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Cancer continues to be a significant global health issue in the 21st century, presenting substantial risks to the health and well-being of individuals worldwide. Although there have been improvements in comprehending the molecular processes of the disease and creating treatments that specifically target it, a considerable portion of patients continue to encounter difficulties in attaining favorable results. Conventional two-dimensional (2D) cell cultures have been extensively used in cancer research. However, their inability to accurately mimic the intricate characteristics of tumors limits their effectiveness in predicting how anticancer treatments would perform in clinical settings. In order to overcome these restrictions, three-dimensional (3D) cell culture models, specifically multicellular spheroids, have arisen as promising tool for investigating cancer biology and therapeutic response. This work analyzes the development and growth dynamics of spheroids obtained from four distinct cancer cell lines: 9L-GFP, U251-RFP, A431, and FaDu. The stability and growth features of these spheroids were evaluated by culturing them using different cell counts and dilution ratios. Confocal microscopy was used to observe the formation of spheroids and measure their sizes for a duration of seven days. The results of our study reveal clear variations in growth patterns and stability profiles across the investigated cell lines. Notably, the 9L-GFP cell line demonstrates exceptional stability and continuous growth. The statistical analysis demonstrated that spheroids exhibited the most stability when the cell count was 25,000 cells and the dilution ratio was 1:3, as indicated by the high R-squared values. These findings highlight the significance of adjusting the number of cells and dilution ratios to ensure consistent and replicable spheroid formation. In summary, our study emphasizes the capacity of 3D spheroid models as effective instruments in cancer research and medication development, providing vital information about tumor biology and therapeutic responses in a context that closely resembles the physiological conditions.
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