个自动系统，用于在生物物理性质（电阻，电容）的分析， <a href="https://www.jove.com/files/ftp_upload/52204/52204fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Isolation of Primary Murine Brain Microvascular Endothelial Cells at JoVE.com

Isolation of Primary Murine Brain Microvascular Endothelial Cells

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-of-primary-murine-brain-microvascular-endothelial-cells

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32.0K 次观看.  University of Münster. 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. 

视频: Isolation of Primary Murine Brain Microvascular Endothelial Cells

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

代的永生化小鼠脑微血管内皮细胞株作为<em在体外</em血脑屏障型号

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

科研

神经科学

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

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 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 ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

原代小鼠骨骼肌微血管内皮细胞的分离

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

人类诱导多能干细胞脑微血管内皮细胞的衍生、扩张、冷冻保存和特征化

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate pups.