该研究得到了国家自然科学基金（81960226,81760223）、云南省自然科学基金（202001AS070045,202301AY070001-011）和云南省教育厅科研基金（2023Y0784）的资助。

该研究获得了昆明医科大学动物实验伦理委员会（kmmu20220945）的批准。所有实验均遵循既定的动物护理准则。从年龄在6-8周龄、体重在20-25g之间的雄性C57Bl/6J小鼠中收获软脑膜淋巴内皮细胞（LEC）。这些小鼠购自中国昆明的昆明医科大学。整个实验过程在严格的无菌条件下进行。除非另有说明，否则所有离心步骤均在室温下进行。
1.试剂和仪器的制备
注意：所有涉及溶液的步骤都必须在 II 类生物危害柜内进行。
<ol><li>通过将磷酸盐缓冲盐水（PBS）与钙和镁，10%胎牛血清（FBS）和1%青霉素 - 链霉素（P / S）混合来制备洗涤缓冲液（参见 材料表）。将该缓冲液保持在4°C。 必须在使用当天新鲜准备并对缓冲液进行脱气。</li>
	<li>通过将 10 mL PBS（不含钙和镁）与 2 mL 0.25% 胰蛋白酶、1 mL 20 mg/mL 木瓜蛋白酶和 200 μL 1 mg/mL 胶原酶 I 混合来制备消化酶（见 材料表）。 注意： 确保使用适当体积的等分试样，以防止反复冻融循环。将这些等分试样储存在-20°C。</li>
	<li>利用市售的内皮细胞生长培养基试剂盒制备培养基，其中包括VEGF-C（100ng / mL）和1%P / S（参见 材料表）。</li>
	<li>通过用 10% FBS 补充 Dulbecco 改良的 Eagle 培养基 （DMEM） 来制备终止缓冲液以停止消化过程。</li>
	<li>准备无菌手术器械：这些器械应包括剪刀、锯齿尖镊子和细尖镊子。</li>
</ol>2. 烧瓶涂层
<ol><li>用100μg/ mL纤连蛋白溶液包被T25烧瓶（参见 材料表），并在37°C下孵育过夜。 使用前，使用移液管去除包衣溶液。</li>
	<li>用 PBS 洗涤烧瓶三次，随后吸出 PBS，让 T25 烧瓶风干。</li>
</ol>3.软脑膜解离
注意：始终在4°C下使用预冷的缓冲液和溶液。
<ol><li>用过量吸入4%异氟烷麻醉小鼠，然后使用预先用70%乙醇清洁的剪刀迅速斩首处死它们。</li>
	<li>小心地沿着中线切开皮肤，从颅骨后部的开口开始，一直延伸到前部区域。</li>
	<li>小心翼翼地取出头骨，用剪刀轻轻提起，以免损伤软脑膜，确保获得包括软脑膜在内的整个大脑。</li>
	<li>将整个大脑浸入洗涤缓冲液中，轻轻冲洗以去除表面血液。</li>
	<li>将大脑转移到无菌培养皿中，无需切碎，并在显微镜下使用细尖镊子从大脑表面提取软脑膜。</li>
	<li>使用无菌微剪刀将软脑膜组织切成碎片。</li>
</ol>4.软脑膜酶消化
<ol><li>向片段中加入10mL酶混合物（步骤1.2），并在37°C下孵育15分钟。轻轻搅拌以确保碎片从管底部分离。之后，加入 10 mL 终止缓冲液（步骤 1.4）以停止消化。</li>
	<li>在4°C下以300× g 离心5分钟，并使用移液管小心地除去上清液。</li>
	<li>加入 10 mL 冷 PBS，并将混合物通过 70 μm 过滤器进入无菌 50 mL 试管中以过滤掉任何团块。</li>
</ol>5. 细胞扩增
<ol><li>将 1 x 10 5 个细胞/cm 2 接种到装有5 mL 培养基的纤连蛋白包被的 T25 烧瓶（步骤2 ）中。</li>
	<li>将细胞在37°C下与5%CO2 孵育24小时。之后，取出培养基以消除未附着的细胞。</li>
	<li>通过每两天更换 50% 的培养基来维持培养。重复此过程两到三次。</li>
</ol>6.磁芯选择
注意：快速工作，保持细胞冷度，并利用预冷溶液防止非特异性细胞标记。
