Name: a choroid plexus epithelial cell-based model of the human blood-cerebrospinal fluid barrier to study bacterial infection from the basolateral side
Uploaded: 2016-05-06T15:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side at JoVE.com

The authors would like to thank Prof. Hartwig Wolburg for performing the electron microscopy.

1.准备播种HIBCPP细胞细胞培养滤芯在倒立模型系统<ol><li>补充有5微克/毫升胰岛素预暖的DMEM / F12（HAM），100U / ml青霉素，100微克/毫升链霉素和10％胎牛血清（FCS）。 </li><li>使用无菌镊子放置0.33平方厘米的生长区域的细胞培养滤芯为3微米的孔尺寸倒置放入一个12孔板（ 图1E）。 </li><li>填充介质进入细胞培养滤芯的下部隔室（约3毫升）和100微升在过滤器插入件的顶端。淹没板以及与介质的下隔室。吸在这样一种方式，滤波器插入件的下隔室保持填充（ 图1E）的过度介质。 注:建议使用血清吸管用于此步骤。 </li><li>盖上盖12孔板和制备的细胞培养滤芯转移到孵化器，37℃，5％的CO 2，直到加入细胞。 </li></ol> 2.栽培HIBCPP细胞传代<ol><li>制备补充有5微克/毫升，100U / ml青霉素，100微克/毫升链霉素和10％FCS的DMEM / F12（HAM）。 </li><li>预暖介质和PBS在37℃水浴中。从瓶中吸出网上平台。加入10毫升的PBS至烧瓶并乱舞。重复此步骤一次。 </li><li>添加3毫升0.25％胰蛋白酶-EDTA至烧瓶并乱舞。放入培养箱，在37℃，5％的CO 2。 </li><li>大约20分钟后，除去从孵化器的烧瓶中。确保细胞从烧瓶底部脱离，当用显微镜调查显示圆​​形。 注意:细胞不彼此完全分离，并且经常在附聚物中。它建议使用细胞达通道38。 </li><li>要停止胰蛋白酶加入17β-毫升培养基。通过上下吹打重悬细胞并转移该悬浮液到50毫升管中。离心，在50×g离心在室温下10分钟。 </li><li>悬浮细胞培养基中的适当体积，并通过使用血球计数细胞。 注意:悬浮HIBCPP细胞的浓度应为1×10 6个细胞/ ml。维护HIBCPP细胞，建议在10毫升培养基转移1-6×10 6细胞的量到一个T75烧瓶。更换培养基每隔一天。 </li></ol> 3.播种倒细胞培养滤芯与HIBCPP细胞<ol><li>每个反相滤波器插入件（ 即过滤器的底侧）的顶部添加80微升细胞悬浮液（ 即 ，8×10 4个细胞）（ 图1E）的。 注意:确保细胞均匀地播种前颠倒试管分布于停牌。 </li><li>覆盖种子细胞培养滤芯与12孔板，并转移到孵化器的盖，在37℃，5％CO 2。 </li><li>在第一天，填充1ml培养基到24孔板的孔中。通过使用镊子出12孔板的解除细胞培养滤波器的插入，丢弃培养基内，转动过滤器插入并放置在标准定向到准备24孔板（ 图1E）。 </li><li>放置细胞进入新鲜培养基每隔一天。准备24井用新鲜的介质和滤芯转移到它。填用0.5ml新鲜培养基插入。检查TEER每天都在第4节。 </li><li>当在细胞培养插入HIBCPP接种细胞的TEER值超过70Ω点¯x平方厘米（约种植后4天），然后继续在含1％FCS和5微克/毫升胰岛素介质细胞培养物。准备24孔板用每孔1ml培养基。转移滤芯到准备好的井和交换介质上格。 注:汇合后，此血清撤离导致的形成更高的膜电位。 </li><li>从过滤器吸车厢中，转移到准备好，用500微升培养基填充。重复此步骤一次。把细胞过夜进入培养箱，在37℃，5％的CO 2。 </li></ol> 4.跨膜电阻的测量（TEER） <ol><li>沉浸上皮组织voltohmmeter的电极尖端为在80％乙醇15分钟。引擎盖下转移voltohmmeter，让电极干一会儿。放置电极到用于所述细胞另外15分钟以达到平衡各自的培养基。 </li><li>通过这样它每次触及下部隔室的底部的电极的较长臂定位执行测量，较短的臂放置到过滤器插入件隔室。 注:细胞培养滤芯的电阻值，而不在介质中的细胞应作为空白值（（测量值（Ω） - 空白值（Ω））×滤波器表面（ 即 0.33平方厘米））。 </li><li>测量代替电极回80％乙醇15分钟后。存放在干燥管中。 </li></ol> 5.测定细胞旁渗透性<ol><li>在补充有5微克/毫升胰岛素1％FCS的200毫升培养基溶解1g的FITC-菊粉。细胞的感染前应用50μg/ ml的溶液进入上部过滤器室的。 </li><li>感染后收集培养基样品从下面的井，以确定多从菊糖将过滤器如何穿过细胞层。 </li><li>制备的FITC菊粉标准溶液，并执行1:2稀释液，10倍（100％，50％，25％，12.5％，6.25％，3.13％，1.56％，0.78％，0.39％，0.2％和0％） 。在酶标仪所有样品的测量确定荧光。 </li></ol> 6.细胞培养滤芯细菌的HIBCPP感染细胞的制备<ol><li>有一天，在实验前，刮Ť他冷冻N.株脑膜炎掀起了甘油的股票和连胜了与Vitox巧克力琼脂。