| 3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation
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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; ...
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| Retinal Differentiation of Mouse Embryonic Stem Cells
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Author:
Date:
2016-07-05
[Abstract] Groundbreaking studies from Dr. Yoshiki Sasai’s laboratory have recently introduced novel methods to differentiate mouse and human Embryonic Stem Cells (mESCs and hESCs) into organ-like 3D structures aimed to recapitulate developmental organogenesis programs (Eiraku et al., 2011; Eiraku and Sasai, 2012; Nakano et al., 2012; Kamiya et al., 2011). We took advantage of this method to optimize a 3D protocol to efficiently generate retinal progenitor cells and subsequently retinal neurons in vitro. This culture system provides an invaluable platform both to study early developmental processes and to obtain retinal neurons for transplantation approaches. The protocol described here has been successfully applied to several mouse ESC (including the R1, WD44 and ...
[摘要] 来自Yoshiki Sasai博士的实验室的开创性研究最近已经引入了将小鼠和人胚胎干细胞（mESC和hESC）区分为器官样3D结构的新方法，其旨在重现发育器官发生程序（Eiraku等人， ，2011; Eiraku和Sasai，2012; Nakano等人，2012; Kamiya等人，2011）。 我们利用这种方法优化3D协议以有效地生成视网膜祖细胞和随后视网膜神经元体外。 这种文化系统提供了一个宝贵的平台，既研究早期发展过程，并获得视网膜神经元的移植方法。 这里描述的协议已成功应用于几个鼠标ESC（包括R1，WD44和G4细胞系）和小鼠诱导多能干细胞（iPSCs）线。
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| Dissociated Retinal Cell Culture
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Author:
Date:
2014-01-20
[Abstract] The retina is a relatively simple and accessible part of the central nervous system, making it a powerful model to study cell fate specification mechanisms. Multipotent retinal progenitor cells (RPCs) give rise to seven major classes of retinal cell types. Mechanisms regulating cell fate choice in the retina depend on both cell intrinsic and environmental factors, but their relative contribution to specific cell fate decisions remains unclear. Dissociated retinal cell cultures provide a great assay to study this problem. RPCs are cultured in serum-free and extract-free medium, providing the investigator with a control over the environment to address questions related to the effects of a particular molecule on the development of retinal neurons. In addition, dissociated cell cultures can ...
[摘要] 视网膜是中枢神经系统相对简单易用的部分，是研究细胞命运规范机制的有力模型。多能视网膜祖细胞（RPC）产生七大类视网膜细胞类型。调节视网膜细胞命运选择的机制取决于细胞内在和环境因素，但它们对特定细胞命运决定的相对贡献仍不清楚。分离的视网膜细胞培养物提供了一个很好的分析来研究这个问题。 RPC在无血清和无提取物培养基中培养，为研究者提供对环境的控制，以解决与特定分子对视网膜神经元发育的影响有关的问题。此外，分离的细胞培养物可用于通过从其正常环境中分离RPC来研究细胞内在机制的重要性（Cayouette等人，2003; Jensen和Raff，1997）。下述方法适用于RPC的克隆密度培养。在这样的培养物中，RPC彼此分离并且从后神经元分离。它们分化和分化成不同的视网膜细胞类型以形成小菌落或“克隆”。在最近的一项研究中，我们发现这些克隆与细胞数量和细胞类型组成方面在视网膜原位发生的克隆是无法区分的，这表明细胞内机制在视网膜发育中起关键作用（Cayouette et al。 ，2003）。
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