本研究由中国国家自然科学基金委员会（第81673676号资助）和东莞市科技局（第2019622101002号资助）支持。作者感谢玛丽罗斯·沙利文博士（哈佛医学院外科助理教授）的技术咨询。

所有实验协议和动物程序都符合国家研究理事会的道德原则准则。
1. 材料准备
<ol><li>在进行实验之前，使用高压灭菌器对所有仪器和ddH2O进行消毒。仪器包括但不限于手术剪刀、眼科剪刀、钳子、勺子核分隔器、玻璃盘（直径 60-100 mm）和玻璃破碎机。</li>
	<li>准备Krebs解决方案如下（表1）:在使用前将所有化学物质与ddH2O一起溶解。单独溶解 CaCI2， 在搅拌时缓慢加入混合溶液中，以避免沉积物。</li>
	<li>准备冲洗介质，它包括F12介质与10%胎儿牛血清（FBS）和1%抗生素/抗菌（100x）。将 5 毫升 FBS 和 0.5 毫升抗生素/抗菌剂添加到 44.5 mL 的 F12 介质中。</li>
	<li>准备神经元介质A，包括神经巴沙A介质2%B-27，1%FBS，1%L-谷氨酰胺，1%抗生素/抗菌（100x）和0.1%胶质衍生神经营养因子（GDNF）。加入 200 μL B-27、100 μL 的 FBS、100 μL 的 L-谷氨酰胺、100 μL 的抗生素/抗菌剂（100 倍）和 10 μL 的 GDNF （10μg/mL） 到 9.5 毫升神经巴沙 A 介质。 注:在使用后一周内准备神经元介质A，以确保B-27、L-谷氨酰胺和GDNF的新鲜度。</li>
	<li>使用与神经元介质 A 的制备相同的程序准备神经元介质 B，但无需添加 FBS。</li>
	<li>通过溶解 20 毫克拼贴酶 II、6 毫克牛血清白蛋白和 200 μL 抗生素/抗菌 （100x） 溶解 10 mL 氧稳定 Krebs 溶液来准备消化解决方案 1。</li>
	<li>用汉克平衡盐溶液的4mL稀释1mL的0.25%三氯辛来准备消化溶液2。</li>
	<li>准备一个带涂层盖片的盘子。<ol>
<li>将玻璃盖片放入 48 井培养板时，在层压流动长凳中使用钳子。</li>
		<li>稀释多D-莱氨酸与ddH2O浓度为0.1毫克/毫升作为多D-莱氨酸股票。将库存储存在-20°C，并在使用前解冻。</li>
		<li>在每个盖片顶部添加 40μL 的多 D-lysine 库存，并在室温下孵育溶液 10 分钟。 注意:对于具有不同基底区域的不同板，将浓度调整为 4 μg/cm2。例如，如果一口井的基底面积从24井板是2厘米2，那么我们应该转移80μL的多D-化氨酸股票在井里。每厘米2 将包含4微克多D-莱氨酸。</li>
		<li>在 48 井板中去除多 D-lysine，用 ddH2O 三次冲洗盖片。</li>
		<li>将板在层流罩中干燥至少 30 分钟，以确保板无水。</li>
		<li>在层压素涂层前，将盘子存放在4°C，最多1天。将盘子存放在−20°C，对于长期存储更可取。</li>
		<li>在4°C时解冻层蛋白。 稀释层蛋白与ddH2O浓度为50μg/mL作为层压素库存。将库存储存在-20°C，并在使用前解冻在4°C。</li>
		<li>使用移液器将 100 μL 的稀释层蛋白转移到每个盖唇的顶部，并在 4 °C 下孵育层敏 1 小时。 注意:对于具有不同基底区域的不同板，将浓度调整为 5 μg/cm2。例如，如果一口井的基底区域从24井板是2厘米2，然后转移200μL的稀释层蛋白在井中。每厘米2 将包含5μg的层蛋白。</li>
		<li>取出层蛋白溶液，用ddH2O冲洗盖片一次。在盖片边缘执行此操作，以避免刮伤。 注:涂层盖片最多可存储在4°C，最多2周。</li>
	</ol>
</li>
</ol>2. 膀胱收获
<ol><li>获得五周大的斯普拉格-道利老鼠。</li>
	<li>在冰浴中将碳水化合物（95%氧，5%CO 2）注入克雷布斯溶液至少30分钟，以达到稳定的氧气状态和pH水平。</li>