在孵化生长过夜，在37℃下，用5％ 的 CO 2。 </li><li>从过夜培养提取20-30殖民地和转移到装有8毫升蛋白胨培养基中添加0.042％ 碳酸氢钠 ，0.01M的氯化镁和1％Polyvitex管。 </li><li>摇细菌1.25小时，以220rpm，37℃。沉淀通过离心细菌在2684克在室温下10分钟。丢弃上清，悬浮用8ml无血清培养基中的细菌沉淀。涡流以确保均匀悬浮液。 </li><li>以确定培养物密度，在600nm的光密度（OD）测量的1:10的稀释液。调节细菌悬浮液的OD 0.1 600。 注:此悬浮液含有约。 1×10 8 CFU / ml的。 </li><li>确认细菌细胞浓度，稀释该悬浮液逐步（1:10）到一个10 -5稀释和平板接种到巧克力琼脂平板上。 注意:类似的系列稀释需要执行计算细菌生长曲线。 </li></ol>在双重免疫细胞培养滤芯和测定细菌侵入HIBCPP细胞感染7 <ol><li>继续在含有1％FCS和5微克/毫升胰岛素培养基的细胞培养后，使用上皮组织voltohmmeter测量细胞的每一天。如果电池已经达到了大约500Ω点¯x平方厘米TEER，进行感染。 </li><li>感染与制备细菌悬浮液中的细胞在感染10商店感染的细胞在培养箱的（MOI）的多重性，在37℃，5％CO 2的时间的指示的时间。 注意:MOI可以通过考虑在合流（1.21×10 6个细胞/ cm 2）每细胞培养滤芯细胞的数目来计算。 </li><li>停止侵染离子洗涤用500μl无血清培养基含有1％牛血清白蛋白（BSA）的三倍（SFM）施加到过滤器室，将1ml到下​​部隔室。 </li><li>用含有500微升的SFM 1％BSA缓冲液中的过滤器室和1ml在下部隔室20分钟，在室温下，以防止抗体的非特异性结合位点的粘附阻止。 </li><li>用100μl初级抗体抗N.孵育meningtidisα-OMP（1:200），在过滤器室和500微升在在室温下20分钟的下隔室。 注意:如果需要，抗体浓度需要被滴定。 </li><li>通过吸从过滤室中的介质洗涤细胞，滤芯转移到含有1％BSA一个制备好用1ml SFM和填充用含有1％BSA的500μl的SFM的排空过滤隔室。重复此步骤两次。 </li><li>固定用500μl的4％甲醛中的细胞过滤器COMPArtment，将1ml在室温10分钟的下隔室。 </li><li>通过吸从过滤室中的介质洗涤细胞，滤芯转移到准备好用1ml PBS中并填充用500μlPBS中清空过滤隔室。重复此步骤一次。 注:样品可以在4℃保存过夜于PBS中。 </li><li>切割固定细胞培养筛选出插入物，用含有1％BSA的250μl的PBS洗涤。孵育15分钟，细胞在室温下与标记的250微升荧光（激发波长594纳米）的第二抗体鸡抗兔（1:500）染色细胞外细菌。 注意:如果需要，抗体浓度需要被滴定。 </li><li>透用含有1％BSA和0.5％的Triton X-100在室温下1小时，250微升PBS中的细胞。洗涤细胞三次用含有1％BSA的250μl的PBS中。 </li><li>用250微升一抗抗孵育N.脑膜炎 </青霉&gt;α-OMP（1:200），30分钟进行染色细胞内外的细菌。洗涤细胞三次用含有1％BSA的250μl的PBS中。 </li><li>应用荧光标记的（激发波长488纳米）的第二抗体（1:500）的250微升，荧光标记的（激发波长660纳米）鬼笔环肽（1:250）和4&#39;-，6-二脒基-2-苯基二盐酸盐（DAPI）（ 1:50,000）在室温下1小时，染色细胞内外的细菌，肌动蛋白细胞骨架和细胞核。 注意:如果需要，抗体浓度需要被滴定。 </li><li>洗涤细胞三次用含有1％BSA的250μl的PBS中。嵌入安装在4℃介质和存储，直到通过显微镜观察细胞。 </li><li>确定每个预定义字段入侵细菌的数量。通过计算每滤膜20场做到这一点。计算入侵细菌的百分比。 注意:使用面积coeffic乘以20微观领域的平均细菌数ient。结果表示存在于0.33平方厘米的细胞培养滤芯总细菌的量。感染的持续时间期间除以在培养基中生长的细菌的量该值。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+capsular+polysaccharides+indice+inflammatory+responses+via+TLR2+and+TLR4-MD-2.">Neisseria meningitidis capsular polysaccharides indice inflammatory responses via TLR2 and TLR4-MD-2.</a> J Leukoc Biol. 89, 469-480 (2011).</li><li>Yamamoto, M., Yamazaki, S., Uematsu, S., Sato, S., Hemmi, M., Hoshino, K., 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=Regulation+of+Toll/IL-1-receptor+-mediated+gene+expression+by+the+inducible+nuclear+protein+IkappaBzeta.">Regulation of Toll/IL-1-receptor -mediated gene expression by the inducible nuclear protein IkappaBzeta.