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Simplified low-cost methodology to establish, histologically process and analyze three-dimensional cancer cell spheroid arrays.
European Journal of Cell Biology ( IF 6.6 ) Pub Date : 2020-06-10 , DOI: 10.1016/j.ejcb.2020.151095
Giselle de Souza Moraes 1 , Márcia Rosângela Wink 2 , Fábio Klamt 3 , Andrew Oliveira Silva 1 , Marilda da Cruz Fernandes 1
Affiliation  

Differently of two-dimensional cell culture, three-dimensional (3D) multicellular spheroid model allows cells to establish cell-cell/cell-matrix interactions over the entire cell surface, more closely mimicking tumor microenvironments and cellular subpopulations with specific standards of morphology, differentiation and gene expression. Thenceforth several methodologies involving or the 3D cell aggregates generation or its histological processing and analysis have emerged, but in general they are laborious, expensive and complex to set up as a routine technique. Thus, we developed a complete methodology, detailing a simple, accessible and low-cost step by step, including 1) the 3D cell aggregate generation using hanging drop technique; 2) providing a simple way to assess morphological parameters of generated spheroids; followed by 3) a multiple and organized histological processing, keeping several individual spheroids inside an agarose apparatus, maintaining a known order and position of each ones, similar to tissue microarray principle; 4) until the last step, where it is allowed a simultaneous histological composition analysis of several spheroid slices, organized side by side, in a same block section, through conventional stainings or 5) immunostaining against different molecular markers. Therefore, the present methodology aims to popularize 3D cell culture, allowing to make this a regular technique in basic cell biology research, once all steps are performed without using onerous reagents, materials or equipment. In addition to bring the agarose apparatus as a simple low cost novelty, allowing high-throughput analysis of several spheroids simultaneously in an organized manner.



中文翻译:

用于建立,组织学处理和分析三维癌细胞球体阵列的简化低成本方法。

与二维细胞培养不同,三维(3D)多细胞球体模型允许细胞在整个细胞表面上建立细胞-细胞/细胞-基质相互作用,更紧密地模仿肿瘤微环境和具有特定形态学,分化标准的细胞亚群和基因表达。从那时起,出现了涉及3D细胞聚集体生成或组织学处理和分析的几种方法,但是总的来说,它们作为常规技术设置起来费力,昂贵且复杂。因此,我们开发了一套完整的方法,逐步详细介绍了一种简单,可访问且低成本的方法,其中包括:1)使用悬挂滴技术生成3D细胞聚合;2)提供一种简单的方法来评估生成的球体的形态参数;3)多次组织化的组织学处理,在琼脂糖装置中保留几个单独的球体,保持每个球体的已知顺序和位置,类似于组织芯片原理。4)直到最后一步,在此允许通过常规染色在同一块切片中并排组织的几个球形切片的同时组织学组成分析,或5)针对不同分子标记的免疫染色。因此,本方法论旨在普及3D细胞培养,一旦执行所有步骤而无需使用繁重的试剂,材料或设备,就可以使其成为基础细胞生物学研究中的常规技术。除了将琼脂糖装置作为一种简单的低成本新颖产品之外,

更新日期:2020-06-28
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