Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels
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Author:
Date:
2017-04-20
[Abstract] Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial cells and perivascular cells secret and assemble a vascular basement membrane and interact via close cell-cell contacts. To mimic these processes in vitro we have developed a versatile three-dimensional culture system where perivascular cells (PVC) are co-cultured with human umbilical cord vascular endothelial cells (HUVEC) in a collagen type I gel. This co-culture system can be used to determine biochemical and cellular processes ...
[摘要] 血管发生定义了形成现有血管的新血管结构的形成过程,涉及发育过程中的修复过程,如伤口愈合,还与病理变化有关。 在血管生成过程中,内皮细胞建立血管网络并招募血管周围细胞以形成成熟稳定的血管。 内皮细胞和血管周围细胞秘密并组装血管基底膜,并通过细胞间接触进行相互作用。 为了体外模拟这些过程,我们开发了一种通用的三维培养系统,其中血管周围细胞(PVC)与胶原I型凝胶中的人脐带血管内皮细胞(HUVEC)共培养。 这种共培养系统可用于通过广泛的分析选项来确定新生血管生成事件期间的生物化学和细胞过程。 【背景】内皮细胞和血管周围细胞之间的协调相互作用对于根据给定组织内的局部需要形成稳定的血管网是非常重要的。多个分子组分有助于相互作用,但仍然很少了解。需要各种生长因子来吸引内皮细胞到低氧浓度的位点,并建立新的血管,然后被血管周围细胞覆盖。两种细胞类型相互作用以分泌特定的细胞外基质并稳定新形成的血管。在过去已经建立了多个测定法来分析二维基质胶底物上的血管细胞相互作用和血管样网络形成,但是这些测定在三维细胞内提供关于血管内血管周围细胞相互作用和血管基底膜形成的初始步骤的信息是有限的维度微环境。此外,缺乏适合于培养实验的良好表征的血管周围细胞。 我们以前分离出具有血管周围特征的细胞,因为它们表达周细胞特异性标记,产生和分泌细胞外基质蛋白并在体内刺激血管生成过程(Brachvogel等,2005和2007)。这些细胞用于与人脐静脉内皮细胞建立共培养系统,并研究三维微环境中两种细胞类型相互作用后新生血管发生的关键步骤(Pitzler等,2016; ...
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Generation of Tumour-stroma Minispheroids for Drug Efficacy Testing
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Author:
Date:
2017-01-05
[Abstract] The three-dimensional organisation of cells in a tissue and their interaction with adjacent cells and extracellular matrix is a key determinant of cellular responses, including how tumour cells respond to stress conditions or therapeutic drugs (Elliott and Yuan, 2011). In vivo, tumour cells are embedded in a stroma formed primarily by fibroblasts that produce an extracellular matrix and enwoven with blood vessels. The 3D mixed cell type spheroid model described here incorporates these key features of the tissue microenvironment that in vivo tumours exist in; namely the three-dimensional organisation, the most abundant stromal cell types (fibroblasts and endothelial cells), and extracellular matrix. This method combined with confocal microscopy can be a powerful tool to ...
[摘要] 组织中细胞的三维组织及其与相邻细胞和细胞外基质的相互作用是细胞反应的关键决定因素,包括肿瘤细胞如何对应激条件或治疗药物的反应(Elliott和Yuan,2011)。在体内,肿瘤细胞被包埋在主要由成纤维细胞形成的基质中,所述成纤维细胞产生细胞外基质并用血管编织。这里描述的3D混合细胞类型球体模型包括了体内存在肿瘤的组织微环境的这些关键特征;即三维组织,最丰富的基质细胞类型(成纤维细胞和内皮细胞)和细胞外基质。该方法结合共聚焦显微镜可以成为不同肿瘤类型的药物敏感性,血管生成和细胞迁移/侵袭测定的有力工具。
背景 传统的单层细胞培养(二维)强化人造环境,其与体内存在的组织细胞大不相同。最重要的区别之一是在单层培养物中,细胞是极化的,即,面向培养物的细胞表面和暴露于培养基的上细胞表面完全不同,经常反对的信号(Fitzgerald等人,2015)。为了解决细胞极化的问题,肿瘤球状体培养越来越多地用于癌症研究。肿瘤球体可以通过减少其通常发生在单层培养物中的生长培养基的扩散和稀释,通过细胞因子和趋化因子复制存在于组织中的三维细胞 - 细胞相互作用和一定程度的旁分泌信号传导(Lawlor等,2002; ...
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BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry
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Author:
Date:
2016-09-05
[Abstract] Lipid droplets (LDs) are ubiquitous, dynamic organelles and function as a storage depot for neutral lipids, including triglycerides and cholesterol esters (Walther and Farese, 2012). The movement of lipid species into and out of LDs impacts a variety of cellular processes, such as energy homeostasis, lipid-based signaling, and membrane homeostasis (Greenberg et al., 2011). For example, neutral lipid storage is enhanced upon increased synthesis or uptake of lipid species. On the other hand, extracellular signals can enhance the release of lipid species packaged within neutral LDs. Thus, the investigation of topics involving lipid metabolism may require the assessment of cellular neutral lipid content. In this protocol, we describe the use of the fluorescent neutral lipid dye ...
[摘要] 脂滴(LD)是普遍存在的,动态的细胞器,并且作为中性脂质包括甘油三酯和胆固醇酯的储存库(Walther和Farese,2012)。 脂质物质进出LD的运动影响多种细胞过程,例如能量稳态,基于脂质的信号传导和膜内环境稳定(Greenberg等人,2011)。 例如,当增加脂质种类的合成或摄取时,中性脂质储存增强。 另一方面,细胞外信号可以增强包装在中性LD内的脂质物质的释放。 因此,涉及脂质代谢的主题的研究可能需要评估细胞中性脂质含量。 在该方案中,我们描述了荧光中性脂质染料4,4-二氟-1,3,5,7,8-五甲基-4-硼-3a,4a-二氮杂-s-引达省(BODIPY 493/503 ),以通过流式细胞术和通过显微镜观察LD来促进中性脂质含量的定量。
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