Name: organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells
Uploaded: 2017-08-27T13:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

作者想感谢克里斯汀· Auste 的支持, 她的视频录音和 Chalid Ghadban 为他的出色技术援助。Urszula Grabiec 是支持 FKZ 29/18。

动物实验是按照欧洲共同体理事会 2010/63/欧共体欧洲议会指令批准的《关于在神经科学研究中使用动物的政策和政策》进行的。欧洲联盟理事会关于保护用于科学目的的动物的问题. 
 1. 准备仪器和培养基 <ol> <li> 准备 OHSC 使用以下工具集: 两个小剪刀, 两个弯曲的镊子, 一个镊子与罚款提示, 三刀片 (两个大小 11, 一个大小 15),三手术刀夹, 圆形滤纸 (直径:35 毫米), 琼脂, 一刀片, 一个未修改的巴斯德吸管, 和一个没有小费的改良巴斯德吸管。使用前灭菌釜中的所有物料 ( 图 1 ). </li>
	<li> 称5克琼脂, 在100毫升蒸馏水中溶解。在20分钟的121.7 和 #176 消毒解决方案; C 在210.8 人民军在一个高压釜。使用无菌玻璃吸管将3毫升液体琼脂溶液分布在 60 mm 的培养皿中, 使其固化5小时, 用塑料石蜡膜覆盖, 以避免污染并贮存在4和 #176; C 直到进一步使用。在切片过程中, 需要用琼脂块来稳定大脑. </li>
	<li> 媒体 <ol> <li> 使200毫升的制备介质 (pH 7.35) 由198毫升极小必需介质 (记忆) 和2毫升 l-谷氨酰胺溶液 (最终浓度 2 mM) 组成。在媒体准备的当天准备解决方案, 并将其存储在4和 #176; C. </li>
		<li> 准备100毫升的培养基由49毫升的记忆, 25 毫升汉克斯和 #39; 平衡盐溶液 (HBSS), 25 毫升 (v/v) 正常马血清 (NHS), 1 毫升 l-谷氨酰胺溶液 (最终浓度2毫米), 100 和 #181; g 胰岛素, 120 毫克葡萄糖, 10 毫克链霉素, 1万 U 青霉素, 800 和 #181; g 维生素 C 以前报告。加热介质 (37 和 #176; C), 将 ph 值调整为 ph 7.4 和无菌过滤器 (0.2 和 #181; m 孔径大小)。在更改介质之前, 重复该过程 (升温、pH 值调整、 等 ) 每第二天。存储在4和 #176 时最多使用一个星期的介质; C. </li>
	</ol> </li> <li> 用准备介质填充一个 35 mm 的培养皿来存储大脑。放置两个空的培养皿, 用于在工作区的冷却包上收集组织. </li>
</ol> 2。准备和切片用 Vibratome <ol> <li> 使用7-9 天大鼠或4-5 天老老鼠的大脑进行 OHSC 准备, 根据 Stoppini et al 。4 动物斩首后, 用剪刀从头骨上取下皮肤. </li>
	<li> 将一根细剪刀的刀片引入孔内, 并通过沿尾 (后) 侧 (前) 轴切割来打开颅骨。垂直于第一个切口, 使剪刀指向左右耳, 分别 (#34; T 形切口和 #34;). </li>
	<li> 小心地用细钳打开颅骨, 注意不要伤害大脑。使用手术刀 (刀片大小 11), 以削减最侧的部分额杆和小脑. </li>
	<li> 通过刮刀, 移除大脑并将其小心放置在填充有准备介质的培养皿中 ( 图 2 )。将大脑放置在标本架上, 用医用氰胶水固定。使用琼脂片确保机械稳定. </li>
	<li> 在350和 #181 中横向解剖组织, 使用滑动 vibratome OHSC. </li>
	<li> 使用双目显微镜对切片进行光学评估。立即丢弃低质量的 OHSC。这是很重要的, 只有那些完整的细胞从海马体的中间部分分离 (见图3和 4) 之间的背部和腹海马. </li>
	<li> 用手术刀分离海马区和嗅皮质 (圆刀片大小 15; 图 3 )。必须保留 perforant 通路和嗅皮质. </li>
	<li> 六到八 OHSC 从每个大脑中获得。将2-3 切片转换为一个单元格区域性插入 (孔径0.4 和 #181; m), 并将其放置在一个6井培养皿中, 每井含有1毫升培养基. </li>
	<li> 在35和 #176 中孵育6个以上的菜; C 在完全湿润的气氛中, 5% (v/v) CO 2 并每隔一天更改单元培养基. 
	注意: 在6天的 体外 (div) 中进行实验。与切片程序相关的炎症反应在6天消失。在这个阶段, 胶质细胞再次显示出分支形态, 突触连接已经成熟. </li>
</ol> 3。组织质量的评价 <ol> <li> OHSC 中变性神经元的固定、标记和可视化 <ol> 在执行实验后, 用5和 #181 孵化 OHSC; 碘碘 (PI) 在固定前2小时, 在为了使退化神经元的细胞核染色. 
		注意: PI 是一种疑似致癌物, 总是穿戴个人防护用品 (PPE) 如手套. </li>
		<li> 用0.1 米磷酸缓冲液 (pH 7.4) 清洗 OHSC, 并用 4% (v/v) 溶液将其固定在 24 h 的甲醛 (粉煤灰) 中. 
		警告: 粉煤灰是一种有毒和可疑的致癌物质。工作在油烟罩和穿戴 PPE. </li>
		<li> 用1毫升 PBS 清洗 OHSC, 使用工刷将切片与膜分开. </li>
		<li> 用 IB 标记 OHSC 4 在24个井板上染色 ( 图 5 ). </li>
	</ol> </li> <li> 在实验开始前使用荧光标记的单细胞进行肿瘤侵袭实验的传导, 使用荧光染料羧基双乙酸琥珀酰亚胺对肿瘤细胞进行标记酯 (CFDA SE). </li>
		<li> 使用 Neubauer 室分离和计数肿瘤细胞. </li>
		<li> 重中的单元格, 例如10和 #181; 悬浮液的 L 包含所需的单元格数 (通常为5万或10万单元格). </li>
		<li> 应用10和 #181; L 细胞悬浮在切片培养上, 允许细胞侵入3天. </li>
		<li> 在实验结束时, 使用 4% (v/v) 的煤灰为24小时修复切片区域性, 并将 co-cultures 与 PI 标记为另 24 h 以可视化细胞. 
		警告: 粉煤灰是一种有毒和可疑的致癌物质。工作在油烟罩和穿戴 PPE. </li>
		<li> 将 co-cultures 安装到盖板上, 以便使用安装介质进行进一步分析. </li>
	</ol> </li> </ol> 4。OHSC 实验的评价 使用共焦激光扫描显微镜 (CLSM) 对 OHSC 进行分析。用于检测 pi 标记, 变性神经元或 pi 标记细胞使用波长和 #955 的单色光; = 543 nm 和一个发射带通滤波器的波长和 #955; = 585-615 nm。对于 CFDA 标记的肿瘤细胞或 IB 4 小胶质瘤, 使用 #955 的激发波长; = 488 nm。对于这两种实验类型, 记录一个 z 堆栈与2和 #181; m 厚的光学切片, 并用于评估.