	<li>通过颈椎脱位安乐死后，将大鼠浸泡在75%乙醇中30s进行绝育。</li>
	<li>将老鼠放在消毒手术毛巾上，露出腹部。打开腹腔，用一组剪刀和钳子露出膀胱。</li>
	<li>轻轻抬起膀胱，用另一组剪刀和钳子将膀胱从膀胱颈部切开，以避免交叉污染。将膀胱快速置于冷氧稳定 Krebs 溶液中，以提高细胞存活率。 注意:一旦膀胱被切除，快速执行以下操作，以改善神经元生存的前景。</li>
	<li>将 Krebs 溶液添加到三个玻璃盘和玻璃破碎机中。</li>
	<li>在冰浴中用 Krebs 溶液准备玻璃菜肴和玻璃破碎机，进行预冷。</li>
	<li>将这些容器与数字 1–3 进行相应的标记，以防止混淆。</li>
	<li>将每道玻璃盘与钳子和勺子核分隔器配对。</li>
	<li>在玻璃盘1中，用眼科剪刀切开膀胱，用钳子和勺子核分隔器展开膀胱。</li>
	<li>将膀胱冲洗成玻璃断路器1，并将其放在玻璃盘2中。</li>
	<li>使用玻璃盘 2 中的钳子和眼科剪刀消除组织表面的粘附脂肪。</li>
	<li>将膀胱冲洗在玻璃破碎机 2 中，并将其放在玻璃盘 3 中。</li>
	<li>用钳子和勺子核分隔器轻轻刮膀胱到玻璃盘 3 上，以去除外源附件。</li>
	<li>在玻璃断路器 3 中冲洗膀胱，将膀胱转移到 15 mL 离心管，配有 14 mL 的冷 Krebs 溶液，并在 356 x g 和 4 °C 下旋转样品 1 分钟。</li>
	<li>用 Krebs 解决方案在另外两个管中重复前一步两次，以减少污染。</li>
</ol>3. 两步膀胱消化
<ol><li>将膀胱从离心管转移到含有 1 mL 消化溶液 1 的 2 mL 小瓶中。使用眼科剪刀将膀胱切成小块（小于1毫米）溶液。</li>
	<li>将膀胱溶液与 9 mL 的消化溶液 1 混合在无菌细胞培养盘中（直径 100 mm）。在震动孵化器中执行第 1 步消化，在 5%CO 2、37 °C 和 200 rpm 下 1 小时。</li>
	<li>第 1 步消化后，将溶液在 356 x g 在 4 °C 下 8 分钟。</li>
	<li>将消化溶液2放在37°C的水浴中预热。</li>
	<li>离心后，取出含有消化溶液1的超自然物，并收获细胞沉积物。允许使用一些剩余的液体。完全去除溶液可能会促进细胞损失。</li>
	<li>将细胞沉积物与温暖消化液2混合在15mL离心管中，在37°C水浴中消化时摇动混合物5分钟。不要超过第2步消化7分钟，否则神经元将灭亡。</li>
	<li>消化后，立即停用10mL冷冲洗介质混合物中的 trypsin。 注意:在 0 °C=4 °C 下执行以下步骤。 冰浴可以提供这样的条件。</li>
	<li>离心后在 356 x g 在 4 °C 下收获细胞沉积物 8 分钟。尽可能多地去除介质，因为剩余的肌素对细胞生长有害。</li>
	<li>用3mL的神经元介质轻轻补充沉积物。确保含有细胞的溶液中不会产生气泡，以达到较高的存活率。</li>
	<li>通过 70μm 细胞过滤器将混合物介质过滤到 50 mL 离心机管中。</li>
	<li>在冰浴中将滤水器保持在摇床上 30 rpm，持续 30 分钟。此步骤没有必要，但建议。</li>
	<li>通过离心收集细胞在356 x g 在4°C 8分钟，并轻轻地恢复细胞颗粒在1 mL的神经元介质A。</li>
	<li>将 500 μL 的细胞混合物加入准备好的 48 井板的每口井中。</li>
	<li>孵化器中的培养细胞为37°C和5%CO2。</li>
	<li>在 1 小时内用神经元介质 B 替换所有介质，以提供无血清培养。</li>
	<li>每3天更换一半的神经元介质B。 注:神经元在经过5-7天的培养后，已准备好进行免疫细胞化学实验。</li>
</ol>

