作者想承认伊丽莎白. 海伦娜 Bruun, 萨拉. 克里斯汀和 Kristiansen 的技术援助, 以及 Lundbeck 基金会授予号码 R155-2013-14113。

猪脑被作为丹麦食品工业的副产品获得。丹麦的屠宰场受到丹麦环境和食品部的严格监督和观察. 
 用于分离星形胶质细胞的大鼠在当地的动物设施中繁殖和组织, 环境温度为22和 #176; C-23 和 #176; C 和在 12/12 h 黑暗或轻的周期在兽医的检查之下和根据丹麦实验室动物条例。根据《动物道德使用国际准则》 (欧洲共同体理事会1986年11月24日的指示, 86/609/EEC) 和丹麦的准则, 对这些老鼠进行了安乐死。在这些实验中没有使用动物或人体材料的 活体 实验. 
 注意: 下面是一个理事会主协议, 描述 ( 步骤 1 ) 净化, ( 步骤 2 ) 培养和 ( 步骤 3 ) TEER 测量。为建立一个与星形胶质细胞, 一个备用的协议 ( 步骤 4 ) 描述的纯化和培养大鼠和猪星形胶质细胞提出. 
 1. 猪脑毛细血管的纯化 <ol> <li> 从5-6 月大的家养猪中收集8-10 大脑 (如 从附近的屠宰场开始), 并将它们在冰上运送到实验室。我们建议在动物终止后2-3 小时内启动以下纯化程序. </li>
	<li> 将大脑放在无菌的流动工作台中, 用1升 PBS 在冰上的烧杯中轻轻地冲洗它们. </li>
	<li> 小心地从一个大脑中取出脑膜, 一次使用细尖钳, 将无脑膜的大脑转移到另一个 1 L 烧杯中, PBS 放在冰上。延长所采取的时间可能会使搬迁复杂化。每个大脑所用的大约时间应该是10-15 分钟. </li>
	<li> 使用手术刀, 一次从一个大脑中刮掉灰质, 并将隔离的材料转移到培养皿 (8.8 厘米 2 ) 中, 含有20毫升 DMEM 营养混合物 F-12 (DMEM/F-12) 放在冰上。尽可能多地隔离灰质, 而不提取白质物质。对于最初的碎裂, 用50毫升的注射器来运行灰质物质, 不用针头。继续为所有的大脑和汇集收集的灰质物质。延长所采取的时间可能会使搬迁复杂化。每个大脑所用的大约时间应该是10-15 分钟. </li>
	<li> 将隔离的灰质物质转移到 hand-held 组织均质机的磨床管中, 其比例为50/50 与 DMEM/F-12 介质。质的材料, 使8上下中风与一个松散的杵, 其次是8上下中风与紧杵。继续, 直到所有分离的材料已匀浆, 然后转移到一个500毫升的瓶子和稀释与 DMEM/F-12 约450毫升总体积. </li>
	<li> 过滤器使用500毫升蓝帽瓶与过滤器和140和 #181; m 过滤网格, 并通过运行通过过滤器的组织隔离毛细血管。每50毫升匀浆使用一个过滤器, 然后用 DMEM/F-12 洗涤每个过滤器. </li>
	<li> 在培养皿中放置含毛细管的过滤器, 并在 DMEM/F-12 中采用胰蛋白酶/edta (2.5% 胰蛋白酶、0.1 nM edta)、胶原酶 CLS2 (2000 u/毫升) 和 dnasei 1 (3400 u/毫升) 的消解溶液。每3滤网使用1培养皿 (8.8 厘米 2 , 20 毫升溶液). </li>
	<li> 将培养皿放置在37和 #176 上; C 为1小时, 在轨道振动筛上 180 rpm 或轻轻搅拌每10分钟。1小时后, 用1毫升吸管从培养皿中用悬浮液从过滤器中清洗毛细血管. </li>
	<li> 将悬浮液从3碟中分离成 2 50 毫升的管子, 并通过在每50毫升管中加入10毫升 DMEM/F-12 来停止消化. </li>
	<li> 离心机悬浮在 250 x g, 4 和 #176; C 为5分钟抽清和 re-suspend 每粒在10毫升 DMEM/F-12。在每根管子上再加20毫升的 DMEM/F12。重复这两次离心机的步骤. </li>
	<li> 让管冷却在冰上5分钟, 并将解决方案转移到2新的50毫升管. </li>
	<li> 离心机悬浮在 250 x g, 4 和 #176; C 为5分钟, 并且 re-suspend 每个药丸在 8-10 毫升 (大约1毫升/脑子使用) 冷冻解答由10% 亚砜组成在 FBS. </li>
	<li> 将电池悬浮液转移到 cryovials, 使用每 cryovial 1 毫升。平均而言, 一个纯化结果为每个大脑使用1瓶, 平均提供12-16 插入的内皮细胞。将小瓶放在-80 和 #176 的冷冻箱中; C 至少4小时至一夜。然后, 将 cryovials 储存在 cryotank 的液氮中. 
	注意: 液氮的温度极低。请穿戴适当的保护. </li>
</ol> 2。初级 pBECs 的培养 (8-10 天) <ol> <li> 初始区域性 (3-5 天) 日 1 <ol> <li> 以优化附加 pBECs 的条件, 通过添加最终解决方案来执行 T75 烧瓶的涂层在 ddH 2 O 中, 将胶原蛋白 IV (150 和 #181; g/毫升) 和纤维连接蛋白 (50 和 #181; g/毫升) 的浓度放到烧瓶中, 确保溶液覆盖整个表面。每 T75 瓶使用10毫升。孵育在37和 #176; C 为 2 h. 
