研究病原体或药物交叉障碍及其与大脑相互作用的人血脑界面模型
Summary
在这里, 我们提出了一个协议, 描述了设置在纤维素 BBB (血脑屏障)-minibrain 聚酯多孔膜培养插入系统, 以评估生物分子或传染因子在人类 BBB 的运输和对邻近脑细胞的生理影响。
Abstract
早期筛选神经系统药物在一个相关和可靠的蜂窝 BBB 模型, 为他们的渗透和他们与障碍和脑实质的相互作用仍然是一个未得到满足的需求。为了填补这一空白, 我们设计了一个2D 在蜂窝模型, BBB-Minibrain, 通过结合聚酯多孔膜培养插入人类 BBB 模型与由人类脑细胞 (神经元, 星形胶质细胞和微胶质细胞) 形成的三培养形成的微型 Bbb 模型。BBB-Minibrain 允许我们测试神经保护药物候选物 (例如, Neurovita) 通过 BBB 的迁移情况, 以确定这种分子对神经元的特定靶向性, 并表明药物的神经保护性能在之后得到了保留药物已经越过了 BBB。我们还证明了 BBB-Minibrain 是一个有趣的模型, 用于检测病毒颗粒通过内皮细胞屏障的过程, 并监测微博病毒颗粒对微根病毒的感染。BBB-Minibrain 是一个可靠的系统, 易于处理的研究人员在细胞培养技术和预测脑细胞表型治疗或侮辱后。这种对纤维素检测的兴趣将是双重的: 一方面是在药物开发的早期引入了嘲讽步骤, 另一方面减少了动物试验的使用。
Introduction
大脑与全身循环分离, 这种结构是不可渗透的结构, 它限制了大脑实质与血液之间的交流, 称为血脑屏障 (BBB)。BBB 主要由大脑内皮细胞组成, 与星形胶质细胞、血管周围微胶质细胞和邻近脑实质的神经元动态相互作用。Bbb 的三大功能是创造和维持离子稳态的神经元功能, 为大脑提供营养, 并防止有毒伤害或病原体进入1, 2,这有助于维持大脑的稳态及其功能3。这个屏障是如此有效, 只有少数药物可以越过 bbb4,5。目前, 预测分子是否会通过 BBB 并扩散到大脑的现有方法包括对尸检材料的体外研究、MRI (磁共振成像) 或 PET (位置发射) 对人类志愿者大脑中的图像跟踪断层扫描) 或药物动力学和药物动力学在临床前研究动物 6,7,8。这些技术和模型有一些局限性, 如 pet 的分辨率有限, mri6,8的灵敏度较低, 难以量化的分子 (例如, 基于抗体的分子)穿透大脑7, 并为临床前研究他们的高成本和动物试验的手段。
最后一点很重要, 因为根据3R 的规定, (动物试验的替代、减少和细化) 监管部门要求研究人员紧急开发出科学上准确的动物替代品实验9,10,11,12,13, 14,15.