<ol><li>仪器和试剂的制备：将磁选机连接到磁选机支架上（见 材料表）。将选择柱连接到磁选机，并在选择柱顶部放置一个 70 μm 的细胞过滤器。在选择柱下方放置一个 50 mL 试管以收集流量。</li>
	<li>酶消化：一旦细胞达到 80% 汇合，吸出培养基并用 PBS 冲洗细胞。加入0.25%胰蛋白酶分离贴壁细胞，并在37°C孵育5分钟。通过添加停止缓冲液来停止消解。在室温下以300× g 离心细胞5分钟，并除去上清液。</li>
	<li>抗体孵育：将 1 x 107 个细胞重悬于 100 μL PBS 中，并加入 10 μL LYVE-1 抗体（参见 材料表）。充分混合并在4°C下在黑暗中孵育30分钟。<ol><li>以300× g 旋转细胞5分钟，弃去上清液。通过引入 1 mL PBS 并以 300 x g 离心 5 分钟冲洗细胞。完全去除上清液。</li>
	</ol></li>
	<li>微珠标记：将细胞重悬于 100 μL PBS 中，并加入 20 μL 微珠（参见 材料表）。充分混合并在4°C下在黑暗中孵育30分钟。<ol><li>以300× g 旋转细胞5分钟，并倒出上清液。随后，通过以 300 x g 离心 5 分钟，用 1 mL PBS 清洁细胞。最后，完全除去上清液。</li>
	</ol></li>
	<li>磁阴性排除：将细胞重悬于 4 mL PBS 中。将细胞通过70μm细胞过滤器以消除团块。用 3 mL PBS 冲洗来制备选择柱（参见 材料表），然后将细胞悬液施加到选择柱中。<ol><li>用 3 mL PBS 执行洗涤步骤，并将 LYVE-1 阴性细胞收集到位于选择柱下方的 50 mL 试管中，以使其通过。</li>
	</ol></li>
	<li>磁性阳性选择：将 6 mL PBS 移液管入选择柱中。将柱塞用力推入选择柱，立即冲洗出磁性标记的细胞，以获得 LYVE-1 阳性 LEC。 注意： 始终等到选择色谱柱储液槽为空后再进行下一步。</li>
</ol>7. LLECs文化
<ol><li>将LYVE-1阳性LLEC以300× g 离心5分钟，然后小心地除去上清液。</li>
	<li>将 1 x 10 5 个细胞/cm2 接种到装有5 mL 培养基的纤连蛋白包被的 T25 烧瓶中。</li>
	<li>每隔一天更换 50% 的培养基来维持培养。当细胞达到 80% 汇合时，用 0.25% 胰蛋白酶分离细胞并进行细胞传代。在2-3次传代后利用细胞进行后续实验。</li>
</ol>8. 流式细胞术分析
注：流式细胞术分析按照前面描述的程序15 进行。
<ol><li>加入0.25%胰蛋白酶分离贴壁细胞，并在37°C孵育5分钟。然后，加入停止缓冲液以停止消化。</li>
	<li>将细胞以300× g 离心5分钟，并完全吸出上清液。将 1 x 106 个细胞重悬于 100 μL PBS 中。</li>
	<li>加入 1 μL LYVE-1 抗体。充分混合并在室温下在黑暗中孵育 10 分钟。</li>
	<li>将细胞重悬于适量的PBS缓冲液中，使用流式细胞术和FlowJo软件进行分析（参见 材料表）。</li>
</ol>9. 免疫荧光染色
注意：免疫荧光染色按照前面描述的程序进行16.
<ol><li>用PBS洗涤细胞三次。在室温下用4%多聚甲醛（PFA）固定细胞10分钟。弃去PFA并用PBS洗涤细胞三次。</li>
	<li>在室温下应用块缓冲液1小时。从盖玻片中取出模块缓冲液。</li>
	<li>将染色缓冲液中的 LYVE-1 抗体添加到细胞中并在黑暗中孵育。用PBS洗涤细胞三次。</li>
	<li>在染色缓冲液中向细胞中加入适当的二抗（参见 材料表），并在黑暗中孵育。用PBS洗涤细胞三次。</li>
	<li>用 4'，6-二脒基-2-苯基吲哚 （DAPI） 复染。这些细胞现在已准备好进行免疫荧光显微镜检查。</li>
</ol>