		注: 涂层可在使用前或前一周进行, 并与 PBS 一起存放在4和 #176;涂层不能干燥, 否则将不再形成合适的附着物和生长表面. </li>
		<li> 制备16毫升的理事会生长培养基, 由 DMEM/F-12 补充10% 血浆来源的血清 (PDS), 青霉素 (100 毫升), 链霉素 (100 和 #181; g/毫升), 和肝素 (15 u/毫升)。用嘌呤 (4 和 #181; g/毫升) 补充培养基, 选择内皮细胞。请注意, 嘌呤治疗最多只能使用5天. </li>
		<li> 分在两个15毫升的管子中将准备好的培养基分成6毫升和10毫升的体积. </li>
		<li> 从液氮 (步骤 1.13) 中带毛细管, 并通过使用37和 #176 来解冻毛细血管; C 水浴或添加750和 #181; 从 6-mL 分中制备的培养基 L。如果通过加入培养基, 小心吸管向上和向下解冻和质悬浮。解冻时, 将电池悬浮液转移到6毫升介质分. </li>
		<li> 移除冻结介质, 将毛细管向下旋转 250 x g 、4和 #176; C 为 7 min. </li>
		<li> 小心地吸取上清和 re-suspend 1-2 毫升的10毫升分的颗粒。当悬浮时, 将解决方案转移到 10 mL 介质分. </li>
		<li> 将毛细管悬浮液转移到涂覆的 T75 烧瓶中, 并轻轻地倾斜烧瓶, 以确保在表面上均匀分布。将 T75 烧瓶放置在37和 #176; C, 5% CO 2 . 
		 天 2 </li>
		<li> 在夜间孵化后, 准备10毫升理事会生长培养基, 辅以嘌呤 (4 #181; g/毫升), 用于一 T75 烧瓶, 并进行中等变化。请注意, 所有适用于单元格的介质解决方案应在使用前5ml 到37和 #176; C. 
		注: 或者, 当毛细血管应该附着在表面时, 介质的变化可以执行4-6 小时的 post-plating。孵育细胞直到60-95% 汇合达到, 通常3-5 天从解冻。不进行同种w 细胞达到100% 汇合, 以避免接触抑制, 可以阻止进一步的细胞生长. 
		 天 4-6 </li>
		<li> 当达到所需的70-100 时, 将内皮细胞通过渗透膜插入 (见2.2 节)。如果在4天没有达到所需的 70-100, 则执行介质的更改。在最新的6天, 细胞应传代的渗透膜插入。如果所需的70-100 在6天内未实现, 则单元格不适于实验使用. 
		注: 为促进的星形胶质细胞的制备: 在建立共培养模型时, 应在培养基上进行培养基改变, 在传代前的一天, 2-8 周老星形细胞 (见第4节) 为共培育模型做准备。对于每一个星形胶质细胞, 准备1500和 #181; DMEM 低葡萄糖的星形胶质细胞生长培养基中含有 10% FBS、青霉素 (100 毫升) 和链霉素 (100 和 #181; g/毫升), 然后进行培养基变化. </li>
	</ol> </li> <li> 渗透膜上的 pBECs 的生长 (5 天) 日 4-6 <ol> 准备渗透膜插入 (12-井板, 插入 1.12 cm <li> 2 表面积, 0.4 和 #181; m 孔隙)在 ddH 2 中, 用 IV 型胶原 (500 和 #181; g/毫升) 和纤维连接蛋白 (100 和 #181; g/毫升) 的涂层播种理事会, 使用大约300和 #181; L 为每个插入, 并确保解决方案覆盖整个增长的表面。孵育在37和 #176; C, 5% CO 2 , 至少为 2 h. </li>
		<li> 准备30毫升的理事会生长培养基 (用于培养基成分, 参考 2.1.2) 而不嘌呤. </li>
		<li> T75 瓶中从理事会培养液中吸取培养基, 并在室温下用5毫升无菌 PBS 轻轻冲洗两次. </li>
		<li> 处理将2毫升的胰蛋白酶-edta 溶液 (PBS 中的2.5% 胰蛋白酶, 0.1 nM edta) 添加到 T75, 并将烧瓶放在37和 #176; C、5% CO 2, 为 5-7 min. </li>
		<li> 要分离大脑内皮细胞, 用指尖轻轻拍打 T75 烧瓶的一侧, 最终在烧瓶表面留下存活和更强的附着周。目视观察显微镜下的剥离。当80% 支队被观察, 停止 trypsinization 通过增加5毫升培养基. </li>
		<li> 使用10毫升吸管将细胞溶液转移到15毫升管, 并通过离心在 250 x g 、4和 #176 上旋转大脑内皮细胞; C 为7分钟. </li>
		<li> 小心地去除上清和重1mL 介质中的颗粒。暂停时, 再添加2毫升介质, 以使总体积为3毫升 </li> <li> 通过使用单元计数室或自动计数系统手动计算单元数。准备一个 2.2 x 10 5 单元格/mL 介质的单元格解决方案, 最终的播种密度为 1.1 x 10 5 单元格/插入. </li>
		<li> 从渗透膜插入和转移500和 #181 中去除涂层溶液; 每个插入物的 L 细胞悬浮。在其中一个插入, 只添加介质, 并使用此插入作为一个和 #39; 仅筛选和 #39; 控制 TEER 测量. </li>
