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Medicine

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

Published: September 24, 2017
doi:
10.3791/56277
, , , , , , ,
1Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Department of Biomedicine,Aarhus University, 2Institute of Pharmaceutical Science,King’s College London, 3HICoE Centre for Drug Research,Universiti Sains Malaysia

Summary

该协议的目的是提出一个优化的程序, 建立一个体外血脑屏障 (BBB) 模型的基础上猪脑内皮细胞 (pBECs)。该模型具有重现性高、严密性高的优点, 适合于研究药物发现中的转运和胞内贩运。

Abstract

本议定书的目的是为纯化和培养 pBECs, 并建立体外血脑屏障 (BBB) 模型的基础上 pBECs 单培养 (mc), mc 与星形胶质细胞条件培养基 (ACM), 并与猪或大鼠源星形胶质细胞的非接触共培养。pBECs 从5-6 月大的家养猪脑皮质的毛细血管片段中分离和培养。这些碎片通过仔细去除脑膜, 分离和均匀化的灰质, 过滤, 酶消化, 和离心纯化。为了进一步消除污染细胞, 用含嘌呤的培养基培养毛细血管片段。当60-95% 汇合, pBECs 生长从毛细管片段被传代对渗透性膜过滤器插入物和建立在模型。为提高 pBECs 的屏障严密性和血脑屏障特征表型, 对细胞进行了以下分化因素的处理: 膜 permeant 8-CPT-camp (这里缩写阵营), 氢化可的松, 磷酸二酯酶抑制剂, RO-20-1724(RO)。该手术在9-11 天内进行, 在建立该模型时, 星形胶质细胞被提前2-8 周培养。遵守该议定书所述的程序允许建立具有高度限制的细胞渗透性的内皮层, 而该模型显示了1249年±80Ω cm 的平均 transendothelial 电阻 (TEER).2, 和细胞渗透性 (Papp) 为路西法黄色的 0.90 10-6 ± 0.13 10-6 cm 秒-1 (平均± SEM, n=55)。该理事会表型的进一步评价表明, claudin 5、ZO-1、蛋白和黏着结蛋白 p120 蛋白的紧密连接蛋白表达良好。该模型可用于健康和疾病 BBB 的一系列研究, 并具有高度限制性的细胞渗透性, 该模型适用于研究运输和细胞内贩运。

Introduction

血脑屏障的细胞结构与功能

在循环系统和中枢神经系统 (cns) 的界面, BBB 作为 homoeostatic 控制中枢神经系统微环境的关键调控场所, 这对神经系统的正常功能和保护至关重要。血脑屏障的部位是血管腔内衬的内皮细胞。在脑毛细血管, 内皮细胞形成复杂的细胞间紧密连接和强烈极化表达模式的特殊流入和外排转运运输确保高度特异的分子运输之间的血液和大脑1。紧密接合配合物的结构成分包括来自蛋白和 claudin 家族的蛋白质、紧密拼装(祖伊) 蛋白、cingulin 和相关的连接黏附分子 (果酱)。Claudin 5 在细胞连接限制中尤为重要。诱导和维持这一特征血脑屏障内皮表型涉及与周围细胞的动态相互作用, 包括周、星形胶质细胞、神经元和基底膜, 这些都与大脑内皮干细胞一起形成神经血管单元 (NVU)2,3。这些相互作用所涉及的机制尚未完全被理解, 但包括在细胞间交换化学信号, 这允许在短期内调制 bbb 通透性, 并诱发长期 bbb 功能4。星形胶质细胞尤其被称为对脑血管内皮表型的贡献, 是一种调节因子的来源, 如胶质源性神经营养因子 (影响细胞内 cAMP)5, 碱性成纤维细胞生长因子6,氢化可的松7, 并转化生长因子β (TGF β)8。然而, TGF β的影响已经被争论了9。

从体内到体外BBB

在体内研究继续提供有关 BBB 生物学的宝贵信息。然而, 细胞培养模型可以提供更多的见解, 并构成有用的工具, 以了解详细的分子和功能方面的 BBB 在健康和疾病。虽然脑血 BBB 的细胞类型和成分之间的复杂相互作用很难在体外模型中完全实现, 但自从第一次纯化大脑内皮细胞和在单培养中应用后, 已经有了。10,11,12, 广泛开发的净化程序和生长条件的 BBB 细胞培养模型, 导致更大的相似性的在体内屏障。通常使用的体外BBB 模型基于啮齿动物、猪和牛的原细胞, 以及永生细胞系。每个模型都有不同的优点和缺点。为了比较和模型选择, 验证标记, 如血 BBB 酶的表达, 转运蛋白, 受体和结构蛋白质被用来创建当前建立的模型的概述1。