</a> Nature. 430, 218-222 (2004).</li><li>Lorenz, J., Zahlten, J., Pollok, I., Lippmann, J., Scharf, S., N'Guessan, P. 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E., Schoeller, J., Steinmann, U., Borkowski, J., Ishikawa, H., 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=Chemotaxis+of+T-cells+after+infection+of+human+choroid+plexus+papilloma+cells+with+Echovirus+30+in+an+in+vitro+model+of+the+blood-cerebrospinal+fluid+barrier.">Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood-cerebrospinal fluid barrier.</a> Virus Res. 170, 66-74 (2012).</li><li>Chodobski, A., Szmydynger-Chodobska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+plexus:+Target+for+polypeptides+and+site+of+their+synthesis.">Choroid plexus: Target for polypeptides and site of their synthesis.</a> Microsc. Res. Tech. 52, 65-82 (2001).</li><li>Dickson, P. W., Schreiber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+messenger+RNA+for+transthyretin+(prealbumin)+in+human+choroid+plexus.">High levels of messenger RNA for transthyretin (prealbumin) in human choroid plexus.</a> Neurosci. Lett. 66, 311-315 (1986).</li><li>Stylianopoulou, F., Herbert, J., Soares, M. B., Efstratiadis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+insulin-like+growth+factor+II+gene+in+the+choroid+plexus+and+the+leptomeninges+of+the+adult+rat+central+nervous+system.">Expression of the insulin-like growth factor II gene in the choroid plexus and the leptomeninges of the adult rat central nervous system.</a> Proc Natl Acad Sci USA. 85, 141-145 (1988).</li><li>Lim, L., Zhou, H., Costa, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+winged+helix+transcription+factor+HFH-4+is+expressed+during+choroid+plexus+epithelial+development+in+the+mouse+embryo.">The winged helix transcription factor HFH-4 is expressed during choroid plexus epithelial development in the mouse embryo.</a> Proc. Natl Acad. Sci. USA. 94, 3094-3099 (1997).</li><li>Vandenhaute, E., Stump-Guthier, C., Lasierra Losada, M., Tenenbaum, T., Rudolph, H., Ishikawa, H., 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+choroid+plexus+may+be+an+underestimated+site+of+tumor+invasion+to+the+brain:+an+in+vitro+study+using+neuroblastoma+cell+lines.">The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines.</a> Cancer Cell Int. , 15-102 (2015).</li></ol>