		<li> 在 mc, mc 与 ACM, 或在与星形胶质细胞的协商中, 以下列方式建立内皮干细胞: <ol> <li> 用于 mc: 添加1500和 #181; L 理事会生长培养基/ACM 每井的 12-井板下渗透膜插入物。将渗透膜插入在37和 #176; C, 5% CO 2 , 孵育2天. </li>
			<li> : 向井中传送插入星形胶质细胞, 在前一天对其进行更新。将渗透膜插入在37和 #176; C, 5% CO 2 , 孵育2天. 
			注: 注意在三机型的底部井中使用不同的介质类型. 
			 天 6-8 </li>
		</ol> </li> <li> 在2天, 更改具有 pBECs 的渗透膜插入介质。准备500和 #181; L 理事会生长培养基每插入, 并且仔细地执行变动减少细胞层的干扰。孵育细胞两天。请注意, 介质的变化只在插入物上执行, 而不是在井底. </li>
	</ol> </li> <li> 有差异因素的刺激 (1-2 天) 日 8-10 <ol> <li> 对于每个不同的模型, 刺激介质应准备如下: <ol> <li> 为 MC: 为每个插入和井, 准备500和 #181; L 理事会生长培养基, 含有 cAMP (250 和 #181; m), 氢化可的松 (550 nM) 和 RO (17.5 和 #181; m). 
			MC: 另外准备1500和 #181; L 理事会生长培养基包含阵营 (250 和 #181; m), 氢化可的松 (550 毫微米) 和 RO (17.5 和 #181; m). 
			MC 与 acm: 融解1500和 #181; L 的 acm 和补充它与阵营 (250 和 #181; m), 氢化可的松 (550 毫微米) 和 RO (17.5 和 #181; m). </li>
			<li> 用于: 每个插入的 , 准备500和 #181; 含理事会生长培养基 (250 和 #181; m), 氢化可的松 (550 nM) 和 RO (17.5 和 #181; m)。对于每个星形胶质细胞, 准备1500和 #181; DMEM/F-12 补充青霉素 (100 u/毫升) 和链霉素 (100 和 #181; 克/毫升), 肝素 (15 u/毫升), cAMP (250 和 #181; m), 氢化可的松 (550 nM) 和 RO (17.5 和 #181; m). </li>
		</ol> </li> <li> 对于每个不同的模型, 按如下方式执行媒体交换: <ol> <li> 用于 MC: 从井中精心抽出介质, 并将分度介质插入和添加到两个舱室, 使用500和 #181; L 为每个插入和1500和 #181; 我为每一个井。请注意, 插入的任何一侧的中断都可能影响和破坏单元层. </li>
			<li> 用于: 从井中仔细吸取培养基, 并加750和 #181;小心地从插入物中吸取培养基, 并加入500和 #181;然后添加其余的750和 #181; L 区分介质对每个星形胶质细胞. </li>
		</ol> </li> <li> 对于所有模型: 将单元格放置在37和 #176; C, 5% CO 2 , 并以分化培养基孵育, 直到第二天. 
		注意: 要注意, 对于星形胶质细胞, 星形胶质细胞是从这个点保存在无血清培养基中. </li>
	</ol> </li> </ol> 3。TEER 测量 <ol> <li> 在激发具有分化介质的细胞后的第二天, 准备 TEER 测量的组织电阻测量室系统, 用 ddH 2 O 两次冲洗腔, 一次用70% 乙醇5分钟和2次 ddH 2 O. </li>
	<li> 将4毫升的 DMEM/F-12 添加到组织电阻测量室, 将其连接到系统并按照下列设置校准大约30分钟: r = 0, 测试 r = 1000, 模式 = r. </li>
	<li> 通过在组织电阻测量室中仔细放置插入物来执行 TEER 测量。对于每一个插入, 执行测量三份和计算平均和 #937; cm 2 . 
	注: 对于共培养模型, TEER 值预计达到和 #62; 500 和 #937; cm 2 。如果在测量 TEER 的第一天没有达到适当的 TEER 水平, 增加新的分化因素 (阵营 (250 和 #181; m), 氢化可的松 (550 毫微米) 和 RO (17.5 和 #181; m)) 和测量 TEER 第二天。当适当的水平达到时, 模型应该为计划的实验准备好。注意, 在进行 TEER 测量后的实验之前, 细胞应该至少休息3小时。刺激后, 一般 TEER 值在刺激后仍可接受24-48 小时. 
	注: 使用刚性 STX-100C 电极对的另一种快速方法也可以在该模型 81 中记录高 TEER。柔性 STX2 电器ctrode 配对给出的读数不太可靠. </li>
</ol> 4。非接触共培养星形胶质细胞的制备: 替代方法 <ol> <li> 纯化星形胶质细胞 <ol> 为每个使用的鼠脑, 2 T75 烧瓶添加10毫升的聚 l-赖氨酸 (5 和 #181; 克/毫升) 在 ddH <li> 2 O 对每个T75 瓶在37和 #176 孵育烧瓶; C 为 30 min. </li>
		<li> 星形胶质细胞的分离可按如下方式执行: <ol> <li> 大鼠星形胶质细胞: <ol> <li> 斩首 2 1-2 天的老老鼠使用批准的方法. </li>
