这项研究是FMMU新发现的基础和部分美国国立卫生研究院拨款的支持。

1。胶质细胞培养来自不同地区的中枢神经系统神经胶质细胞可以培养。整个过程是在过程图所示。 <img src="/files/ftp_upload/2111/2111process.jpg" alt="过程1" />第1天涂层培养板和盖玻片<ol><li>干燥灭菌的玻璃显微镜下一轮盖玻片高压灭菌器。 </li><li>板到灭菌的24孔培养板消毒盖玻片。 </li><li>外套与多聚赖氨酸和根的温度下2小时孵化的盖玻片。 </li><li>大衣6步骤3相同的方式培养板。 </li><li>两次洗净的盖玻片和6孔培养板，用蒸馏水和空气干燥文化引擎盖。 </li><li>在6孔板，每孔在24孔板和2毫升，每孔加200μLDMEM培养基（含10％胎牛血清）和37度下，将它们放入孵化器5 ％的CO 2 </li></ol>第2天的隔离皮层和神经胶质细胞培养<ol><li>消毒正流夹层罩。 </li><li>紫外线灯开启20分钟。 </li><li>喷雾用70％乙醇的所有表面和使用前等待15分钟。 </li><li>改变紧张:由低温麻醉幼鼠（P2 - P6），并在framen使用操作系统剪刀的万能基地斩首。 </li><li>打开头盖骨沿矢状使用虹膜剪和关闭颅骨剥离缝合。 </li><li>删除前脑和他们放进冷冻L15中等，体视显微镜下，仔细，清洁血管硬膜和软膜，隔离皮层和L15的中型洗几次。 </li><li>显微剪刀切成小块的皮质。 </li><li>添加0.125％胰蛋白酶EDTA制备成皮质件，并培育与L15的介质，在37度15分。 </li><li>组织块转移到20ML的DMEM培养基含20％FBS，于50ml试管中，脱氧核糖核酸酶原液加入终浓度为10ug/mL，吸组织解决方案，并与火抛光的玻璃Pasteure吸管的20倍，收集的单细胞悬浮液。 </li><li>洗细胞悬液一次DMEM培养基，重新暂停含10％胎牛血清的DMEM细胞，计数细胞与血球，种子细胞，在显微镜下5000-10000/cm 2 。细胞保持在37度5％的孵化器，改变介质的每周2次的一半。 </li></ol> 2。背根神经节神经元的分离，培养和纯化第1天准备文化的物质<ol><li>外套与多聚赖氨酸，洗盖玻片消毒盖玻片两次提取水和空气干燥，放入24孔板。 </li><li> 100μLNeurobasal培养基添加2％B27与2.5S神经生长因子（50毫微克/毫升），放入37度5％的CO 2板。 </li></ol>第2天，从胚胎分离的背根节<ol><li>消毒胶质细胞培养相同的方式流罩。 </li><li>过量的CO 2，由70％乙醇消毒腹部安乐死一个孕鼠（E15），胚胎从子宫中分离到L15的冷冻介质。 </li><li>隔离脊髓背根神经节在体视显微镜和转让下连接与L15的冷冻介质为35mm菜。 </li><li>收集单细胞悬液和用NBF培养基（Neurobasal培养基含2％B27与2.5S神经生长因子（50毫微克/毫升））。 </li></ol>第3天净化DRG神经元<ol><li> 18H - 24H，神经元的播种后，添加1毫米的集中FUDR股票解决方案的神经元文化的终浓度为20微米和200μL的最终体积。 </li><li> 72小时后，更换Neurobasal含2％B27和2.5S神经生长因子（50ng/mL）的培养基没有FUDR中期的一半。之后，改变培养基隔日。 </li></ol> 3。共培养背根神经节神经元与神经胶质细胞<ol><li>当神经胶质细胞培养达到合流（播种后20天左右），他们准备与神经元共培养。 </li><li> 24小时前加入纯化的神经元，神经胶质细胞的培养基改变Neurobasal培养基中含有2％B27与2.5S神经生长因子（50毫微克/毫升）。 </li><li>纯净的神经元是从文化的井，单细胞悬浮液获得通过火抛光Pasteure吸管机械传递。 </li><li>计数后，接种于融合的神经胶质细胞神经元在Neurobasal培养基含2％B27与2.5S神经生长因子（50ng/mL）500/cm 2 。 </li><li>神经细胞粘附和神经胶质细胞神经​​突起的生长，可以在不同时间点记录和图像分析软件和使用的细胞类型特异性抗体的免疫细胞化学分析。 </li></ol> 4。代表性的成果<ol><li> 胶质细胞培养:播种后，胶质细胞将成为融合为20天左右。在第一阶段对比显微OPE，这是很容易识别，不同形态的神经胶质细胞亚群形成了不同的增长模式的子结构和GFAP阳性星形胶质细胞占90％以上immuocytochemisty技术（图1，图2）所示。 </li><li> DRG神经元的文化 :在我们的方法，FUDR治疗72小时后，DRG神经元的纯度将达到6天之内高达99％高，DRG神经元表现出其独特的形态和高密度突起的生长（图3，图4）。 </li><li> 胶质细胞和神经元共培养 DRG细胞的粘附和轴突生长发生在播种后4小时内的神经胶质细胞容易，仔细观察发现，神经细胞的粘附和突起的生长是由神经胶质细胞亚群，形成了特殊的经济增长模式的子结构的影响，这可以很容易地确定相差显微镜下，免疫细胞化学法（图5，图6）。 </li></ol><img src="/files/ftp_upload/2111/2111fig1.jpg" alt="图1" /> 图1。融合的神经胶质细胞的形态。皮质神经胶质细胞，接种于多聚赖氨酸涂布盖玻片培养20天，请注意不同的经济增长模式的神经胶质细胞，细胞辐射的方式排列在左侧。 <img src="/files/ftp_upload/2111/2111fig2.jpg" alt="图2" /> 图2。聚合胶质细胞胶质纤维酸性蛋白（GFAP）抗体标记注意GFAP（红色），在右侧的阳性细胞的辐射安排。 <img src="/files/ftp_upload/2111/2111fig3.jpg" alt="图3" /> 图3。背根神经节神经元生长在体外无FUDR治疗 。污染的细胞形成背根神经节神经元"的背景。 <img src="/files/ftp_upload/2111/2111fig4.jpg" alt="图4" /> 图4。背根神经节神经元增长FUDR治疗后在体外 。背景污染的细胞已经完全消除。 <img src="/files/ftp_upload/2111/2111fig5.jpg" alt="图5" /> 图5。背根神经节神经胶质细胞生长的神经元的神经细胞粘附和突起的生长受到抑制，并在右侧的神经胶质细胞对辐射的细胞排列有限。 <img src="/files/ftp_upload/2111/2111fig6.jpg" alt="图6" /> 图6。背根神经节神经元的神经丝蛋白抗体标记的神经胶质细胞生长 。突起（绿色）是抑制辐射安排左侧的神经胶质细胞，并在右侧的限制，所有的神经胶质细胞标记GFAP抗体（红色）。

