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

<ol>
	<li>Shibata-Germanos, S., 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=Structural+and+functional+conservation+of+non-lumenized+lymphatic+endothelial+cells+in+the+mammalian+leptomeninges.">Structural and functional conservation of non-lumenized lymphatic endothelial cells in the mammalian leptomeninges.</a> Acta Neuropathologica. 139 (2), 383-401 (2020).</li><li>Su&#225;rez, I., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+with+many+talents:+lymphatic+endothelial+cells+in+the+brain+meninges.">Cells with many talents: lymphatic endothelial cells in the brain meninges.</a> Cells. 10 (4), 799 (2021).</li><li>Jordan-Williams, K. L., Ruddell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+purifies+murine+lymph+node+lymphatic+endothelium.">Culturing purifies murine lymph node lymphatic endothelium.</a> Lymphatic Research and Biology. 12 (3), 144-149 (2014).</li><li>Jones, B. E., Yong, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+characterization+of+bovine+mesenteric+lymphatic+endothelium.">Culture and characterization of bovine mesenteric lymphatic endothelium.</a> In vitro Cellular &amp; Developmental Biology. 23 (10), 698-706 (1987).</li><li>Podgrabinska, S., 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=Molecular+characterization+of+lymphatic+endothelial+cells.">Molecular characterization of lymphatic endothelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (25), 16069-16074 (2002).</li><li>Jablon, K. L., 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=Isolation+and+short-term+culturing+of+primary+lymphatic+endothelial+cells+from+collecting+lymphatics:+A+techniques+study.">Isolation and short-term culturing of primary lymphatic endothelial cells from collecting lymphatics: A techniques study.</a> Microcirculation. 30 (2-3), e12778 (2023).</li><li>Lapinski, P. E., King, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+lymphatic+endothelial+cells+from+lung+tissue.">Isolation and culture of mouse lymphatic endothelial cells from lung tissue.</a> Methods in Molecular Biology. 2319, 69-75 (2021).</li><li>Lokmic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Identification,+and+culture+of+human+lymphatic+endothelial+cells.">Isolation, Identification, and culture of human lymphatic endothelial cells.</a> Methods in Molecular Biology. 1430, 77-90 (2016).</li><li>Thiele, W., Rothley, M., Schmaus, A., Plaumann, D., Sleeman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry-based+isolation+of+dermal+lymphatic+endothelial+cells+from+newborn+rats.">Flow cytometry-based isolation of dermal lymphatic endothelial cells from newborn rats.</a> Lymphology. 47 (4), 177-186 (2014).</li><li>Lokmic, Z., 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=Isolation+of+human+lymphatic+endothelial+cells+by+multi-parameter+fluorescence-activated+cell+sorting.">Isolation of human lymphatic endothelial cells by multi-parameter fluorescence-activated cell sorting.</a> Journal of Visualized Experiments. 99, e52691 (2015).</li><li>Janson, C., Romanova, L., Hansen, E., Hubel, A., Lam, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+functional+characterization+of+rat+arachnoid+cell+lines.">Immortalization and functional characterization of rat arachnoid cell lines.</a> Neuroscience. 177, 23-34 (2011).</li><li>Romanova, L. G., Hansen, E. A., Lam, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+preliminary+characterization+of+immortalized+cell+line+derived+from+rat+lymphatic+capillaries.">Generation and preliminary characterization of immortalized cell line derived from rat lymphatic capillaries.</a> Microcirculation. 21 (6), 551-561 (2014).</li><li>Park, T. I., 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=Routine+culture+and+study+of+adult+human+brain+cells+from+neurosurgical+specimens.">Routine culture and study of adult human brain cells from neurosurgical specimens.</a> Nature Protocols. 17 (2), 190-221 (2022).</li><li>Okuda, K. S., 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=lyve1+expression+reveals+novel+lymphatic+vessels+and+new+mechanisms+for+lymphatic+vessel+development+in+zebrafish.">lyve1 expression reveals novel lymphatic vessels and new mechanisms for lymphatic vessel development in zebrafish.</a> Development. 139 (13), 2381-2391 (2012).</li><li>Bokobza, C., 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=Magnetic+Isolation+of+microglial+cells+from+neonate+mouse+for+primary+cell+cultures.">Magnetic Isolation of microglial cells from neonate mouse for primary cell cultures.</a> Journal of Visualized Experiments. 185, e62964 (2022).</li><li>Wang, J. M., Chen, A. F., Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+mouse+aortic+endothelial+cells.">Isolation and primary culture of mouse aortic endothelial cells.</a> Journal of Visualized Experiments. 118, e52965 (2016).</li><li>Breiteneder-Geleff, S., 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=Angiosarcomas+express+mixed+endothelial+phenotypes+of+blood+and+lymphatic+capillaries:+podoplanin+as+a+specific+marker+for+lymphatic+endothelium.">Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.</a> The American Journal of Pathology. 154 (2), 385-394 (1999).</li><li>Petrova, T. V., Koh, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+lymphatic+vasculature:+From+development+to+pathophysiology.">Organ-specific lymphatic vasculature: From development to pathophysiology.</a> The Journal of Experimental Medicine. 215 (1), 35-49 (2018).</li><li>Wilting, J., 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=The+transcription+factor+Prox1+is+a+marker+for+lymphatic+endothelial+cells+in+normal+and+diseased+human+tissues.">The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.</a> The FASEB Journal. 16 (10), 1271-1273 (2002).</li><li>Yin, X., 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=Lymphatic+endothelial+heparan+sulfate+deficiency+results+in+altered+growth+responses+to+vascular+endothelial+growth+factor-C+(VEGF-C).">Lymphatic endothelial heparan sulfate deficiency results in altered growth responses to vascular endothelial growth factor-C (VEGF-C).</a> The Journal of Biological Chemistry. 286 (17), 14952-14962 (2011).</li><li>DeSisto, J., 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=Single-cell+transcriptomic+analyses+of+the+developing+meninges+reveal+meningeal+fibroblast+diversity+and+function.">Single-cell transcriptomic analyses of the developing meninges reveal meningeal fibroblast diversity and function.</a> Developmental Cell. 54 (1), 43-59 (2020).</li><li>Chen, Z., 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+method+for+eliminating+fibroblast+contamination+in+mouse+and+human+primary+corneal+epithelial+cell+cultures.">A method for eliminating fibroblast contamination in mouse and human primary corneal epithelial cell cultures.</a> Current Eye Research. , 1-11 (2023).</li><li>Starzonek, C., 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=Enrichment+of+human+dermal+stem+cells+from+primary+cell+cultures+through+the+elimination+of+fibroblasts.">Enrichment of human dermal stem cells from primary cell cultures through the elimination of fibroblasts.</a> Cells. 12 (6), 949 (2023).</li><li>Lam, C. H., Romanova, L., Hubel, A., Janson, C., Hansen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+fibroblast+on+the+arachnoid+leptomeningeal+cells+in+vitro.">The influence of fibroblast on the arachnoid leptomeningeal cells in vitro.</a> Brain Research. 1657, 109-119 (2017).</li></ol>