				<li> 将头骨上的皮肤从颈部向鼻部切开, 用矢状切口切开骨. </li>
				<li> 用弯曲钳打开颅骨, 取出大脑并将其放在15毫升管 (管 1) 中, 11 毫升的 DMEM 低葡萄糖补充 10% FBS 和硫酸庆大霉素 (125 和 #181; g/毫升). </li>
				<li> 使用细尖钳将脑膜取出, 用1毫升吸管将大脑材料暂停. </li>
			</ol> </li> <li> 猪星形胶质细胞: <ol> <li> 从一个5-6 月大的家养猪获得1的大脑. </li>
				<li> 用细尖钳和/或手小心地从大脑中取出脑膜. </li>
				<li> 从大脑中收集4克灰质, 并将1-2 毫升 DMEM 低葡萄糖补充 10% FBS 和硫酸庆大霉素 (125 和 #181; g/毫升) 在培养皿中。如果是大块的, 用手术刀或镊子剁碎. </li>
				<li> 使用1毫升吸管将隔离材料挂起, 将其移至15毫升管 (管 1), 并用 DMEM 低葡萄糖补充 10% FBS 和硫酸庆大霉素 (125 和 #181; g/毫升), 总容量为11毫升. </li>
				<li> 继续将隔离材料与10毫升注射器相连的长针进行均匀化, 并将其挂起3次。等待, 直到大件已落户在底部的管, 然后收集7毫升介质从顶部的管 (管 1). </li>
				<li> 通过40和 #181 将7毫升的电池悬浮液过滤到一个新的50毫升管 (管 2). </li>
				<li> 增加7毫升培养基到管1与大脑。继续进行同质化和过滤从步骤 4.1. 2.2. 5-6 直到管2中过滤细胞悬浮液的体积约为35毫升. </li>
			</ol> </li> </ol> </li> <li> 从 T75 烧瓶中删除涂层解决方案 [步骤 4.1.1]。用多聚 l-赖氨酸孵育后快速冲洗5毫升 PBS, 可以确保细胞不会受到涂层溶液的任何毒性作用的伤害. </li>
		<li> 将孤立的星形胶质细胞溶液在 T75 烧瓶中均匀分离和播种。在37和 #176 孵育烧瓶; C, 5% CO 2 , 3-5 天. </li>
	</ol> </li> <li> 培养星形胶质细胞, 并在 3-5 天的孵育后, 在3周后, <ol> <li> 初始区域性 , 通过仔细的吸气介质和添加10毫升进行介质变化DMEM 低葡萄糖辅以 10% FBS 和硫酸庆大霉素 (125 和 #181; g/毫升). 
			注: 第一次介质变化后, 介质应每5天更换一次。在最初的2周内, 在改变培养基的时候, 用 PBS 彻底摇动烧瓶, 用清水冲洗细胞。这有助于消除污染的小胶质细胞. </li>
			<li> 经过3周的培养, 处理在每个 T75 烧瓶的底部加入2毫升的胰蛋白酶-EDTA 溶液, 并将其孵育在37和 #176; C、5% CO 2 为 5-7 min. </li>
			<li> 停止 trypsinization 通过添加5毫升培养基, 将细胞溶液转移到15毫升管, 并将星形胶质细胞在 250 x g、 4 & #176; C 表示 7 min. </li>
			<li> 小心移除上清液, re-suspend 在 FBS 中由10% 个亚砜组成的冷冻溶液中的细胞. </li>
			<li> 计算单元格数并准备 4.0 x 10 6 单元/mL 的单元格解决方案。冻结 cryovials 中的细胞, 每瓶加1毫升. </li>
		</ol> </li> <li> 渗透膜插入系统底部井 (2-12 周) <ol> <li> 为星形胶质细胞生长用1毫升的聚 l-赖氨酸 (5 和 #181; g/毫升) 在 ddH 2 O 中的每口井准备 12-井板。把盘子放在37和 #176; C 为 2 h. </li>
			<li> 对于每个 12-井板, 准备25毫升的星形胶质细胞生长培养基 (DMEM 低血糖补充 10% FBS 和青霉素 (100 U/毫升) 和链霉素 (100 和 #181; 克/毫升)). </li>
			<li> 将星形胶质细胞从液氮中拿出来, 并通过加入750和 #181 使其解冻; L DMEM 低葡萄糖培养基。小心吸管向上和向下融化和质的悬浮。解冻时, 将细胞悬浮液转移至15毫升的管子, 并将培养基添加到总容积7毫升. 
			注意: 液氮的温度极低, 所以戴上手套等个人防护. </li>
			<li> 移除冻结介质, 将毛细管向下旋转 250 x g 、4和 #176; C 为 7 min. </li>
			<li> 小心地吸气上清液, re-suspend 细胞. </li>
			<li> 从12井板的井中取出涂层, 并将电池悬浮液转换为每井1毫升。孵育细胞在37和 #176; C, 5% CO 2 . </li>
			<li> 每隔三天刷新一次培养基, 在使用细胞与内皮细胞共培养之前, 至少要在2周内培育细胞。在解冻后的12周内, 细胞可用于共培养, 最佳年龄为2-8 周. </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

