Influence of experimental variables on spheroid attributes.

The adoption of three-dimensional (3D) cell culture systems represents a critical advancement in biomedical research, better mimicking complex 3D tissue environments than traditional two-dimensional (2D) models. However, variability in experimental outcomes has limited their reproducibility and clinical translation. Here, we systematically analyzed 32,000 spheroid images to identify key parameters influencing 3D model reliability. Our large-scale analysis revealed that oxygen levels significantly affect spheroid size and necrosis, while media composition (e.g., glucose and calcium concentrations) and serum levels (0-20%) critically regulate cell viability and structural integrity. For instance, spheroids cultured in 3% oxygen exhibited reduced dimensions and increased necrosis, whereas serum concentrations above 10% promoted dense spheroid formation with distinct necrotic and proliferative zones. By integrating single-cell RNA sequencing and automated image analysis, we uncovered dynamic gene expression patterns linked to spheroid maturation and hypoxia. These findings provide actionable guidelines for standardizing 3D culture protocols, addressing critical reproducibility challenges. Our work establishes a robust framework to enhance the reliability of 3D models in drug testing, personalized medicine, and tumor biology, facilitating their broader adoption in translational research.