使用磁性细胞选择建立体外 LLEC 培养物

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作者声明他们没有利益冲突需要披露。

以前没有报道过在 体外 收获和培养LLEC的现有方案。本研究介绍了一种可重复的多程序方案，用于从小鼠软脑膜中收获和培养 LEC。
虽然这种多程序方案是可重复的，但有几个关键考虑因素。例如，纤连蛋白包被的 T25 烧瓶通过消除非贴壁细胞来促进 LEC 的粘附和功能，从而确保更均匀的细胞群。此外，解离软脑膜的手术过程的持续时间和温度是影响细胞活力的关键因素。因此，将持续时间保持在 1 小时内并保持缓冲液足够低是至关重要的。另一个临界点与软脑膜中红细胞的存在有关，血红蛋白的释放可能会损害 LLECs。为了减轻这种情况，必须轻轻冲洗和清洗以去除红细胞，防止其细胞毒性作用。最后，LLEC 需要用 VEGF-C20 刺激，尤其是在 100 ng/mL 的浓度下。在这种刺激下，LLEC表现出特征性的内皮样形态，而不是形成管（补充图3A-C）。因此，本研究采用含有 100 ng/mL VEGF-C 的培养基来诱导细胞群扩增并保持均一性。
本文中描述的多过程协议附带故障排除步骤和一些限制。首先，该协议需要 2-3 周才能完成，因此频繁重复是不切实际的。其次，外科手术和酶促程序可能导致死细胞的存在，这些死细胞在接种前没有被消除，可能会影响细胞培养。这种可能性将在未来的协议实施中探索。第三，虽然收获的 LEC（VEGFR-3 和 PDPN 阳性细胞）的纯度超过 95%，但一些异质细胞群仍然存在，包括 CD31 阳性细胞，其占不到 5%（补充图 4A-C）。这是原代细胞培养中的常见挑战。来自苏木精-伊红染色的高分辨率图像表明，LLECs显示出特征性的内皮样形状，但不表达CD31（补充图5A，B）。最后，CCK-8 测定结果表明，与第 2 和第 5 传代相比，第 1 和第 6 传代时 LEC 的增殖速率显着降低（补充图 6）。由于抗体处理和分选过程中的物理应力，传代 1 可能导致 LLEC 损伤，而传代 6 随着传代次数的增加，增殖减少。因此，传代6后的后续细胞不适合进行相关实验。鉴于成纤维细胞在脑膜中的分布21，大约 5% 的受污染细胞可能含有成纤维细胞。为了解决这个问题，添加遗传素和免疫磁性细胞分选可用于去除受污染的成纤维细胞22,23。监测 LEC 每次传代中成纤维细胞的污染率将有助于消除成纤维细胞对 LEC 生长的影响24.考虑到小鼠和人类之间的异质性，应追求原代人类 LEC 的收获和培养，以便在未来的研究中具有潜在的临床应用。
总之，目前尚无既定 的体外收获和培养LLEC的方案。本研究引入了一种可重复的多程序方案，用于从小鼠软脑膜中收获 LEC，并随后 在体外建立其原代培养物。这项工作将有助于进一步探索LLEC的细胞功能和潜在的临床应用。

