Name: in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules
Uploaded: 2016-08-22T15:00:00.000+00:00
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Description: 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

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>

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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><tbody><tr><td>Rat Brain Microvascular Endothelial Cells&amp;nbsp;</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&amp;nbsp;</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 &amp;deg;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 &amp;deg;C water bath before use</td></tr><tr><td>Fluorescein isothiocyanate-dextran 40,000</td><td>Sigma</td><td>FD40S</td><td>protect from light</td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>158127</td><td>diluition in chemical hood</td></tr><tr><td>Dulbecco&#039;s phosphate buffer saline w/o Ca and Mg</td><td>EuroClone</td><td>ECB4004L</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>bovine serum albumin&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>goat serum&amp;nbsp;</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&amp;rsquo; ,6-diamidino-2-phenylindole)&amp;nbsp;</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&amp;nbsp;</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 cm&lt;sup&gt;2&lt;/sup&gt;; pore size 0.4 &amp;micro;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&amp;nbsp;&amp;nbsp;&amp;nbsp;</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>&amp;nbsp;inverted microscope for live cells with camera&amp;nbsp;</td></tr><tr><td>Confocal Microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>Spectrofluorimeter</td><td>Jasco</td><td>FP-8000</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 ...

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

癌症研究

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 ...

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

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

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

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

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

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

医学

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

神经科学

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules

Source: Le Joncour, V. et al. <a href="https://www.jove.com/v/59384/predicting-vivo-payloads-delivery-using-blood-brain-tumor-barrier">Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish.</a> J. Vis. Exp. (2019)
This video describes a permeability assay to evaluate the permeability of in vitro blood-brain barrier using sodium fluorescein molecules.

血脑屏障模拟物通透性测定:一种评估 BBB 模拟物对示踪分子通透性的体外测定

Source: Le Joncour, V. et al. Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish. J. Vis. Exp. (2019)
This video describes a ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Studying the Permeation of FITC and FITC-Ferritin through an In Vitro Blood-Brain Barrier Model

Source: Fiandra, L., et al. <a href="https://app.jove.com/v/54279">In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules.</a> J. Vis. Exp. (2016).
This video demonstrates a method to study permeation through an in vitro blood-brain barrier (BBB) model using astrocytes and endothelial cells on a membrane insert, with free FITC and FITC-loaded ferritin. FITC-ferritin bound to receptors on endothelial cells and crossed the membrane barrier to the lower chamber while free FITC showed restricted permeation and remained in the upper chamber.

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

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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

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

利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

改进方法对血脑屏障的人体细胞为主，接触模型的制备

设置了一个

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

内皮细胞转吞测定作为体外模型评估内部血-视网膜屏障通透性

血脑屏障模拟物通透性测定:一种评估 BBB 模拟物对示踪分子通透性的体外测定

通过体外血脑屏障模型研究 FITC 和 FITC-Ferritin 的渗透

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

摘要

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此视频中的章节

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

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

利用血脑肿瘤屏障在盘中进行活体有效载荷传递预测

改进方法对血脑屏障的人体细胞为主，接触模型的制备

设置了一个

FITC 标记白蛋白静脉滴注对神经退行性疾病模型血脑屏障通透性的评价

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

内皮细胞转吞测定作为 体外 模型评估内部血-视网膜屏障通透性

血脑屏障模拟物通透性测定:一种评估 BBB 模拟物对示踪分子通透性的体外测定

通过体外血脑屏障模型研究 FITC 和 FITC-Ferritin 的渗透

内皮细胞转吞测定作为体外模型评估内部血-视网膜屏障通透性