Name: post-differentiation replating of human pluripotent stem cell-derived neurons for high-content screening of neuritogenesis and synapse maturation
Uploaded: 2019-08-28T12:30:00
Description: Watch this Scientific Journal Video about Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation at JoVE.com

这项工作是国家合作重新编程细胞研究小组（NCRCRG）研究精神疾病的组成部分，并得到了NIH2015-0644年U19MH1资助。初步工作也得到了NIH赠款NS070297的支持。我们感谢Salk研究所的卡罗尔·马尔切托博士和弗雷德·盖奇博士提供了WT 126线的神经祖细胞，以及加州大学圣地亚哥分校的尤金·杨博士和劳伦斯·戈德斯坦博士提供了CVB WT24线的神经祖细胞。我们感谢黛博拉·普雷在安妮·邦博士的实验室，桑福德·伯纳姆·普雷比斯医学发现研究所，有益的讨论。

1. 再镀层前的分化期
<ol><li>在10厘米的菜肴上区分神经元，使用选择7，8的协议，直到神经元已经形成了一个厚厚的网络与他们的过程，并表达不仅早期的神经元标记，如MAP2或TuJ1，但也后期标记，如NeuN。</li>
	<li>
在神经元分化过程中，每4天改变一半选择的介质。 注：更广泛或频繁的介质变化会稀释必要的营养因素，并可能不利于成熟?...

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我们展示了一种直接的人类神经元培养物的再悬浮和再镀层程序，以与高含量筛选平台兼容的方式优化生存能力、分化成功和亚细胞成像，和其他与药物发现有关的测定。图 6说明了整个工作流和此类应用程序的示例。
虽然在这里，我们专注于hiPSC衍生的神经元，这是针对皮质神经元的命运，我们期望这种方法应同样适用于人类胚胎干细胞衍生神经元，?...