作者没有透露

本议定书描述了 OHSC 的准备工作。这个模型允许测试的内在能力和脑组织的反应后, 应用的生理和病理刺激。除了对电生理参数的分析外, OHSC 可以损伤, 并可确定损伤对所有细胞类型的影响。治疗与不同的物质和详细描述毁损过程或愈合在没有巨噬细胞和淋巴细胞是可能的。
成功的准备和培养的最关键的步骤是: 1) 在无菌条件下工作, 2) 正确准备介质, 考虑温度和 pH 值, 和 3) ?...

OHSC 是一个良好的体外模型, 研究神经元、星形胶质细胞和小鼠的生理和病理性质,1。对细胞外环境进行控制, 对各种刺激后的细胞学和形态学变化进行监测是很容易的。在准备2,3之后, 海马神经元的组织及其连接保存完好。在一些优势, OHSC 允许监测脑损伤和肿瘤侵袭没有动物手术。六至八 OHSC 可从单个啮齿动物的大脑中获得。因此, OHSC 有助于显著减少动物数量, 并允许在同一动物体内检测多种药物浓度、基因操纵或不同的损伤模型。在 slice-based 测定中, 可以精确控制实验条件。此外, 时间依赖性发展的病理条件, 如次生损害, 可以很容易地监测的延时成像。
在给定的协议中, 最初由 Stoppini et al.建立4, 描述了准备步骤, 并突出显示了选择适当切片的重要形态学标志。我们建议在后天准备7-9 大鼠或产后日4-5 只小鼠。在这些时期, OHSC 表现出对机械创伤的强大抵抗力和神经元回路重组的高电位。相比之下, 从胚胎或成年大鼠的准备工作迅速改变其结构, 并失去其器官形态学在种植期间, 因此更不适合研究长期的过程中的基础研究5,6,7,8,9,10,11. OHSC 生存率的另一个关键点是切片本身的厚度, 作为扩散, 因而养分供应是有限的12,13,14。