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...

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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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。

The newly discovered leptomeningeal lymphatic endothelial cells (LLECs) form a meshwork of individual cells within the leptomeninges, exhibiting a distinct distribution pattern when compared to peripheral lymphatic endothelial cells1,2. The cellular functions and clinical implications associated with LLECs remain largely uncharted territory. In order to pave the way for functional research on LLECs, it is imperative to establish an in vitro model for their study. Therefore, this study has devised a comprehensive protocol for the isolation and primary culture of LLECs.
Mice are the preferred animal model due to their suitability for genetic manipulation in disease research. Previous studies have successfully isolated lymphatic endothelial cells from various mouse tissues, including lymph nodes3, mesenteric tissue4, dermal tissue5, collecting lymphatics6, and lung tissue7. These isolation procedures have primarily relied on techniques such as magnetic-activated cell sorting (MACS) and flow cytometry sorting8,9,10. Additionally, research efforts have led to the establishment of rat arachnoid cell lines and rat lymphatic capillary cell lines11,12. Despite the existence of explant culture techniques for leptomeninges13, there exists an urgent need for a standardized protocol for the isolation and culture of LLECs. Consequently, this study has successfully harvested and cultured LLECs by meticulously dissociating leptomeninges under the guidance of a microscope and promoting LLECs expansion through the use of vascular endothelial growth factor-C (VEGF-C). The distinctive marker for lymphatic endothelial cells is lymphatic vessel hyaluronic receptor-1 (LYVE-1)14. This multi-step protocol selectively isolates LYVE-1-positive LLECs using MACS and subsequently verifies their purity through flow cytometric analysis and immunofluorescent staining.
The primary steps of this multi-step protocol can be summarized as follows: flask coating, dissociation of leptomeninges, enzymatic digestion of leptomeninges, cell expansion, magnetic cell selection, and subsequent culture of LLECs. Finally, the purity of the isolated LLECs is confirmed through flow cytometric analysis and immunofluorescent staining. The overarching aim of this study is to present a reproducible, multi-step protocol for the isolation of LLECs from mouse leptomeninges and their subsequent in vitro culture. This protocol is poised to greatly facilitate investigations into the cellular functions and clinical implications of LLECs.

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的收获和原代培养

This protocol is designed for areas of by other researchers only interested where established the multi procedure to and cultural primary areas Our protocol ultimately led to the establishment of our primary culture of area CS with a purity level exceeding 95%There is currently no existing protocol for harvesting and culturing Our results paved the way for further investigating the similar function of LLECS in virtual. At RUC, our recent discovery intracranial cellular population, the biological significance of RUC We will conduct further research on the cellular function and review its clinical implication.

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（...

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

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。

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。

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Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

858 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。