The authors acknowledge Assessorato alla Sanit&#224;, Regione Lombardia and Sacco Hospital (NanoMeDia Project) for research funding.

1.建立血脑屏障模型注意:对于建立血脑屏障模型，我们建议您使用市售的主RBMECs和RCA接头。所有步骤都必须用无菌的试剂和耗材，在层流罩处理来执行。 <ol><li>细胞培养<ol><li>用聚L-赖氨酸100微克/毫升（在RT 1小时）或纤连蛋白50微克/毫升（在37℃下1小时）的外套细胞培养烧瓶，以促进合会或RBMECs的附件，分别。然后，解冻1×10 6个 RCA接头和5×10 5 RBMECs在内皮细胞培养基，补充有5％胎牛血清，1％的内皮细胞生长补充和1％青霉素/链霉素（SECM）。种子合会在在10毫升SECM在20ml SECM和RBMECs在T75烧瓶中T175烧瓶中。 注意:解冻和RCA接头和RBMECs的播种通道必须进行调整，在培养细胞密度和时间方面，根据与血脑屏障模型被测试的实验条件的数目。通过启动用1瓶1×10 6个 RCA接头和5×10 5 RBMECs 1小瓶ING，有可能获得高达20承载血脑屏障插入。 </li><li>保持在37℃和5％的CO 2在大约6天潮湿气氛的细胞，直到大约80％汇合为RCA接头和用于RBMECs超过90％汇合。分离使用胰蛋白酶-EDTA溶液（胰蛋白酶1:250）的细胞5分钟（1 ml胰蛋白酶为T75烧瓶和2ml为T175烧瓶）。停止与SECM胰蛋白酶活性（2:1），在750×g下5分钟离心细胞悬浮液和悬浮颗粒在60ml SECM。 </li><li>播种到插入之前拆分成RBMECs 3 T175瓶和文化SECM另外3天。计数活RCA接头，通过观察稀释1细胞悬浮液的总数量:在Burker室中在光学显微镜下1用锥虫蓝。 </li></ol></li><li>细胞种植到刀片<ol><li> 6多井transpar的对待聚对苯二甲酸的一侧（PET）膜用聚L-赖氨酸100微克/毫升，而另一侧与纤连蛋白50微克/毫升，耳鼻喉科插入以允许RCA接头和BMECs的附着。处理与镊子插入件，以避免与PET膜接触。 <ol><li>放置插入到一个6孔板中并添加纤连蛋白溶液（最小500微升）到上部室中。在37℃孵育1小时后，取出纤维连接蛋白的解决方案，脱下多孔板嵌件，并把它们倒挂在150 平方厘米的培养皿底部，这是这里用作无菌支持已经涂层刀片。 </li><li>轻轻添加800微升多聚-L-赖氨酸的上刀片的底侧（如在图1A中示出;称为RCA接头播种），并保持在插入的溶液在室温1小时。 </li><li>吸出溶液，并让插入在RT干燥15-30分钟。插入件现在已准备好细胞接种，但也可以存储在多孔板数天在4℃，用血脑屏障结构，然后再继续。 注:记住要保持至少3涂层刀片不含细胞，用作控制由FD40通量测量，以验证BBB建立。 </li></ol></li><li>种子合会（35,000 /厘米2）到聚-L-赖氨酸包被的插入物的底侧，滴加800微升细胞悬液到倒置插入物（ 图1A）。留在插入RCA的悬浮液在室温4小时，以便使细胞的膜的有效附着。 </li><li>吸出残留溶液，插入件放入含2ml SECM的井和在湿润的气氛中保持在37℃和5％的CO 2的多井板，改变SECM每2天。 </li><li>在3天之后，当合会已涂覆的刀片的下表面，种子RBMECs到插入件的上侧，以下步骤: <ol><li>从T175瓶分离RBMECs和计数总活细胞，据报道在步骤1.1.2和1.1.3。 </li><li>种子RBMECs（60,000 /厘米2）到所述插入件的中SECM（1,000微升）（ 图1B）的上表面上，留下3插入仅与星形胶质细胞，也可以用作TEER背景。放置在标准培养条件下的多孔板。维护系统来培养至少3天，改变SECM在内侧和下部腔室，每2天。 </li></ol></li></ol></li></ol> 2. BBB验证<ol><li> TEER测量<ol><li>上共培养的第 3天，通过将承载血脑屏障插入到一个适当的腔室检查TEER（具有盖和其中两个腔室和盖包含一对电压感测和电流电极的），填充有4 SECM毫升的。连接到上皮组织伏特/欧姆表。 </li><li>连同细胞血脑屏障的系统的TEER测量，记录3插入轴承合会第的TEER值在将从与商务改善局获得的值中减去。通过插入（4.2厘米2）的表面上，以表达结果作为ΩX招商局2相乘所得的TEER值。 </li><li>从联合培养的第 3天，每天测量TEER。