Laboratório de Pesquisa em Patologia, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento Leite, 245, Porto Alegre - Rio Grande do Sul, Brazil.
Laboratório de Pesquisa em Biologia Celular, Universidade Federal de Ciências da Saúde de Porto Alegre, Rua Sarmento Leite, 245, Porto Alegre - Rio Grande do Sul, Brazil.
Eur J Cell Biol. 2020 Jun;99(5):151095. doi: 10.1016/j.ejcb.2020.151095. Epub 2020 Jun 10.
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.
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，更紧密地模拟肿瘤微环境和具有特定形态、分化和基因表达标准的细胞亚群。此后，出现了几种涉及 3D 细胞聚集体生成或其组织学处理和分析的方法，但总体而言，它们作为常规技术建立起来既费力、昂贵又复杂。因此，我们开发了一种完整的方法，详细说明了一种简单、易于访问且低成本的分步方法，包括 1）使用悬滴技术生成 3D 细胞聚集体；2）提供一种简单的方法来评估生成的球体的形态参数；随后是 3）多次组织学处理，将几个单独的球体保存在琼脂糖装置中，保持每个球体的已知顺序和位置，类似于组织微阵列原理；4）直到最后一步，允许对几个球体切片进行同时的组织学组成分析，这些切片并排组织在同一块切片中，通过常规染色或 5）针对不同分子标志物的免疫染色。因此，本方法旨在普及 3D 细胞培养，使这成为基础细胞生物学研究中的常规技术，因为所有步骤都是在不使用繁琐的试剂、材料或设备的情况下完成的。此外，将琼脂糖装置作为一种简单的低成本创新引入，允许以有组织的方式同时对多个球体进行高通量分析。