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作者声明没有重大的利益冲突。

板制备 在 6、12 或 48 井培养板中使用玻璃盖片进行免疫荧光或钙成像实验是一种经济且抽样操作。在初级细胞培养的准备过程中，细胞在没有盖片的板中生长良好。因此，盖片在实验中是可有可无的，如西方斑点或聚合酶链反应。此外，涂层是电镀细胞之前的必要步骤，有或没有盖片。拉米宁和多D-lysine是涂层神经元的常见选择，尤其是层氨酸，这对神经元生长至关重要。
媒体准备 第一次中等置换后，细胞分离需要无血清的介质，因为血清刺激细胞分裂，导致神经元生长空间有限。因此，神经元生长因子至关重要。B27和GDNF的质量可能因不同批次而异，对神经元生长产生重大影响。因此，当神经元产量差时，建议检查介质的批次数。同时，新媒体股票至关重要:在更换媒体之前，每次应提前计算和准备所需金额。
动物 这种方法中使用了斯普拉格-道利大鼠。C57BL/6 小鼠在本实验中也是可以接受的。因此，尽管形态和神经元电路存在一些变化，但也可以采用其他菌株的鼠或小鼠。在不同的动物模型方面，研究人员应该开发一个优化和有针对性的方案。此外，在应用这种方法之前，应始终考虑幼年动物。
组织治疗 在实验中，除了消化过程外，将组织保持在低温下对于提高细胞存活率至关重要，这可以减少细胞代谢并避免能量不足。氧气水平，营养和pH度也会影响细胞产量11。此外，对于其他组织，我们建议研究人员使用这种方法进行调整消化条件。
细胞培养 接种疫苗时神经元的一个显著特征是它们对涂层板20的快速粘连。在这种情况下，建议在1小时培养后更换介质以获得高比例的神经元。此外，当大多数细胞开始生长伪足细胞时，根据介质的颜色和细胞状态，可以适当减少改变介质的频率。处于良好状态的初级细胞培养显示具有明亮边框的黑色索马。
大多数从膀胱中分离出的神经细胞是膀胱内膜结节，它由膀胱13的内向和自主的发泡组成。此外，收获的组织中没有主要的骨盆结节。它分布在膀胱颈部下方21。
限度 这是分离和培养神经元和胶质的初步研究。已经进行了许多尝试，如细胞拉宾治疗或密度梯度离心。然而，所需细胞的比例仍然不理想，甚至出现了更多的细胞损失。此外，本协议中的传统消化条件，如37°C，可能会杀死一些敏感的神经元类型，并导致潜在的基因表达文物22。
总之，这个协议提供了一种方法来培养神经元和胶质从大鼠膀胱。隔离易于重复，时间效率高，涉及最小的微生物污染。虽然提高神经元的纯度是必要的，但我们希望这种方法有助于LUTNS的研究。

