Name: primary culture of neurons isolated from embryonic mouse cerebellum
Uploaded: 2019-10-26T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum at JoVE.com

这些研究得到了自然科学和工程研究理事会（HM：NSERC发现赠款 –RGPIN-2018-06040）和马尼托巴儿童医院研究所（HM：格兰特#320035）和ALS加拿大-脑加拿大Arthur J的资助。哈德逊翻译团队格兰特（JK，HM）。

所有动物程序均按照机构条例和加拿大动物护理理事会的《实验动物护理和使用指南》进行，并已获得地方当局的批准（"班纳特恩校园动物护理"）委员会"）。已作出一切努力，尽量减少使用的动物的数量和痛苦。观察操作和脚趾捏或角膜反射相关的呼吸速率没有变化，从而证实了麻醉的充分深度。
1. 准备
注：根据研究计划，为受孕后E12_E18安排有时位怀?...

<ol>
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使用初级培养物是一种众所周知的方法，适用于所有类型的神经元17，18，19。在提出的协议中，我们解释如何分离小脑神经元，并保持其生存能力与最佳生存在体外最多3周。在E15+E18中分离的小脑细胞的主要培养，证实了三类大神经元的集合：PC、Golgi细胞和CN。在培养19、21的21天内?...

几十年来，细胞系在临床前研究和生物学研究中被广泛用作高通量工具。成本效益高、生长快和减少活体动物使用是使用这些细胞的一些好处。然而，基因改变和现象变化积累后，在体外几次通道1。细胞系的误认和原发细胞的遗传差异会导致不可重复的实验和错误的结论2，3，4，5。因此，尽管与分化的细胞（如神经元（如神经递质、电道、受体和其他神经元特异性蛋白质）有一些相似之处，但神经元细胞系无法复制神经元的全部表型。使用成熟的神经元是另一种选择;然而，这些细胞是非分裂的后密细胞，很难在培养中繁殖。此外，重新进入细胞周期可能沉淀凋亡6。
三维 （3D） 细胞培养、组织切片培养和有机体培养物培养已经开发出来，以提供一个环境，使细胞可以排列成模仿体内环境的 3D 形式。因此，可以研究细胞与细胞的沟通、迁移、肿瘤细胞侵入周围组织以及血管生成。然而，使用额外的细胞基质（ECM）蛋白质或合成水凝胶作为床上用品的额外费用、成像难度以及与高通量筛选仪器的兼容性是3D细胞培养的相当缺点。器官组织切片培养的一个主要缺点是使用大量的动物和斧头的不良影响，这导致无法获取轴xons的目标和生长因子，从而导致神经元死亡8。
因此，另一种避免细胞系问题、成熟细胞生长困难和组织复杂性的另一种方法是未成熟的原细胞在体外成熟。原细胞直接来自人类或动物组织，并使用酶和/或机械方法分离9。无论组织来源如何，培养培养基中分离、播种和维护的主要原理都相似。然而，促进增殖和成熟所必需的营养因子是高度细胞特异性6。
了解每个小脑细胞类型的"出生日期"是设计初级培养实验的先决条件。一般来说，Purkinje细胞（PC）和小脑细胞核（CN）的神经元在较小的细胞之前出生，包括内神经元（如篮子、硬质细胞）和颗粒细胞。在小鼠中，PC 在胚胎日 （E）10_E13 之间出现，而 CN 神经元在大约 E9_E1210之间出现。
其他小脑神经元的出生时间要晚得多。例如，在小鼠中，内神经元的Golgi亚群由VZ在（+E14+E18）生成，分子层中剩余的内神经元（篮子细胞和硬质细胞）从早期白质中的祖细胞分裂出来产后 （P）0=P711.颗粒细胞产生于外部生殖区（EGZ），这是一个从红斑唇衍生的次生菌区，出生后通过终端分裂。但是，在它们的前体从E13_E16的菱形唇出现之前，细胞已经沿着pia表面沿皮面沿波拉迁移，在小脑的背表面产生一层薄薄的细胞。非神经性巨胶质细胞，如星形细胞和寡核苷酸细胞，起源于心室神经皮皮，分别诞生于E13.5+P0和P0+P7，分别为11、12、13、14 ， 15.微胶质来自E8+E10之间的蛋黄囊原始骨髓祖细胞，入侵中枢神经系统后可以通过E916在小鼠大脑中检测到。
本文介绍的方法基于Furuya等人和Tabata等人最初开发的方法，即17、18，该方法针对从威斯塔大鼠Cerebella衍生的Purkinje细胞进行初级培养进行了优化。我们现在已经适应了这种方法，并仔细修改了它，以研究小鼠小脑神经元的生长19。与新协议不同，冷解剖介质是在Furuya协议17中添加播种介质之前，在解剖和分离步骤中使用的主要洗涤缓冲液。此缓冲液缺乏营养、生长因子和激素（所有在 Dulbecco 的改良 Eagle 介质中：营养混合物 F-12 [DMEM/F12]）是支持上述步骤中细胞生长和存活所必需的。此外，根据我们对鼠原小脑培养的丰富经验，我们在每个井中使用了500μL的培养基（而不是1 mL），并将三碘氨酸浓度提高到0.5纳克/mL，从而改善神经元细胞的生长。尤其是那些具有Purkinje细胞表型，并促进培养中树突状分支的生长。本文介绍的主要方法可广泛应用于胚胎发育过程中的其他小啮齿动物（如松鼠和仓鼠），并可用于研究不同胚胎阶段的小啮齿动物神经发生和分化，不同物种。

