一个<em>体外</em>血 - 脑屏障使用阻抗谱型号：对T细胞，内皮细胞的相互作用为中心
Summary
Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.
Abstract
Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not naïve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.
Introduction
血-脑屏障分开的中枢神经系统（CNS）的1系统循环– 3。它提供了一种抑制细胞的自由流动和水溶性分子的扩散，保护从病原体和潜在的有害物质的大脑的物理屏障。除了它的屏障功能，血脑屏障能使脑实质，这确保了神经元组织的适当运作的输送氧和营养物质。血脑屏障的功能特性深受其细胞和非细胞成分调节，具有高度专业化的内皮细胞是其主要结构件。血脑屏障的内皮细胞是由紧密连接（TJ）配合，缺乏穿孔的，极低的胞饮的活性，并永久主动转运机制的存在表征。嵌入内皮细胞，星形胶质细胞最终脚=相关比肩BBB的其他组成部分的EC基底膜，周细胞与神经元和小胶质6和一起形成神经血管单元（NVU），这使得能够在CNS 7适当运作– – enchymal基底膜也有助于血脑屏障2,4开发，维护和功能9。
在多种神经性疾病，如神经变性，炎症或传染病的，血脑屏障的功能受到损害2,5,10。 TJ配合物和分子运输机制的失调导致增加的血脑屏障通透性，白细胞外渗，炎症和神经元损伤。为了研究这种病理生理条件下的BBB特性， 各种体外 BBB模型已经建立9,11,12。他们共同提供了宝贵的见解屏障的完整性，透气性以及传输机制的变化。这些模型使用人，小鼠，大鼠，猪或b的内皮细胞羊产地13 – 18;原代内皮细胞或细胞系中培养或者作为单一或一起，以便在体内 19更接近地模拟血脑屏障周细胞和/或星形胶质细胞– 25。近年来，跨内皮电阻（TEER）的测量变得评估内皮屏障性能26,27被广泛接受的工具。
TEER反映阻抗跨细胞单层的离子通量及降低提供损害内皮屏障完整性并因此增加渗透性的敏感量度。各种TEER测量系统已经开发，包括上皮Voltohmmeter（EVOM），电细胞-基底阻抗传感（ECIS），以及实时细胞分析15,28 – 30。 TEER反映至（旁路线）相邻内皮之间的离子通量的电阻和成正比的屏障的完整性。在阻抗谱27,31，复合总阻抗（Z）的测定，其提供了关于通过测量CCL的屏障完整性的其他信息。 CCL涉及通过细胞膜（跨细胞途径）的电容电流：细胞层的作用就像在等效电路的电容器，分离在膜的两侧的收费及成反比的屏障完整性。当插入渗透性增加，内皮细胞附着，增殖，扩散到微孔膜。此抵抗刀片的背景电容电流（其本身的作用就像一个电容器），并直到它到达其最低水平导致降低的电容。这之后是建立TJ的络合物密封关闭相邻内皮之间的空间。这限制了通过细胞旁路径离子通量，和TEER增加，直到它到达平台期。下炎性病症，然而，endotheli人屏障被破坏：TEER降低作为TJ络合物得到破碎和CCl增加作为插入的电容成分再次上升。
我们TEER测量采用自动化小区监控系统，32：它遵循阻抗谱的原则，并扩展了其以前的应用程序。在这里，我们描述了在体外血脑屏障模型，使阻隔性能的研究，包括与免疫细胞脑内皮的相互作用;特别是活化的T细胞。 37 –这样的病理生理条件在CNS的自身免疫疾病，如多发性硬化和其的动物模型实验性自身免疫性脑脊髓炎33观察到。这里，一个重要的步骤是穿过BBB脑炎，髓鞘特异性T细胞的轮回。这之后是其在血管周围空间以及进入脑实质激活，在那里他们招募其他免疫细胞和箱diate炎症和随后脱髓鞘1,35,38。然而，这样的T细胞和内皮细胞之间的相互作用的分子机制，血脑屏障的主要成分，不能得到很好的理解。我们的协议旨在填补这一空白，并给予新的见解对内皮细胞（ 即屏障的完整性和渗透性）在他们的直接接触和复杂的相互作用与活化的T细胞的后果。
这里描述的协议利用主鼠标脑微血管内皮细胞，生长成与微孔膜渗透插入一个单层的。内皮细胞共培养的CD4 + T细胞，其可以预先激活或多克隆或在抗原特异性的方式。用预活化，但不幼稚T细胞MBMECs的共培养诱导的TEER降低和增加CCL，它提供了MBMEC功能障碍和屏障破坏的定量量度。该技术是非侵入性的：它使用内置代替筷子电极，其防止欧共体单层主要障碍;它可以被用于监测屏障功能，而无需使用细胞标记物的。它使得以自动方式连续测量并且使两个阻挡参数（TEER和CCL）随着时间的推移同时进行独立的评估。该方法也是不够敏感不同水平的T细胞活化的和内皮这样的T细胞的作用之间进行区分。
它可以在很宽范围的官能试验中使用：在炎症过程中涉及不同的细胞因子和/或趋化因子可以添加到MBMECs和T细胞的共培养;针对关于EC或T细胞侧粘附分子阻断抗体可以使用;并且可以在T细胞引发或它们的共培养内皮细胞的过程中加入的T细胞的活化标记物或它们的溶细胞性的抑制剂。该法还对T细胞轮回有用测定法，因为它可以作为前加入T细胞的MBMEC单层完整性的质量控制。这一切都使得这种方法的灵活和可靠的工具来研究BBB 体外 ，重点是激活的T细胞对EC单层完整性的影响。这是特别重要的对于理解血脑屏障破坏的机制在自体免疫疾病，如MS和其动物模型EAE，其中自反应，致脑炎的T细胞穿过BBB和引起炎症和神经元损伤的发病机理。
Protocol
Representative Results
Discussion