下尿道有两个主要功能:定期尿液储存和麦克风1。下尿道神经系统 （LUTNS） 控制这些功能，并且细腻且容易受到许多神经病变的影响， 可与生俱来（肺），获得（莱姆病），继发疾病状态（糖尿病细胞病变），药物诱导（出血性膀胱炎），手术引起的（腹部手术切除），或伤害造成的（创伤性脊髓损伤）2，3，4，5，6，7。在生理/病理学研究中，体内和体外实验同样重要。虽然在体内对LUTNS的研究已经在器官，细胞和分子水平进行了一段时间，体外研究的主要神经元从尿囊几乎不存在8，9。虽然目前的研究是有限的，但我们希望在这一领域率先进行研究，以便其他研究人员能够改进这一研究。通过这种方式，这种共同培养可能导致细胞对表型（如膀胱神经元功能障碍）中生理功能障碍的理解。
与具有明确方向性的肠道肌肉成离散层相比，膀胱的肌肉是无组织的10。因此，这种方法建议消化整个膀胱，以减少操作难度，缩短高细胞存活率的预处理时间，而不是剥落膀胱的外层。
按照这种方法，我们可以获得神经元和其他细胞的混合培养。其他细胞是必不可少的，因为它们的存在模仿了体内环境11。此外，这种细胞提供在介质中不可用的物质。
此方法涉及两个消化步骤。首先，拼贴酶II型用于水解胶原蛋白，其次是肌氨酸，将组织分离成细胞10。通过这种方式，膀胱组织被分散到单个细胞中，然后相对独立地生长。当神经元培养成熟时，神经元可用于成像或功能检测。

<table><tbody><tr><td>0.25% trypsin</td><td>Gibico</td><td>15050065</td><td>Enzyme digestion</td></tr><tr><td>48-well culture plate</td><td>Corning</td><td>3548</td><td>Coating dish</td></tr><tr><td>antibiotic/antimycotic</td><td>Gibico</td><td>15240062</td><td>Culture media/Rinse media</td></tr><tr><td>Anti-Glial Fibrillary Acidic Protein Antibody</td><td>Santa Cruz</td><td>sc-33673</td><td>ICC</td></tr><tr><td>B-27</td><td>Gibico</td><td>17504044</td><td>Culture media</td></tr><tr><td>BSA Fraction V</td><td>Gibico</td><td>332</td><td>Enzyme digestion</td></tr><tr><td>Choline Acetyltransferase Antibody</td><td>Abcam</td><td>ab18736</td><td>ICC</td></tr><tr><td>CO2 Incubator</td><td>Heraeus</td><td>B16UU</td><td>Cells culture</td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>2593923</td><td>Enzyme digestion</td></tr><tr><td>DMEM/F-12</td><td>Gibico</td><td>11330032</td><td>Rinse media</td></tr><tr><td>DYNLL2 Antibody</td><td>Santa Cruz</td><td>sc-13969</td><td>ICC</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibico</td><td>10100147</td><td>Culture media/Rinse media</td></tr><tr><td>Forceps</td><td>Shanghai Jin Zhong Medical Devices</td><td>1383</td><td>10 cm; Sterile operation</td></tr><tr><td>Glass breakers</td><td>Huan Qiu Medical Devices</td><td>1101</td><td>50 ml; Sterile operation</td></tr><tr><td>Glass coverslips</td><td>WHB Scientific</td><td>WHB-48-CS</td><td>Coating dish</td></tr><tr><td>Glass dishes</td><td>Huan Qiu Medical Devices</td><td>1177</td><td>100 mm; Sterile operation</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td><td>Southernbiotech</td><td>3050-30</td><td>ICC</td></tr><tr><td>Hoechst 33342</td><td>BD</td><td>561908</td><td>ICC</td></tr><tr><td>Laminar flow bench</td><td>Su Jie Medical Devices</td><td>CB 1400V</td><td>Sterile operation</td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td>Coating dish</td></tr><tr><td>L-glutamine</td><td>Gibico</td><td>25030081</td><td>Culture media</td></tr><tr><td>MAP-2 Antibody</td><td>Affinity</td><td>AF5156</td><td>ICC</td></tr><tr><td>Murine GDNF</td><td>Peprotech</td><td>AF45044</td><td>Culture media</td></tr><tr><td>Neurobasal-A Medium</td><td>Gibico</td><td>10888022</td><td>Culture media</td></tr><tr><td>Ophthalmic scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21010</td><td>12.5 cm; Sterile operation</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000240</td><td>100-1000 ul; Reagent and sample pipetting</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>3120000267</td><td>10-100 ul; Reagent and sample pipetting</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P7280</td><td>Coating dish</td></tr><tr><td>Refrigerated centrifuge</td><td>Ping Fan Instrument</td><td>TGL-16A</td><td>Enzyme digestion</td></tr><tr><td>Shaking incubator</td><td>Haimen Kylin-Bell Lab Instruments</td><td>T8-1</td><td>Enzyme digestion</td></tr><tr><td>SLC17A9 Antibody</td><td>MBL International</td><td>BMP079</td><td>ICC</td></tr><tr><td>Spoons nucleus divider</td><td>Shanghai Jin Zhong Medical Devices</td><td>YZR030</td><td>12 cm; Sterile operation</td></tr><tr><td>Substance P Antibody</td><td>Santa Cruz</td><td>sc-58591</td><td>ICC</td></tr><tr><td>Surgical scissors</td><td>Shanghai Jin Zhong Medical Devices</td><td>J21130</td><td>16 cm; Sterile operation</td></tr><tr><td>Surgical towel</td><td>Fu Kang Medical Devices</td><td>5002</td><td>40 x 50 cm; Sterile operation</td></tr><tr><td>Synapsin-1 Antibody</td><td>CST</td><td>5297T</td><td>ICC</td></tr><tr><td>Tubulin beta Antibody(&amp;beta;-III-tubulin)</td><td>Affinity</td><td>AF7011</td><td>ICC</td></tr></tbody></table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

