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）。

实验方案是按照DL一千九百九十二分之一百一十六和热那亚（普罗特大学的指导方针获得了欧洲动物保护立法（2010年/ 63 / EU），由卫生意大利外交部。N. 13130​​，五月2011）。所作的所有努力减少动物项目的数量，并尽量减少他们的痛苦。 1.准备材料，并支持<ol><li>构建模具由CNC（计算机数控）磨粉机的方式来构建PDMS（聚二甲基硅氧烷）的约束。这种模具被实现在聚碳酸酯与聚四氟乙烯中央筒（PTFE）。这种材料允许容易提取荫罩一旦与PDMS已硬化。 注:该模具的设计已经通过计算机辅助-设计（CAD）中进行，然后输送到数控铣床。 </li><li>准备PDMS弹性体。 PDMS是一种有机聚合物。它是由两个元件，固化剂和聚合物。通过1:10的体积比混合使用它们，一个部分固化剂，9份聚合物。把两种组分在培养皿，摇并插入真空室中的混合物10分钟，以消除气泡。 3克PDMS是足够的一个制约因素。 </li><li>构建PDMS约束。将PDMS材料进入成型机，并把它放入烤箱30分钟，在120℃。将PDMS约束具有理想缸筒用的22.0和3.0毫米，分别外部和内部的直径和650微米的高度的形状。 </li><li>电镀的前一天，夫妇由镊子的装置在立体显微镜下对掩模的微电极阵列（MEAs）中的有效区域和消毒的PDMS掩模在烘箱中在120℃。 </li><li>消毒玻璃微珠（40±2微米的标称直径; 42.3±1.1微米的认证平均直径）在70％乙醇处理2小时在锥形瓶中并旋转EVERY 30分钟，揭露所有的微珠。 </li><li>除去从管形瓶中的乙醇溶液和冲洗微珠两次用无菌水。 </li><li>调节所述MEA面积由PDMS掩模在0.05微克/毫升分隔（直径等于3.0毫米），24微升聚-D-赖氨酸层粘连蛋白和混合溶液的中心部分。 </li><li>外套与粘附蛋白，层粘连蛋白和聚-D-赖氨酸的微珠在0.05微克/毫升和离开他们过夜，在培养箱中在37℃，以获得稳定和持久的神经元网络。 </li><li>既从MEA的玻璃表面，并从该玻璃微珠除去粘附因子。抽吸用移液器涂布溶液的约95％，应用的无菌水的小体积 - 24μl-在MEA的表面。抽吸再次超过95％的水，让层流罩1小时下在MEA干镀细胞之前。 注意:在微珠的情况下，而不是，吸出涂层溶液并进行第一次洗涤用无菌水，而第二个与基础培养基和其补充如B27，以调节所述的玻璃表面，其中神经元将被电镀。不要让微珠干。其保留在悬浮液中的小瓶内培养基中。 </li><li>分发，通过吸管-200μl-，处理微珠的悬浮液（32000）的方式到多孔板与膜插入（孔径0.4微米），他们将自组装形成均匀的一层。 </li><li>填充膜的每个孔与0.5培养基中，并把它放在孵化器（T = 37.0℃，CO 2 = 5％），直到神经元是准备镀（3.2）。 </li></ol> 2.解剖胚胎和组织的解离<ol><li>房子成年雌性大鼠（200-250克）中的正则明暗时间表的恒定温度（22±1℃）和相对湿度（50％）（光7 AM-7 PM）中的动物设施。确保食物和水都是免费的。 </li><li>后用3％异氟醚18天发展（E18）麻醉怀孕的雌性大鼠。然后，通过颈椎脱位牺牲动物。 </li><li>从每个大鼠胚胎中取出海马并将其放置在冰冷的汉克的无钙 ，镁平衡盐溶液2+。在第18天发育海马和皮层组织的非常柔软，不需要被切成小块。进一步的细节可以发现18,19。 </li><li>离解在含有DNA酶的0.05％为18-20分钟胰蛋白酶/汉克溶液0.125％的组织，在37℃。 </li><li>除去用巴氏吸管将上清液溶液并用5分钟加入培养基，用10％胎牛血清（FBS）停止酶消化。 </li><li>用FBS除去介质并用与它的补充，1％L-谷氨酰胺，庆大霉素10μg/ ml的培养基洗一次。用巴斯德点子除去再次介质ETTE并再次用生长培养基少量（500微升）与它的补充，1％L-谷氨酰胺，庆大霉素10μg/ ml的填充它。 </li><li>具有窄巴斯德吸管机械解离组织粒料直到细胞的乳状悬浮液是显而易见的。它没有必要离心细胞悬浮液。 </li><li>稀释小体积的细胞悬浮液与生长培养基，得到2.0 ml的终体积。计数与血球室所得到的细胞浓度。为了得到600-700个细胞/微升的期望的细胞浓度为5:稀释该浓度为1。 </li></ol> 3.细胞电镀<ol><li>板的细胞以约2000个/ mm 2的密度到MEA的有源区域，由PDMS约束定义来创建2D神经网络。 注意:各海马包含约5×10 5个细胞中，（1×10 6，用于单个胚胎）。解剖6胚胎获得的6×10的总量6细胞在2ml，和3000个细胞/微升的估计浓度。为了获得所需的细胞浓度和板大约600-700个细胞/微升5:稀释该浓度为1。如果在MEA面积由PDMS约束分隔为约7.065毫米2，并铺板细胞总数600个细胞/微升×24微升= 14400细胞，2038个/ mm 2的最终密度。 </li><li>放置在MEA器件与湿润的 CO 2气氛（5％）培养箱，在37℃。 </li><li>分发160微升的悬浮液用600-700个细胞/微升（约100,000个细胞）的细胞浓度到微珠单层放置在多孔板内的表面，以完成三维培养的结构的初步步骤。把多孔板在培养箱加湿的 CO 2气氛（5％），在37℃。 </li></ol> 4. 3D神经元网络建设<ol><li> 6-8小时的电镀后，从多孔板非常仔细转移悬浮液（微珠与神经元的）分隔由PDMS约束通过使用移液管为约30-40微升的体积设置的区域内。每次传输后，等待半分钟左右，以使微球自组装的六边形结构紧凑。 </li><li>一旦所有的层被沉积并自发地聚集，填充用一大滴的约300微升培养基由PDMS约束限定的区域的顶部。 </li><li>把耦合到MEA的3D结构中的保育箱（T = 37.0℃，CO 2 = 5％）48小时加入生长培养基培养物的最终体积（约1毫升）与它的补充之前。 注意:考虑的珠子上的多孔板与膜插入件（30,000）和珠上的单一层上的MEA装置（6000）的数量的总数。将所得的三维结构是由5层英里的crobeads和细胞。考虑到所述三维神经网络不完美几何，所得到的三维结构可以由5-8层。 </li><li>在天铺板后，小心地添加培养基（约900微升）在MEA环内的最终体积。保持在湿润的 CO 2气氛（5％）的3D培养，在37℃下进行4-5周。更换每周一次培养基的一半。 </li></ol> 5.共聚焦显微镜采集<ol><li>固定的生物样品，以在支架的显微镜阶段。设置激光（氩，496-555纳米），扫描速度（400赫兹），在此获得PMT的图像，所述增益和偏移（-0.3％）为每个图像捕获正确的带宽发射滤波器，并为了以避免饱和，并提高信​​噪比。结果部分报告用于获取图4的图像值。 </li><li>对于此次收购的 Z -stack序列，冷杉圣选择，顶部和样品的底层的z位置的值，然后选择步长，使采集。 </li></ol>

