该议定书得到了大韩民国科学、信息和通信技术和未来规划部赠款 NRF-2015R1A2A1A01003410 的支持。

所有涉及动物的实验都得到了 Ajou 大学医学院动物保育和使用委员会的批准。
1. 游离成人背根神经节神经元的培养制备
<ol><li>在建立培养之前, 先用聚 d-赖氨酸和层粘连蛋白对6井板进行预涂。在37°c 孵化一个6井板, 0.01% 聚 d-赖氨酸, 在2小时或在4°c 上过夜。然后, 用蒸馏水洗两次盘子。</li>
	<li>在室温下, 用层粘连蛋白溶液在3µg/毫升浓度下孵育板材, 然后用蒸馏水冲洗两次盘子。至少在室温下将板材干燥1小时。</li>
	<li>弄死与压缩 co2气瓶相连的透明 co2腔中的鼠标。用手术刀片切开椎柱上的皮肤, 并通过双边解剖旁的肌肉以暴露椎体的骨骼。使用狭窄的外科钳, 在 DRGs 完全暴露之前, 小心切除椎体骨骼。 注: 成年 DRGs 神经元由成年 C57BL6 雄性小鼠, 年龄介于8周至12周之间。</li>
	<li>使用虹膜剪刀和细尖钳在解剖显微镜下, 从 S1 一直到 C1 水平 DRGs。尽量从 DRGs 中切下根部, 以尽量减少雪旺细胞或成纤维细胞的污染。</li>
	<li>将解剖过的 DRGs 保存在60毫米培养皿中, 其中5毫升冷 Dulbecco 的修饰鹰培养基 (DMEM) 在冰上, 直到所有的 DRGs 都被收集。 注意: 尽快解剖 DRGs 是至关重要的。所有 DRGs 从一只老鼠的收集不应超过30分钟30分钟后, 安乐死, 停止收集 DRGs, 并继续下一步, 即使仍然有剩余的 DRGs。</li>
	<li>使用蓝色吸管尖端 (1000 µL) 将 DRGs 转移到1.5 毫升离心管, 并切断末端。使用微型离心机, 在快速旋转几秒钟后, 取出 DMEM。添加1毫升的 DMEM 含有 125 U/毫升类型的西胶原酶和孵化管90分钟以温和的旋转 (35-40 rpm) 使用扭振器在37°c 孵化器。用胶带固定在振动筛地板上的管子。</li>
	<li>放弃含胶原酶的 DMEM, 加入1毫升新鲜 DMEM。等待, 直到漂浮的 DRGs 完全下沉到底部, 然后删除 DMEM 与组织碎片。重复这个洗涤步骤至少六次。 注意: 不要试图完全丢弃上清。小心不要删除任何 DRGs 与上清 DMEM。</li>
	<li>使用蓝色吸管尖端 (1000 µL) 将 DRGs 转移到15毫升圆锥管, 并切断末端。然后用蓝尖 (1000 µL) 轻轻地向上和向下吸15次, 使均匀的细胞悬浮。避免制造气泡, 尽量不要用吸管尖触碰锥形管的底部。</li>
	<li>离心管在 239 x g 3 分钟, 并小心地丢弃上清上漂浮的碎片。添加1毫升的 Neurobasal 培养基, 辅以 B27 (2.0% 伏/v), 并并用重悬细胞颗粒轻轻吹打向上和向下5到10倍。</li>
	<li>通过 70-µm 细胞过滤器覆盖在50毫升圆锥管顶部的细胞悬浮。等待2分钟, 然后用吸管控制器将 Neurobasal/B-27 介质倒入过滤器上。</li>
	<li>板所有收集的背根神经节神经元 (总共约 2 x 106背节神经元从一只老鼠) 到两个井的6井板。一只成年小鼠的背根神经节在6井板上覆盖两口井。然后将该板块放置在37°c 的 co2孵化器中, 直到巨噬细胞共培养。</li>
</ol>2. P 原发性腹腔巨噬细胞的共培养
注: 建立离型背根神经节神经元初始电镀后4小时的共培养
<ol><li>原发腹腔巨噬细胞由成年 C57BL6 雄性小鼠在8周至12周龄之间制备。弄死在 CO2腔中的动物。细致切开腹部皮肤, 暴露腹膜, 避免切开腹膜, 防止灌洗液渗漏。</li>