作者报告没有利益冲突。

理事会的纯化与增殖
在纯化过程中, 关键步骤包括快速有效地去除脑膜和白、灰物质的分离, 这对纯化产量和纯度以及正确建立模型具有重要意义。为提出的体外BBB 模型使用 pBECs, 我们改进和简化了基于机械均匀化的分离灰物质的纯化过程, 微血管分离的大小选择过滤, 胶原酶消化, dnasei 和胰蛋白酶, 具有微血管片段的初始培养。一般而言, 净化和培养原细胞的主要挑战之?...

血脑屏障的细胞结构与功能
在循环系统和中枢神经系统 (cns) 的界面, BBB 作为 homoeostatic 控制中枢神经系统微环境的关键调控场所, 这对神经系统的正常功能和保护至关重要。血脑屏障的部位是血管腔内衬的内皮细胞。在脑毛细血管, 内皮细胞形成复杂的细胞间紧密连接和强烈极化表达模式的特殊流入和外排转运运输确保高度特异的分子运输之间的血液和大脑1。紧密接合配合物的结构成分包括来自蛋白和 claudin 家族的蛋白质、紧密拼装(祖伊) 蛋白、cingulin 和相关的连接黏附分子 (果酱)。Claudin 5 在细胞连接限制中尤为重要。诱导和维持这一特征血脑屏障内皮表型涉及与周围细胞的动态相互作用, 包括周、星形胶质细胞、神经元和基底膜, 这些都与大脑内皮干细胞一起形成神经血管单元 (NVU)2,3。这些相互作用所涉及的机制尚未完全被理解, 但包括在细胞间交换化学信号, 这允许在短期内调制 bbb 通透性, 并诱发长期 bbb 功能4。星形胶质细胞尤其被称为对脑血管内皮表型的贡献, 是一种调节因子的来源, 如胶质源性神经营养因子 (影响细胞内 cAMP)5, 碱性成纤维细胞生长因子6,氢化可的松7, 并转化生长因子β (TGF β)8。然而, TGF β的影响已经被争论了9。
从体内到体外BBB
在体内研究继续提供有关 BBB 生物学的宝贵信息。然而, 细胞培养模型可以提供更多的见解, 并构成有用的工具, 以了解详细的分子和功能方面的 BBB 在健康和疾病。虽然脑血 BBB 的细胞类型和成分之间的复杂相互作用很难在体外模型中完全实现, 但自从第一次纯化大脑内皮细胞和在单培养中应用后, 已经有了。10,11,12, 广泛开发的净化程序和生长条件的 BBB 细胞培养模型, 导致更大的相似性的在体内屏障。通常使用的体外BBB 模型基于啮齿动物、猪和牛的原细胞, 以及永生细胞系。每个模型都有不同的优点和缺点。为了比较和模型选择, 验证标记, 如血 BBB 酶的表达, 转运蛋白, 受体和结构蛋白质被用来创建当前建立的模型的概述1。
议定书的目的
BBB 的一个重要特点是屏障严密性和高 TEER, 然而大量可用的模型并没有很好地反映在体内的水平。将开发和优化的贡献从几个实验室, 该协议的目的是提出一种方法, 建立一个高 TEER体外BBB 模型的基础上 pBECs 在 MC 与或不含 ACM, 或在与初级大鼠或猪源星形胶质细胞。该模型的应用程序和建立包括努力消除污染细胞和改善 pBECs 分化为 BBB 表型。这项工作的结果是建立了可靠的, 高 TEER 模型低细胞渗透性和良好的功能表达的关键紧密连接蛋白, 转运, 和受体。然而, 由于星形胶质细胞是一个因素, 以脑细胞表型, 三不同的文化条件代表三不同的表现型脑内皮细胞。该模型对于研究药物发现、运输研究和细胞内贩运的某些专门机制, 以及对血脑屏障功能最大表达的细胞间相互作用的调查是特别有用的。有利.
议定书的起源和历史
这里描述的理事会模型主要基于在卫材实验室开发的猪模型 (伦敦) 由 Dr. 路易斯摩根和同事, 是基于成功的早先牛脑内皮细胞模型13。原细胞制备方法是用尼龙网格捕捉微血管的两级过滤, 其次是传代步骤以提高纯度。在该方法的早期发展中, 通过补充培养基 (包括 ACM) 的生长, 达到了最佳 BBB 表型和屏障的致密性。对方法的进一步修改由 r. 斯金纳在 Rothwell 的实验室在曼彻斯特英国14,15中进行。该方法是通过雅培实验室, 氯化钾伦敦, 在那里 Patabendige 使它大大简化准备通过避免使用星形胶质细胞或 ACM 和消除污染细胞, 如周与嘌呤。第一篇论文证实 MC 模型保留了在体内BBB 的几个重要特征, 包括有效的紧密连接, 膜传输系统和受体介导的 transcytosis16,17,18,19,20. 后来 s. 尤索夫再次测试星形胶质细胞共培养, 发现它显著改善了 TEER, 所以这是目前在 Abbott 实验室21中使用的首选变体。该模型现已成功地转移到了奥胡斯的 m. 尼尔森实验室, 在那里进行了进一步的修改 (本议定书), 包括简化灰色物质提取, 只使用一个滤网过滤步骤, 和一个单一的过滤涂层步骤结合胶原蛋白和纤维连接蛋白。猪星形胶质细胞分离的应用程序 (本议定书) 是基于奥尔堡的 t Moos 实验室开发的协议, 由汤姆森et al22描述。在伦敦和奥胡斯所产生的模型的 TEER 和其他性质是相似的, 这使人们相信模型很容易在实验室之间转移, 并能很好地对方法步骤的仔细观察和合理化作出反应。事实上, 尤索夫已经建立了一个热带国家 (马来西亚)23的 MC 模型, 它涉及对当地条件和组织来源的进一步调整。