人类多能干细胞（hiPSC）衍生的神经元在基础研究、药物开发和再生医学领域越来越相关。工作流程和程序，以优化他们的文化和维护，提高效率分化成特定的神经元亚型，正在迅速演变1，2。为了提高人类干细胞衍生神经元的效用和成本效益，作为在药物发现和目标验证中适应高含量分析的模型系统，减少成熟、功能性生成所需的培养时间是有益的神经元，同时保持最大的鲁棒性、可重复性和表型相关性。虽然三维有机体培养物正在推动神经发育研究的突破3，但二维单层培养物由于其最小的组织厚度，与基于自动成像的应用特别兼容。
然而，根据人类神经和神经发育疾病模型调整基于成像的筛查方法面临重大挑战。人类神经系统在体内成熟的漫长时间要求延长培养时间，以适应基因表达的自然程序，实现神经元成熟。
冗长的神经元分化程序的一个实际后果是，hiPSC衍生的单层培养物的维持必须持续数周，以实现足够的突触成熟。在此期间，保持未分化的神经祖体继续分裂。这些可以迅速过度生长的培养和篡夺所需的营养成分，以保持可行的后分体神经元。大力分割神经祖细胞（NPC）也可以与神经元竞争生长基质。这会使这些培养物受到粘附不良问题的影响，这种情况不适合基于成像的测定。此外，许多研究者发现，培养体积越小，维持健康人群的分化神经元的难度越大，足以观察神经元分化的晚期。换句话说，使用高含量筛选 （HCS） 方法进行突触成熟检测对于人类衍生的神经元来说可能非常具有挑战性。
为了规避其中一些问题，使用了重新悬浮和重新镀层以前分化的hiPSC衍生神经元的过程。首先，它允许研究神经质外生长（或更准确地说，神经质再生）在完全承诺的神经元群体。其次，将先前分化的神经元从大体积格式（如 10 厘米板或更大）重新调出，再到小体积格式（如 HCS 兼容的 96 或 384 孔微小质板），可显著减少总培养时间。小体积条件。这有利于研究突触组装和随后在体外几周的成熟。
然而，已经建立长神经酸盐和复杂连接网络的成熟神经元的重新电化带来了几个挑战，其中之一是细胞死亡率有时很高且可变。在这里，我们描述了一个再电化过程，它能产生优异的细胞存活率和可重复性。通常，神经元暴露于蛋白水解酶短的潜伏期（通常±3-10分钟），以便在三次三次分离之前从基质分离细胞。这个短暂的蛋白解时间通常用于重新悬浮和传递许多类型的分裂细胞，包括非神经细胞和未分化的祖细胞4，5，6。然而，对于具有长、相互连接的神经质的分化神经元，不仅要从基底分离细胞，而且必须破坏树突状和斧状网络，以便分离单个细胞，同时尽量减少损伤。事实上，神经元的厚网状通常倾向于从基底分离为单个表，而不是单个细胞。如果不注意放松厚厚的神经酸盐网络，神经元不仅在三聚过程中变得不可逆转地受损，而且其中许多神经元无法通过用于去除团块的过滤器，导致细胞产量差。下面我们描述了对广泛使用的蛋白酶孵化程序的简单修改，以对抗这些困难。
在下面描述的协议中，神经元用温和的蛋白酶（如蛋白酶）孵育40-45分钟，如蛋白酶（例如，Accutase）。在加入酶后的前 5-10 分钟，神经元网络从基底以片形上升出。在进行温和的三聚和过滤之前，使用蛋白酶孵育30-40分钟。这种额外的孵育时间有助于确保材料的消化放松细胞间网络，从而确保随后的三次三次培养产生单个细胞的悬浮。此过程在重新镀层时最大化细胞分布的均匀性，同时最大限度地减少细胞死亡。我们已经成功地将这种再电化方法应用于由各种分化协议7、8和从HiPSCs各行生成的hiPSC衍生神经元培养物。该程序名义上适用于大多数或所有干细胞衍生神经元的行。我们观察到，延长蛋白酶孵育时间对于从小尺寸板（例如直径35毫米）重新镀层培养物并非绝对必要;然而，正如我们在这里展示的，它提供了一个显著的好处，当从大直径板（例如，直径10厘米或更大），可能是因为神经酸盐在此类板可以延长很长的过程，并形成一个密集互连的阵列。
在这里，我们演示了这种方法，并简要说明了它在早期神经生成和突触成熟测定中的应用，这涉及到沿树突和斧头聚集前和后发蛋白，然后它们后来在突触部位进行联合定位。这些示例强调了该协议在保持细胞活力和可重复性方面的优势。首先，它允许研究者研究人类神经发生的早期步骤。实验环境类似于常用的啮齿动物皮质或海马神经元的主要培养物，其中细胞从晚期胎儿或产后早期大脑提取，在温和的蛋白酶治疗后通过三聚体分离，并允许启动神经酸盐或再生在程序9，10中被切断的中微子。与这种啮齿动物原神经元类似，hiPSC衍生的神经元在重新镀层后几小时内开始形成或再生其神经质，允许在最佳环境中对生长锥和神经质形态进行成像，而较少周围未分化的细胞。我们观察到，与神经元首次开始从祖体群中分化时看到的可变延迟和不同的生长速率相比，神经质外生长更加同步。此外，重新镀层能够检测神经元表达神经元亚型标记，通常出现在稍后的神经发育，如皮质层5/6转录因子CTIP2（鸡奥巴平上游启动子转录因子相互作用蛋白2），或泛神经元标记NeuN1。重新镀层方法的一个特别有用的特征是突触发生在与HCS兼容的时限内进行。