所描述的协议的几个步骤是一个成功的实验是必不可少的。在初始MBMEC分离和培养，这是至关重要的工作尽可能无菌条件下进行，以防止细胞培养物的污染，真菌孢子或细菌。为了获得内皮细胞的纯培养物，推荐使用含有嘌呤霉素的前三天的介质，使内皮细胞存活，但不是其他细胞类型（特别是周细胞）41,42。值得特别注意的另一个关键步骤是添加预活化的T细胞的正确时间点的识别。期间…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们是对TEER测量有益的讨论感谢安妮卡Engbers和弗兰克·库尔特的出色的技术支持和马库斯·舍费尔博士（nanoAnalytics有限公司）。这项工作是由德意志研究联合会（DFG），SFB1009项目A03汉王和LK，CRC TR128，项目支持A08; Z1和B01，以LK和硬件，以及跨学科临床研究中心（医学院明斯特）的授权号KL2 / 2015/14 LK。
Materials
| cellZscope | nanoAnalytics GmbH | www.nanoanalytics.com | including: 24-well Cell Module, Controller, PC with cellZscope software v2.2.2 |
| Ultracentrifuge | Thermo Scientific | www.thermoscientific.com | SORVALL RC 6+; rotor F21S-8x50y; for MBMEC isolation |
| flow cytometer | Beckman Coulter | www.beckmancoulter.com | for analysis of T cell transmigration |
| FlowJo7.6.5 software | Tree Star | www.flowjo.com | for analysis of T cell transmigration |
| Oak Ridge centrifuge tubes, PC | Thermo Fisher Scientific | 3118-0050 | 50 ml; for MBMEC isolation |
| Transwell membrane inserts – pore size 0.4 µm | Corning | 3470 | for TEER measurement as the main readout |
| Transwell membrane inserts – pore size 3 µm | Corning | 3472 | for TEER measurement as the quality control prior to T-cell transmigration assay |
| 24-well cell culture plate | Greiner | 650 180 | flat-bottom; for MBMEC culture |
| 96-well cell culture plate | Costar | 3526 | round-bottom; for immune cell culture |
| QuadroMACS Separator | Miltenyi Biotec | 130-090-976 | for T cell and B cell isolation; supports MACS LS columns |
| OctoMACS Separator | Miltenyi Biotec | 130-042-109 | for dendritic cell isolation; supports MACS MS columns |
| Neubauer counting chamber | Marienfeld | MF-0640010 | for cell counting |
| Cell strainer, 70 µm | Corning | 352350 | for immune cell isolation |
| Cell strainer, 40 µm | Corning | 352340 | for immune cell isolation |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | for immune cell isolation |
| MACS LS separation columns | Miltenyi Biotec | 130-042-401 | for T cell and B cell isolation |
| MACS MS separation columns | Miltenyi Biotec | 130-042-201 | for dendritic cell isolation |
| Mouse CD4 MicroBeads | Miltenyi Biotec | 130-049-201 | for CD4+ T cell isolation |
| Mouse CD19 MicroBeads | Miltenyi Biotec | 130-052-201 | for B cell isolation |
| Mouse CD11c MicroBeads | Miltenyi Biotec | 130-052-001 | for dendritic cell isolation |
| Collagen type IV from human placenta | Sigma | C5533 | for MBMEC coating solution |