<ol>
	<li>Van Pelt, J., Corner, M. A., Wolters, P. S., Rutten, W. L. C., Ramakers, G. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+and+developmental+changes+in+spontaneous+network+burst+firing+patterns+in+dissociated+rat+cerebral+cortex+cell+cultures+on+multi-electrode+arrays.">Long-term stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multi-electrode arrays.</a> Neurosci. Lett. 361, 86-89 (2004).</li><li>Rolston, J. D., Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precisely+timed+spatiotemporal+patterns+of+neural+activity+in+dissociated+cortical+cultures.">Precisely timed spatiotemporal patterns of neural activity in dissociated cortical cultures.</a> Neuroscienc. 148, 294-303 (2007).</li><li>Frey, U., Egert, U., Heer, F., Hafizovic, S., Hierlemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectronic+system+for+high-resolution+mapping+of+extracellular+electric+fields+applied+to+brain+slices.">Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices.</a> Biosens. Bioelectron. 24, 2191-2198 (2009).</li><li>Macis, E., Tedesco, M., Massobrio, P., Raiteri, R., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+microdrop+delivery+system+for+neuronal+network+patterning+on+microelectrode+arrays.">An automated microdrop delivery system for neuronal network patterning on microelectrode arrays.</a> J. Neurosci. Meth. 161, 88-95 (2007).</li><li>Kanagasabapathi, T. T., Ciliberti, D., Martinoia, S., Wadman, W. J., Decre, M. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+compartment+neurofluidic+system+for+electrophysiological+measurements+in+physically+segregated+and+functionally+connected+neuronal+cell+culture.">Dual compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture.</a> Front. Neuroeng. 4, (2011).</li><li>Kanagasabapathi, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+and+dynamics+of+cortical-thalamic+networks+co-cultured+in+a+dual+compartment+device.">Functional connectivity and dynamics of cortical-thalamic networks co-cultured in a dual compartment device.</a> J. Neural. Eng. 9, (2012).</li><li>Cullen, D. K., Wolf, J. A., Vernekar, V., Vukasinovic, J., LaPlaca, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering+and+biohybridized+microsystems+for+neurobiological+investigation+in+vitro+(Part+1).">Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (Part 1).</a> Crit. Rev. Biomed. Eng. 39, 201-240 (2011).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Bursting+in+Cortical+Cultures+with+Closed-Loop+Multi-Electrode+Stimulation.">Controlling Bursting in Cortical Cultures with Closed-Loop Multi-Electrode Stimulation.</a> J. Neurosci. 25, 680-688 (2005).</li><li>Shany, B., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+primary+hippocampal+neuronal+tissue+on+an+aragonite+crystalline+biomatrix.">Growth of primary hippocampal neuronal tissue on an aragonite crystalline biomatrix.</a> Tiss. Eng. 11, 585-596 (2005).</li><li>Schmidt, C. E., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering:+strategies+for+repair+and+regeneration.">Neural tissue engineering: strategies for repair and regeneration.</a> Ann. Rev. Biomed. Eng. 5, 293-347 (2003).</li><li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+stem+and+progenitor+cell+differentiation+into+functional+neuronal+circuits+in+three-dimensional+collagen+gels.">CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels.</a> Exp. Neurol. 190, 276-288 (2004).</li><li>Baranes, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interconnected+network+of+ganglion-like+neural+cell+spheres+formed+on+hydrozoan+skeleton.">Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.</a> Tissue En. 13, 473-482 (2007).</li><li>Lee, J., Cuddihy, M. J., Kotov, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+matrices:+state+of+the+art.">Three-dimensional cell culture matrices: state of the art.</a> Tissue engineering. Part B, Review. 14, 61-86 (2008).</li><li>Schuz, A., Palm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+of+neurons+and+synapses+in+the+cerebral+cortex+of+the+mouse.">Density of neurons and synapses in the cerebral cortex of the mouse.</a> J. Comp. Neurol. 286, 442-455 (1989).</li><li>Pautot, S., Wyart, C., Isacoff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloid-guided+assembly+of+oriented+3D+neuronal+networks.">Colloid-guided assembly of oriented 3D neuronal networks.</a> Nat. Method. 5, 735-740 (2008).</li><li>Frega, M., Tedesco, M., Massobrio, P., Pesce, M., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+dynamics+of+3D+engineered+neuronal+cultures:+a+new+experimental+model+for+in-vitro+electrophysiology.">Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology.</a> Sci. Rep. 4, (2014).</li><li>Tang-Schomer, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+functional+brain-like+cortical+tissue.">Bioengineered functional brain-like cortical tissue.</a> Proc Natl Acad Sci U S. 111, 13811-13816 (2014).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cell. , (1998).</li><li>Hales, C. M., Rolston, J. D., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+culture,+record+and+stimulate+neuronal+networks+on+Micro-electrode+arrays+(MEAs).">How to culture, record and stimulate neuronal networks on Micro-electrode arrays (MEAs).</a> J. Vis. Exp. (39), (2010).</li><li>Jayakumar, R., Prabaharan, M., Nair, S. V., Tamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+chitin+and+chitosan+nanofibers+in+biomedical+applications.">Novel chitin and chitosan nanofibers in biomedical applications.</a> Biotech. Adv. 28, 142-150 (2010).</li><li>Crompton, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polylysine-functionalised+thermoresponsive+chitosan+hydrogel+for+neural+tissue+engineering.">Polylysine-functionalised thermoresponsive chitosan hydrogel for neural tissue engineering.</a> Biomaterial. 28, 441-449 (2007).</li><li>Frega, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+engineered+neural+networks+coupled+to+Micro-Electrode+Arrays:+Development+of+an+innovative+in-vitro+experimental+model+for+neurophysiological+studies.">3D engineered neural networks coupled to Micro-Electrode Arrays: Development of an innovative in-vitro experimental model for neurophysiological studies.</a> , 957-960 (2013).</li></ol>