注意:一个初始阶段，其中TEER增加后，所记录的值应为连续至少两天保持稳定（通常为5 个和共培养的第 7天之间）。在该点所述BBB准备验证（第2.2节）和/或用于反式血脑屏障实验的第二步骤。 注意:在2.1节中描述的过程可以在专门用于以下渗透性实验（第3节）相同的BBB系统来执行。在无菌条件下进行操作。 </li></ol></li><li>反式BBB助焊剂FITC右旋糖酐40（FD40） <ol><li>测量从上部的FD40通量相比，在3个空的嵌入式BBB模型的下腔根据下列步骤（见本说明部分1.2.1）: <ol><li>加入1毫克/毫升FD40（在SECM稀释）插入商务改善局的上隔室和后1,2和3小时从下室撤回200微升SECM和由分光光度计测量的荧光强度（λEX 488纳米，λ 青霉 515纳米，狭缝5）。 </li><li>获得SECM背景值fluorescenceby使用相同的分析参数测量500微升处女介质。减去所有FD40荧光值的SECM背景荧光。 </li><li>通过与溶于500微升SECM的荧光示踪剂的已知浓度（ 即 0.312，0.625，1.25,2.5微克/毫升）所产生的校准曲线所观察到的荧光值的比较确定渗透FD40的量。 </li><li>计算从根据平均通量值的表观渗透系数（P 应用 ）: P = 应用焦耳/ AC <br/&gt;其中J是分子（摩尔/秒）的通量，A是渗透面积（cm 2）和C是在上隔室（摩尔/厘米3）的分子的浓度。 注:三个或更多个已达到适当的TEER值血脑屏障的系统中，必须将专门讨论此验证过程，并且不应当被用于以下渗透性实验（第3节）。育不是强制性的。 </li></ol></li></ol></li></ol> 3.跨BBB FITC加载铁蛋白渗透（FNN） 注:人类铁蛋白的重组变异体（FN），在大肠杆菌中产生并聚集在纳米笼（FNN）针对不同的荧光分子的封装，可提供从Prosperi教授的NanoBioLab（大学米兰比可卡，意大利）。 FNN装载用FITC，根据先前描述的方案13和Fn和加载的分子的浓度准确determin编辑。 <ol><li> FITC的跨BBB通量和FITC-FNN <ol><li>测量从上所述的FITC FNN助熔剂验证BBB模型（通常是在第5 次 -共培养的第 7天）的下室中，相对于该后孵化的7和24小时的自由染料，根据以下步骤: <ol><li>添加FITC标记FNN（50微克/毫升FNN，1.1μM，FITC）或等量的游离的FITC成每个制剂至少6血脑屏障系统的上腔室，以便从下部腔室退出的2ml SECM溶液至少3插入后7小时，并从其他3插入物24小时后的下腔室。 </li><li>通过分光光度计测量的500微升收集的样品的荧光强度（λEX 488纳米，λEM 515纳米，狭缝5）。 </li><li>通过使用相同的分析参数测定500μl的培养基中获得的SECM背景荧光。减去SECM背景荧光所有FITC或FITC-FNN荧光值。 </li><li>通过将得到的荧光值与溶解在500微升SECM游离或nanoformulated染料已知浓度（ 即 6.87，13.75，27.5，55纳米）产生两种不同的校准曲线比较确定渗透的FITC或FITC-FNN的浓度。 </li></ol></li></ol></li><li> FITC-FNN本土化RBMECs <ol><li>从血脑屏障系统的上腔室中移除SECM，用PBS洗，插入和通过在上隔室中加入500μl的多聚甲醛（于磷酸盐缓冲液盐水PBS中的4％）10固定在至少两个插入每个实验组的RBMECs分钟在室温。 </li><li>用PBS洗插入三次以除去残余的多聚甲醛。 注意:从此时起，不育是没有必要的。 </li><li>切割在PET膜的合适的片，并且具有根据以下步骤的单元的immunodecoration继续: <ol><li> Permeabi丽泽RBMECs用0.1％的Triton X-100的PBS 10分钟。在RT进行1小时封闭步骤用含2％牛血清白蛋白（BSA），2％山羊血清的PBS溶液中。在1:20稀释孵育与抗血管性血友病因子（vWF）的样品中，在室温下2小时。 </li><li>在用PBS洗涤三次后，在室温下暴露在细胞2小时至2％的BSA，含有适当的二级抗体以1 2％山羊血清溶液:300稀释以揭示抗VWR，和DAPI在一个1:1500稀释的核检测。 </li></ol></li><li>安装插片上使用防褪色安装解决方案（每插入片段一滴）显微镜玻片，然后用盖玻片关闭样品。使用油浸镜头放大40倍，1.5变焦和1024x1024个像素分辨率的激光共聚焦显微镜分析。 </li></ol></li></ol>