优于替代方法和现有模型的优势
与牛和啮齿类动物的脑内皮细胞相比, pBECs 提供了在隔离状态下的在体内BBB 表型的损失率较低的优势.此外, pBECs 能够形成相对严密的内皮屏障, 即使在 MC (800 Ω cm2)16中生长, 与通常报告的细胞系的单分子层 (如弯曲. 5 和弯曲) 相比. 3 (50 Ω cm2)25,26, 27, cEND (300-800 Ωcm2)282930和 cerebEND (500 Ω cm 2)2931,32和主脑小鼠的内皮细胞 (100-300 Ω厘米2)ss = "xref" > 33,34,35,36和 rat (100-300 Ω cm2)37,38。然而, TEER 已经显示了对纯化和培养程序的依赖性。在大多数情况下, 增加的 ACM 或共培养与星形胶质细胞显示出差异的影响, 血管内皮细胞膜和加强致密的内皮层1。然而, 在优化培养条件的努力下, 只有 bovine-based 模型显示了与 porcine-based 模型相似的 TEER 值 (MC 中的800ω cm2的平均值 (在星形胶质细胞共育中高达2500ω cm2 )13 ,39,40,41,42,4344,45。由于基于初级牛脑内皮细胞的模型显示出很大的变化, 两个实验室之间和内部的14,45,46,47,48,重现性可能是个问题。在这里报告的理事会模型中, 提供的实验室已经达到了非常相似的 TEER 和细胞渗透率的低变异性, 在实验室和之间。因此, 其他实验室应该可以利用这里提出的方法建立一个低变异性的鲁棒模型。除了形成致密的内皮层, 模型与 pBECs 以前已经通过表达紧密连接蛋白, 功能 BBB 转运体, 受体和酶, 并证明适合的一系列研究15,16,17,19,20,22,49,50,51,52,53,54,55,56,57,58,59. 此外, pBECs 的 co-cultures 上未发布的转录数据显示了 BBB 转运体和受体的预期分布 (未发表的结果, 尼尔森et al.)。
porcine-based BBB 模型具有更大的优势, 因为猪的基因组、解剖学、生理学和疾病进展比其他建立的模型60更能反映人类生物学的程度, 这是制药业。由于猪的大脑是肉类工业的常见 by-product, 它们构成了一个易于获取的脑血管内皮细胞来源, 使实验所需的动物数量减少, 并提供了一个猪脑的高纯度的产量。虽然纯化和培养初级细胞是有点费时, 需要专门知识的标准化建立模型, 初级细胞产生最可靠的 BBB 模型。永生化细胞系不能替代, 如屏障严密性、转运蛋白表达谱和微环境调节等重要特性不反映实验结果在体内61,62.体外模型提供了更高分辨率的活细胞成像的优势, 通过允许对取样或观察到的细胞进行近距离接近, 从而实现了细胞内过程的可视化, 使用目标具有更高的放大倍数和更好的光学质量63。这不是使用双光子显微镜在活体动物中的情况。此外,体外模型提供了染细胞的能力, 允许可视化标记蛋白质和调查其贩运。
模型的应用
血脑屏障功能不固定, 可以在生理和病理两方面进行动态调节。在许多神经系统疾病, 包括神经退行性、炎症和传染性疾病, 扰乱和提高 BBB 的渗透性被观察64,65,66,67.为了减少和防止疾病进展和随后的损害, 识别和定性的分子机制的基础上的调制的 BBB 是重要的。在这种情况下, 可靠的体外模型在制药工业中需求量很大, 而且在预测药物对中枢神经系统的 BBB 通透性方面发挥了重要作用。任何体外模型都应显示限制性的细胞通路、生理上逼真的细胞结构和传送器机制的功能表达式68。在先前的研究中演示了16,17,57, 并通过细胞的渗透率和 TJ 和 AJ 蛋白的表达, 提出的模型符合所有这些标准, 适用于 BBB 的范围在正常生理学和病理学方面的研究。所提出的纯化和培养方法的优点包括简单性和重现性的结合, 并能够包括星的影响与产生的强大和可靠的高 TEER在体外BBB 模型。为此, 已证明猪和大鼠的星形胶质细胞以类似的方式增强了 pBECs 的 BBB 表型.22。

