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>

<ol>
	<li>Fowler, C. J., Griffiths, D., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+control+of+micturition.">The neural control of micturition.</a> Nature Reviews Neuroscience. 9 (6), 453-466 (2008).</li><li>Golbidi, S., Laher, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+in+diabetes+mellitus.">Bladder dysfunction in diabetes mellitus.</a> Frontiers in Pharmacology. 1, 136 (2010).</li><li>Iwasawa, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Effects+of+Intravenous+Cyclophosphamide+in+Combination+with+Mesna+Provided+Intravenously+and+via+Bladder+Perfusion+in+a+Patient+with+Severe+Multifocal+Motor+Neuropathy.">Long-term Effects of Intravenous Cyclophosphamide in Combination with Mesna Provided Intravenously and via Bladder Perfusion in a Patient with Severe Multifocal Motor Neuropathy.</a> Internal Medicine. 56 (14), 1893-1896 (2017).</li><li>Lange, M. M., van de Velde, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urinary+and+sexual+dysfunction+after+rectal+cancer+treatment.">Urinary and sexual dysfunction after rectal cancer treatment.</a> Nature Reviews. Urology. 8 (1), 51-57 (2011).</li><li>Lin, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+function+and+dysfunction+in+acute+intermittent+porphyria.">Nerve function and dysfunction in acute intermittent porphyria.</a> Brain. 131, 2510-2519 (2008).</li><li>Rantell, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+utility+of+urodynamics,+including+ambulatory,+and+24+h+monitoring,+in+predicting+upper+urinary+tract+damage+in+neuro-urological+patients+and+other+lower+urinary+tract+dysfunction?+ICI-RS+2017.">What is the utility of urodynamics, including ambulatory, and 24 h monitoring, in predicting upper urinary tract damage in neuro-urological patients and other lower urinary tract dysfunction? ICI-RS 2017.</a> Neurourology and Urodynamics. 37, 25-31 (2018).</li><li>Halperin, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+Lyme+neuroborreliosis.">Diagnosis and management of Lyme neuroborreliosis.</a> Expert Review of Anti-Infective Therapy. 16 (1), 5-11 (2018).</li><li>Walter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+supraspinal+correlates+to+lower+urinary+tract+stimulation+in+healthy+participants+-+A+fMRI+study.">Reliability of supraspinal correlates to lower urinary tract stimulation in healthy participants - A fMRI study.</a> Neuroimage. 191, 481-492 (2019).</li><li>Leitner, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+infusion-drainage+device+to+assess+lower+urinary+tract+function+in+neuro-imaging.">A novel infusion-drainage device to assess lower urinary tract function in neuro-imaging.</a> BJU International. 119 (2), 305-316 (2017).</li><li>Smith, T. H., Ngwainmbi, J., Grider, J. R., Dewey, W. L., Akbarali, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in-vitro+preparation+of+isolated+enteric+neurons+and+glia+from+the+myenteric+plexus+of+the+adult+mouse.">An in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse.</a> Journal of Visualized Experiments. (78), e50688 (2013).</li><li>Gordon, J., Amini, S., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+overview+of+neuronal+cell+culture.">General overview of neuronal cell culture.</a> Methods in Molecular Biology. 1078, 1-8 (2013).</li><li>Roppongi, R. T., Champagne-Jorgensen, K. P., Siddiqui, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Density+Primary+Hippocampal+Neuron+Culture.">Low-Density Primary Hippocampal Neuron Culture.</a> Journal of Visualized Experiments. (122), e55000 (2017).</li><li>Arms, L., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropeptides+in+lower+urinary+tract+function.">Neuropeptides in lower urinary tract function.</a> Handbook of Experimental Pharmacology. (202), 395-423 (2011).</li><li>Moriyama, Y., Hiasa, M., Sakamoto, S., Omote, H., Nomura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+nucleotide+transporter+(VNUT):+appearance+of+an+actress+on+the+stage+of+purinergic+signaling.">Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling.</a> Purinergic Signal. 13 (3), 387-404 (2017).</li><li>Chaudhury, A., He, X. D., Goyal, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Va+plays+a+key+role+in+nitrergic+neurotransmission+by+transporting+nNOS&#945;+to+enteric+varicosity+membrane.">Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOS&#945; to enteric varicosity membrane.</a> American journal of physiology. Gastrointestinal and Liver Physiology. 301 (3), 498-507 (2011).</li><li>Le Berre-Scoul, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+enteric+neuron-glia+coculture+system+reveals+the+role+of+glia+in+neuronal+development.">A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.</a> The Journal of Physiology. 595 (2), 583-598 (2017).</li><li>Hayashi, H., Yamada, M., Kumai, J., Takagi, N., Nomizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+laminin-111-derived+peptide-chitosan+matrices+in+a+primary+culture+of+rat+cortical+neurons.">Biological activities of laminin-111-derived peptide-chitosan matrices in a primary culture of rat cortical neurons.</a> Archives of Biochemistry and Biophysics. 648, 53-59 (2018).