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			<td height="21" style="height:21px;width:304px;">Post Replating Media</td>
			<td style="width:276px;"></td>
			<td style="width:119px;"></td>
			<td style="width:377px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">L-Ascorbic Acid</td>
			<td style="width:276px;">Sigma</td>
			<td>A4403</td>
			<td>Add 1ml of 200mM stock to 1L of N2B27 media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dibutyryl-cAMP</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>Add 1 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Human BDNF</td>
			<td style="width:276px;">Peprotech</td>
			<td style="width:119px;">450-02</td>
			<td>10 ng/ml final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">B27 (50X)</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td style="width:119px;">17504044</td>
			<td>Add 20 ml to 1L N2B27 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DMEM/F12 with Glutamax</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>31331093</td>
			<td>Add N2 and distribute in 50 mL conicals; parafilm wrap lids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Human GDNF</td>
			<td style="width:276px;">Peprotech</td>
			<td>450-10</td>
			<td>10 ng/ml final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Glutamax</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>35050038</td>
			<td>Add 10 ml to 1L N2B27 media; glutamine supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Mouse Laminin</td>
			<td style="width:276px;">Sigma</td>
			<td>P3655-10mg</td>
			<td>Add 100 &#181;l to 50 mL N2B27</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">MEM Nonessential Amino Acids</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td style="width:119px;">11140035</td>
			<td>Add 5ml to 1L N2B27 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">N2 (100X) Supplement</td>
			<td style="width:276px;">Life Technologies</td>
			<td>17502048</td>
			<td>Add 5ml to 500mL media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Neurobasal A Media</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>10888022</td>
			<td>Combine with DMEM/F12 to generate N2B27 media for CVB wt cells; neural basal A media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Neurobasal Media</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>21103049</td>
			<td>for WT126 cells; neural basal media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">SM1 Supplement</td>
			<td style="width:276px;">StemCell Technologies</td>
			<td>5711</td>
			<td>Add 1:50 to media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">sodium bicarbonate</td>
			<td style="width:276px;">Thermofisher Scientific</td>
			<td>25080-094</td>
			<td>Add 10ml to 1L N2B27 media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Plate Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">10cm Tissue Culture Dishes</td>
			<td style="width:276px;">Fisher Scientific</td>
			<td>08772-E</td>
			<td>Plastic TC-treated dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">6-well Tissue Culture Dishes</td>
			<td style="width:276px;">Thomas Scientific</td>
			<td>1194Y80</td>
			<td>NEST plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Mouse Laminin</td>
			<td style="width:276px;">Life Technologies</td>
			<td>23017-015</td>
			<td>Add 1:400 on plastic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Poly-Ornithine</td>
			<td style="width:276px;">Sigma</td>
			<td>P3655-10mg</td>
			<td>Add 1:1000 on plastic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">UltraPure Distilled Water</td>
			<td style="width:276px;">Life Technologies</td>
			<td style="width:119px;">10977-015</td>
			<td>To dilute Poly-L-Ornithine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Replating Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">100mM Cell Strainer</td>
			<td style="width:276px;">Corning</td>
			<td style="width:119px;">431752</td>
			<td>Sterile, individually wrapped</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">384-well plate, uncoated</td>
			<td style="width:276px;">PerkinElmer</td>
			<td>6007550</td>
			<td>Coat with PLO and Laminin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DPBS</td>
			<td style="width:276px;">Life Technologies</td>
			<td style="width:119px;">14190144</td>
			<td>Dulbecco&#39;s phosphate-buffered saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Poly-D-Lysine-Precoated 384-well Plates</td>
			<td style="width:276px;">PerkinElmer</td>
			<td>6057500</td>
			<td>Rinse before coating with laminin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">StemPro Accutase</td>
			<td style="width:276px;">Life Technologies</td>
			<td>A1110501</td>
			<td>Apply 1mL/10cm plate for 30-45 minutes; proteolytic enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fixation Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">37% Formaldehyde</td>
			<td style="width:276px;">Fisher Scientific</td>
			<td style="width:119px;">F79-1</td>
			<td>Dissolved in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Sucrose</td>
			<td style="width:276px;">Fisher Scientific</td>
			<td style="width:119px;">S5-12</td>
			<td>0.8 g per 10 ml of fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Immunostaining Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 488 Goat anti-mouse</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-11001</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 568 Goat anti-chicken</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-11041</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 647 Goat anti-chicken</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-21449</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Alexa Fluor 561 Goat anti-rat</td>
			<td style="width:276px;">Invitrogen</td>
			<td>A-11077</td>
			<td>secondary antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DAPI</td>
			<td style="width:276px;">Biotium</td>
			<td>40043</td>
			<td>visualizes DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">mouse antibody against b3-tubulin (TuJ-1)</td>
			<td style="width:276px;">Neuromics</td>
			<td>MO15013</td>
			<td>early stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rat antibody against CTIP2</td>
			<td style="width:276px;">Abcam</td>
			<td>ab18465</td>
			<td>layer 5/6 cortical neurons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">chicken antibody against MAP2</td>
			<td style="width:276px;">LifeSpan Biosciences</td>
			<td>LS-B290</td>
			<td>early stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">chicken antibody against NeuN</td>
			<td style="width:276px;">Millipore</td>
			<td>ABN91</td>
			<td>late stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rabbit antibody against MAP2</td>
			<td style="width:276px;">Shelley Halpain</td>
			<td>N/A</td>
			<td>early stage neuronal marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">mouse antibody against PSD-95</td>
			<td style="width:276px;">Sigma</td>
			<td>P-246</td>
			<td>post-synaptic marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">rabbit antibody against Synapsin 1</td>
			<td style="width:276px;">Millipore</td>
			<td>AB1543</td>
			<td>pre-synaptic marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Bovine serum albumin (BSA)</td>
			<td style="width:276px;">GE Healthcare Life Sciences</td>
			<td>SH30574.02</td>
			<td>10% in PBS for blocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Titon X-100</td>
			<td style="width:276px;">Sigma</td>
			<td style="width:119px;">9002931</td>
			<td>Dilute to 0.2% on PBS for permeabilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Viability Markers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Vivafix 649/660</td>
			<td style="width:276px;">Biorad</td>
			<td style="width:119px;">135-1118</td>
			<td>cell death marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Calcium Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Name of Reagent/ Equipment</td>
			<td style="width:276px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AAV8-syn-jGCAMP7f-WPRE</td>
			<td>THE SALK INSTITUTE, GT3 Core Facility</td>
			<td style="width:119px;">N/A</td>
			<td>calcium reporter in a viral delivery system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">hiPSC-derived NPCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">WT 126 (Y2610)</td>
			<td style="width:276px;">Gage lab</td>
			<td style="width:119px;">N/A</td>
			<td>Marchetto et al., 2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">CVB WT24</td>
			<td style="width:276px;">Yeo and Goldstein labs</td>
			<td style="width:119px;">N/A</td>
			<td>unpublished</td>
		</tr>
	</tbody>
</table>