下尿道有两个主要功能，即定期尿液储存和麦克风:这些功能通过中央和外围神经调节进行调解。虽然已经对下尿道神经系统进行了广泛的研究，但大多数研究都集中在初级文化上。该协议引入了一种从斯普拉格-道利大鼠分离和培养膀胱神经元和胶质的方法。在这种方法中，神经元和胶质在 37 °C、5% CO2 孵化器中孵育 5–7 天。因此，它们成长为适合相关后续免疫荧光实验的成熟形状。使用光学显微镜观察细胞的形态学。神经元、突触囊泡和胶质分别被β-III-图布林和MAP-2、突触-1和GFAP染色识别。同时，对几种神经递质相关蛋白质进行了免疫细胞化学，如胆碱乙酰转移酶、DYNLL2和SLC17A9。

在原细胞培养过程中，获得的细胞在附加状态之前具有明亮而清晰的边界。随着神经元的生长，树突和轴突开始变得与众不同。经过5-7天的培养，神经元达到一个成熟的形式与长期投影，这是理想的成像或功能研究。虽然由于介质变化，大部分杂质和细胞碎片可以清除，但可见附着在聚D-赖氨酸和层氨酸涂层上的某些残留物（图1）。
经过适当的培养，神经元可以通过典型的β-III-图布林和MAP-2免疫维持10，12来识别。此外，通过GFAP免疫保持10专门鉴定了胶质。成熟的神经元发展出突触性脊柱，接近突触蛋白制造者突触-1（图2）12的免疫消毒所识别的先天性特异性。这些结果表明，成熟细胞具有发达的突触是通过这种方法获得的。这一结果表明它在今后的函数研究中发挥着重要作用。
同时，通过免疫细胞化学实验（图3）识别出几个神经元亚型。肽神经元，其中含有各种神经肽，免疫与物质P13。通过SLC17A9染色14识别具有表达囊细胞核苷酸输送器的纯能神经元。硝基神经元与DYNLL-2可视化，DYNLL-2将nNOS与神经元15中的运动蛋白连接起来。胆碱神经元对胆碱乙酰转移酶16免疫。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61177/61177fig1.jpg"> 图1。电镀后1天、3天和7天从大鼠膀胱培养中分离出的原细胞的相位对比图像（分别为A、B、C）。比例杆:50微米。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61177/61177fig1large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61177/61177fig2.jpg"> 图2。从大鼠膀胱中分离出的主要细胞的免疫荧光图像。 共焦显微镜分析显示，神经元细胞球菌蛋白染色（β-III-图布林，RRID:AB_2827688，1:200）在初级培养神经元（A）和整个坐骑膀胱制备（B）。在初级培养神经元中，神经元磷蛋白免疫染色（MAP 2，RRID:AB_2827689，1:200）也可视化（C）。格利亚是通过胶质纤维酸性蛋白染色（D:瑞德:AB_627673，1:50）。突触素通过突触蛋白染色（Synapsin-1，RRID:AB_2798146，1:200）在细胞（E）和组织（F）水平可视化。使用的次要抗体如下:亚历克萨氟488（绿色，山羊抗兔lgG，1:200），亚历克萨氟555（红色，山羊抗鼠标lgG，1:200）。该原子核使用霍奇斯特33342（A、C、D、E;蓝色、1μg/mL）可视化。比例杆:50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61177/61177fig2large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61177/61177fig3.jpg"> 图3。原发神经元的几个神经元亚型的免疫荧光图像。 肽神经元被免疫含有物质P（A:瑞德:AB_785913，1:50）。通过SLC17A9染色（B:瑞德:AB_10597575，1:200）。硝基神经元通过 DYNLL-2 染色（C;瑞德:AB_654147，1:50）。胆碱神经元对胆碱乙酰转移酶（D）免疫。瑞德:AB_2244867，1:100）。使用的次要抗体如下:亚历克萨氟488（绿色，山羊抗兔lgG，1:200），亚历克萨氟555（红色，山羊抗鼠标lgG，1:200）。该原子核使用霍奇斯特33342（A、B、C、D 、蓝色、1μg/mL）可视化。比例杆:50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61177/61177fig3large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<table border="1" fo:keep-together.within-page="1"><tbody>