</li><li>Bottenstein, J. E., Sato, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+a+rat+neuroblastoma+cell+line+in+serum-free+supplemented+medium.">Growth of a rat neuroblastoma cell line in serum-free supplemented medium.</a> Proceedings of the National Academy of Sciences of the United States of America. 76 (1), 514-517 (1979).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Survival+of+Hippocampal+Neurons+in+B27-Supplemented+Neurobasalm,+a+New+Serum-free+Medium+Combination.">Optimized Survival of Hippocampal Neurons in B27-Supplemented Neurobasalm, a New Serum-free Medium Combination.</a> Journal of Neuroscience Research. 35 (5), 567-576 (1993).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1 (5), 2406-2415 (2006).</li><li>Georgas, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+illustrated+anatomical+ontology+of+the+developing+mouse+lower+urogenital+tract.">An illustrated anatomical ontology of the developing mouse lower urogenital tract.</a> Development. 142 (10), 1893-1908 (2015).</li><li>Adam, M., Potter, A. S., Potter, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychrophilic+proteases+dramatically+reduce+single-cell+RNA-seq+artifacts:+a+molecular+atlas+of+kidney+development.">Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.</a> Development. 144 (19), 3625-3632 (2017).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0" style="width:1061px;" width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.25% trypsin</td>
			<td style="width:279px;">Gibico</td>
			<td style="width:119px;">15050065</td>
			<td style="width:296px;">Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48-well culture plate</td>
			<td>Corning</td>
			<td style="width:119px;">3548</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibiotic/antimycotic</td>
			<td>Gibico</td>
			<td>15240062</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Gibico</td>
			<td>17504044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA Fraction V</td>
			<td>Gibico</td>
			<td>332</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choline Acetyltransferase Antibody</td>
			<td>Abcam</td>
			<td>ab18736</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator</td>
			<td>Heraeus</td>
			<td>B16UU</td>
			<td>Cells culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type II</td>
			<td>Sigma</td>
			<td>2593923</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">DMEM/F-12</td>
			<td>Gibico</td>
			<td>11330032</td>
			<td>Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DYNLL2 Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-13969</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gibico</td>
			<td>10100147</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>1383</td>
			<td>10 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass breakers</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1101</td>
			<td>50 ml; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>WHB Scientific</td>
			<td>WHB-48-CS</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1177</td>
			<td>100 mm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>BD</td>
			<td>561908</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow bench</td>
			<td>Su Jie Medical Devices</td>
			<td>CB 1400V</td>
			<td>Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Antibody</td>
			<td>Affinity</td>
			<td>AF5156</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murine GDNF</td>
			<td>Peprotech</td>
			<td>AF45044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal-A Medium</td>
			<td>Gibico</td>
			<td>10888022</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21010</td>
			<td>12.5 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>100-1000 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000267</td>
			<td>10-100 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Ping Fan Instrument</td>
			<td>TGL-16A</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments</td>
			<td style="width:119px;">T8-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SLC17A9 Antibody</td>
			<td>MBL International</td>
			<td>BMP079</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spoons nucleus divider</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>YZR030</td>
			<td>12 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substance P Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-58591</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21130</td>
			<td>16 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical towel</td>
			<td>Fu Kang Medical Devices</td>
			<td>5002</td>
			<td>40 x 50 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synapsin-1 Antibody</td>
			<td>CST</td>
			<td>5297T</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubulin beta Antibody(&#946;-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，?...

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

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

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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

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 ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

从胚胎小鼠小脑分离的神经元的主要培养

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...