Article 57

3D spheroid culture; microwell array; immunohistochemistry; pathology; glioblastoma; breast adenocarcinoma

This study presents an improved method for obtaining spheroids microwell arrays for histological processing and analysis, focusing on glioblastoma (U87 MG) and breast adenocarcinoma (MCF-7) tumor models. By transitioning from traditional 2D cell cultures to 3D systems, this approach overcomes the limitations of 2D cultures by more accurately replicating the tumor microenvironment. The method consists of producing homotypic and heterotypic spheroids using low-adherence agarose-coated wells, embedding these spheroids in agarose microwell arrays, and conducting immunohistochemistry (IHC) to analyze cellular and molecular profiles. Morphological analyses were performed using OrganoSeg software, and IHC staining confirmed marker expressions consistent with respective tumor types. The study details the workflow from 2D cell culture to IHC analysis, including agarose well coating, spheroid embedding, and IHC staining for markers such as EMA, p53, Ki-67, ER, PR, and HER2. Results demonstrated compact, round U87 MG spheroids and fibroblast-stabilized MCF-7 spheroids, with both types exhibiting specific marker expressions. This innovative approach significantly enhances the efficiency of producing and analyzing large volumes of spheroids, making it both quick and cost-effective. It offers a robust drug screening and cancer research platform, maintaining spheroid traceability even in bulk workflow conditions. Furthermore, this methodology supports advances in personalized medicine by providing a more physiologically relevant model than 2D cultures, which is crucial for investigating tumor behavior and therapeutic responses through IHC.