<tr>
<td>成分</td>
			<td>莫拉利蒂 （mM）</td>
		</tr>
<tr>
<td>纳西</td>
			<td>120</td>
		</tr>
<tr>
<td>KCI</td>
			<td>5.9</td>
		</tr>
<tr>
<td>纳赫科3
</td>
			<td>25</td>
		</tr>
<tr>
<td>娜2HPO4[12H2O</td>
			<td>1.2</td>
		</tr>
<tr>
<td>MgCI2+6H2O</td>
			<td>1.2</td>
		</tr>
<tr>
<td>卡奇2
</td>
			<td>2.5</td>
		</tr>
<tr>
<td>葡萄糖</td>
			<td>11.5</td>
		</tr>
</tbody></table>表1。克雷布斯解决方案组成

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

该协议试图建立一个可重复的协议，为初级神经元和胶质隔离从大鼠膀胱进一步细胞实验。

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.8K 次观看.  Guangzhou University of Chinese Medicine. 该协议试图建立一个可重复的协议，为初级神经元和胶质隔离从大鼠膀胱进一步细胞实验。

视频: Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

科研

发育生物学

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

免疫与感染

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 ...

神经科学

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

背根神经节分离与原代培养对神经递质释放的研究

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

从成人人类脑组织获取人类微胶质

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Isolation and Culture of Primary Neurons and Glia from a Rat Urinary Bladder

Source: Wang, R. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61177">Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for generating a primary culture of neurons and glia from a rat&#39;s urinary bladder. It outlines the steps involved in tissue preparation, enzymatic digestion to obtain single cells, and culturing the cells in a neuron-specific medium to establish a primary neuronal and glial culture.

大鼠膀胱原代神经元和神经胶质细胞的分离和培养

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

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

从成年大鼠的脊髓脑室周围区域分离神经干细胞祖细胞

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...