post-differentiation replating of human pluripotent stem cell-derived neurons for high-content screening of neuritogenesis and synapse maturation

在二维培养中与人类多能干细胞衍生神经祖细胞（NPC）区分的神经元，是探索疾病机制和进行高含量筛选（HCS）以询问化合物的强大模型系统库或识别基因突变表型。然而，随着人类细胞从NPC过渡到功能，成熟的神经元需要几个星期。突触通常在单层培养中分化 3 周后开始形成，并且几种神经元特异性蛋白质（例如后来表达的泛神经元标记 NeuN 或第 5/6 层脑皮质神经元标记 CTIP2）开始表达分化后约4-5周。这种较长的区分时间可能与用于小容量多井 HCS 平台的最佳培养条件不兼容。许多挑战包括需要粘附良好、均匀分布的细胞，并实现最小的细胞聚类，以及培养促进长期活力和功能突触成熟的过程。一种方法是以大体积格式区分神经元，然后在 HCS 兼容多孔的稍后时间点重新加板。使用这种再镀层方法时，由于树突状和斧状网络的紧张中断，一些主要的挑战涉及可重复性和细胞活力。在这里，我们演示了酶重新悬浮人类诱导多能干细胞（hiPSC）衍生神经元的一个详细和可靠的程序，在它们分化4-8周后，以大体积的形式，将它们转移到384孔微蒂板，并培养他们进一步1-3周与优秀的细胞生存。这种人类神经元的再光，不仅允许在两周内研究突触组装和成熟，而且还能研究神经素再生和生长锥体特性。我们使用384井平台为神经生成和突触发生提供可扩展的测定示例。

重插出经过数周差异化的hiPSCs衍生神经元具有若干优点。然而，分离和重新镀灭具有长、相互连接的树突和斧头的分化神经元（图1A）可能导致大量不可逆转的受损神经元。
如协议部分所述，我们使用培养与蛋白解酶分离神经元从基质。通常，由于中微石的厚网状（图1A），细胞往往一次性分离为单个同步...