The authors have nothing to disclose.

CP的上皮细胞形成分隔从血液2,3脑脊液的BCSFB。我们最近成立了HIBCPP细胞系作为BCSFB的功能人体模型。细胞显示在体外 BCSFB的重要屏障功能，包括一个高的膜电位的发展，一个低渗透性的大分子，以及紧密连接5的连续股线的存在。的TJ蛋白向细胞的顶/底外侧极性。极性是表面受体的定向定位特定的转运蛋白高的重要性，以及。我们已经证明了受体ECAD和Met以及ATP结合盒的部件...

血-脑脊髓液屏障（BCSFB）是血液和脑1之间三个屏障站点之一。其形态相关成分是脉络丛（CP）2,3-上皮细胞，内皮上皮回旋，这强烈血管和位于脑的脑室。将CP用来产生脑脊液（CSF），以及后者从血液中分离出来。为了实现屏障功能，在CP的上皮细胞显示出低的吞饮活性，表达特定转运，并且通过紧密连接（紧密连接）2,3-的连续网络密集连接。 人类脉络丛乳头状瘤（HIBCPP）细胞，从日本女子4的恶性脉络丛乳头状瘤衍生的，用于构建在 BCSFB的体外模型的功能。 HIBCPP细胞显示一对夫妇的功能BCSFB的特点，为TJ形成股线，可以判断为跨上皮电阻（TEER）高跨上皮膜电位的发展，以及对大分子次要渗透率。此外，HIBCPP细胞表达的特点转运，这可能会起到调节离子微环境，并显示心尖/基底极性5,6,7。 该BCSFB已显示作为一个条目的网站的病原体（细菌，病毒和真菌）进入中枢神经系统（CNS）8。病原体，包括脑膜炎奈瑟氏球菌 （ 脑膜炎奈瑟氏球菌 ），一种革兰氏阴性细菌，入侵能引起严重的疾病如脑膜炎。 证据表明，它克服了CP的保护上皮屏障是由组织病理学观察患者支持与脑膜炎球菌性疾病表现出增加的血管和CP上皮细胞9,10-脑膜炎球菌的量。要进入宿主细胞巴cteria经常挟持内吞机制，其介导或由位于宿主细胞特异性表面受体触发。由于具有这些受体的病原体的相互作用可以是物种特异性11，动物模型只能协商，以一个受限制的程度。该HIBCPP细胞系提供了机会，研究入侵过程中，以及在人体模型系统的分子机制。采用细胞培养插入使我们能够分析病原体相互作用与宿主细胞从两个不同的细胞两侧。许多细菌，包括N。脑膜炎 ，强烈受重力的期间感染测定法的影响。对于与检测期间HIBCPP细胞病原体最佳交互，该细菌最初添加到细胞培养滤芯系统的上隔室。到从心尖或基底细胞侧，分别在体外系统的两种变化已经埃斯塔使感染blished:在标准系统HIBCPP细胞接种到过滤器插入件的上部隔室，模仿情况时微生物位于CSF的侧，并获得与所述细胞（ 图1A，C）的顶面接触。与此相反，使用HIBCPP细胞在倒置的细胞培养滤芯系统反映当细菌进入血流的条件。微生物传播在从基底外侧侧（ 图1B，D）的血液和遭遇的CP上皮细胞。值得注意的是，在该模型系统，已经表明，细菌侵入HIBCPP细胞极性的方式专门从基底细胞侧5,7。 随后向CP的感染，所述入侵病原体可通过结扎先天免疫系统对模式识别受体（的PRRs）识别。蓝耳的良好描述成员属于Toll样受体（TLR）家族。 Toll样受体能斌d，来感染性微生物的特性的结构，其被称为病原体相关分子模式（PAMP）。受体的结扎导致触发的细胞因子和趋化因子12，其反过来横跨BCSFB 13,14刺激免疫细胞的轮回的表达宿主细胞的活化信号级联。它已经显示HIBCPP细胞表达在mRNA水平数的TLR和感染N.脑膜炎导致多种细胞因子和趋化因子，包括CXCL1-3，IL6，IL8和TNFα15,16的分泌。 在这里，我们描述了一个模仿BCSFB倒置细胞培养插管系统种植和人细胞系HIBCPP的感染。此模型系统使得来研究与体内的相关基底细胞侧以及随后的细胞反应的病原体的相互作用。