	<li>用一根22G 针的注射器刺穿腹膜, 将10毫升的冰冻磷酸盐缓冲 salin (PBS) 注入腹腔。轻轻按摩腹膜1-2 分钟。然后, 拔出针头, 通过穿刺部位挤出 PBS, 在50毫升锥形管中收集灌洗液。 注: 使用前, 把注射器放在冰上, 以保持 PBS 的寒冷。</li>
	<li>离心冲洗液在 239 x g 10 分钟在4°c, 以颗粒的细胞成分。并用重悬3毫升的红细胞 (红细胞) 裂解缓冲液在室温下为3分钟。然后离心细胞悬浮在 239 x g 10 分钟在4°c。并用重悬颗粒与3毫升 PBS 和离心机细胞悬浮再次删除任何剩余的红细胞溶解缓冲。 注意: 确保红细胞溶解缓冲液完全清除。否则剩余的红细胞溶解缓冲液可能对培养的巨噬细胞有毒。</li>
	<li>并用重悬1毫升 Neurobasal/B-27 培养基中的颗粒细胞。所有收集到的巨噬细胞的一半 (总共 3 x 106到 6 x 106单元格) 在单元格区域性插入与有效面积 4.2 cm2, 它被放置在分离的背根的井的顶部。 注: 在共培养期, 巨噬细胞生长在 Neurobasal/B-27 神经元培养基中。在这种情况下证实了巨噬细胞的充分存活。</li>
</ol>3. 治疗 Db 营和收集巨噬细胞 CM
注: 在神经元-巨噬细胞联合培养后开始4小时的 db 营治疗。
<ol><li>添加2µL 100 µM db 阵营解决方案的神经元-巨噬细胞共培养。为控制实验添加相同体积的 PBS。</li>
	<li>24小时后, 用1毫升的巨噬细胞培养培养基在同一6井板中填充空井。用巨噬细胞培养培养基将神经元-巨噬细胞共培养物中的胞培养物转移到空井。保持细胞在相同的条件下72小时, 而不改变培养基。 注: 在 cm 收集过程中, 我们只添加1毫升的巨噬细胞培养基, 以使浓缩厘米。在 CM 收集, 如果需要, 我们增加了更多的完全覆盖在插入的巨噬细胞。</li>
	<li>在 72 h 以后, 离心机巨噬细胞 CM 在 239 x g 为5分钟去除细胞组分。通过0.2 µm 过滤器将上清液移除其余的细胞碎片。将收集的 CM 保存在-70 摄氏度, 直到使用。</li>
</ol>4. 用收集到的 CM 突起生长试验
<ol><li>在建立一个单独的成人背根神经节神经元培养之前, 在1.1 和1.2 中所描述的相同的过程中, 预先涂上8井室滑块。</li>
	<li>用从1.3 到1.10 的相同方法获得 Neurobasal 培养基中游离的成人背根神经节神经元, 辅以 B27。板 5 x 104单元格, 每个井到预涂8井室滑动。</li>
	<li>放置在37°c 孵化器2小时的房间幻灯片, 允许细胞附着在底部。然后, 用37摄氏度预热的解冻厘米替换培养基。 注意: 移除培养基并添加收集的 CM 时, 小心不要触及8井室滑动的底部。</li>
	<li>15 h 在初始电镀后, 取出培养基, 用 PBS 清洗细胞一次。然后, 加入200µL 冰冷4% 多聚甲醛溶液的水井, 并孵化细胞与多聚甲醛溶液20分钟在4°c 注: 突起的根背节神经元培养应精确限制为15小时。在这种情况下, 没有可观的突起。</li>
	<li>用冷的 PBS 洗三次, 然后用10% 正常的山羊血清 (anti-Tuj-1) 与0.1% 的海卫 x 30 分钟进行阻断. 孵育固定细胞, 并以 10% (浓度为1µg/毫升) 稀释 4 h 在房间内温度或隔夜在4°c。</li>
	<li>用 PBS 冲洗三次, 然后在室温下用二级抗体 (山羊-抗小鼠) 溶液孵化细胞, 2 小时。然后, 用 PBS 清洗两次, 并使用安装解决方案安装盖玻片 (24 x 50 mm) 的文化幻灯片。</li>
	<li>使用荧光显微镜拍摄图像, 以可视化突起的产物。</li>
</ol>

