Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models

•Platform for generation of multicellular tumor spheroids in stirred-tank culture systems.•High number of spheroids per run for feeding high-throughput screenings in pre-clinical research.•Cells from distinct oncopathologies successfully aggregated and cultured by standardized protocols.•Tumor spheroids with different morphologies (spherical and irregular) and size can be produced by manipulation of hydrodynamic conditions in the stirred-tank culture systems.•Co-cultures with fibroblasts were also demonstrated with or without physical support from hydrogels. Currently there is an effort toward the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000–1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.