议定书的目的

BBB 的一个重要特点是屏障严密性和高 TEER, 然而大量可用的模型并没有很好地反映在体内的水平。将开发和优化的贡献从几个实验室, 该协议的目的是提出一种方法, 建立一个高 TEER体外BBB 模型的基础上 pBECs 在 MC 与或不含 ACM, 或在与初级大鼠或猪源星形胶质细胞。该模型的应用程序和建立包括努力消除污染细胞和改善 pBECs 分化为 BBB 表型。这项工作的结果是建立了可靠的, 高 TEER 模型低细胞渗透性和良好的功能表达的关键紧密连接蛋白, 转运, 和受体。然而, 由于星形胶质细胞是一个因素, 以脑细胞表型, 三不同的文化条件代表三不同的表现型脑内皮细胞。该模型对于研究药物发现、运输研究和细胞内贩运的某些专门机制, 以及对血脑屏障功能最大表达的细胞间相互作用的调查是特别有用的。有利.

议定书的起源和历史

这里描述的理事会模型主要基于在卫材实验室开发的猪模型 (伦敦) 由 Dr. 路易斯摩根和同事, 是基于成功的早先牛脑内皮细胞模型13。原细胞制备方法是用尼龙网格捕捉微血管的两级过滤, 其次是传代步骤以提高纯度。在该方法的早期发展中, 通过补充培养基 (包括 ACM) 的生长, 达到了最佳 BBB 表型和屏障的致密性。对方法的进一步修改由 r. 斯金纳在 Rothwell 的实验室在曼彻斯特英国14,15中进行。该方法是通过雅培实验室, 氯化钾伦敦, 在那里 Patabendige 使它大大简化准备通过避免使用星形胶质细胞或 ACM 和消除污染细胞, 如周与嘌呤。第一篇论文证实 MC 模型保留了在体内BBB 的几个重要特征, 包括有效的紧密连接, 膜传输系统和受体介导的 transcytosis16,17,18,19,20. 后来 s. 尤索夫再次测试星形胶质细胞共培养, 发现它显著改善了 TEER, 所以这是目前在 Abbott 实验室21中使用的首选变体。该模型现已成功地转移到了奥胡斯的 m. 尼尔森实验室, 在那里进行了进一步的修改 (本议定书), 包括简化灰色物质提取, 只使用一个滤网过滤步骤, 和一个单一的过滤涂层步骤结合胶原蛋白和纤维连接蛋白。猪星形胶质细胞分离的应用程序 (本议定书) 是基于奥尔堡的 t Moos 实验室开发的协议, 由汤姆森et al22描述。在伦敦和奥胡斯所产生的模型的 TEER 和其他性质是相似的, 这使人们相信模型很容易在实验室之间转移, 并能很好地对方法步骤的仔细观察和合理化作出反应。事实上, 尤索夫已经建立了一个热带国家 (马来西亚)23的 MC 模型, 它涉及对当地条件和组织来源的进一步调整。

