用于体外研究缺血性中风的人血脑屏障的三重原代细胞培养模型
Summary
在这里，我们描述了基于原代人脑微血管内皮细胞，星形胶质细胞和周细胞建立血脑屏障三重细胞培养模型的方法。该多细胞模型适用于 体外缺 血性卒中期间神经血管单元功能障碍的研究或候选药物的筛选。
Abstract
缺血性中风是全世界死亡和残疾的主要原因，治疗选择有限。缺血性中风的神经病理学特征是大脑血液供应中断，导致细胞死亡和认知功能障碍。在缺血性卒中期间和之后，血脑屏障（BBB）功能障碍会促进损伤进展，并导致患者恢复不良。目前的BBB模型主要包括内皮单培养和与星形胶质细胞或周细胞的双重共培养。
这种模型缺乏完全模仿动态大脑微环境的能力，这对于细胞间通信至关重要。此外，常用的BBB模型通常包含永生化的人内皮细胞或动物源性（啮齿动物，猪或牛）细胞培养物，这些细胞培养物具有翻译局限性。本文描述了一种基于插入良好的新型BBB模型，该模型仅包含原代人细胞（脑微血管内皮细胞，星形胶质细胞和脑血管周细胞），能够在 体外研究缺血性脑损伤。
通过被动通透性、经内皮电阻（TEER）测量和缺氧细胞直接可视化评估氧-葡萄糖剥夺（OGD）对屏障完整性的影响。所提出的方案在 模拟体内BBB的细胞间环境方面具有明显的优势，可作为更现实的 体外 BBB模型，用于在缺血性脑损伤的情况下开发新的治疗策略。
Introduction
中风是全球死亡和长期残疾的主要原因之一1。卒中的发病率随着年龄的增长而迅速增加，55岁以后每10年翻一番2.缺血性卒中是由于血栓和栓塞事件引起的脑血流中断而发生的，占所有卒中病例的80%以上3。即使是现在，可用于减少缺血性中风后组织死亡的治疗选择也相对较少。确实存在的治疗方法对时间敏感，因此并不总是导致良好的临床结果。因此，迫切需要研究缺血性脑卒中影响脑卒中后恢复的复杂细胞机制。
BBB是血液和脑实质之间分子交换的动态界面。在结构上，BBB由脑微血管内皮细胞组成，这些细胞由基底膜包围的连接复合物，周细胞和星形胶质细胞尾足4相互连接。周细胞和星形胶质细胞通过分泌形成强而紧密的连接所必需的各种因子在维持BBB完整性方面起着至关重要的作用5，6。BBB的分解是缺血性中风的标志之一。与脑缺血相关的急性炎症反应和氧化应激导致紧密连接蛋白复合物的破坏和星形胶质细胞、周细胞和内皮细胞之间的串扰失调，从而导致 BBB7 的细胞旁溶质通透性增加。BBB功能障碍进一步促进脑水肿的形成，增加出血转化的风险8。考虑到上述所有因素，人们对了解缺血性中风期间和之后BBB水平发生的分子和细胞变化非常感兴趣。
尽管近几十年来已经开发了许多体外BBB模型并用于各种研究，但它们都不能完全复制体内条件9。虽然一些模型基于在插入良好的渗透性载体上单独培养或与周细胞或星形胶质细胞组合培养的内皮细胞单层，但只有最近的研究引入了三重细胞培养模型设计。几乎所有现有的三重培养BBB模型都包含原代脑内皮细胞以及从动物物种中分离的星形胶质细胞和周细胞或来自人类多能干细胞的细胞10，11，12，13。
认识到需要在体外 更好地概括人BBB，我们建立了由人脑微血管内皮细胞（HBMEC），原代人星形胶质细胞（HA）和原代人脑血管周细胞（HBVP）组成的三细胞培养 体外 BBB模型。该三重培养BBB模型设置在孔径为0.4μm的6孔板聚酯膜插入物上。这些孔插入物为细胞附着提供了最佳环境，并可轻松进入顶端（血液）和基底外侧（脑）隔室，以进行培养基采样或化合物应用。通过测量模拟缺血性中风的体 外OGD后的TEER和细胞旁通量来评估该三细胞培养BBB模型的特征，通过使用加湿的密封室实现氧气（<1%O2）和营养物质（通过使用无葡萄糖培养基）。此外，该模型中诱导的缺血样条件通过缺氧细胞的直接可视化得到准确验证。
Protocol
Representative Results
Discussion
在该协议中，我们描述了一种建立可靠的三重内皮细胞 – 周细胞 – 星形胶质细胞培养BBB模型的方法，用于研究 体外缺血性中风情况下的BBB功能障碍。考虑到周细胞是 体内 内皮细胞的最近邻，HBVP在该模型16中接种在孔插入物的下侧。虽然这种配置缺乏星形胶质细胞和内皮细胞之间的直接细胞间通讯，但这种排列允许通过分泌的可溶性因子 在 细胞类型之间进?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（NIH）资助MH128022，MH122235，MH072567，MH122235，HL126559，DA044579，DA039576，DA040537，DA050528和DA047157的支持。
Materials
| 24 mm Transwell with 0.4 µm Pore Polyester Membrane Insert | Corning | 3450 | |
| 35 mm Glass Bottom Dishes | MatTek Life Sciences (FISHERSCI) | P35GC-1.5-14-C | |
| Astrocyte Medium | Science Cell | 1801 | |
| Attachment Factor | Cell Systems (Fisher Scientific) | 4Z0-201 | |
| BD 60 mL Syringe | BD | 309653 | |
| BrainPhys Imaging Optimized Medium | STEMCELL Technologies | 5791 | |
| Complete Classic Medium With Serum and CultureBoost | 4Z0-500 | Cell Systems | |
