SpheroidSync as edge cutting transfer strategy for uniform and robust MCF7 spheroids in 3D culture

Three-dimensional spheroid models play a crucial role in cancer stem cell (CSC) research and drug screening. However, traditional methods often struggle with issues such as inconsistent shapes, difficulties in nutrient diffusion, and technical complexities. In this study, we introduce the SpheroidSync (SS) method, an innovative approach that combines conventional techniques to create uniform and size-adjustable MCF7 breast cancer spheroids at a very low cost, without the need for special growth factors or supplements. Our research involved culturing MCF7 cells using both the standard methods and the new SS method. We meticulously assessed various factors, including spheroid shape and cell viability, through fluorescent staining, colony formation assays, and gene expression analysis. The results revealed that the SS method produced spheroids that were not only uniform but also showed better structural integrity and maintained viability over time compared to traditional methods. Fluorescent viability tests indicated that SS spheroids consistently exhibited healthy cell viability as evidenced by sustained intracellular esterase activity throughout more extended culture periods. In contrast, spheroids generated through conventional methods exhibited declining viability, characterized by core deterioration and uneven staining. Furthermore, gene expression analysis showed a significant increase in CSC markers in SS spheroids, with CD44 levels rising over 40-fold, ALDH1 increasing more than threefold, CD24 decreasing, and HIF-1α elevating over 11-fold when compared to two-dimensional cultures. This establishes the typical breast CSC characteristics and confirms that a hypoxic environment was effectively created. Notably, as esterase activity declined, we observed an increase in stem cell populations, indicating a successful shift towards a quiescent, stem-like state. In summary, SpheroidSync represents a significant advancement in three-dimensional cancer modeling. It enables the production of uniform spheroids in a cost-effective manner, while ensuring long-term viability and enriching CSC populations.