新发现的软脑膜淋巴内皮细胞 （LEC） 在软脑膜内形成单个细胞的网状结构，与外周淋巴内皮细胞相比，表现出不同的分布模式 1,2。与LLEC相关的细胞功能和临床意义在很大程度上仍然是未知的领域。为了为LLEC的功能研究铺平道路，必须建立体外模型进行研究。因此，本研究设计了一种用于 LEC 分离和原代培养的综合方案。
小鼠是首选的动物模型，因为它们适合在疾病研究中进行基因操作。先前的研究已成功从各种小鼠组织中分离出淋巴内皮细胞，包括淋巴结3、肠系膜组织4、真皮组织5、收集淋巴管6 和肺组织7。这些分离程序主要依赖于磁活化细胞分选 （MACS） 和流式细胞术分选等技术 8,9,10。此外，研究工作还导致了大鼠蛛网膜细胞系和大鼠淋巴毛细血管细胞系的建立11,12。尽管存在针对软脑膜13 的外植体培养技术，但迫切需要一种标准化的方案来分离和培养 LLECs。因此，本研究通过在显微镜引导下细致地解离软脑膜，并通过使用血管内皮生长因子-C（VEGF-C）促进LLECs扩增，成功地收获和培养了LEC。淋巴内皮细胞的独特标志物是淋巴管透明质酸受体-1 （LYVE-1）14。该多步骤方案使用 MACS 选择性分离 LYVE-1 阳性 LEC，随后通过流式细胞术分析和免疫荧光染色验证其纯度。
该多步骤方案的主要步骤可以总结如下：烧瓶包被、软脑膜解离、软脑膜酶消化、细胞扩增、磁性细胞选择和随后的 LEC 培养。最后，通过流式细胞术分析和免疫荧光染色确认分离的LLECs的纯度。本研究的总体目标是提出一种可重复的多步骤方案，用于从小鼠软脑膜及其随后 的体外 培养中分离 LLEC。该协议有望极大地促进对 LEC 的细胞功能和临床意义的研究。

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		<tr>
			<td>Block buffer</td>
			<td>Beyotime</td>
			<td>P0102</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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		<tr>
			<td>Collagenase I</td>
			<td>Solarbio</td>
			<td>C8140</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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			<td>DAPI</td>
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			<td>P0131</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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		<tr>
			<td>DMEM</td>
			<td>Solarbio</td>
			<td>11995</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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			<td>D-PBS</td>
			<td>Solarbio</td>
			<td>D1041</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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		<tr>
			<td>EGM-2 MV Bullet Kit</td>
			<td>Lonza</td>
			<td>C-3202</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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		<tr>
			<td>FBS</td>
			<td>Solarbio</td>
			<td>S9010</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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			<td>Fibronectin</td>
			<td>Solarbio</td>
			<td>F8180&#160;</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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		<tr>
			<td>FlowJo Software</td>
			<td>BD Biosciences</td>
			<td>V10.8.1</td>
			<td></td>
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			<td>LYVE-1 antibody</td>
			<td>eBioscience</td>
			<td>12-0443-82</td>
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			<td>Miltenyi</td>
			<td>130-042-302</td>
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			<td>Miltenyi</td>
			<td>130-042-303</td>
			<td>Sterile before use</td>
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			<td>130-048-801</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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			<td>P1400</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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			<td>Papain</td>
			<td>Solarbio</td>
			<td>G8430-25g</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>D1040</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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			<td>PDPN antibody</td>
			<td>Santa</td>
			<td>sc-53533</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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			<td>PFA</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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		<tr>
			<td>PROX1 antibody</td>
			<td>Santa</td>
			<td>sc-81983</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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			<td>Selection column&#160;</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td>Sterile before use</td>
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		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200072</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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		<tr>
			<td>VEGF-C&#160;</td>
			<td>Abcam</td>
			<td>ab51947</td>
			<td>Store aliquots at &#8211;20 &#176;C</td>
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			<td>VEGFR-3 antibody</td>
			<td>Santa</td>
			<td>sc-514825</td>
			<td>Store aliquots at &#8211;4 &#176;C</td>
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harvest and primary culture of leptomeningeal lymphatic endothelial cells