<table><tbody><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Collagen IV</td><td>Sigma-Aldrich</td><td>C5533</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P1524</td><td></td></tr><tr><td>DMEM/F-12</td><td>Lonza</td><td>BE12-719F</td><td></td></tr><tr><td>DMEM/Low Glucose</td><td>Sigma-Aldrich</td><td>D6046</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco Invitrogen</td><td>15140</td><td></td></tr><tr><td>Plasma derived serum (PDS)</td><td>First Link UK Ltd.</td><td>60-00-89</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10-270-106</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Gibco Invitrogen</td><td>15090-046</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H4001</td><td></td></tr><tr><td>8-CPT-cAMP</td><td>Biolog</td><td>C010</td><td></td></tr><tr><td>RO 20-1724</td><td>Sigma-Aldrich</td><td>B8279</td><td></td></tr><tr><td>Gentamicin Sulfate</td><td>Lonza</td><td>17-518Z</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>34896</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>EtOH</td><td>VWR</td><td>20,824,296</td><td>Mix the 70 % solution from the 96 % EtOH</td></tr><tr><td>DNAse 1</td><td>Sigma-Aldrich</td><td>D4513</td><td></td></tr><tr><td>Collagenase CLS2</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Made with Elga System</td></tr><tr><td>T75 flasks</td><td>Thermo Scientific</td><td>156499</td><td></td></tr><tr><td>Costar Transwell inserts (Cell permeable membrane inserts)</td><td>Costar</td><td>CLS3401</td><td>12-well plate, 12 mm diameter, 0.4 &amp;mu;m polycarbonate membrane</td></tr><tr><td>15 ml centrifuge tubes</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>Cellstar</td><td>227261</td><td></td></tr><tr><td>Petri dishes</td><td>Thermo Scientific</td><td>150350</td><td></td></tr><tr><td>Cryo vials</td><td>Thermo Scientific</td><td>377224</td><td></td></tr><tr><td>500 ml bottle</td><td>Thermo Scientific</td><td>159910/159920</td><td></td></tr><tr><td>Scalpels</td><td>Swann-Morten</td><td>REF0211</td><td>Type 24</td></tr><tr><td>Tissue homogenizer</td><td>Sigma</td><td>D9188</td><td></td></tr><tr><td>140 &amp;mu;m filters</td><td>MERCK</td><td>NY4H04700</td><td></td></tr><tr><td>40 &amp;mu;m filters</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>EndOhm chamber system</td><td>World Precision Instruments</td><td>ENDOHM-12</td><td>EndOhm chamber for 12mm Culture Cups</td></tr><tr><td>EVOM2 electrode system</td><td>World Precision Instruments</td><td>300523+STX100C</td><td>TEER measurement system with rigid STX-100C electrode pair</td></tr><tr><td>Long needle</td><td>Sigma</td><td></td><td>Attach to a syringe</td></tr><tr><td>Fine-tip curved forceps</td><td>KLS Martin</td><td>12-409-12-07</td><td></td></tr><tr><td>Broad tip forceps</td><td>VWR</td><td>82027-390</td><td></td></tr><tr><td>Filter holder</td><td>MERCK Milipore</td><td>Swinnex-47</td><td></td></tr><tr><td>50 ml syringe</td><td>Braun</td><td>4617509F</td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SSt20ESI</td><td></td></tr><tr><td>Anti-Occludin antibody</td><td>Abcam</td><td>ab31721</td><td>1:100</td></tr><tr><td>Anti-p120 Catenin antibody</td><td>BD Transduction laboratories</td><td>610133</td><td>1:200</td></tr><tr><td>Anti-ZO-1 antibody</td><td>Invitrogen</td><td>61-7300</td><td>1:200</td></tr><tr><td>Anti-Claudin 5 antibody</td><td>Sigma-Aldrich</td><td>SAB4502981</td><td>1:100</td></tr><tr><td>Donkey anti rabbit IgG conjugated with Alexa Flour 568</td><td>Thermo Scientific</td><td>A10042</td><td>1:500</td></tr><tr><td>Donkey anti mouse IgG conjugated with Alexa Flour 488</td><td>Thermo Scientific</td><td>A21202</td><td>1:500</td></tr><tr><td>Sucrose</td><td>Perkin Elmer</td><td>NEC100X250UC</td><td>0.15&amp;micro;l/ml final working conc</td></tr><tr><td>Lucifer Yellow</td><td>Sigma</td><td>L0144</td><td>10 &amp;micro;g/ml final working conc</td></tr></tbody></table>