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作者没有什么可透露的。

这一共同文化体系的产生有几个关键步骤。重要的是要确保小鼠背节神经元和腹腔巨噬细胞的准备新鲜和健康。当所有的 DRGs 的解剖都超过30分钟时, 我们经历了突起的 CM 生长活动。此外, 腹腔巨噬细胞中血液成分的污染也导致了突起在 CM 中的生长活动减少。为了通过 db 阵营诱发强健的神经元巨噬细胞相互作用, 彻底冲洗也将是确保完全清除组织碎片和消除可能残留的细胞毒成分, 如胶原酶或红细...

在脊髓或脑损伤后, 各种研究试图增强中枢神经轴突再生。炎症反应, 不可避免地伴随着神经系统的损伤, 传统上被认为参与继发病理过程导致有害结果1,2。事实上, 能抑制炎症反应的甲基强的甲泼尼松龙是唯一经批准的治疗急性脊髓损伤的方法3。然而, 最近的研究表明, 有代表性的炎症细胞类型的巨噬细胞可以参与再生或修复受损神经系统的4,5,6。例如, 在晶状体损伤后浸润巨噬细胞产生支持再生分子, 促进视网膜神经节神经元的再生7,8。此外, 移植的背根神经节神经元增加轴突生长的区域, 巨噬细胞由酵母多糖9激活.此外, 病变部位的巨噬细胞可以为受损的周围神经创造一个生长许可的环境, 如10。
我们的研究也提供了强有力的证据, 证明巨噬细胞能促进相邻神经元轴突再生的能力。我们已经表明, 在背根神经节中的巨噬细胞活化是重要的, 以增强再生能力的节神经的感觉神经元后, 预适应周围神经系统损伤11。另一实验室12独立报告了类似的研究。我们还表明, intraganglionic 注射 dibutyryl 循环放大器 (db 阵营), 这是一个众所周知的分子, 以提高轴突再生能力的13, 诱导激活的巨噬细胞。巨噬细胞的失活消除了 db 阵营对突起生长活动的影响。随后的工程鉴定了损伤诱导神经元中 CCL2 的表达, 作为刺激细胞的信号, 可再生表型14,15。
基于上述实验结果, 我们建立了一个体外模型, 类似于在 DRGs 后发生的分子事件, 在预适应损伤模型11,14。在这个模型中, db 阵营被应用于神经元-巨噬细胞共培养诱导细胞间信号, 导致激活的巨噬细胞与支持再生表型。在这里, 我们描述了详细的协议, 我们可以生成巨噬细胞分泌分子因子促进突起的产生 (图 1)。这个实验模型说明了一个概念, 即巨噬细胞可以被刺激或诱导, 以支持轴突再生后的神经系统损伤。我们的模型也将有助于研究细胞间信号的机制, 导致激活的促再生巨噬细胞。