<ol>
	<li>Rossignol, S., Schwab, M., Schwartz, M., Fehlings, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+time+to+move?.">Spinal cord injury: time to move?.</a> J Neurosci. 27 (44), 11782-11792 (2007).</li><li>Yiu, G., He, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+inhibition+of+CNS+axon+regeneration.">Glial inhibition of CNS axon regeneration.</a> Nat Rev Neurosci. 7 (8), 617-627 (2006).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Matyash, V., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+astrocyte+morphology+and+physiology.">Heterogeneity in astrocyte morphology and physiology.</a> Brain Res Rev. , (2009).</li><li>Wanner, I. B., Deik, A., Torres, M., Rosendahl, A., Neary, J. T., Lemmon, V. P., Bixby, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+in+vitro+model+of+the+glial+scar+inhibits+axon+growth.+Glia.">A new in vitro model of the glial scar inhibits axon growth. Glia.</a> 56 (15), 1691-1709 (2008).</li><li>Kuffler, D. P., Sosa, I. J., Reyes, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+chondroitin+sulfate+proteoglycan+inhibits+dorsal+root+ganglion+neuron+neurite+outgrowth+and+substrate+specificity+via+a+soma+and+not+a+growth+cone+mechanism.">Schwann cell chondroitin sulfate proteoglycan inhibits dorsal root ganglion neuron neurite outgrowth and substrate specificity via a soma and not a growth cone mechanism.</a> J Neurosci Res. 87 (13), 2863-2871 (2009).</li><li>Kleitman, N., Wood, P. M., Bunge, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+method+for+the+study+of+myelination.">Tissue culture method for the study of myelination.</a> Culturing nerve cells. , P559-P559 (1998).</li></ol>

这个实验协议的目的是要达到两个目标，以研究神经胶质细胞，神经细胞粘附和突起的生长，特别是星形胶质细胞的异质性的影响。第一个目标是保持星形胶质细胞的异质性，尽可能在本实验中，胶质细胞的融合文化，丰富的星形胶质细胞混合小学文化，无任何化学处理和消化的传播，这可能会进一步破坏细胞，并诱导星形胶质细胞的损伤反应。最后的星形胶质细胞纯度为90％以上，GFAP阳性污染?...