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>4&amp;acute;,6 diamidino-2-phenylindole (DAPI)</td><td>Life Technologies</td><td>D1306</td><td></td></tr><tr><td>12-well plates</td><td>Starlab</td><td>CC7682-7512</td><td></td></tr><tr><td>24-well plates</td><td>Starlab</td><td>CC7682-7524</td><td></td></tr><tr><td>Anti Neisseria meningitidis &amp;alpha;-OMP</td><td></td><td></td><td>This antibody was a gift from Drs. H. Claus and U. Vogel (University of W&amp;uuml;rzburg, Germany)</td></tr><tr><td>Alexa Fluor 488 (chicken anti rabbit)</td><td>Invitrogen</td><td>A21441</td><td></td></tr><tr><td>Alexa Fluor 594 (chicken anti rabbit)</td><td>Invitrogen</td><td>A21442</td><td></td></tr><tr><td>Alexa Fluor 660 Phalloidin</td><td>Invitrogen</td><td>A22285</td><td></td></tr><tr><td>Bovine serum albumine (BSA)</td><td>Calbiochem</td><td>12659</td><td></td></tr><tr><td>Chocolate agar plates</td><td>Biomerieux</td><td>43109</td><td></td></tr><tr><td>Cytochalasin D</td><td>Sigma</td><td>C8273</td><td></td></tr><tr><td>DMEM/F12 + L-Glut + 15 mM HEPES</td><td>Gibco</td><td>31330-095</td><td></td></tr><tr><td>DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred</td><td>Gibco</td><td>11039-047</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Life Technologies</td><td>10270106</td><td></td></tr><tr><td>FITC-Inulin</td><td>Sigma</td><td>F3272</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>19278</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>2393</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>55761</td><td></td></tr><tr><td>PBS + Mg + Ca</td><td>Gibco</td><td>14040-174</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>MP Biomedicals</td><td>1670049</td><td></td></tr><tr><td>Polyvitex</td><td>Biomerieux</td><td>55651</td><td></td></tr><tr><td>Proteose peptone</td><td>BD</td><td>211684</td><td></td></tr><tr><td>Serum-free medium</td><td>Gibco</td><td>10902-096</td><td></td></tr><tr><td>Thincert cell culture inserts for 24-well plates, pore size 3 &amp;micro;m</td><td>Greiner</td><td>662630</td><td></td></tr><tr><td>Tissue culture flask 75 cm&amp;sup2; red cap sterile</td><td>Greiner</td><td>658175</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Volt-Ohm Meter Millicell-ERS2 with MERSSTX01 electrode</td><td>Millipore</td><td>MERSSTX00</td><td></td></tr></tbody></table>

a choroid plexus epithelial cell-based model of the human blood-cerebrospinal fluid barrier to study bacterial infection from the basolateral side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