<ol>
	<li>Banks, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+compounds+that+cross+the+blood-brain+barrier.">Characteristics of compounds that cross the blood-brain barrier.</a> BMC Neurol. 9 (1), 3 (2009).</li><li>L&#246;scher, W., Potschka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+resistance+in+brain+disease+and+the+role+of+efflux+transporters.">Drug resistance in brain disease and the role of efflux transporters.</a> Nat Rev Neurosci. 6, 591-602 (2005).</li><li>Xu, G., Mahajan, S., Roy, I., Yong, K. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theranostic+quantum+dots+for+crossing+blood-brain+barrier+in+vitro+and+providing+therapy+of+HIV-associated+encephalopathy.">Theranostic quantum dots for crossing blood-brain barrier in vitro and providing therapy of HIV-associated encephalopathy.</a> Front Pharmacol. 15 (4), 140 (2013).</li><li>Masserini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+for+Brain+Drug+Delivery.">Nanoparticles for Brain Drug Delivery.</a> ISRN Biochem. , (2013).</li><li>Sagar, V., Pilakka-Kanthikeel, S., Pottathil, R., Saxena, S. K., Nair, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+nanomedicines+for+neuroAIDS.">Towards nanomedicines for neuroAIDS.</a> Rev. Med. Virol. 24 (2), 103-124 (2014).</li><li>Kreuter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+to+the+central+nervous+system+by+polymeric+nanoparticles:+What+do+we+know.">Drug delivery to the central nervous system by polymeric nanoparticles: What do we know.</a> Adv. Drug Deliver. Rev. 71, 2-14 (2014).</li><li>Arosio, P., Ingrassia, R., Cavadini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferritins:+a+family+of+molecules+for+iron+storage,+antioxidation+and+more.">Ferritins: a family of molecules for iron storage, antioxidation and more.</a> Biochim. Biophys. Acta. 1790 (7), 589-599 (2009).</li><li>J&#228;&#228;skel&#228;inen, A., Soukka, T., Lamminm&#228;ki, U., Korpim&#228;ki, T., Virta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+denaturation/renaturation-based+production+process+for+ferritin+nanoparticles.">Development of a denaturation/renaturation-based production process for ferritin nanoparticles.</a> Biotechnol. Bioeng. 102 (4), 1012-1024 (2009).</li><li>Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C., Mason, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+receptor+on+endothelium+of+brain+capillaries.">Transferrin receptor on endothelium of brain capillaries.</a> Nature. 312, 162-163 (1984).</li><li>Deli, M. A., Abraham, C. S., Kataoka, Y., Niwa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+studies+on+in+vitro+blood-brain+barrier+models:+physiology,+pathology,+and+pharmacology.">Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology.</a> Cell. Mol. Neurobiol. 25 (1), 59-127 (2005).</li><li>Wilhelm, I., Fazakas, C., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier.">In vitro models of the blood-brain barrier.</a> Acta Neurobiol. Exp. (Wars). 71, 113-128 (2011).</li><li>Wilhelm, I., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier+for+the+study+of+drug+delivery+to+the+brain.">In vitro models of the blood-brain barrier for the study of drug delivery to the brain.</a> Mol Pharm. 11 (7), 1949-1963 (2014).</li><li>Bellini, M., 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=Protein+nanocages+for+self-triggered+nuclear+delivery+of+DNA-targeted+chemotherapeutics+in+Cancer+Cells.">Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells.</a> J. Control. Release. 196, 184-196 (2014).</li><li>Fiandra, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoformulation+of+antiretroviral+drugs+enhances+their+penetration+across+the+blood+brain+barrier+in+mice.">Nanoformulation of antiretroviral drugs enhances their penetration across the blood brain barrier in mice.</a> Nanomedicine. 11 (6), 1387-1397 (2015).</li><li>Molino, Y., Jab&#232;s, F., Lacassagne, E., Gaudin, N., Khrestchatisky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting-up+an+in+vitro+model+of+rat+blood-brain+barrier+(BBB):+a+focus+on+BBB+impermeability+and+receptor-mediated+transport.">Setting-up an in vitro model of rat blood-brain barrier (BBB): a focus on BBB impermeability and receptor-mediated transport.</a> J. Vis. Exp. , e51278 (2014).</li><li>Perri&#232;re, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+in+vitro+model+of+rat+blood-brain+barrier+for+molecular+analysis+of+efflux+transporters.">A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters.</a> Brain Res. 1150, 1-13 (2007).</li></ol>