<table>
	<tbody>
		<tr>
			<td>Adobe Photoshop CS5 Version 12</td>
			<td>Adobe Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sodium Channel (SCN)PN4 (Nav1.6)</td>
			<td>Sigma</td>
			<td>S0438</td>
			<td>3 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>A3608</td>
			<td></td>
		</tr>
		<tr>
			<td>CALB1</td>
			<td>Swant Swiss Antibodies (Polyclonal)</td>
			<td>CB38</td>
			<td>1/5000 dilution</td>
		</tr>
		<tr>
			<td>CALB1</td>
			<td>Swant Swiss Antibodies (Monoclonal)</td>
			<td>300</td>
			<td>1/1000 dilution</td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside or Cytosine Arabinoside (Ara-C)</td>
			<td>Millipore Sigma</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I from bovine pancreas</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing Forceps</td>
			<td>Delasco</td>
			<td>DF-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM-F12)</td>
			<td>Lonza</td>
			<td>12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses: Circles</td>
			<td>fisher scientific</td>
			<td>12-545-81</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Millipore sigma</td>
			<td>I5500, I6634, I1882, and I4011</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Scissor</td>
			<td>Stoelting</td>
			<td>52134-38</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Metallized Hemacytometer</td>
			<td>Hausser Bright-Line</td>
			<td>3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissection Forceps</td>
			<td>Merlan</td>
			<td>624734</td>
			<td></td>
		</tr>
		<tr>
			<td>Pattern 5 Tweezer</td>
			<td>Dixon</td>
			<td>291-9454</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher BioReagents</td>
			<td>BP399-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Millipore Sigma</td>
			<td>P4638</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesteron (P4)</td>
			<td>Millipore sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>PVALB</td>
			<td>Swant Swiss Antibodies</td>
			<td>235</td>
			<td>1/1500 dilution</td>
		</tr>
		<tr>
			<td>Samll Scissor</td>
			<td>WPI Swiss Scissors, 9cm</td>
			<td>504519</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Millipore Sigma</td>
			<td>S9133</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Millipore Sigma</td>
			<td>T8158</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-iodothyronine (T3)</td>
			<td>Millipore Sigma</td>
			<td>T2877</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Fluorescence microscope</td>
			<td>Zeiss</td>
			<td>Z2 Imager</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary culture of neurons isolated from embryonic mouse cerebellum

使用原发性细胞培养物已成为研究体外神经系统的主要工具之一。使用这种简化的模型系统的最终目标是提供受控的微环境，并在体外条件下尽可能保持分离神经元和非神经元细胞的高存活率和自然特征。在本文中，我们演示了一种将原神经元与发育中的小鼠小脑分离的方法，将它们置于体外环境中，确定其生长，并监测其生存能力和分化数周。该方法适用于胚胎间12-18之间与小脑分离的胚胎神经元。

根据小脑神经元亚型的不同出生日期，E12-E18小鼠胚胎的培养物产生了不同的细胞类型。大型投影神经元，如CN神经元（E9+E12）和PC（E10+E13），在小脑发育过程中早期出现。在小鼠中，颗粒和Golgi细胞在[E13]E18之间出现，并经历了端子分裂，一长达4周。
在体外（DIV）7天用新鲜培养基II取代旧的培养基I，最终将防止胶质细胞增殖。分子层的内神经元，如篮子和硬质细胞，在出生?...

Watch this Scientific Journal Video about Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum at JoVE.com

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

进行体外实验以尽可能充分地反映体内状况并非易事。使用原发细胞培养物是了解整个生物体中细胞生物学的重要一步。提供的协议概述了如何成功生长和培养胚胎小鼠小脑神经元。

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 ...