<table><tbody><tr><td>6-Well</td><td>Falcon</td><td>35-3046</td><td></td></tr><tr><td>Agar</td><td>Fluka</td><td>5040</td><td></td></tr><tr><td>Autoclav</td><td>Systec</td><td>DX-45</td><td></td></tr><tr><td>CFDA</td><td>&amp;nbsp;Thermo Fisher</td><td>V12883</td><td></td></tr><tr><td>Confocal laser scanning microscope (CLSM) LSM700</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Eagle&amp;acute;s Minimal Essential Medium&amp;nbsp;</td><td>Invitrogen</td><td>32360-034</td><td></td></tr><tr><td>Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I</td><td>Vector Labs</td><td>FL-1101</td><td></td></tr><tr><td>Glucose</td><td>Merk</td><td>1083371000</td><td></td></tr><tr><td>Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>24020-133</td><td></td></tr><tr><td>Hank&amp;acute;s Balanced Salt Solution (without Ca&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Invitrogen</td><td>14170-138</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>I5500</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma Aldrich</td><td>A5960</td><td></td></tr><tr><td>L-Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>LN229</td><td>Cell-Lines-Service</td><td>300363</td><td></td></tr><tr><td>Medical cyanoacrylate glue (Histoacryl glue)</td><td>B.Braun</td><td>1050052</td><td></td></tr><tr><td>Millicell Culture Inserts</td><td>Millipore</td><td>PICMORG50</td><td></td></tr><tr><td>NMDA N-methyl-D-aspartic acid</td><td>Sigma Aldrich</td><td>M3262</td><td></td></tr><tr><td>Normal Horse Seum</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Petri dishes (all sizes)</td><td>Greiner</td><td>627160/664160/628160</td><td></td></tr><tr><td>PFA</td><td>Roth</td><td>0335.1</td><td>toxic</td></tr><tr><td>Propidium iodid (PI)</td><td>Sigma Aldrich</td><td>81845-25MG</td><td>toxic</td></tr><tr><td>U138</td><td>ATCC</td><td>HTB-14</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>Leica VT 1200</td><td></td></tr></tbody></table>

organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells

在器官海马切片培养 (OHSC) 中, 神经元和胶质细胞的形态和功能特征得到了很好的保存。该模型适用于处理不同的研究问题, 涉及神经保护、神经元电生理实验、神经元网络或肿瘤侵袭等研究。multisynaptic 回路中的海马结构和神经元活动在 OHSC 中很好保存, 尽管切片程序本身最初会损伤并导致胶质瘢痕的形成。瘢痕的形成可能改变了小分子的力学性质和扩散行为,等。切片允许在没有动物手术的情况下监测脑损伤后的时间依赖性过程, 并研究各种脑源性细胞类型的相互作用, 即在生理和病理上条件.这个模型的一个矛盾的方面是缺乏血流和免疫血细胞。在神经元损伤过程中, 从血液中迁移免疫细胞起着重要作用。当这些细胞在切片中丢失时, 就可以观察到文化中的内在过程, 而不受外界干扰。此外, 在 OHSC 中, 中外部环境的组成是精确控制的。这个方法的一个进一步好处是被牺牲的动物的数量比标准准备更低。几个 OHSC 可以获得从一个动物做同时研究与多个治疗在一个动物可能。由于这些原因, OHSC 很适合分析新的保护疗法在组织损伤或肿瘤侵袭过程中的作用。
这里介绍的协议描述了一种 OHSC 的制备方法, 它允许产生高重复性的、保存完好的切片, 可用于各种实验研究, 如神经保护或肿瘤侵袭研究。

神经保护研究:为了确定神经元损伤, 计数的 PI 阳性核和 IB4阳性小胶质细胞在每三个光学部分的颗粒胞层 (协鑫) 的齿状回 (DG) 的数量。对于肿瘤侵袭实验, 采用最大强度 z 投影法计算肿瘤细胞覆盖面积, 作为入侵的测量手段, 并对不同的入侵模式进行可视化 (图 6)。
在未经治疗的控制 O...

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

器官海马切片培养 (OHSC) 代表一个体外模型, 它能很好地模拟体内的情况。在这里, 我们描述一个 vibratome-based 改进的切片协议, 以获得高质量的切片, 用于评估新物质的神经保护潜能或肿瘤细胞的生物学行为。

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

organotypic-hippocampal-slice-cultures-as-model-to-study

Research

JoVE Journal

Neuroscience

12.8K 次观看.  Martin Luther University Halle-Wittenberg. 器官海马切片培养 (OHSC) 代表一个体外模型, 它能很好地模拟体内的情况。在这里, 我们描述一个 vibratome-based 改进的切片协议, 以获得高质量的切片, 用于评估新物质的神经保护潜能或肿瘤细胞的生物学行为。

视频: Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

器官切片文化为产后神经再生的研究

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

科研

发育生物学

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

与器官型脑片和癌细胞的3D球体共培养系统

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

医学

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

神经血管重塑的分析Entorhino，海马脑切片培养

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

器官细胞生存在器官细胞切片培养

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

莱纳尔皮质-希波坎普斯有机切片文化中的癫痫发生模型

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Source: Chadwick, E. J. et al. <a href="https://app.jove.com/v/53304">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> J. Vis. Exp. (2015).
This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

在器官型切片共培养模型中建立脑肿瘤

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plating the Slices onto the ...