Simplified low-cost methodology to establish, histologically process and analyze three-dimensional cancer cell spheroid arrays.

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.