The authors declare that they have no competing financial interests.

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从电活性组织测量细胞外信号的集成阵列中。典型的记录的信号由尖峰（即单超阈值电压变化代表一个或多个神经元的电活动）和脉冲串（ 即，高度填充尖峰序列常常occurrin的克同时在几个通道）。神经元网络活动与MEA为基础的系统的记录产生大量的数据 （例如，一个4小时的实验具有60个通道MEA产生约15 GB的记录）的。这意味着需要有高效率的工具来处理和分析数据，特别是在需要长期的研究记录的情况下。在一般情况下，是由一直接外转导与神经元培养物，并采集的电压信号而获得的电生理信号的记录。后的扩增和滤波级，信号采样10-50千赫和存储。原始信号然后峰值检测由定制开发的软件工具。记录系统包括:（1）MEA放大器，（2）个人计算机配有A / D采集板，（3）记录的软件，（D）的加热系统和温度控制器，（4）CO 2气氛波保持和蒸发防止系统。 该三维神经网络的建设经过两个关键步骤:第一个是对多孔板与膜插入件表面珠粒的正确量的分布，第二个是微珠与来自多孔粘附神经元的悬浮液的传递板的膜插入到:（i）所述2D神经网络镀到MEA（第一层）的有源区; （二）已经沉淀层。这种操作应重复多次，需要的层数。在培养的第一个星期，神经炎扩展快速增长需要大约属于在不同层中的微珠的地方。神经元和微珠之间的密切联系稳定生物样品，并允许从孵化器移动以改变/更换培养基并无需担心破坏它与MEA放大器来记录电活动。 由于全网的电生理活动工作只从底层（即，一个直接耦合到MEA）中，活性区域必须覆盖通过常规的2D网络记录。其它层将被添加步步在2D 1，和远程连接将这些层之间传播。实际上，其它层是由罢免神经元和微珠的混合物中加入6-8小时后比2D之一。此时间窗的选择允许的2D层中的几个突触连接的形成，并保证建立连接到上层太的可能性。 两个因素使用多孔板的与膜插入必要实现三维网络:（ⅰ）因为神经元和微珠本不同沉淀速度，使用多孔板的与膜插入，保证的神经元上的微珠的顶部均匀分布:平均每个微珠可以由神经元的相同数目的包围。 （二）乙ý利用该多孔板与膜插入件的弹性性质，微珠耦合到神经元的溶液的反冲洗比使用固体表面如聚碳酸酯多井更容易。 所呈现的工作如下提出Pautot的方法和同事15:在该研究中使用的硅球提供了一种生长表面为神经元细胞体粘附足够大并为他们的过程生长，成熟和产生前和突触后特。一个有效的替代方案的使用珠子支架来自水凝胶基质或生物活性和由天然生物​​聚合物像壳聚糖生物可降解的珠。几丁质和脱乙酰其衍生物，脱乙酰壳多糖，是无毒的，抗菌的，生物相容和生物可降解的生物聚合物。这种生物降解过程的时间常数是与网络长大20,21兼容。 这项研究可能构成一个参考WORK代表了先进的3D神经元模型系统的未来发展有系统地研究网络动态与结构之间的相互作用。在很短的时间，我们希望能够转移三维网络结构的新一代设备22。的制造技术包括生产中的微柱在50至350微米的高度的变量的形式的电极。