| Fibronectin from bovine plasma | Sigma | F1141-5MG | for MBMEC coating solution |
| Collagenase 2 (CSL2) | Worthington | LS004176 | for MBMEC isolation |
| Collagenase/Dispase (C/D) | Roche | 11097113001 | for MBMEC isolation |
| DNase I | Sigma | DN25 | for MBMEC isolation |
| Fetal Bovine Serum (FBS) | Sigma | F7524 | for MBMEC isolation |
| Bovine Serum Albumin (BSA) | Amresco | 0332-100G | for MBMEC isolation |
| Percoll | Sigma | P1644-1L | for MBMEC isolation |
| DMEM (+ GlutaMAX) | Gibco | 31966-021 | for MBMEC isolation and MBMEC culture medium |
| Penicillin/Streptomycin | Sigma | P4333 | for MBMEC isolation and MBMEC culture medium |
| Phosphate-Buffered Saline (PBS) | Sigma | D8537 | for MBMEC and immune cell isolation |
| Heparin | Sigma | H3393 | for MBMEC culture medium |
| Human Basic Fibroblast Growth Factor (bFGF) | PeproTech | 100-18B | for MBMEC culture medium |
| Puromycin | Sigma | P8833 | for MBMEC culture medium; only for the first three days |
| 0.05% Trypsin-EDTA | Gibco | 25300-054 | for harvesting MBMECs |
| Collagenase Type IA | Sigma | C9891 | for dendritic cell isolation |
| Trypan Blue solution, 0.4% | Thermo Fisher Scientific | 15250061 | for cell counting |
| EDTA | Sigma | E5134 | for immune cell isolation |
| IMDM + 1% L-Glutamin | Gibco | 21980-032 | for T cell culture medium |
| X-VIVO 15 | Lonza | BE04-418Q | protect from light; for B cell culture medium |
| β-mercaptoethanol | Gibco | 31350-010 | for B cell culture medium |
| L-Glutamine (100x Glutamax) | Gibco | 35050-061 | for B cell culture medium |
| mouse MOG35—55 peptide | Biotrend | BP0328 | for antigen-specific T cell activation |
| purified anti-mouse CD3 Ab | BioLegend | 100302 | clone 145-2C11; for polyclonal T cell activation |
| purified NA/LE anti-mouse CD28 Ab | BD Pharmingen | 553294 | clone 37.51; for polyclonal T cell activation |
| Recombinant Murine IFN-γ | PeproTech | 315-05 | for T-cell transmigration assays |
| Recombinant Murine TNF-α | PeproTech | 315-01A | for T-cell transmigration assays |
| NA/LE purified anti-mouse IFN-γ antibody | BD Biosciences | 554408 | clone XMG1.2; recommended final concentration: 20 µg/ml |
| Granzyme B Inhibitor II | Calbiochem | 368055 | recommended final concentration: 10 µM |
| PE anti-mouse CD4 antibody | Biolegend | 116005 | clone RM4-4; for analysis of T cell transmigration |
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