在过去的几十年里, 通过在滤膜上培养小鼠、大鼠、牛和猪等不同物种的内皮细胞, 提出了几种 bovine 体外模型。就人类物种而言, 原代细胞的稀缺和困难促使研究人员开发了基于永生脑内皮细胞或人类干细胞19,20的人类模型。 21岁这些屏障是适当的 BBB 体外代药, 只要它们表达内皮细胞标记, 紧密的连接标记, 外流转运, 溶质载体, 受体, 并对内皮刺激20的反应. 一些使用滤膜的 bbb 模型与内皮细胞和其他细胞类型 (即星形胶质细胞、神经元或周细胞22,23,24) 进行了检测。这些共培养的目的是利用星形胶质细胞分泌可溶性因子或过氧化物酶来增加 BBB 的物理特性。
然而, 这些模型都不包括脑实质, 以研究和预测药物候选人一旦通过障碍后的命运。因此, 我们的目标是建立一个蜂窝血大脑界面, BBB-Minibrain, 结合 bbb 模型和混合脑细胞的培养成一个单一的套件。BBB-Minibrain 使用的培养系统由插入多井细胞培养板井中的多孔过滤器组成。该过滤器涂有 hcmece/d3 细胞, 这是一种人类大脑内皮细胞系, 已被证明对 bbb 药物检测25、26、27非常可靠, 以形成 bbb。Minibrain 是一种共同分化的人类神经元和星形胶质细胞, 其来源来自 nterae/cl2 细胞系28、29与人类微胶质细胞系 chmey-cl530的比率与微胶质细胞与神经星形胶质细胞的大脑31的比率, 是在盘的底部培养良好的。
除了研究药物在 BBB 上的传播及其在实质中的命运外, 纤维素模型中的血脑界面可以成为解决病原体进入大脑 (神经入侵)、扩散到大脑 (神经性) 的有力工具以及它们对脑实质细胞的毒性 (神经毒力)。神经毒力和神经侵袭性研究将受益于纤维素模型中高效的开发, 并有利于取代动物模型。使用 bbb-minibrain 工具包 32, 我们展示了在法国神经质病毒黄热病毒株 (即 fnv-yfv33,34) 中积累的罕见病毒突变体的神经侵入型表型, 用于制备停止活 YFV 疫苗和神经生成和神经保护生物分子的通过称为神经免疫 (下称 NV 在手稿中)35。由于 NV 既不自然穿过细胞膜, 也不自然穿过 BBB, 因此 NV 与 Llama 单链抗体的可变部分 (VHH) 融合在一起, 该抗体穿过包括 BBB 在内的生物膜, 并起到细胞穿透分子 (CPM)36的作用。VHH 的 CPM 特性似乎取决于等电点和 VHH37 的长度.
在蜂窝测试中, 这应该可以在动物进行药代动力学和药效学分析之前对可能横穿 BBB 的分子进行分类, 最好同时进行, 以便能够预测它们在神经中的行为实质。该系统在生物学上相关, 易于由在 26、29、30、38受过良好培训的专业人员建立和处理。这种对纤维素检测的兴趣将有两个方面: 一方面降低临床前检测的成本, 另一方面减少动物试验的使用。
Protocol
1. Ntera/CL2 的细胞培养工作。D1 准备有丝分裂后的 hNeurons 和 Hastrocyes (NT2-NNA) 的共培养 注: 这是微型的组成部分 (图 1)。 培育 nterasne2. d1 从液氮罐中取出一小瓶冷冻细胞。继续冰上。 在37°c 的水浴中快速解冻细胞。 将细胞转移到含有10毫升完整 DMEM F12 培养基的 15 mL 管中 (Dulbecco 的改良鹰介质: 营养混合物 F-12 培养基), 辅以10% …
Representative Results
BBB-Minibrain 是血脑界面的蜂窝实验模型。 BBB-Minibrain 设置在聚酯膜培养插入系统上, 以模拟上层的血室和血脑界面下层的大脑隔间 (图 2a, b)。它由一个腔隔间与 hCMEC/D3 内皮细胞在过滤器形成 BBB 和一个腔室, 其中包含微型人类三培养的脑细胞 (神经元, 星形细胞和微胶质细胞)。Minibr…
Discussion
在这篇文章中, 我们演示了如何建立一个在蜂窝血/大脑界面, BBB-Minibrain, 结合 bbb 模型和混合脑细胞 (Minibrain) 的培养成一个单一的套件。该系统在生物学上是相关的, 易于建立和处理的实验者在细胞培养方面受过良好的训练。
对于 BBB 的任何其他体外模型, 如果对屏障的密封性进行严格控制, 都能获得可靠的结果。应仔细测试插入物的渗透率, 并应丢弃任何渗透率值不足的刀片 (…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了巴斯德研究所内部赠款的支持, 包括一项煽动赠款 (PTR 435) 和赛诺菲巴斯德向巴斯德研究所提供的 “Contrat de Soutien la Recherche” 赠款。A. da Costa 得到 Sanofi-Pasteur 赠款的支持, Florian Bakoa 接受了国家研究和技术协会提供的博士赠款。我们感谢 Pr Pierre-Olivier Couraud 博士和 Florence miller 博士进行了有益的讨论。
Materials
| 12 well plates | Corning | 3336 | |
| 5-fluoro-2’deoxyuridine | Merck-Sigma Aldrich | F0503 | |
| 85mm Petri Dish | Sarstedt | 83-3902-500 | |
| Anti-Nf200 | Merck-Sigma Aldrich | N4142 | |
| β-mercapto-ethanol | Merck-Sigma Aldrich | M3148 | |
| CHME/Cl5 | Unité de Neuroimmunologie Virale | On request to Dr Lafon | |
