接口3D工程神经细胞微电极阵列：一种创新<em>体外</em>实验模型
Summary
In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.
Abstract
Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 µm in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.
Introduction
体外二维（2D）神经耦合到微电极阵列（MEAs）的网络正像空通过研究神经元动力学和底层连接之间的相互作用黄金标准实验模型。在发展过程中，神经细胞再造的复杂网络，其显示效果也很好definedspatio，temporalpatternsof活性1,2（ 即阵阵，网络阵阵，随机扣球活动）。多边环境协定记录的电生理活动从许多网站（从几十到几千微电极），使表达的动态在网络层进行详细的调查。另外，使用解离的培养物的不可能性使得设计工程-网络。这是比较容易用这种方式来理解和所记录的电生理活动之间的功能关系样细胞密度3，模块化4,5的程度，异构神经流行的存在的网络组织的参数ulations 6等。然而，所有在体外研究上解离的培养细胞是基于二维的神经元网络。这种方法导致过分简单化相对于所述体内 （本质三维，三维）系统：（i）在二维模型中，胞体和生长锥变平及轴突-树突生长不能散布在所有方向7。 （二）2D 体外网络表现出刻板通过爆破涉及大部分的网络8的神经元的活动为主的电动力学。
最近，不同的解决方案已经开发了允许在体外构建3D解离的神经元网络。在共同的理念在于创造一个支架，其中神经元可以在一个三维的方式成长。这样的支架可以实现与聚合物凝胶和固体多孔基质9-13通过利用聚合物的机械性能，有可能以嵌入内采取这些细胞Ë结构定义神经球11的3D文化的统一块。这种方法的主要特征是神经球9,12的刚性的机械性能。然而，这些材料具有有限的孔隙率，并且他们不保证基质内细胞迁移。为了克服这个缺点，一种可能的解决方案包括在切片矩阵变成'单元'的模块。不幸的是，颗粒的大小和形状的多样性会妨碍包装成规则的层状结构。 7，Cullen和同事设计内的生物活性细胞外基质为基础的支架由神经元和/或星形胶质细胞的神经元的三维结构。这种工程化神经组织允许在体外的调查研究和操作中的3D微环境的神经生物学反应。这个模型包括神经元和神经胶质细胞在整个细胞外基质（ECM）和/或水凝胶支架（500-600微米厚）分布的。在这种合作ndition，最佳细胞存活率（大于90％），发现通过在约3750的最终密度电镀细胞- 5000 细胞 / mm3。必须指出的是，这样的密度值小于一个在体内条件下，其中小鼠大脑皮质的细胞密度为约90000个细胞/ mm 3的14低得多。为了克服这个限制Pautot和同事15实现其中的细胞密度和网络连接被控制在体内条件类似于同时使网络的实时成像一个三维的体外系统 。实际上，该方法是基于解离培养的神经元是能够生长在硅胶上微珠的概念。这些珠子提供生长表面的神经元胞体坚持和他们arborizations成长，成熟，扩展和定义突触联系到其他神经元足够大。这种方法利用了单分散珠自发组装性能FORM 3D分层含不同层，不同的珠子神经元之间的约束连接神经元的不同的子集六角形阵列。用这种方法所达到的细胞密度为约75000个/ mm 3。
最近，我们已适应Pautot的方法来多边环境16：所获得的结果表明，该三维电生理学活性呈现比所述一个二维网络表示活动更宽剧目。 3D成熟的文化表现出增强的动态，其中两个网络的突发和随机秒杀活动并存。同样，汤-Schomer和同事17实现可维持原代皮层体外培养数月丝蛋白质基多孔支架，并通过一个钨电极的装置记录的电生理活性。
在这项工作中，实验步骤建立联接到多边环境三维神经元网络进行说明。
Protocol
Representative Results
Discussion
在这项工作中，一个新的实验体外平台由三维设计的耦合到多边环境为网络电已提交的神经元培养物。使用微珠为支架，以允许沿 z轴的神经炎生长已针对与平面MEA被集成。以这种方式，所得到的微系统导致有效和可靠的体外三维模型来研究所述紧急电动力学16。
MEA记录的建立
MEA设备包括一个文化室的底物嵌入式微电极能够?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢乔治卡烈尼在制定限制结构和多特的技术支持。的Ornella LoBrutto对稿件的彻底的修改。研究导致这些结果已收到的资金来自欧盟的7 个框架计划（ICT-FET FP7 / 2007-2013年FET年轻探险家计划）下拨款协议284772脑弓（www.brainbowproject.eu）。
Materials
| Laminin | Sigma-Aldrich | L2020 | 0.05 µg/ml |
| Poly-D-lysine | Sigma-Aldrich | P6407 | 0.05 µg/ml |
| Trypsin | Gibco | 25050-014 | 0.125% diluted 1:2 in HBSS wo CA++, MG++ |
| DNAase | Sigma-Aldrich | D5025 | 0.05% diluted in Hanks solution |
| Neurobasal | Gibco Invitrogen | 21103049 | culture medium |
| B27 | Gibco Invitrogen | 17504044 | 2% medium supplent |
| Fetal bovine serum (FBS) | Sigma-Aldrich | F-2442 | 10% |
| Glutamax-I | Gibco | 35050038 | 0.5 mM |
| gentamicin | Sigma-Aldrich | G.1272 | 5mg/liter |
| Poly-Dimethyl-Siloxane (PDMS) | Corning Sigma | 481939 | curing agent and the polymer |
| Micro-Electrode Arrays | Multi Channel Systems (MCS) | 60MEA200/30-Ti-pr | MEA with: Electrode grid 8×8; Electrode spacing and diameter 200 and 30 µm, respectively; plastic ring without thread |
| Microbead | Distrilab-Duke Scientific | 9040 | 1gr Glass Part.Size Stds 40 µm |
| Transwell | Costar Sigma | CLS 3413 | multiwell plates with membrane insert (6.5 mm diameter porous 0.4 µm) |
| HBSS wo Ca++ ,Mg++ | Gibco Invitrogen | 14175-052 | |
| Hanks Buffer Solution | Sigma | H8264 | |
| Teflon | Sigma | 430935-5G | polytetrafluoroethylene |
| Rat | Sprague Dawley | Wistar Rat | |
| Confocal Microscopy Upright | Leica | TCS SP5 AOBS | |
| 20.0×0.50 WATER objective | Leica | Leica HCX APO L U-V-I | |
| 40.0×0.80 WATER objective | Leica | Leica HCX APO L U-V-I | |
| 25.0×0.95 WATER objective | Leica | HCX IRAPO L |
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