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&#39;单元&#39;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。

<table><tbody><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>0.05 &amp;micro;g/ml</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>0.05 &amp;micro;g/ml</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25050-014</td><td>0.125% diluted 1:2 in HBSS w\o CA&lt;sup&gt;++&lt;/sup&gt;, MG&lt;sup&gt;++&lt;/sup&gt;</td></tr><tr><td>DNAase</td><td>Sigma-Aldrich</td><td>D5025</td><td>0.05% diluted&amp;nbsp; in Hanks solution</td></tr><tr><td>Neurobasal</td><td>Gibco Invitrogen</td><td>21103049</td><td>culture medium</td></tr><tr><td>B27</td><td>Gibco Invitrogen</td><td>17504044</td><td>2% medium supplent</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F-2442</td><td>10%</td></tr><tr><td>Glutamax-I&amp;nbsp;</td><td>Gibco</td><td>35050038</td><td>0.5 mM</td></tr><tr><td>gentamicin</td><td>Sigma-Aldrich</td><td>G.1272</td><td>5 mg/L</td></tr><tr><td>Poly-Dimethyl-Siloxane (PDMS)</td><td>Corning Sigma</td><td>481939</td><td>curing agent and the polymer</td></tr><tr><td>Micro-Electrode Arrays</td><td>Multi Channel Systems (MCS)</td><td>60MEA200/30-Ti-pr</td><td>MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 &amp;micro;m, respectively; plastic ring without thread</td></tr><tr><td>Microbead</td><td>Distrilab-Duke Scientific</td><td>9040</td><td>&amp;nbsp;1 gr Glass Part. Size Stds 40 &amp;micro;m</td></tr><tr><td>Transwell</td><td>Costar Sigma</td><td>CLS 3413</td><td>multiwell plates with membrane insert (6.5 mm diameter porous 0.4 &amp;micro;m)</td></tr><tr><td>HBSS w\o Ca&lt;sup&gt;++&lt;/sup&gt; ,Mg&lt;sup&gt;++&lt;/sup&gt;&amp;nbsp;</td><td>Gibco Invitrogen</td><td>14175-052</td><td></td></tr><tr><td>Hanks Buffer Solution&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>H8264</td><td></td></tr><tr><td>Teflon</td><td>Sigma</td><td>430935-5G</td><td>polytetrafluoroethylene</td></tr><tr><td>Rat</td><td>Sprague Dawley</td><td>Wistar Rat</td><td></td></tr><tr><td>Confocal Microscopy Upright</td><td>Leica</td><td>TCS SP5 AOBS</td><td></td></tr><tr><td>20.0x0.50 WATER objective</td><td>Leica</td><td>Leica HCX APO L U-V-I&amp;nbsp;</td><td></td></tr><tr><td>40.0x0.80 WATER objective</td><td>Leica</td><td>Leica HCX APO L U-V-I&amp;nbsp;</td><td></td></tr><tr><td>25.0x0.95 WATER objective</td><td>Leica</td><td>HCX IRAPO L</td><td></td></tr></tbody></table>

interfacing 3d engineered neuronal cultures to micro-electrode arrays: an innovative in vitro experimental model

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 &#181;m 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.

