间接神经元的星形胶质细胞共培养试验：一个<em>体外</em>设置 - 为神经元的神经胶质细胞相互作用的详细调查
Summary
该协议描述了神经元的神经胶质细胞相互作用的条块分析间接神经元的星形胶质细胞共培养。
Abstract
正确的神经细胞的发育和功能是发展中国家和成人大脑的先决条件。然而，复杂的神经元网络的高度控制形成和维护背后的机制尚未完全迄今理解。关于健康和疾病的神经元的开放式问题是多种多样的，从理解基本的发展，人类的调查相关疾病， 如阿尔茨海默氏症和精神分裂症深远。可以在体外进行的神经元的最详细的分析。然而，神经元细胞，要求和需要为他们的长期生存的额外支持星形胶质细胞。此细胞异质性是在与目标解剖神经元和星形胶质细胞的分析冲突。我们在这里提出一个细胞培养测定法，其允许纯原代神经元和星形胶质细胞，其共享相同的化学上确定的培养基的长期共培养，而在物理上分开。在此设置中，培养物存活多达四个星期，该测定是适于关于神经元的神经胶质细胞的相互作用研究的多样性。
Introduction
纵观几十年来，神经胶质细胞功能的一般性解释已经从一个单纯的支持对有关神经功能1积极调节作用的归属演变。因为对健康和疾病的2脑稳态其突出的影响，星形胶质细胞是科学界特别感兴趣的。在过去的几年中，研究一个多样性集中于神经胶质细胞的相互作用中体内和体外 3。然而，大多数的培养系统的不允许这两种细胞类型的单独分析和各自secretomes的。
有几种方法利用神经元和神经胶质细胞的直接共培养，实现长期持久的生存与生理有关的神经网络的发展4-6。本协议达到同样的目标，同时保持物理上分离的7两种细胞类型。相比conditioneD培养基接近8,9，我们的系统可以让研究神经细胞和星形胶质细胞之间的双向通信。分泌的信号分子的表达可以在细胞中的共享介质maturate进行监测。这个机会是特别相关的，因为星形细胞释放可溶性因子，如细胞因子，生长因子和细胞外基质分子10,11，从而调节神经元生长和功能7,12。因此，已经证明，另外在体外血小板反应蛋白视网膜神经节细胞的诱导突触13的形成。然而，其他未知因素是必要的，以使突触功能13。此外，通过星形胶质细胞释放分子具有以了解神经胶质细胞的相互作用的基础上被识别。
从小鼠和大鼠原代神经元和星形胶质细胞的培养先前已14-16所述。在这里，我们提出了一种优雅和多功能的工具，这两种细胞类型中的一种间接共培养方法结合起来。由于两个培养物物理分离但共享相同的介质，神经元，星形细胞和可溶性分子的影响，可以分别进行分析，从而为神经胶质细胞的相互作用的研究的强有力的工具。
Protocol
小鼠实验是按照德国法律和德国社会为畜牧业的神经科学的指导方针。在波鸿鲁尔大学的动物保护与利用委员会授予相应的许可证。 1.准备和皮层星形胶质细胞的培养注：进行下一个步骤，有准备的神经元前的星形胶质细胞的文化应该发展成融合单层之前完成这些步骤，该协议至少7天的。原发性星形胶质细胞从各地日龄（P），0-3的乳鼠获得的混合胶质细?…
Representative Results
通过间接共培养系统中的神经元培养物的分析是多方面的，可以在培养的成熟的不同阶段进行。由于这样的事实，该细胞可被保持长达4周，培养物的长期的调查是可能的。 在图1中的中间左侧面板概略说明了共培养设置。与使用该系统，可以执行这两种细胞类型的活细胞成像，由星形胶质细胞单层（左上面板）和?…
Discussion
当前协议的主要目的是完全独立的神经元和星形细胞培养物，同时保持它们在共享介质。由于这个原因，所得到的培养物的纯度应在过程的开始进行验证。我们推荐使用神经元特异性微管蛋白，神经丝或蛋白质的NeuN神经元的标记，GFAP作为星形细胞标记，O4抗原少突胶质细胞前体标记，并Iba1蛋白识别小胶质细胞。
执行该协议的一个关键步骤的时候要特别注意，如由步骤说明之…
Declarações
The authors have nothing to disclose.
Acknowledgements
The present work was supported by the German research foundation (Deutsche Forschungsgemeinschaft DFG: GRK 736, Fa 159/22-1; the research school of the Ruhr University Bochum (GSC98/1) and the priority program SSP 1172 “Glia and Synapse”, Fa 159/11-1,2,3).
Materials
| Reagents | |||
| B27 | Gibco (Life Technologies) | 17504-044 | |
| Cell culture grade water | MilliQ | ||
| Cell culture grade water | MilliQ | ||
| Cytosine-ß-D arabinofuranoside (AraC) | Sigma-Aldrich | C1768 | CAUTION: H317, H361 |
| DMEM | Gibco (Life Technologies) | 41966-029 | |
| DNAse | Worthington | LS002007 | |
| Gentamycin | Sigma-Aldrich | G1397 | CAUTION: H317-334 |
| Glucose | Serva | 22700 | |
| HBSS | Gibco (Life Technologies) | 14170-088 | |
| HEPES | Gibco (Life Technologies) | 15630-056 | |
| Horse serum | Biochrom AG | S9135 | |
| L-Cysteine | Sigma-Aldrich | C-2529 | |
| MEM | Gibco (Life Technologies) | 31095-029 | |
| Ovalbumin | Sigma-Aldrich | A7641 | CAUTION: H334 |
| Papain | Worthington | 3126 | |
| PBS | self-made | ||
| Poly-D-lysine | Sigma-Aldrich | P0899 | |
| Poly-L-ornithine | Sigma-Aldrich | P3655 | |
| Sodium pyruvate | Sigma-Aldrich | S8636 | |
| Trypsin-EDTA | Gibco (Life Technologies) | 25300054 | |
| Equipment | |||
| 24 well plates | Thermoscientific/Nunc | 142475 | |
| 24-wells plate (for the indirect co-culture) | BD Falcon | 353504 | |
| Binocular | Leica | MZ6 | |
| Cell-culture inserts | BD Falcon | 353095 | |
| Centrifuge | Heraeus | Multifuge 3S-R | |
| Counting Chamber | Marienfeld | 650010 | |
| Forceps | FST Dumont (#5) | 11254-20 | |
| glass cover slips (12 mm) | Carl Roth (Menzel- Gläser) | P231.1 | |
| Incubator | Thermo Scientific | Heracell 240i | |
| Micro tube (2 ml) | Sarstedt | 72,691 | |
| Microscope | Leica | DMIL | |
| Millex Syringe-driven filter unit | Millipore | SLGV013SL | |
| Orbital shaker | New Brunswick Scientific | Innova 4000 | |
| Parafilm | Bemis | PM-996 | |
| Petri dishes (10 cm) | Sarstedt | 833,902 | |
| pipette (1 ml) | Gilson | Pipetman 1000 | |
| Sterile work bench | The Baker Company | Laminar Flow SterilGARD III | |
| Surgical scissors | FST Dumont | 14094-11 | |
| Syringe | Henry Schein | 9003016 | |
| T75 flask | Sarstedt | 833,911,002 | |
| tube (15 ml) | Sarstedt | 64,554,502 | |
| Water bath | GFL | Water bath type 1004 |
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Citar este artigo
Gottschling, C., Dzyubenko, E., Geissler, M., Faissner, A. The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions. J. Vis. Exp. (117), e54757, doi:10.3791/54757 (2016).