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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A., Urwin, R., 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=Genetic+analysis+of+meningococci+carried+by+children+and+young+adults.">Genetic analysis of meningococci carried by children and young adults.</a> J Infect Dis. 191, 1263-1271 (2005).</li><li>Tenenbaum, T., Papandreou, T., Gellrich, D., Friedrichs, U., Seibt, A., Adam, R., 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=Polar+bacterial+invasion+and+translocation+of+Streptococcus+suis+across+the+blood-cerebrospinal+fluid+barrier+in+vitro.">Polar bacterial invasion and translocation of Streptococcus suis across the blood-cerebrospinal fluid barrier in vitro.</a> Cell Microbiol. 11, 323-336 (2009).</li><li>Laflamme, N., Echchannaoui, H., Landmann, R., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperation+between+toll-like+receptor+2+and+4+in+the+brain+of+mice+challenged+with+cell+wall+components+derived+from+gram-negative+and+gram-positive+bacteria.">Cooperation between toll-like receptor 2 and 4 in the brain of mice challenged with cell wall components derived from gram-negative and gram-positive bacteria.</a> Eur J Immunol. 33, 1127-1138 (2003).</li><li>Laflamme, S., Rivest, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4:+the+missing+link+of+the+cerebral+innate+immune+response+triggered+by+circulating+gram-negative+bacterial+cell+wall+components.">Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components.</a> FASEB J. 15, 155-163 (2001).</li><li>Zughaier, S. 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. D., 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=Legionella+pheumophila-induced+IkappaBzeta-dependent+expression+of+interleukin-6+in+lung+epithelium.">Legionella pheumophila-induced IkappaBzeta-dependent expression of interleukin-6 in lung epithelium.</a> Eur Respir J. 37, 648-657 (2011).</li><li>Jaerve, A., Muller, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+CNS+injury+and+repair.">Chemokines in CNS injury and repair.</a> Cell Tissue Res. 349, 229-248 (2012).</li><li>Schneider, H., Weber, C. 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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin-EDTA</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#180;,6 diamidino-2-phenylindole (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well plates</td>
			<td>Starlab</td>
			<td>CC7682-7512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well plates</td>
			<td>Starlab</td>
			<td>CC7682-7524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti Neisseria meningitidis &#945;-OMP</td>
			<td></td>
			<td></td>
			<td>This antibody was a gift from Drs. H. Claus and U. Vogel (University of W&#252;rzburg, Germany)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 (chicken anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A21441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 594 (chicken anti rabbit)</td>
			<td>Invitrogen</td>
			<td>A21442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 660 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A22285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumine (BSA)</td>
			<td>Calbiochem</td>
			<td>12659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chocolate agar plates</td>
			<td>Biomerieux</td>
			<td>43109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytochalasin D</td>
			<td>Sigma</td>
			<td>C8273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 + L-Glut + 15 mM HEPES</td>
			<td>Gibco</td>
			<td>31330-095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 + L-Glut + 15 mM HEPES w/o Phenolred</td>
			<td>Gibco</td>
			<td>11039-047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum (FCS)</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-Inulin</td>
			<td>Sigma</td>
			<td>F3272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin</td>
			<td>Sigma</td>
			<td>19278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma</td>
			<td>2393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Sigma</td>
			<td>55761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS + Mg +Ca</td>
			<td>Gibco</td>
			<td>14040-174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>MP Biomedicals</td>
			<td>1670049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvitex</td>
			<td>Biomerieux</td>
			<td>55651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteose peptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serum-free medium</td>
			<td>Gibco</td>
			<td>10902-096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thincert cell culture inserts for 24-well plates, pore size 3 &#181;m</td>
			<td>Greiner</td>
			<td>662630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture flask 75 cm&#178; red cap sterile</td>
			<td>Greiner</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

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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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

利用经颅磁刺激，以研究人类的神经肌肉系统

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel stenting are crucial for translational approaches1,2.
The commonly used animal models include mice, rats, rabbits, and pigs3-5. However, the translation of these models into clinical settings remains difficult, since those biological processes are already studied in animal vessels but never performed before in human research models6,7. In this video we demonstrate a new humanized model to overcome this translational gap. The shown procedure is reproducible, easy, and fast to perform and is suitable to study the development of intimal hyperplasia and the applicability of diverse stents.
This video shows how to perform the stent technique in human vessels followed by transplantation into immunodeficient rats, and identifies the origin of proliferating cells as human.

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

人乳内动脉（IMA）移植及内支架置入术:人体模型支架内再狭窄的研究开发

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

一个人力<em&gt;前体内</em&gt;动脉粥样硬化斑块模型研究病变的生物学

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

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 ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

干细胞基工程免疫抗HIV感染的人性化小鼠模型

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...