优于替代方法和现有模型的优势

与牛和啮齿类动物的脑内皮细胞相比, pBECs 提供了在隔离状态下的在体内BBB 表型的损失率较低的优势.此外, pBECs 能够形成相对严密的内皮屏障, 即使在 MC (800 Ω cm2)16中生长, 与通常报告的细胞系的单分子层 (如弯曲. 5 和弯曲) 相比. 3 (50 Ω cm2)25,26, 27, cEND (300-800 Ωcm2)282930和 cerebEND (500 Ω cm 2)2931,32和主脑小鼠的内皮细胞 (100-300 Ω厘米2)ss = “xref” > 33,34,35,36和 rat (100-300 Ω cm2)37,38。然而, TEER 已经显示了对纯化和培养程序的依赖性。在大多数情况下, 增加的 ACM 或共培养与星形胶质细胞显示出差异的影响, 血管内皮细胞膜和加强致密的内皮层1。然而, 在优化培养条件的努力下, 只有 bovine-based 模型显示了与 porcine-based 模型相似的 TEER 值 (MC 中的800ω cm2的平均值 (在星形胶质细胞共育中高达2500ω cm2 )13 ,39,40,41,42,4344,45。由于基于初级牛脑内皮细胞的模型显示出很大的变化, 两个实验室之间和内部的14,45,46,47,48,重现性可能是个问题。在这里报告的理事会模型中, 提供的实验室已经达到了非常相似的 TEER 和细胞渗透率的低变异性, 在实验室和之间。因此, 其他实验室应该可以利用这里提出的方法建立一个低变异性的鲁棒模型。除了形成致密的内皮层, 模型与 pBECs 以前已经通过表达紧密连接蛋白, 功能 BBB 转运体, 受体和酶, 并证明适合的一系列研究15,16,17,19,20,22,49,50,51,52,53,54,55,56,57,58,59. 此外, pBECs 的 co-cultures 上未发布的转录数据显示了 BBB 转运体和受体的预期分布 (未发表的结果, 尼尔森et al.)。

porcine-based BBB 模型具有更大的优势, 因为猪的基因组、解剖学、生理学和疾病进展比其他建立的模型60更能反映人类生物学的程度, 这是制药业。由于猪的大脑是肉类工业的常见 by-product, 它们构成了一个易于获取的脑血管内皮细胞来源, 使实验所需的动物数量减少, 并提供了一个猪脑的高纯度的产量。虽然纯化和培养初级细胞是有点费时, 需要专门知识的标准化建立模型, 初级细胞产生最可靠的 BBB 模型。永生化细胞系不能替代, 如屏障严密性、转运蛋白表达谱和微环境调节等重要特性不反映实验结果在体内61,62.体外模型提供了更高分辨率的活细胞成像的优势, 通过允许对取样或观察到的细胞进行近距离接近, 从而实现了细胞内过程的可视化, 使用目标具有更高的放大倍数和更好的光学质量63。这不是使用双光子显微镜在活体动物中的情况。此外,体外模型提供了染细胞的能力, 允许可视化标记蛋白质和调查其贩运。

模型的应用

血脑屏障功能不固定, 可以在生理和病理两方面进行动态调节。在许多神经系统疾病, 包括神经退行性、炎症和传染性疾病, 扰乱和提高 BBB 的渗透性被观察64,65,66,67.为了减少和防止疾病进展和随后的损害, 识别和定性的分子机制的基础上的调制的 BBB 是重要的。在这种情况下, 可靠的体外模型在制药工业中需求量很大, 而且在预测药物对中枢神经系统的 BBB 通透性方面发挥了重要作用。任何体外模型都应显示限制性的细胞通路、生理上逼真的细胞结构和传送器机制的功能表达式68。在先前的研究中演示了16,17,57, 并通过细胞的渗透率和 TJ 和 AJ 蛋白的表达, 提出的模型符合所有这些标准, 适用于 BBB 的范围在正常生理学和病理学方面的研究。所提出的纯化和培养方法的优点包括简单性和重现性的结合, 并能够包括星的影响与产生的强大和可靠的高 TEER在体外BBB 模型。为此, 已证明猪和大鼠的星形胶质细胞以类似的方式增强了 pBECs 的 BBB 表型.22。

Protocol

猪脑被作为丹麦食品工业的副产品获得。丹麦的屠宰场受到丹麦环境和食品部的严格监督和观察. 用于分离星形胶质细胞的大鼠在当地的动物设施中繁殖和组织, 环境温度为22和 #176; C-23 和 #176; C 和在 12/12 h 黑暗或轻的周期在兽医的检查之下和根据丹麦实验室动物条例。根据《动物道德使用国际准则》 (欧洲共同体理事会1986年11月24日的指示, 86/609/EEC) 和丹麦的准则, 对这些老?…

Representative Results

BBB体外模型的建立 在所提出的优化方法, 培养 pBECs 和建立的渗透膜插入系统与 MC 或不含 ACM 或全国星形胶质细胞 (图 1) 进行了为期9-11 天 (图 2)。对于内皮细胞的选择, 初步培养的纯化毛细管片段与嘌呤治疗相结合, 最多5天, 消除了大多数污染细胞, 促进了纺锤形内皮细胞的生长?…