在此实验过程中，相应的作用是通过定义机械支架的三维神经网络的生长微珠起到图1A和B显示了微珠（排列的40±2微米的标称直径;认证平均直径42.3±1.1微米），在平面中。这种结构的特点是，微珠自发自组装限定紧凑六角形形状（图1B 和C）。如此生成的支架保证了持续增长的表面突起和胞体的代谢交换足够大的空隙空间。这种微珠的尺寸被证明是一个合理的妥协间质性间距和最终神经元密度。考虑到PDMS结构再现一个"理想的"气缸，并且由于紧凑六方结构的影响因子（HPF）等于0.74，珠粒的最大数量（直径4081;米）含有PDMS约束内是约100,000，其对应于每每层约6000微珠。正如在协议部分所解释的，这些微珠是预调节与粘附因子（即，层粘连蛋白和聚-D-赖氨酸）。这个过程保证了神经元坚持微珠。 图1D示出了其中3神经元被耦合到一个直径为40微米的微珠的一个例子。 <img alt="图1" src="/files/ftp_upload/53080/53080fig1.jpg" /> 图用于创建支架的神经网络的微珠1.图像（A，B）一个多孔板与膜插入件的表面上的微珠单层的两个例子。在重力作用下安定（C）的微球和自发地组装成二维六角形阵列。一旦第一层被完全填满，其它珠相加，建立荷兰国际集团具有相同的六边形对称性的第二有序层。另外除了微珠导致包装的3D组件的建设。微珠之间的自由空间是由神经元突起和胞体填充的。（D）三个神经元是固定单一微珠表面上，以前与粘附因子（层粘连蛋白和聚维赖氨酸）空调。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53080/53080fig1large.jpg" target="_blank">请点击这里查看一个更大的版本这个数字。</a> 微珠和神经元与MEA的有源区的接口是由于弹性体约束（图2C），实现了与图 2A 和B中所描绘的模具。 图2A示出的布局和所选择的尺寸。 MEA的有效区域是通过将图2C的 PDMS结构与MEA衬底（ 图2D分隔）。以这种方式，神经元被强制坚持MEA的有效电极面积。随后，将玻璃微珠 ​​堆叠粘连的神经元将被安置在该第一层上。 图2D示出了最终的结构中，其中PDMS结构的作用，可以清楚地理解的一个横截面。通过PDMS结构限定的空间中含有微珠和神经元的混合物（白色粉末在图2D的底部面板）。 PDMS结构具有内置与描绘在图2B中具有以下尺寸的模具中的圆筒的形状:22.0外部和内部的直径和3.0的分别毫米和650微米的高度。模具是用聚碳酸酯和PTFE其中成为一个理想的光滑，耐热材料，以促进弹性体mask.The PDMS约束显示寿命更短的提取建，由于每次使用后的PDMS降低粘性性质。通常情况下<html>中，每个PDMS结构可以成功地使用三次。 <img alt="图2" src="/files/ftp_upload/53080/53080fig2.jpg" /> 图2.材料构建3D神经元网络。 （A）的设计与（B）的图像来创建物理约束的模具。灰色3毫米直径筒对应于（C）中所描绘的约束。（C）的 PDMS约束置于微阵列电极（MEA）的有源区的孔。使用这样的物理屏障允许含有的目标区域内的微珠（约7mm 2）并限制所述三维网络的发展。（D）在MEA系统和约束的横截面。微珠（由在右上角的显微镜示出）被填充到由PDMS约束（底部面板）限定的空间。080fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 MEA的有效面积的由PDMS掩模手段的限制后，协议继续如在图3的面板所描绘的，描述的主要步骤来实现的神经元和微珠的组装。 第一预备步骤包括电镀解离的神经元上的MEA表面这是以前的预涂覆有粘附分子层粘连蛋白和聚-D-赖氨酸（ 图3A）。至关重要的是，该二维神经网络直接附着到MEA的有效面积:只有在这些条件下，电生理信号（即，峰值和脉冲串）的记录是可行的。该协议的这个第一部分对应于用于板2d的神经元的标准程序。三维系统的实际结构在图3B，C和D被描绘</strong&gt;。首先，微珠被定位成多孔板与膜插入载体（图3B）;之后，一个160微升悬浮液用600-700个细胞/微升（约100,000个细胞）的细胞浓度分配在微珠单层（ 图3C）。考虑到细胞可以占据一半胎圈区域，总自由表面为75毫米2。对微珠单层细胞密度为约1500个/ mm 2。所利用的多孔板与膜插入件呈现多孔膜具有33.6毫米2的表面上。通过考虑一个虚拟圆柱的形状，其底面积等于所述多孔膜表面至40μm（珠直径）的高度，30000微珠构成的均匀的层结合到多孔板与膜的插入。微珠和神经元的，以便实现悬架撑杆的多孔板与膜插入物内约6-8小时。一度完成上述步骤后，三维网络的结构可以启动。此时，神经元和微珠的混合物从多孔板与膜的插入取出，置于预先镀到由PDMS约束（图3D）定义的区域中的神经元的2D网络。在此过程中，发生的约20-30％的神经元的损失，由于（ⅰ）机械操作和（ii）向膜外转移。