在这里，我们描述了一个倒置的细胞培养插管系统培养和HIBCPP细胞的感染。此模型使我们能够研究入侵机制和潜在的分子信号传导通路从基底细胞侧，再现细菌传播并且经由血液流进入上皮细胞（ 图1）的生理状况。 该HIBCPP细胞显示一定的屏障功能，这使他们能够限制分子交换到最低水平。这是通过粘附连接（AJ?...

Watch this Scientific Journal Video about A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side at JoVE.com

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

人体血液，脑脊液屏障的脉络丛上皮基于Cell的产品型号从基底外侧面研究细菌感染

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...

a-choroid-plexus-epithelial-cell-based-model-human-blood

Research

JoVE Journal

Medicine

14.1K 次观看.  Heidelberg University. The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

视频: A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

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

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

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 ...

神经科学

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

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) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

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

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

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) ...

医学

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

微生物通过微血管内皮细胞对血脑屏障渗透性分析

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

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 ...

Microdissection and Whole Mount Scanning Electron Microscopy Visualization of Mouse Choroid Plexus

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in neuroscience. As it is becoming increasingly clear that this tiny structure plays a crucial role in health and disease of the central nervous system (CNS), it is of utmost importance to properly dissect the CP out of the brain ventricles in a way that allows downstream processing, ranging from functional to structural analysis. Here, isolation of the lateral and fourth brain ventricle mouse CP without the need for specialized tools or equipment is described. This isolation technique preserves the viability, function, and structure of cells within the CP. On account of its high vascularization, the CP can be visualized floating inside the ventricular cavities of the brain using a binocular microscope. However, transcardial perfusion required for downstream analysis can complicate the identification of the CP tissue. Depending on the further processing steps (e.g., RNA and protein analysis), this can be solved by visualizing the CP via transcardial perfusion with bromophenol blue. After isolation, the CP can be processed using several techniques, including RNA, protein, or single cell analysis, to gain further understanding on the function of this special brain structure. Here, scanning electron microscopy (SEM) on whole mount CP is used to get an overall view of the structure.

The choroid plexus (CP), an understudied tissue in neuroscience, plays a key role in health and disease of the central nervous system. This protocol describes a microdissection technique for isolating the CP and the use of scanning electron microscopy to obtain an overall view of its cellular structure.

小鼠脉络丛的显微切割和全安装扫描电子显微镜可视化

The choroid plexus (CP), a highly vascularized structure protruding into the ventricles of the brain, is one of the most understudied tissues in ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

使用具有可编程输出频率的自制伏特安度计执行 TEER 测量的简单方法

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

生物学

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

模拟人血脑屏障的三重培养细胞系统

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection

Source: da Costa, A., et al. <a href="https://app.jove.com/v/59220">A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain.</a> J. Vis. Exp. (2019).
This video demonstrates an in vitro model to study pathogens crossing the blood-brain barrier (BBB). Microvascular endothelial cells, which express tight junctions, form an endothelial layer in the upper chamber, while neurons, astrocytes, and microglia represent the brain parenchyma in the lower chamber, separated by a porous membrane. Virus infection in the BBB model mimics the pathogen crossing from blood to the brain. Upon introducing a neurotropic virus, it crosses the BBB via transcytosis, infects neural cells, replicates, and releases viral progeny, demonstrating successful neuroinvasion.

Source: da Costa, A., et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the ...