Discussion

理事会的纯化与增殖

在纯化过程中, 关键步骤包括快速有效地去除脑膜和白、灰物质的分离, 这对纯化产量和纯度以及正确建立模型具有重要意义。为提出的体外BBB 模型使用 pBECs, 我们改进和简化了基于机械均匀化的分离灰物质的纯化过程, 微血管分离的大小选择过滤, 胶原酶消化, dnasei 和胰蛋白酶, 具有微血管片段的初始培养。一般而言, 净化和培养原细胞的主要挑战之?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者想承认伊丽莎白. 海伦娜 Bruun, 萨拉. 克里斯汀和 Kristiansen 的技术援助, 以及 Lundbeck 基金会授予号码 R155-2013-14113。

Materials

Fibronectin Sigma-Aldrich F1141
Collagen IV Sigma-Aldrich C5533
Poly-L-lysine Sigma-Aldrich P1524
DMEM/F-12 Lonza BE12-719F
DMEM/Low Glucose Sigma-Aldrich D6046
Penicillin/Streptomycin Gibco Invitrogen 15140
Plasma derived serum (PDS) First Link UK Ltd. 60-00-89
Fetal bovine serum (FBS) Gibco Invitrogen 10-270-106
Trypsin/EDTA Gibco Invitrogen 15090-046
Heparin Sigma-Aldrich H3393
Puromycin Sigma-Aldrich P8833
Hydrocortisone Sigma-Aldrich H4001
8-CPT-cAMP Biolog C010
RO 20-1724 Sigma-Aldrich B8279
Gentamicin Sulfate Lonza 17-518Z
DMSO Sigma-Aldrich 34896
PBS Sigma-Aldrich D8537
EtOH VWR 20,824,296 Mix the 70 % solution from the 96 % EtOH
DNAse 1 Sigma-Aldrich D4513
Collagenase CLS2 Sigma-Aldrich C6885
ddH2O Made with Elga System
T75 flasks Thermo Scientific 156499
Costar Transwell inserts (Cell permeable membrane inserts) Costar CLS3401 12-well plate, 12 mm diameter, 0.4 μm polycarbonate membrane
15 ml centrifuge tubes Cellstar 188271
50 ml centrifuge tubes Cellstar 227261
Petri dishes Thermo Scientific 150350
Cryo vials Thermo Scientific 377224
500 ml bottle Thermo Scientific 159910/159920
Scalpels Swann-Morten REF0211 Type 24
Tissue homogenizer Sigma D9188
140 μm filters MERCK NY4H04700
40 μm filters Corning 431750
EndOhm chamber system World Precision Instruments ENDOHM-12 EndOhm chamber for 12mm Culture Cups
EVOM2 electrode system World Precision Instruments 300523+STX100C TEER measurement system with rigid STX-100C electrode pair
Long needle Sigma Attach to a syringe
Fine-tip curved forceps KLS Martin 12-409-12-07
Broad tip forceps VWR 82027-390
Filter holder MERCK Milipore Swinnex-47
50 ml syringe Braun 4617509F
10 ml syringe Terumo SSt20ESI
Anti-Occludin antibody Abcam ab31721 1:100
Anti-p120 Catenin antibody BD Transduction laboratories 610133 1:200
Anti-ZO-1 antibody Invitrogen 61-7300 1:200
Anti-Claudin 5 antibody Sigma-Aldrich SAB4502981 1:100
Donkey anti rabbit IgG conjugated with Alexa Flour 568 Thermo Scientific A10042 1:500
Donkey anti mouse IgG conjugated with Alexa Flour 488 Thermo Scientific A21202 1:500
Sucrose Perkin Elmer NEC100X250UC 0.15µl/ml final working conc
Lucifer Yellow Sigma L0144 10 µg/ml final working conc
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Blood-brain BarrierPorcine Brain Endothelial CellsIn Vitro ModelPrimary Cell IsolationCapillary IsolationCell DigestionCell CultureTEERPermeability

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Nielsen, S. S. E., Siupka, P., Georgian, A., Preston, J. E., Tóth, A. E., Yusof, S. R., Abbott, N. J., Nielsen, M. S. Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells. J. Vis. Exp. (127), e56277, doi:10.3791/56277 (2017).
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