<table><tbody><tr><td>Cell culture insert transparent PET membrane 0.4&amp;mu;m pore size</td><td>Corning,Falcon</td><td>353090</td><td>Transparent PET membrane with 0.4-&amp;mu;m pore size, for 6-well plate</td></tr><tr><td>70-&amp;mu;m nylon cell strainer</td><td>Corning, Falcon</td><td>352350</td><td></td></tr><tr><td>8-well culture slide</td><td>Biocoat</td><td>354632</td><td>with a uniform application of Poly-D-Lysine</td></tr><tr><td>Red blood cell lysis buffer</td><td>Qiagen</td><td>158904</td><td></td></tr><tr><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>C9407-100MG</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermo Fisher Scientific, Gibco</td><td>21103-049</td><td>Containing 1% glutamax and 1% penicilin-streptomycin</td></tr><tr><td>B-27 supplement, serum free</td><td>Thermo Fisher Scientific, Gibco</td><td>17504-044</td><td>extracellular solution</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific, Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo Fisher Scientific, Gibco</td><td>15140-122</td><td></td></tr><tr><td>Poly-D-lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td></td></tr><tr><td>Laminin</td><td>Thermo Fisher Scientific, Invitrogen</td><td>23017-015</td><td></td></tr><tr><td>Adenosine 3&#039;, 5&#039;-cyclic monophosphate, N&lt;sup&gt;6&lt;/sup&gt;,O2&#039;-dibutyryl-, sodium salt</td><td>Merck Millipore Corporation, Calbiochem</td><td>28745</td><td></td></tr><tr><td>10% Normal Goat Serum</td><td>Thermo Fisher Scientific</td><td>16210072</td><td></td></tr><tr><td>Triton-X-100</td><td>Daejung Chemical and Metal Co</td><td>8566-4405</td><td></td></tr><tr><td>Anti &amp;beta; III tubulin (Tuj-1)</td><td>Promega Corporation</td><td>G7121</td><td>Mouse monoclonal antibody</td></tr><tr><td>Goat anti-Mouse IgG (H+L) secondary antibody</td><td>Thermo Fisher Scientific, Invitrogen</td><td>A11005</td><td></td></tr><tr><td>Hemacytometer</td><td>Marienfeld-Superior</td><td>N/A</td><td></td></tr><tr><td>Cell culture CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Panasonic</td><td>N/A</td><td></td></tr><tr><td>Dissecting stereomicroscope</td><td>Carl Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>Twist shaker</td><td>FINEPCR</td><td>Tw3t</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Sorvall</td><td>N/A</td><td></td></tr><tr><td>Confocal microscope</td><td>Olympus America Inc</td><td>IX71</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>VWR International, Hyclone</td><td>SH30919.03</td><td></td></tr><tr><td>Friedman Pearson Rongeurs</td><td>FST (Fine Science Tools)</td><td>16021-14</td><td>Stainless steel, 14cm, curved, single joint action</td></tr><tr><td>Fine Scissors - Tungsten Carbide &amp;amp; ToughCut</td><td>FST (Fine Science Tools)</td><td>14558-11</td><td>sharp, serrated</td></tr><tr><td>Dumont #7 Forceps</td><td>FST (Fine Science Tools)</td><td>11272-30</td><td>Dumoxel, 0.07 x 0.04mm, curved</td></tr><tr><td>Vannas Spring Scissors</td><td>FST (Fine Science Tools)</td><td>15000-00</td><td>straight, 3mm cutting-edge, sharp</td></tr><tr><td>Qualitron DW-41 Micro-Centrifuge</td><td>Artisan Technology Group</td><td>DW-41</td><td>Input Voltage: 115VAC</td></tr></tbody></table>

neuron-macrophage co-cultures to activate macrophages secreting molecular factors with neurite outgrowth activity

有确凿的证据表明巨噬细胞可以参与损伤神经系统的再生或修复。在这里, 我们描述了一个协议, 其中诱导巨噬细胞产生条件培养基 (CM), 促进突起的生长。成人背根神经节神经元是尖锐的离解和电镀。在神经元稳定附着后, 腹腔巨噬细胞在同一井上被贴上的单元培养物上进行共培养。Dibutyryl 循环放大器 (db 阵营) 适用于24小时的共培养, 之后, 细胞培养插入含有巨噬细胞被移动到另一个井收集 CM 为72小时。CM 从联合文化处理与 db 阵营, 当应用于一个单独成人背根神经节神经元文化, 展示结实突起生长活动。由单一细胞类型组成的单胞型、背根神经节或腹膜巨噬细胞所获得的突起的 CM 没有表现出其生长活性。这表明, 神经元和巨噬细胞之间的相互作用是必不可少的活化巨噬细胞分泌分子因子与突起生长活动成厘米。因此, 我们的共同培养范式也将有助于研究神经元-巨噬细胞相互作用中的细胞间信号, 以刺激巨噬细胞被赋予可再生的表型。