通过考虑约束（7.06毫米2）和178.56微米（5层的珠粒）的高度的基区，和分裂细胞的数量通过由5层的珠粒所形成的理想的圆柱体的体积，所得到的三维神经网络的细胞密度约80000个/ 立方毫米。 <img alt="图3" src="/files/ftp_upload/53080/53080fig3.jpg" /> 图3.素描的基本步骤来创建2D和3D的神经网络。 （一）电镀Ø˚F神经元上的MEA的有效面积。神经元的悬浮液铺板于在MEA表面，预先调节与粘附因子和由PDMS约束分隔。此步骤可以实现:一）一个经典的2D神经网络连接到MEA; ii）一种三维网络从该电生理活动将被记录的第一层（B）的微珠和 （C）的神经元6-8小时后输送到多孔板与膜插入件的表面上。（D）的悬浮液神经元和微珠从多孔板与膜插入到2D网络移动。此步骤几次重复允许定义一个致密的多层三维结构。（E）当三维结构完成后，一滴培养基加入，将得到的结构被存储在培养箱中48小时。（F ）48小时后，在3D培养完全与介质的最终体积再填充。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53080/53080fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 一旦第一层已被定位，则重复相同的操作，以获得一个压缩三维合奏。所得三维结构重组本身限定了胶体晶体是自我装配在一个结构紧凑由约5-8层微珠和神经元（ 图4）。一旦所有的层形成，该三维网络必须用一滴加入300μl培养基（图3E）被重新填充。在此阶段，其耦合到多边环境三维网络可以存储在孵化48小时。在此之后，将1ml培养基加入到填充的MEA（图3F）的环。 一旦所有的层已经被组装时，突起增长，在微珠支架到达最终细胞约80,000个细胞/ mm 3的密度。值得诺蒂奇荷兰国际集团所获得的价值是不远处的92000 个 / mm3，平均神经元密度在小鼠大脑皮质14。 图4显示连接到共聚焦显微镜的方式直立拍摄的多边环境协定3D网络的三个例子。在图4A中，DIC（微分干涉对比度）图像显示了珠粒耦合到MEA表面的单层;在显微镜加上20.0 NA 0.50水的目标并获得了图像传输的PMT包括显微镜和激光氩（488nm处）。该图片显示得很清楚，电极的空间布局（黑点）和微珠的定期空间组织。 在图4B中，免疫荧光染色图-2（红色信号）显示树突arborizations的周围直接耦合到ME的电极平面微珠的神经元的分布和胞体甲（黑接触的存在可观察到的）。 图4B是与步长一个Z-堆栈序列（108微米）的最大投影等于1.53微米。图像被收购40.0 NA 0.80水的目标;激励是氩激光（488纳米），发射带宽496nm-等于655nm。 最后 ，图4C显示一个三维海马培养的 Z -stack序列（187.79微米）的最大凸起（步长为2.27微米）。 在 Z -stack序列获得25.0 NA 0.95水的目标。对于绿色信道，一个氩激光488nm处和发射滤波器带宽499nm-551nm被使用。对于红色信道，543 nm激光和发射滤波器带宽为555nm-645nm被使用。这些通道是在连拍模式下获得的。一个高度互联的网络出现在这里的神经元被标记为的NeuN表达成熟的有丝分裂后的神经元（红色信号）和加巴（绿色信号，YEL低合并）。加巴抗体透露了一个积极的抑制性神经元无论是在胞体和突起。 <img alt="图4" src="/files/ftp_upload/53080/53080fig4.jpg" /> 图4.激光共聚焦显微镜拍摄的3D网络的三个例子。 （A）中的DIC（微分干涉对比度）的图象，显示的珠粒耦合到MEA表面的单层;电极（黑点）的空间布局是可观察到的。（B）的免疫荧光染色为图2（红色信号）显示树突arborizations的分布和胞体周围直接联接到MEA的电极平面微珠的神经元。（ C）三维文化显示一个高度互联的网络中的神经元被标记为的NeuN 的 Z -stack序列（187.79微米）的最大突出表现在成熟的有丝分裂后的神经元（红色信号）和加巴（绿色信号，黄色合并）。加巴抗体转ealed一个积极的无论是在胞体和突起抑制性神经元。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53080/53080fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 理解一个三维结构的存在是否调节网络动态，所以产生的电生理活动进行比较生长在多边环境常规的2D神经网络，它代表参考模型。 <img alt="图5" src="/files/ftp_upload/53080/53080fig5.jpg" /> 图5.由三维（左列）和二维（右栏）海马培养显示出的网络动态之间的比较在每个时间尺度（A:1200秒以下 ，B:300秒; C:60秒; D:10秒）。， 3D组件显示各种活动的签名（ 即短期和长期的网络爆发，随机扣球活动中，同步突发），而那些2D显示，只有高同步爆发更明显的刻板的活动。