原发性神经元细胞培养是研究体外培养分离神经元的理想模型系统。使用此方法的优点是能够保持分离细胞的细胞特征，如机理、功能和体外形态条件，尽可能接近体内条件几个星期。将圆形盖板放在生物安全柜下的 24 井板中。在每个盖滑上添加 90 微升聚 L- 奥尼辛。关闭盖子，轻轻地将盘子放在37度培养箱中两天。准备培养介质和播种介质，并保持在四摄氏度。在 DMEM/F12 中准备工作尝试素溶液，并保持在四度。将培养培养培养物和播种培养在37度的培养箱中。把 24 井板从培养箱中取出。用双蒸馏水清洗盖滑三次。将盖滑至少保持两个小时，以完全干燥。准备三个装满冷 PBS 的培养皿、三个装满冷 HBSS 的培养皿和五个装满冰冷解剖介质的培养皿。切除子宫角，在冰面上用冷PBS清洗三次。从这里，所有步骤应在冰上进行，以尽量减少组织损伤。在最后的洗涤步骤后，将幼崽与HSS中的子宫分离，并转移到冷解剖介质中。在解剖介质中，用剪刀斩首幼崽。打开从前空大号到轨道腔的劣等边界的卡路里。使用一对细钳，取出头骨底座，将头骨从大脑上剥开。小心地从中小脑和庞的横向表面开始切除小脑的脑膜。切开两个小脑，把小脑和大脑的其他部分分开。立即将收集的脑管放在充满DMEM/F12的无菌15mL锥形管中。在 DMEM/F12 中，以 4 度 1000 g 将管以 1，000 g 离心一分钟。重复三次，每次用移液器轻轻去除上流液，并在新鲜的 DMEM/F12 中重新悬浮颗粒。加入两毫升预热的三辛和轻轻地移液器混合在一起。将管子放在37度水浴中12分钟。孵育后，将管子带到安全柜，并加入10mMEM/F12，使试管灭活。将混合物在1，200克中离心5分钟。丢弃上经，再用新鲜介质重新暂停，然后离心三次。使用 DMEM/F2 预湿无菌塑料移液器。在同一管中加入3.5毫升的DNAse工作溶液。用移液器多次缝合组织，直到液体达到均匀的乳色。将 10 mL 的冷 DMEM/F12 添加到混合物中，然后进入离心机。将样品在4度时以1200克离心5分钟。小心地取出上一液，而不干扰颗粒。从培养箱中取出播种介质。加入500微升预预热播种介质，并使用移液器混合。使用血细胞计计算细胞。用播种介质稀释细胞悬浮液，使每毫升50万细胞的密度。将混合物的90微升添加到盖玻片中心的每一个井中。将板放在37度的培养箱中，三到四个小时。孵育后，每井中加入500微升预谋培养的培养。将板以 37 度更换到培养箱中。七天后，用新鲜培养基二、准备一个单独的24井板，并为每个井添加100微升PFA4%。在培养中达到所需天数后，轻轻从原始培养板的井中取下盖滑，并将其放入上一步中描述的 PFA 板的相应孔中。将 PFA 添加到孔中，以完全浸入盖滑。将 PFA 板保持 4 度 30 至 120 分钟。孵育后，将板恢复到室温。用 PBS 轻轻清洗盖滑五分钟三次。文本中的图 2 显示了从 E18 开始的小脑细胞培养。卡宾丁的免疫荧光显示Purkinje神经元与气突扩展三天体外。在体外7至10天，树突过程是显而易见的。在21天，复杂的树突分支存在。文本中的图三显示了从各种胚胎期开始的小脑细胞培养。卡宾丁的免疫荧光检测显示Purkinje神经元在体外18天后才具有早期轴突。Purkinje 神经元培养从 E14 和 E15 开始，将发展树突，但从未像 E18 成为起点时那样复杂。文本中的图四显示，在体外 21 天后，从 E18 培养物中发展出不同的细胞类型。卡尔宾丁绿色免疫荧光显示Purkinje神经元。B 和 C 中帕伐布明的红色免疫荧光显示抑制性内性。E和F中钠电压门通道的红色免疫荧光显示颗粒神经元。本协议具有成本效益，易于执行。在视频的末尾，您将能够收集和培养小脑神经元，并在所需的DIV上修复它们。

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

Anatomy of the Central and Peripheral Nervous System

SCIENTISTS IN ACTION

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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 ...

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

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

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

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 ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

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

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 ...