我们描述了一种能产生能分泌突起的分子因子的巨噬细胞的协议。用 db 阵营处理的共培养物获得的巨噬细胞 CM 在应用于单独的背根神经节神经元培养 (图 2A) 时产生了强健的突起。相比之下, 从 PBS 处理的共培养中获得的 CM 并没有在我们的15小时文化持续时间内诱导突起的产生 (图 2B)。当 db 阵营单独对待与巨噬细胞的文化时, 从...

Watch this Scientific Journal Video about Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity at JoVE.com

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

神经元-巨噬细胞共培养活化巨噬细胞分泌突起活性的分子因子

目前的协议提供了实验程序, 以刺激培养的巨噬细胞, 有能力释放分子因子, 促进突起的生长。对神经元-巨噬细胞共培养的治疗, 诱导巨噬细胞产生具有强突起活性的条件培养基。

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

neuron-macrophage-co-cultures-to-activate-macrophages-secreting

Research

JoVE Journal

Neuroscience

8.7K 次观看.  Ajou University School of Medicine. 目前的协议提供了实验程序, 以刺激培养的巨噬细胞, 有能力释放分子因子, 促进突起的生长。对神经元-巨噬细胞共培养的治疗, 诱导巨噬细胞产生具有强突起活性的条件培养基。

视频: Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

科研

癌症研究

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

免疫与感染

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 ...

神经科学

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

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

发育生物学

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia in health and disease. Furthermore, iPSC-derived microglia can be maintained in co-culture with iPSC-derived cortical neurons, which enable investigations of microglia-neuron interactions that are hypothesized to be dysregulated in a number of neuropsychiatric disorders. Human iPSCs were differentiated to generate microglia using an adapted version of a protocol developed by the Fossati group, and the iPSC-derived microglia were validated with marker analysis and real-time PCR. Human microglia generated using this protocol were positive for the markers CD11C, IBA1, P2RY12, and TMEM119, and expressed the microglial-related genes AIF1, CX3CR1, ITGAM, ITGAX, P2RY12, and TMEM119. Human iPSC-derived cortical neurons that had been differentiated for 30 days were plated with microglia and maintained in co-culture until day 60, when experiments were undertaken. The density of dendritic spines in cortical neurons in co-culture with microglia was quantified under baseline conditions and in the presence of pro-inflammatory cytokines. In order to examine how microglia modulate neuronal function, calcium imaging experiments of the cortical neurons were undertaken using the calcium indicator Fluo-4 AM. Live calcium activity of cortical neurons was obtained using a confocal microscope, and fluorescence intensity was quantified using ImageJ. This report describes how co-culturing human iPSC-derived microglia and cortical neurons provide new approaches to interrogate the effects of microglia on cortical neurons.

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

The ability to generate microglia from human induced pluripotent stem cells (iPSCs) provides new tools and avenues for investigating the role of microglia ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

 用于 CNS 相互作用研究的原代小胶质细胞和神经元共培养方案

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

作者聚焦:用于研究疾病条件下小胶质细胞-神经元相互作用的体外共培养模型

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

神经元-巨噬细胞共培养活化巨噬细胞分泌突起活性的分子因子

摘要

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此视频中的章节

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

培养小胶质细胞的新生和成年的中枢神经系统

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

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

研究对牙髓寄生虫信号反应的神经质生长的共同培养方法

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

生成和共培养小鼠原代小胶质细胞和皮质神经元

生成和共培养小鼠原代小胶质细胞和皮质神经元

生成和共培养小鼠原代小胶质细胞和皮质神经元