improved method for the establishment of an in vitro blood-brain barrier model based on porcine brain endothelial cells

本议定书的目的是为纯化和培养 pBECs, 并建立体外血脑屏障 (BBB) 模型的基础上 pBECs 单培养 (mc), mc 与星形胶质细胞条件培养基 (ACM), 并与猪或大鼠源星形胶质细胞的非接触共培养。pBECs 从5-6 月大的家养猪脑皮质的毛细血管片段中分离和培养。这些碎片通过仔细去除脑膜, 分离和均匀化的灰质, 过滤, 酶消化, 和离心纯化。为了进一步消除污染细胞, 用含嘌呤的培养基培养毛细血管片段。当60-95% 汇合, pBECs 生长从毛细管片段被传代对渗透性膜过滤器插入物和建立在模型。为提高 pBECs 的屏障严密性和血脑屏障特征表型, 对细胞进行了以下分化因素的处理: 膜 permeant 8-CPT-camp (这里缩写阵营), 氢化可的松, 磷酸二酯酶抑制剂, RO-20-1724(RO)。该手术在9-11 天内进行, 在建立该模型时, 星形胶质细胞被提前2-8 周培养。遵守该议定书所述的程序允许建立具有高度限制的细胞渗透性的内皮层, 而该模型显示了1249年±80Ω cm 的平均 transendothelial 电阻 (TEER).2, 和细胞渗透性 (Papp) 为路西法黄色的 0.90 10-6 ± 0.13 10-6 cm 秒-1 (平均± SEM, n=55)。该理事会表型的进一步评价表明, claudin 5、ZO-1、蛋白和黏着结蛋白 p120 蛋白的紧密连接蛋白表达良好。该模型可用于健康和疾病 BBB 的一系列研究, 并具有高度限制性的细胞渗透性, 该模型适用于研究运输和细胞内贩运。

BBB体外模型的建立
在所提出的优化方法, 培养 pBECs 和建立的渗透膜插入系统与 MC 或不含 ACM 或全国星形胶质细胞 (图 1) 进行了为期9-11 天 (图 2)。对于内皮细胞的选择, 初步培养的纯化毛细管片段与嘌呤治疗相结合, 最多5天, 消除了大多数污染细胞, 促进了纺锤形内皮细胞的生长?...

Watch this Scientific Journal Video about Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells at JoVE.com

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

该协议的目的是提出一个优化的程序, 建立一个体外血脑屏障 (BBB) 模型的基础上猪脑内皮细胞 (pBECs)。该模型具有重现性高、严密性高的优点, 适合于研究药物发现中的转运和胞内贩运。

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

improved-method-for-establishment-an-vitro-blood-brain-barrier-model

Research

JoVE Journal

Medicine

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

14.7K 次观看.  Aarhus University. 该协议的目的是提出一个优化的程序, 建立一个体外血脑屏障 (BBB) 模型的基础上猪脑内皮细胞 (pBECs)。该模型具有重现性高、严密性高的优点, 适合于研究药物发现中的转运和胞内贩运。

视频: Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

科研

生物工程

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 ...

神经科学

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

设置了一个<em&gt;体外</em大鼠血脑屏障&gt;模型（BBB）：在BBB抗渗性和受体介导的运输为中心

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

医学

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

发育生物学

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 ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号:对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

免疫与感染

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

研究病原体或药物交叉障碍及其与大脑相互作用的人血脑界面模型

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

源自人类多能干细胞的 3D 血脑屏障模型

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

作者聚焦:使用体外血脑屏障模型研究阿尔茨海默病的个性化方法 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

体外血脑屏障重建，模拟和治疗神经系统疾病

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

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此视频中的章节

改进方法对血脑屏障的人体细胞为主，接触模型的制备

代的永生化小鼠脑微血管内皮细胞株作为

设置了一个

生成基于人类 iPSC 的血脑屏障芯片

原代鼠脑微血管内皮细胞的分离

一个

新鲜人脑组织中毛细血管的分离

研究病原体或药物交叉障碍及其与大脑相互作用的人血脑界面模型

体外血脑屏障重建，模拟和治疗神经系统疾病

体外血脑屏障重建，模拟和治疗神经系统疾病