| Establishing an Adult Mouse Brain Hippocampal Organotypic Slice Culture System that Allows for Tracing and Pharmacological Manipulation of ex vivo Neurogenesis
|
|
Author:
Date:
2021-01-05
[Abstract] The function of the hippocampus depends on the process of adult hippocampal neurogenesis which underpins the exceptional neural plasticity of this structure, and is also frequently affected in CNS pathologies. Thus, manipulation of this process represents an important therapeutic goal. To identify potential strategies, organotypic adult brain slices are emerging as a valuable tool. Over the recent years, this methodology has been refined and here we present a combined protocol that brings together these refinements to enable long-term culture of adult hippocampal slices. We employ a sectioning technique that retains essential afferent inputs onto the hippocampus as well as serum-free culture conditions, so allowing an extended culture period. To sustain the neurogenic potential in the ...
[摘要] [摘要]海马的功能取决于成年海马神经发生的过程，该过程是这种结构异常的神经发育的基础，并且在中枢神经系统病理中也经常受到影响。因此，对该过程的操纵代表了重要的治疗目标。为了确定潜在的策略，器官型成人大脑切片正在成为一种有价值的工具。近年来，此方法已得到完善，在此我们提出一种组合协议， 汇集了这些改进，以实现成人海马切片的长期培养。我们采用了一种切片技术，可将必要的传入输入保留在海马上以及无血清培养条件下，因此可以延长培养时间。为了维持切片中的神经源性潜力，我们利用神经胶质生成抑制剂吲哚美辛。使用EdU保留分析使我们能够评估药理干预对神经发生的影响。通过这些改进，我们建立了一种简单可靠的方法来研究小分子/药物对离体增殖和神经元形成的影响，这将有助于未来发现驱动的药物筛选。
[背景技术]海马是具有高度的可塑性作为整个生命齿状回中正在进行的神经发生的结果，脑的独特区域。成年海马神经发生的这一过程始于在亚颗粒区（SGZ）中神经干细胞（NSC）的不对称分裂，该过程保留了干细胞池并生成了准备用于神经元分化的祖细胞（Kempermann等人，2004；Anacker和Hen ，2017； ...
|
|
|
| 3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
|
|
Author:
Date:
2018-06-05
[Abstract] Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, ...
[摘要] 细胞在固体表面生长为单层的传统二维细胞培养仍然代表了药物检测癌症研究的标准方法。然而，在2D培养物中生长的细胞缺乏相关的细胞基质和细胞 - 细胞相互作用，并且忽略实体肿瘤的真实三维解剖结构。在2D中培养的细胞也可经历细胞骨架重排并获得与异常基因表达相关的人造极性（Edmondson等人，2014）。最近开发出更好地模拟体内情况的3D文化系统。用于研究癌症干细胞的3D体外肿瘤模型（肿瘤球体）在该领域已经获得了越来越多的普及（Weiswald等人，2015）。使用基质嵌入或封装的球体，悬滴培养的球体，磁悬浮系统或3D打印方法的系统已经广泛用于研究和新药筛选。在本文中，我们描述了测试shRNA介导的基因沉默对肿瘤球体形成和生长的影响的详细方案。这种方法允许研究人员测试基因敲低对肿瘤起始细胞生长的影响。正如我们实验室所证实的那样，该方案也可用于直接从肿瘤组织中分离3D癌细胞系。
【背景】3D体外肿瘤细胞模型代表了细胞系与体内生长的肿瘤之间的桥接实验方法（Pampaloni等人，2007; ...
|
|
|
| Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs
|
|
Author:
Date:
2017-11-20
[Abstract] According to the World Health Organization (WHO), at least 50% of emerging viruses endowed with pathogenicity in humans can infect the Central Nervous System (CNS) with induction of encephalitis and other neurologic diseases (Taylor et al., 2001; Olival and Daszak, 2005). While neurological diseases are progressively documented, the underlying cellular and molecular mechanisms involved in virus infection and dissemination within the CNS are still poorly understood (Swanson and McGavern, 2015; Ludlow et al., 2016). For example, measles virus (MeV) can infect neural cells, and cause a persistent brain infections leading to lethal encephalitis from several months to years after primary infection with no available treatment (Reuter and Schneider-Schaulies, 2010; Laksono et ...
[摘要] 根据世界卫生组织（WHO）的统计，至少有50％的新发病毒具有致病性，可感染中枢神经系统（CNS），并诱发脑炎和其他神经系统疾病（Taylor et al。 >，2001; Olival和Daszak，2005）。虽然神经系统疾病逐渐被记录下来，但涉及病毒感染和在CNS内传播的潜在细胞和分子机制仍然知之甚少（Swanson and McGavern，2015; Ludlow等人，2016）。例如，麻疹病毒（MeV）可以感染神经细胞，并在原发感染后几个月至数年导致持续的脑感染，导致致死性脑炎，而没有可用的治疗（Reuter和Schneider-Schaulies，2010; Laksono等人。，2016）。器官型脑文化（OBC）是病毒学领域的一个合适的模型，以更好地理解中枢神经系统感染。实际上，它不仅可以研究中枢神经系统内嗜神经病毒的感染和传播，而且还可以作为创新性抗病毒策略或分子的筛选模型，如我们最近发表的有关融合抑制肽和HSP90伴侣蛋白活性抑制剂的研究， 17-DMAG（Welsch等人，2013; ...
|
|
|