软脑膜淋巴内皮细胞 （LEC） 是最近发现的颅内细胞群，具有与外周淋巴内皮细胞明显不同的独特分布。它们的细胞功能和临床意义在很大程度上仍然未知。因此，LLEC供应的可用性对于进行体外功能研究至关重要。然而，目前尚无现成的 体外收获和培养 LEC 的方案。
本研究使用多步骤方案成功收获了 LEC，其中包括用纤连蛋白包被烧瓶、在显微镜的帮助下解剖软脑膜、酶促消化软脑膜以制备单细胞悬浮液、用血管内皮生长因子-C （VEGF-C） 诱导 LLEC 扩增，以及通过磁活化细胞分选 （MACS） 选择淋巴管透明质酸受体-1 （LYVE-1） 阳性细胞。这个过程最终导致了一种主要文化的建立。通过免疫荧光染色和流式细胞术分析确认了LLECs的纯度，纯度超过95%。该多步方案已证明具有可重复性和可行性，这将极大地促进探索LLEC的细胞功能和临床意义。

本研究提出了一种可重复的多步骤方案，用于从小鼠身上收获淋巴内皮细胞 （LEC） 并随后 在体外建立其原代培养物。关键步骤包括烧瓶制备和纤连蛋白包被、软脑膜解离、通过酶消化获得单细胞悬浮液以及用 VEGF-C 诱导 LLECs 扩增。然后使用磁活化细胞分选 （MACS） 选择性分离 LYVE-1 阳性 LEC。最后，进行免疫荧光染色和流式细胞术分析以评估 LLEC 纯度，MTT 测定结果显示 LLEC 生长速率稳健（补充图 1）。该多程序协议的主要步骤如流程图所示，整个过程大约需要 2-3 周（图 1）。
为了收获 LLECs，必须解离软脑膜并促进 LEC 扩增，同时通过有效的手术技术和保持寒冷的环境来保持细胞活力（图 2A-E）。在无菌条件下获得整个大脑和软脑膜后，进行温和的冲洗步骤以去除血红细胞（图2F）。接下来，在显微镜下小心地从大脑表面提取软脑膜（图2G）。这些软脑膜主要由胶原纤维组成，因此它们被碎片化以加速酶消化（图2H）。随后，对这些片段进行酶消化，并通过70μm过滤器过滤任何剩余的团块，以消除较大的胶原纤维簇（图2I）。最后，将细胞接种在纤连蛋白包被的T25烧瓶中，并加入VEGF-C以诱导扩增（图2J）。24小时后，除去培养基以消除未附着的细胞。这些步骤有助于从软脑膜中收获细胞并促进扩增，这对于在该过程的后期获得足够的 LYVE-1 阳性 LEC 至关重要。
虽然 VEGF-C 诱导 LLEC 扩增，但异质细胞群仍然可以一起生长。为了分离纯 LYVE-1 阳性 LEC，使用 MACS，因为 LYVE-1 是淋巴内皮细胞的公认标志物。通过流式细胞术评估LLEC的纯度，结果表明，MACS后第2代LYVE-1阳性细胞的百分比与第3代没有显着差异，纯度大于95%（图3A）。为了进一步验证 LYVE-1 阳性 LEC 的特异性，另外三种淋巴内皮细胞标志物——鬼臼蛋白 （PDPN）、血管内皮生长因子受体-3 （VEGFR-3） 和 Prospero 相关同源盒 1 （PROX1） 用于鉴定17,18,19。免疫荧光染色证实LYVE-1与这三种标记物共染色（图3B）。此外，在生理条件下，在脑切片中建立了 LEC 的身份，显示 LYVE-1 与 PROX1、VEGFR-3 和 PDPN 共染色（补充图 2A）。LYVE-1阳性细胞不表达F4/80和血小板衍生生长因子β（PDGFR-β），有效地将LLEC与巨噬细胞和成纤维细胞区分开来（补充图2B）。总之，该协议允许收获能够进行体外培养的高纯度LLECs。
MACS前的软脑膜细胞表现出异质性，与淋巴内皮细胞的集落不同。它们的形态范围从单个圆形球体到熔融纤维形状（图4A-D）。然而，在MACS后，LLEC表现出典型的纺锤体和鹅卵石状内皮样特征（图4E-H）。基于这些结果，LLEC通过这种多程序方案有效地收获并保持在健康的生长状态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65872/65872fig01.jpg"> 图 1：多程序协议的示意图。 该流程图说明了 LLEC 收获和培养的多程序方案。 <a href="https://www.jove.com/files/ftp_upload/65872/65872fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65872/65872fig02.jpg"> 图2：软脑膜细胞的收获 。 （A） 无菌手术器械的制备。（B） 通过 吸入 4% 异氟烷麻醉小鼠，随后在安乐死后用 70% 乙醇清洁。（C） 将小鼠斩首并小心地切开皮肤中线，从颅骨后部开始向额部开始。（D） 精细切除颅骨，以保持软脑膜的完整性。（E） 取回含有软脑膜的整个大脑。（F） 在缓冲溶液中清洗整个大脑，轻轻冲洗以去除表面血液。（G） 使用细尖镊子从大脑表面解剖软脑膜。（H）用无菌微剪刀将软脑膜切成碎片，然后加入10mL酶混合物并在37°C下孵育15分钟。 （I）将沉淀重悬于10mL PBS中，并通过70μm过滤器过滤团块。（J） 将细胞接种到包被的 T25 烧瓶中。 <a href="https://www.jove.com/files/ftp_upload/65872/65872fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65872/65872fig03.jpg"> 图 3：MACS 后 LLEC 纯度的评估 。 （A） 流式细胞术分析的代表性直方图，显示 MACS 后第 2 和第 3 代中 LYVE-1 阳性细胞的表达。MACS后LYVE-1阳性细胞的百分比超过95%。数据代表了三个独立实验（平均± SEM）。误差条表示 SEM。 （B） 代表性免疫荧光图像，显示 LYVE-1 与 PDPN、VEGFR-3 和 PROX1 的共染色。比例尺 = 50 μm。 <a href="https://www.jove.com/files/ftp_upload/65872/65872fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65872/65872fig04.jpg"> 图4：软脑膜细胞和LLEC的生长特征。 （A）代表性图像中捕获的软脑膜细胞的形态。比例尺 = 50 μm。 （B-D） 描述软脑膜细胞在 MACS 前第 1 天、第 2 天和第 3 天形态的代表性图像。比例尺 = 100 μm。 （E） 代表性图像中捕获的 LEC 的形态。比例尺 = 50 μm。 （F-H） 说明MACS后第1天、第2天和第3天LLEC形态的代表性图像。比例尺 = 100 μm。 <a href="https://www.jove.com/files/ftp_upload/65872/65872fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图1：LLEC和SVECs4-10的增长率。 MTT 测定结果显示，与 SVEC4-10 组相比，第 1 代 LLEC 的生长速率显着降低。与SVEC4-10组相比，第2代和第3代LLECs组之间没有显著差异。数据代表了六个独立实验的结果（平均± SEM）。误差线表示 SEM。 <a href="https://www.jove.com/files/ftp_upload/65872/Supplementary%20Figure%201.png" target="_blank">请点击这里下载此文件。</a>
补充图2：脑切片中LLEC的特征和鉴别。 （A） 代表性免疫荧光图像，说明生理条件下脑切片中 LYVE-1 与 PROX1、VEGFR-3 和 PDPN 的共染色。比例尺 = 2 μm。 （B） 代表性免疫荧光图像，表明 LYVE-1 在生理条件下不与脑切片中的 F4/80 和 PDGFR-β 共染色。比例尺 = 20 μm。 <a href="https://www.jove.com/files/ftp_upload/65872/Supplementary%20Figure%202.png" target="_blank">请点击这里下载此文件。</a>
补充图3：VEGF-C刺激下LLEC的生长。 （A） 1 ng/mL VEGF-C 刺激下 LLEC 的代表性图像。（B） 100 ng/mL VEGF-C 刺激下 LLEC 的代表性图像。（C） 500 ng/mL VEGF-C 刺激下 LLEC 的代表性图像。比例尺 = 50 μm。 <a href="https://www.jove.com/files/ftp_upload/65872/Supplementary%20Figure%203.png" target="_blank">请点击这里下载此文件。</a>
补充图4：收获后CD31、PDPN和VEGFR-3的表达。 （A） 流式细胞术分析的代表性直方图显示 CD31 阳性细胞小于 5%。（B） 流式细胞术分析的代表性直方图表明 PDPN 阳性细胞超过 95%。（C） 流式细胞术分析的代表性直方图表明 VEGFR-3 阳性细胞超过 95%。 <a href="https://www.jove.com/files/ftp_upload/65872/Supplementary%20Figure%204.png" target="_blank">请点击这里下载此文件。</a>
补充图5：LLEC的形态和与CD31的区别。 （A） 苏木精-伊红染色显示的LLECs形态的代表性图像。比例尺 = 20 μm。 （B） 具有代表性的免疫荧光图像表明 LYVE-1 和 PDPN 不与 CD31 共染。比例尺 = 50 μm。 <a href="https://www.jove.com/files/ftp_upload/65872/Supplementary%20Figure%205.png" target="_blank">请点击这里下载此文件。</a>
补充图6：LLEC的增殖率。 CCK-8 测定的代表性结果显示，与第 2 和第 5 传代相比，第 1 代和第 6 代 LLEC 的增殖率显着降低。数据代表了六个独立实验的结果（平均± SEM）。误差线表示 SEM。 <a href="https://www.jove.com/files/ftp_upload/65872/Supplementary%20Figure%206.png" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Unveiling Cellular Functions and Potential Clinical Implications of Leptomeningeal Lymphatic Endothelial Cells at JoVE.com