Watch this Scientific Journal Video about Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation at JoVE.com

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

该协议描述了一个详细的程序，用于重新悬浮和培养人类干细胞衍生神经元，这些神经元以前在体外与神经祖细胞分离了数周。该程序有助于以与光显微镜和高含量筛选相兼容的格式对神经亮物、突触和晚表达神经元标记进行基于成像的检测。

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

该协议有助于将人类多能干细胞衍生神经元用于高含量筛选应用。它特别适合检测突触，在人类神经元中需要长时间的培养。我们演示神经元从大格式盘到 HCS 兼容的多孔，以保持其生存能力的方式。相反，我们表明，在重新补充和重放之前延长蛋白酶的孵化可以改善神经元存活率。人类多能干细胞衍生神经元在基础研究、药物开发和退行医学领域越来越重要。这种温和的滴定方法可用于重新暂停任何具有厚过程网络的细胞大单层。除了人类 iPSC 之外，它还可用于重新镀显原神经元的培养，或重新用于事实排序或单细胞测序。必须仔细遵循协议步骤，并经常监测神经元从板的蛋白酶中调解分离。最佳孵育时间可能因培养而有所不同。视觉演示使即使是没有经验的调查人员也能够重现此过程。首先用PBS轻轻冲洗分化神经元的板。将 PBS 分散到板壁上，而不是直接分散到电池上，以避免破坏它们。从盘子边缘吸出PBS，同时翻倒它，注意不要触摸细胞。每10厘米板至少应用一毫升蛋白解酶。将细胞返回培养箱40至45分钟。在孵育过程中，在相控显微镜上检查神经元，并继续蛋白酶治疗，直到神经网络完全脱离板并开始分离。当神经元分离时，停止消化，每增加五毫升新的 DEMEM 介质，每一毫升蛋白酶。使用血清移液器将细胞轻轻滴定在板上五到八次，确保不要施加太大的压力。通过100微米网格将细胞应变成50毫升的圆锥管，一滴一滴。并用额外的五毫升新鲜DMEM介质冲洗过滤器。使用台式离心机将细胞以 1000 次 G 旋转五分钟。离心后，将圆锥管返回生物安全柜并吸气大部分介质，留下 250 微升来保持细胞湿润。轻轻地将细胞重新在两毫升新鲜DMEM介质中，然后通过五毫升血清移液器的末端。最后，反转管子两到三次。使用血细胞计和三脚架蓝色来计算可行的细胞。然后 DMEM 达到所需的浓度。根据特定细胞系的要求，在管中加入适当的补充剂，通过倾斜锥形管两到三次轻轻混合细胞。从 24 井板中吸层压涂层，然后用 PBS 冲洗一次。吸气 PBS 。并在图八运动中将细胞溶液应用于每一个井，以避免出现咯咯声。电镀完电池后，将板返回37摄氏度和5%二氧化碳的培养箱。简要地涡旋早期细胞死亡记者的库存溶液，并按照手稿指示将其添加到井中。等待 20 分钟。然后成像玻璃底部多孔上的活细胞和死细胞。延长蛋白酶孵育允许释放细胞内神经元网络。这导致分离时细胞死亡减少，并导致在以下几天内更有效地恢复。使用延长的酶孵育程序，培养物在重放后几天内表现出大约两倍的细胞生存能力，并且死亡或死亡细胞的密度较低。神经元分化的过渡阶段在几天内发生，在此期间，细胞逐渐表达晚期神经元标记。这些标记在经过四周的预分化后重新镀金的培养中立即被检测到。扩展蛋白酶协议也适度增强中性酯生长。总中性酯长度和树突生长均得到改善。重放也可用于研究突触。在重放一周后，观察到突触前和突触后蛋白质的标记。此外，使用钙成像或多电位阵列可检测自发去极化和突触驱动电流的电活动。此重放过程可以适应多种类型的应用。除了高含量筛选，以确定潜在的治疗化合物，它可用于成像生长锥或多电子阵列录音。已经形成长过程的神经元的机械分离是有压力的。拉长酶孵育和细胞的温和滴定是这一程序成功的基本步骤。

Research

JoVE Journal

Neuroscience

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

科研

神经科学

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

活细胞 Sod1 突变蛋白聚集物高含量定量测定方法的建立

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

预组装塑料微流体芯片中人类干细胞衍生神经元的分段化

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...