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>ddH2O</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 &#956;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 &#956;m filters</td>
			<td>MERCK</td>
			<td>NY4H04700</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;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&#181;l/ml final working conc</td>
		</tr>
		<tr>
			<td>Lucifer Yellow</td>
			<td>Sigma</td>
			<td>L0144</td>
			<td>10 &#181;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

Universidad de Cartagena UDEC

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

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

科研

医学

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

作为脑微血管内皮细胞（CEND）的伸展<em&gt;体外</em创伤性脑损伤模型血脑屏障

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

缺氧缺糖再给氧作为<em&gt;在体外</em&gt;缺血再灌注损伤模型的研究血脑屏障功能障碍

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

用于评估椎弓根螺钉的麻醉，手术和收获方法<em&gt;体内</em&gt;猪腰椎模型

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

评估血脑屏障网状血管形成和破坏的体外测定

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

摘要

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此视频中的章节

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

允许有针对性的大鼠模型血 - 脑屏障破坏神经血管治疗

作为脑微血管内皮细胞（CEND）的伸展

设置了一个

脑Microslices的兴奋性刺激作为

缺氧缺糖再给氧作为

在小鼠气管插管快速的改进方法

用于评估椎弓根螺钉的麻醉，手术和收获方法

评估血脑屏障网状血管形成和破坏的体外测定

猪静脉移植病模型的建立与评价