器官细胞生存在器官细胞切片培养
Summary
组织切片培养是研究神经发育或退行/再生过程的有力工具。在这里，我们描述了一个协议，模型在小鼠小脑切片培养中的Purkinje细胞的神经发育死亡。该方法有利于神经保护药物发现的研究。
Abstract
组织切片培养是一种强大的体外模型，它比分离的原细胞培养更密切地模拟体内条件。在产后早期发育中，已知小脑Purkinje细胞会经历一个脆弱的时期，在此期间，它们经历程序性细胞死亡。在这里，我们提供了一个详细的协议，以执行小鼠器官小脑切片培养在此关键时间。进一步标记切片，以评估Purkinje细胞存活率和神经保护治疗的有效性。这种方法对于筛选新的神经活性分子非常有价值。
Introduction
体外建模是生物医学研究的重要工具。它允许调查人员研究和严格控制受限制细胞类型或隔离系统/器官中的特定机制。组织切片培养是一种广泛应用的体外技术，特别是在神经科学领域。该方法最初由Göhwiler建立，他使用辊管技术2培养脑切片，后来由山本等人修改，他介绍了使用微孔膜进行皮质切片培养3。与原发细胞培养相比，有机切片培养具有保存组织细胞结构以及组织部分平面中的原生细胞-细胞连接的优势。
有机切片是从中枢神经系统的许多部位培养的，如海马体4，皮层5，纹状体6，小脑4，7，脊髓8，9等。它们已被证明是药物发现研究中的有力工具。神经活性分子的影响可以通过多种方式进行评估：使用免疫染色和生物化学分析的生存和神经退化，神经元回路形成，或使用电生理学和活成像中断。
这项工作的目的是描述一种简单的方法来执行器官小脑切片培养，这是已知的一个相关的模型，以模拟在体外小脑发育。特别是，我们专注于Purkinje细胞发育死亡的研究。在体内，Purkinje细胞在第一个产后周发生凋亡，在产后第3天（P3）11达到峰值。在小脑切片培养中也观察到同样的模式，当从P1和P8之间的动物身上取走脑细胞后，Purkinje神经元死于凋亡，峰值为P34，12。使用器官小脑切片培养物已经允许识别几个神经保护分子7，13，以及了解参与这个程序细胞死亡14，15，16的一部分机制。在这里，我们描述了一个基于在海马区Stoppini等人17号的研究，并适应小脑的Dusart等人的协议4它包括快速解剖和切除了产后脑切除术;将培养物切片到含有微孔膜的细胞培养插件上，无论是否进行神经保护治疗;和免疫荧光染色，以评估神经元存活率。
Protocol
所有涉及动物的实验都是根据西北大学动物研究委员会进行的。 1. 在器官小脑切片培养之前的准备 在事先喷洒 70% 乙醇的细胞培养罩中，在连接到无菌瓶接收器的 250 mL 瓶顶真空过滤器中制备 200 mL 培养基。加入100 mL的底中鹰（BME），50 mL的汉克斯平衡盐溶液（HBSS），50 mL的热灭活马血清，1 mL的200 mM L-谷氨酰胺（最终浓度1 mM），和5 mL的200克/升葡萄糖（最终浓度5毫克/?…
Representative Results
如图4所示，该协议产生有机细胞小脑切片培养物，其中Purkinje细胞存活率可按照免疫荧光和图像采集步骤进行评估。Purkinje细胞被标记为抗卡宾丁D-28K（稀释1/200）和Alexa594抗小鼠（稀释1/300）抗体的组合。通过显微镜采集软件（NIS-元素）自动进行图像拼接，以获得整个小脑切片的图片。在棱镜8软件中输入了每个切片的Purkinje细胞数，以生成图表并执行统计分析。在P6，Purkinje?…
Discussion
切贝拉尔切片培养是研究产后发育过程中程序化的Purkinje细胞死亡的有力工具。该技术可用于快速筛选候选分子的神经保护电位。主要优点是设置简单且经济高效，只需对设备进行适度投资（振动器的成本是纸巾切碎器的 3 倍）。此外，从一只小鼠小狗可以生成10到15个健康的切片，允许使用一种动物并行进行不同的检测。
为了获得一致且可重复的结果，尽可能高效地执行文?…
Declarações
The authors have nothing to disclose.
Acknowledgements
成像工作在西北大学高级显微镜中心进行，由NCI CCSG P30 CA060553慷慨支持，授予罗伯特·赫·卢里综合癌症中心。我们感谢肖恩·麦克德莫特的技术援助和支持，感谢玛雅·爱泼斯坦的手工绘制的插图，如图1所示。
Materials
| Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody | ThermoFisher scientific | A21203 | |
| Basal Medium Eagle (BME) | ThermoFisher scientific | 21010046 | |
| Biosafety cabinet Class II, Type A2 | NuAire | NU-540-400 | |
| Bovine serum albumin | Millipore Sigma | A2153 | |
| Brush | |||
| anti-Calbindin D-28K antibody (CB-955) | Abcam | ab82812 | |
| CO2 Incubator | NuAire | NU-5700 | |
| Corning Costar Flat Bottom 6-well Cell Culture Plates | Fisher Scientific | 07-200-83 | |
| Coverslips, 22 x 50 mm | Fisher Scientific | 12-545-E | |
| Dressing forceps, straight | Harvard Apparatus | 72-8949 | |
| Double edge blades | Fisher Scientific | 50949411 | |
| Ethanol 200 proof | Decon Labs, Inc | 2701 | |
| Eye Scissors, straight | Harvard Apparatus | 72-8428 | |
| Fine forceps | Fisher Scientific | 16-100-127 | |
| L-Glutamine 100X | ThermoFisher scientific | 25030149 | |
| Glucose solution | ThermoFisher scientific | A2494001 | |
| Hanks’ Balanced Salt Solution | ThermoFisher scientific | 14025092 | |
| Hoechst 33342, Trihydrochloride, Trihydrate | Fisher Scientific | H21492 | |
| Horse Serum, heat inactivated, New Zealand origin | ThermoFisher scientific | 26050088 | |
| ImageJ | |||
| McIlwain Tissue Chopper | Fisher Scientific | NC9914528 | |
| Microprobes | Fisher Scientific | 08-850 | |
| Millicell Cell Culture Inserts | Millipore Sigma | PICM0RG50 | |
| Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL | ThermoFisher scientific | 168-0045 | |
| Nikon A1R confocal laser microscope system | Nikon | ||
| NIS-Elements Imaging Software | Nikon | ||
| Paraformaldehyde | Acros Organics | 41678-0010 | |
| Pasteur pipets | Fisher Scientific | 13-678-20D | |
| Potassium Chloride | Fisher Scientific | BP366-500 | |
| ProLong Gold Antifade Mountant | ThermoFisher scientific | P10144 | |
| Operating Scissors, straight | Harvard Apparatus | 72-8403 | |
| Orbital shaker Belly Dancer | IBI Scientific | BDRLS0003 | |
| Prism 8 | GraphPad | ||
| Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
| Tissue Culture Dish, 60 mm w/ grip ring | Fisher Scientific | FB012921 | |
| Tissue culture plate, 24 well | Falcon/Corning | 353047 | |
| Transfer pipettes, sterile | ThermoFisher scientific | 13-711-21 | |
| Triton X-100 | ThermoFisher scientific | BP151-500 |
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Citar este artigo
Rakotomamonjy, J., Guemez-Gamboa, A. Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures. J. Vis. Exp. (154), e60353, doi:10.3791/60353 (2019).