<table><tbody><tr><td>DMEM(high glucose)</td><td>Invitrogen</td><td>10313-039</td><td></td></tr><tr><td>L15 medium</td><td>Invitrogen</td><td>11415-114</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10437-077</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Invitrogen</td><td>21103-049</td><td></td></tr><tr><td>poly-lysine</td><td>Sigma-Aldrich</td><td>P4832</td><td>culture grade</td></tr><tr><td>NGF(2.5S)</td><td>Invitrogen</td><td>13257-019</td><td></td></tr><tr><td>B27 supplyment</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>0.25% trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>FUDR</td><td>Sigma-Aldrich</td><td>F0503</td><td></td></tr><tr><td>neurofilament antibody</td><td>Abcam</td><td>ab24575</td><td></td></tr></tbody></table>

study glial cell heterogeneity influence on axon growth using a new coculture method

在中央的所有哺乳动物的中枢神经系统，切断损伤后的轴突无法再生到原来的目标和功能恢复是非常差的1。轴突再生的失败是几个因素，包括敌对的神经胶质细胞环境，抑制髓鞘相关分子和减少内在的神经元的再生能力2的综合作用的结果。星形胶质细胞是最突出的中枢神经系统的神经胶质细胞类型，并在轴突功能的生理和病理条件3下发挥重要作用。对比同源少突胶质细胞，星形胶质细胞是一个由不同的星形胶质细胞亚群具有不同的形态和基因表达的 4组成的异质细胞群。这种异质性的功能意义，如对轴突的生长影响，在很大程度上是未知的。 为了研究神经胶质细胞，尤其是在星形胶质细胞神经​​元行为的异质性的功能，我们建立了合作培养高纯度的背根与从大鼠皮层获得的神经胶质细胞神经​​节神经元的一种新方法。通过这项技术，我们能够直接比较不同的星形胶质细胞亚群在相同条件下神经细胞的粘附和轴突的生长。 在这份报告中，我们给这种方法的星形胶质细胞的分离和文化，背根神经节神经元的分离和提纯，和背根神经节神经元与星形胶质细胞共培养的详细协议。这种方法也可以扩展到其他脑区，研究细胞或地区的特定神经元和神经胶质细胞之间的相互作用。

Watch this Scientific Journal Video about Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method at JoVE.com

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

在这个协议中，我们描述了一个新的方法来研究与轴突生长的神经胶质细胞的异质性的影响<em&gt;在体外</em&gt;共培养体系。大鼠皮层神经胶质细胞培养汇合和高度纯化的大鼠背根神经节神经元共培养。在相同的文化，不同的神经胶质细胞对神经细胞的粘附和轴突的生长影响比较直接。这种方法提供了一种新的方式直接研究胶质细胞对神经细胞粘附和轴突生长的异质性影响。

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

study-glial-cell-heterogeneity-influence-on-axon-growth-using-new

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.2K 次观看.  Cedars Sinai Medical Center, UCLA. 在这个协议中，我们描述了一个新的方法来研究与轴突生长的神经胶质细胞的异质性的影响在体外共培养体系。大鼠皮层神经胶质细胞培养汇合和高度纯化的大鼠背根神经节神经元共培养。在相同的文化，不同的神经胶质细胞对神经细胞的粘附和轴突的生长影响比较直接。这种方法提供了一种新的方式直接研究胶质细胞对神经细胞粘附和轴突生长?...

视频: Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

二层共培养小学的大鼠皮层神经元和神经胶质

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

科研

神经科学

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

癌症研究

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

一个多室中枢神经系统的神经胶质细胞共培养的微流体平台

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

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 ...

发育生物学

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

背根神经节神经元和区分脂肪干细胞:一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

间接神经元的星形胶质细胞共培养试验:一个<em&gt;体外</em&gt;设置 - 为神经元的神经胶质细胞相互作用的详细调查

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

An Indirect Neuron-Astrocyte Co-Culture Technique for Studying Neuron-Glia Interactions

Source: Gottschling, C. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54757">The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions.</a>&#160;J. Vis. Exp.&#160;(2016)
This video illustrates a method for establishing an indirect neuron-astrocyte co-culture using a compartmentalized approach. In this setup, neurons and astrocytes are spatially separated but share a conditioned medium. Neurotrophic factors released by astrocytes promote neuron differentiation and the formation of synaptic connections, suggesting neuron-glia interactions.

一种用于研究神经元-胶质细胞相互作用的间接神经元-星形胶质细胞共培养技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

研究胶质细胞轴突的生长异质性的影响，使用一个新的共培养法

摘要

探索更多视频

此视频中的章节