| CMC | Calbiochem | 217274 | |
| Cytosine β-D-arabinofuranoside | Merck-Sigma Aldrich | C1768 | |
| Dark 96 well plates | Corning | 3915 | |
| DMEM F12 | Thermofisher Scientific | 31330-038 | |
| DMSO | Merck-Sigma Aldrich | D2650 | |
| Endogro IV | Millipore | SCME004 | endothelial cell medium |
| Ethanol | Carlo Erba | 529121 | |
| FBS | Hyclone | SV30015-04 | |
| Formaldehyde | Merck-Sigma Aldrich | 252549 | |
| GIEMSA | RAL Diagnostic | 320310 | |
| Goat-Anti Mouse | Jackson Immuno Research | 115-545-003 | |
| Goat-Anti Rabbit | Thermofisher Scientific | R37117 | |
| HBSS with Ca2+-Mg2+ | Thermofisher Scientific | 14025-100 | |
| hCMEC/D3 | Cedarlane | CLU512 | |
| Hepes 1M | Thermofisher Scientific | 15630-070 | |
| Hoescht 33342 | Merck-Sigma Aldrich | 33263 | |
| Laminine | Merck-Sigma Aldrich | L6274 | |
| L-glutamin | Thermofisher Scientific | 25030-024 | |
| Lucifer Yellow | Merck-Sigma Aldrich | L0259 | |
| MEM 10X | Thermofisher Scientific | 21430 | |
| MEM 1X | Thermofisher Scientific | 42360 | |
| Ntera/Cl2D.1 | ATCC | CRL-1973 | |
| Paraformaldehyde | Electron Microscopy Sciences | 15714 | |
| PBS without Ca2+-Mg2+ | Thermofisher Scientific | 14190 | |
| PBS-Ca2+-Mg2+ | Thermofisher Scientific | 14040-091 | |
| Pen/Strep | Eurobio | CXXPES00-07 | |
| Poly-d-Lysine | Merck-Sigma Aldrich | P1149 | |
| Prolong Gold | Thermofisher Scientific | P36930 | |
| Qiashredder | QIAGEN | 79656 | |
| Rat Collagen I | Cultrex | 3443-100-01 | |
| Retinoic Acid All-Trans | Merck-Sigma Aldrich | R2625 | |
| RNA purification kit | QIAGEN | 74104 | |
| SDS | Merck-Sigma Aldrich | L4509 | |
| Sodium bicarbonate 5.6% | Eurobio | CXXBIC00-07 | |
| Sodium Pyruvate | Thermofisher Scientific | 11360 | |
| T75 Cell+ Flask | Sarstedt | 83-1813-302 | Tissue culture polystyrene flask with specific surface treatment (Cell+) for sensitive adherent cells |
| Transwell | Corning | 3460 | polyester porous membrane culture inserts |
| Trypsin-EDTA | Merck-Sigma Aldrich | T3924 | |
| Ultra Pure Water | Thermofisher Scientific | 10977-035 | |
| Uridine | Merck-Sigma Aldrich | U3750 | |
| Versene | Thermofisher Scientific | 15040-033 | EDTA |
| YFV-FNV | IP Dakar | Vaccine vial |
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Cite This Article
da Costa, A., Prehaud, C., Bakoa, F., Afonso, P., Ceccaldi, P., Lafaye, P., Lafon, M. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain. J. Vis. Exp. (146), e59220, doi:10.3791/59220 (2019).