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53080/53080fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图5显示了在第四周体外在3D（左小图）和二维（右图）海马培养进行两个代表性实验2光栅地块。这些图显示记录在所有有源电极网络的电活动性（ 即 ，具有的燃烧率大于0.1尖峰/ s的电极）。 二维网络的动态显示，主要的网络的脉冲串组成的准同步活性。这个充满活力的签名是同步的事件涉及最受期间穿插录音通道，可以在几乎没有任何活动记录的存在（见右侧面板图5D）。这种行为可以理解，在不同的时间尺度，为不同层次的放大倍率下划线。 在一个三维网络的情况下，网络动力学的签名由活动更宽剧目的特征在于:（ⅰ）小于全球同步，（ⅱ）更多的随机尖峰活性，（ⅲ）期间，其中的子网络（即，微电极的子集）呈现与网络突发的发生更同步活动。此外，网络的脉冲串的持续时间是更变量:有网络突发与一个类似于在2D网络观察的持续时间的存在，但也有更长的网络突发。通过使用这种方法获得的紧急三维动力学的完整和定量表征可以 16找到。正如预期的那样，在3D神经元网络的展品也有显著"随机扣球"和非同步爆破一ctivity。由这些三维结构所产生的动力学的可信度已校对与一些控制实验（例如，耦合到一个MEA用栈裸微珠和三维网络组装到微珠与裸MEA的二维网络的存在下），作为报道16。

Watch this Scientific Journal Video about Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model at JoVE.com

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

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.

接口3D工程神经细胞微电极阵列:一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

interfacing-3d-engineered-neuronal-cultures-to-micro-electrode-arrays

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

10.0K 次观看.  University of Genova. 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.

视频: Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

科研

发育生物学

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

如何以文化，记录和刺激神经网络的微电极阵列（多边环境协定）

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

生物学

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

神经科学

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

螺旋神经节神经元外植体培养及电多电极阵列

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

生物工程

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

iPSC 来源的神经元和星形胶质细胞的 3D 生物打印共培养物

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

作者聚焦:推进 3D 细胞建模 – 一种用于神经共培养的高通量方法 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

接口3D工程神经细胞微电极阵列:一种创新

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诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

如何以文化，记录和刺激神经网络的微电极阵列（多边环境协定）

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

螺旋神经节神经元外植体培养及电多电极阵列

微电极阵列接口微流体研究神经元通信和轴向信号传播

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用

人 iPSC 衍生的神经元-星形胶质细胞共培养物的三维生物打印用于筛选应用