The authors declare that they have no competing financial interests.

此处描述的体外方法是一项有用的验证方法来研究在nanoformulation与纳米颗粒的荧光分子中的反式血脑屏障输送。在这里，我们使用FNN，它代表一个很好的候选来研究穿过BBB货物分子的易位。 FNN被认为是金nanovector为反式血脑屏障递送药物/剂的，因为它是专门通过TFR1受体，这是在BMECs的管腔膜表达并介导使用受体介导的内化途径中的纳米颗粒的摄取识别。此外，模糊神经网络是一种天?...

中枢神经系统（CNS）的疾病（ 即肿瘤，癫痫症，抑郁症，精神分裂症和HIV相关的神经障碍）耐药物疗法是由于各种不同的机制，包括跨越血脑屏障艰巨药物渗透（BBB） 。血脑屏障是隔离在血液中循环的物质的脑组织的边界。在这个障碍，脑微血管内皮细胞（BMECs），通过周细胞和星形胶质细胞endfeet支持层，是负责对血脑屏障的高选择性的那些水溶性药物的分子量高于400达1。另一个与药物有关的耐药机制被链接到上BMECs存在药物外排转运（P-糖蛋白和多药耐药蛋白），其共同操作以减少药物渗透到中枢神经系统和从大脑2促进其挤出。 在过去十年中，大量的纳米技术的方法已被开发，以满足穿过BBB 3-6递送药物的临床和生物学的挑战。在此背景下，铁蛋白纳米球（FNN）代表一种完全创新的和可行的解决方案。 FNN是24自组装铁蛋白（FN）单体，其被布置在8nm的内径的中空球状结构12纳米球体。铁蛋白亚基可以在酸性pH下被拆卸并通过使pH值至中性，允许被包封各种有机分子中的形状记忆的方式重新组合。因此，FNN代表多功能药物递送系统7,8的发展的一个有趣的模型。此外，FNN可以与BMECs由于转铁蛋白受体（铁蛋白）1，这是对这些细胞9的管腔膜表达的特异性识别相互作用。 到目前为止， 在血脑屏障的体外模型不同已在奥德开发R以阐明反式BBB通透性对各种药物，毒性朝向血脑屏障，或流出转运分子的相互作用。的确，这些模型被认为是体外有效与体内研究在继续之前接近为活性分子的快速筛选。这些模型由BMECs或共培养BMECs和星形胶质细胞（更罕见周细胞）的单个内皮细胞层，从动物（大鼠，小鼠，猪和牛），或人细胞系10,11,12得到的。的跨内皮电阻（TEER），并具有限定分子量示踪剂的表观渗透率（P 应用 ）表示用于确定在体外模型的质量的两个关键参数。在这里，我们描述了就业BBB 体外模型的基础上，大鼠BMECs（RBMECs）的共同的文化和大鼠皮层星形胶质细胞（合会）研究纳米笼铁的跨BBB渗透封装荧光isothiocyanate（FITC）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rat Brain Microvascular Endothelial Cells&#160;</td>
			<td>Innoprot</td>
			<td>P10308</td>
			<td>isolated from Sprague Dawley rat brain tissue, cryopreserved at passage one and delivered frozen</td>
		</tr>
		<tr>
			<td>Cortical Astrocytes&#160;</td>
			<td>Innoprot</td>
			<td>P10202</td>
			<td>isolated from 2 days rat brain tissue, cryopreserved at passage one and delivered frozen.</td>
		</tr>
		<tr>
			<td>Endothelial Cell Medium kit</td>
			<td>Innoprot</td>
			<td>P60104</td>
			<td>ECM (500 ml) and fetal bovin serum (25 ml), endothelial cell growth supplement (5 ml) and penicillin/streptomycin (5 ml). Warm in 37 &#176;C water bath before use and protect from light</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA without Phenol Red</td>
			<td>EuroClone</td>
			<td>ECM0920D</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran 40000</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>diluition in chemical hood</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline w/o Ca and Mg</td>
			<td>EuroClone</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>goat serum&#160;</td>
			<td>EuroClone</td>
			<td>ECS0200D</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Von Willebrand Factor</td>
			<td>Dako</td>
			<td>M0616</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 546-conjugated antibody against mouse IgGs</td>
			<td>ThermoFischer Scientific</td>
			<td>A-11003</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>DAPI (4&#8217; ,6-diamidino-2-phenylindole)&#160;</td>
			<td>ThermoFischer Scientific</td>
			<td>D1306</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFischer Scientific</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Hydrobromide</td>
			<td>Sigma</td>
			<td>P1274</td>
			<td>the same solution can be used several times</td>
		</tr>
		<tr>
			<td>fibronectin from bovine plasma&#160;</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>the same solution can be used several times</td>