| Corning 50 mL PP Centrifuge Tubes (Conical Bottom with CentriStar Cap | VWR | 430829 | |
| Corning 75cm² U-Shaped Canted Neck Not Treated Cell Culture Flask | Corning | 431464U | |
| Corning CellBIND 96-well Flat Clear Bottom Black Polystyrene Microplates | Corning | 3340 | |
| Countes Cell Counting Chamber Slides | Thermo Fisher Scientific | C10228 | |
| Countess II FL Automated Cell Counter | Thermo Fisher Scientific | ZGEXSCCOUNTESS2FL | |
| Decon CiDehol 70 Isopropyl Alcohol Solution | Fisher Scientific | 04-355-71 | |
| Disposable Petri Dishes | VWR | 25384-088 | |
| DMEM Medium (No glucose, No glutamine, No phenol red) | ThermoFisher | A14430-01 | Glucose-free medium |
| DPBS (No Calcium, No Magnesium) | ThermoFisher | 14190250 | |
| EBM Endothelial Cell Growth Basal Medium, Phenol Red Free, 500 mL | Lonza | CC-3129 | |
| EVOM2 Epithelial Volt/Ohm (TEER) Meter with STX2 electrodes | World Precison Instruments | NC9792051 | Epithelial voltohmmeter |
| Fluorescein isothiocyanate–dextran (wt 20,000) | Millipore Sigma | FD20-250MG | |
| Fluorescein isothiocyanate–dextran (wt 70,000) | Millipore Sigma | FD70S-250MG | |
| Fluorview FV3000 Confocal Microscope | Olympus | FV3000 | |
| Gas Tank (95% N2, 5% CO2) | Airgas | X02NI95C2003071 | |
| HBSS (No calcium, No magnesium, no phenol red) | Thermofisher | 14025092 | |
| Hoechst 33342, Trihydrochloride, Trihydrate – 10 mg/mL Solution in Water | ThermoFisher | H3570 | |
| Human Astrocytes | Science Cell | 1800 | |
| Human Brain Vascular Pericytes | Science Cell | 1200 | |
| Hypoxia Incubator Chamber | STEMCELL Technologies | 27310 | |
| Image-iT Green Hypoxia Reagent | ThermoFisher | I14834 | |
| Pericyte Medium | Science Cell | 1201 | |
| Primary Human Brain Microvascular Endothelial Cells | ACBRI 376 | Cell Systems | |
| Rocking Platform Shaker, Double | VWR | 10860-658 | |
| Single Flow Meter | STEMCELL Technologies | 27311 | |
| SpectraMax iD3 Microplate Reader | Molecular Devices | 75886-128 | |
| Syringe Filter, 25 mm, 0.22 μm, PVDF, Sterile | NEST Scientific | 380121 | |
| TPP Mutli-well Plates (6 wells) | MidSci | TP92406 | |
| TPP Tissue Culture Flasks T-75 Flasks | MidSci | TP90075 | Flasks with activated surface for cell adhesion |
| Trypsin-EDTA (0.25%), phenol red | ThermoFisher | 25200056 | |
| UltraPure Distilled Water | Invitrogen (Life Technologies) | 10977-015 | |
| Uno Stage Top Incubator- | Oko Lab | UNO-T-H-CO2-TTL |
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