作者聚焦：揭示软脑膜淋巴内皮细胞的细胞功能和潜在临床意义 

软脑膜淋巴内皮细胞 （LEC） 是一种最近发现的颅内细胞类型，其功能知之甚少。本研究提出了一种可重复的方案，用于从小鼠收获 LLEC 并建立 体外 原代培养物。该协议旨在使研究人员能够深入研究 LEC 的细胞功能和潜在临床意义。

软脑膜淋巴内皮细胞的收获和原代培养

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

该协议是为其他研究人员设计的，只对建立多程序和文化主要区域感兴趣的区域我们的协议最终导致了纯度超过95%的区域CS的初级培养物的建立目前没有现有的收获和培养协议我们的结果为进一步研究LLECS在虚拟中的类似功能铺平了道路。在RUC上，我们最近发现的颅内细胞群，RUC的生物学意义我们将对细胞功能进行进一步的研究，并回顾其临床意义。

harvest-primary-culture-leptomeningeal-lymphatic-endothelial

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

1.3K 次观看.  The First Affiliated Hospital of Kunming Medical University. 该协议是为其他研究人员设计的，只对建立多程序和文化主要区域感兴趣的区域我们的协议最终导致了纯度超过95%的区域CS的初级培养物的建立目前没有现有的收获和培养协议我们的结果为进一步研究LLECS在虚拟中的类似功能铺平了道路。在RUC上，我们最近发现的颅内细胞群，RUC的生物学意义我们将对细胞功能进行进一步的研究，并回顾其临床意义。

视频: 作者聚焦：揭示软脑膜淋巴内皮细胞的细胞功能和潜在临床意义 

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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 ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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 ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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 ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

从胚胎小鼠小脑分离的神经元的主要培养

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...