		</tr>
		<tr>
			<td>Polyethylene terephthalate (PET) inserts</td>
			<td>Falcon</td>
			<td>F3090</td>
			<td>Transparent Polyethylene terephthalate (PET) membranes; surface area: 4.2 cm2; pore size 0.4 &#181;m/surface area</td>
		</tr>
		<tr>
			<td>T75 Primo TC flask</td>
			<td>EuroClone</td>
			<td>ET7076</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 Primo TC flask</td>
			<td>EuroClone</td>
			<td>ET7181</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2 Epithelial Tissue Volt/Ohmmeter&#160;&#160;&#160;</td>
			<td>World Precision Instruments Germany</td>
			<td>EVOM2</td>
			<td></td>
		</tr>
		<tr>
			<td>Endohm- 24SNAP cup</td>
			<td>World Precision Instruments Germany</td>
			<td>ENDOHM-24SNAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Light/fluorescence microscope with camera</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED Fluo/ ICC50 W Camera Module</td>
			<td>&#160;inverted microscope for live cells with camera&#160;</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SPE</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

在建立血脑屏障模型，细胞附着和生长的插入物可以使用光学显微镜由于在PET膜的透明性进行监测。合会，在35,000个细胞/ cm 2时，有效地连接到所述插入件的底侧之后在RT（ 图2A）孵育4小时的密度接种并生长到覆盖膜表面在3天，采取纺锤形的形态（ 图2B）。 RBMECs，在60,000个细胞/ cm 2的密度接种，在37℃下（ 图2C）明?...

Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透:一个模型来研究Nanoformulated分子的交付

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

in-vitro-permeation-fitc-loaded-ferritins-across-rat-blood-brain

Research

JoVE Journal

Bioengineering

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

7.8K 次观看.  Università di Milano. A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

视频: In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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 ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal hernia typically requires implantation of a material to serve as a mechanical bridge across the defect in the abdominal wall. Biomaterials, such as porcine small intestinal submucosa (SIS), also serve as a lattice for cell growth into the implant and can naturally incorporate into the host tissue. Development of such repair materials benefits from use of animal models in which experimental abdominal wall defects are easily created and are amenable to repair in a reproducible fashion. The method offered here describes surgical creation and repair of ventral abdominal hernia in a rat model. When SIS is used to repair an experimental ventral abdominal hernia in this model, it is rapidly incorporated into host tissue within 28 days of implantation. Histologically, incorporation of their implanted material into host tissue is characterized by a robust fibrovascular response. Future refinements and applications of the rat abdominal hernia model may likely involve diabetic and/or obese animals as a means to more closely mimic common co-morbidities of man.

This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

大鼠模型在腹腹疝修补术中的应用研究

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

微流控装置在极小空间中研究尖端生长植物细胞的伸长能力

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...