The aim of this master’s thesis was to optimize the formation and cultivation of mouse osteosarcoma K7M2 spheroids with respect to initial cell number and culture duration, and to evaluate the applicability of the PrestoBlue™ and ATPlite 3D metabolic assays for monitoring spheroid growth and cellular viability. Spheroids represent a three-dimensional tumor model that mimics the spatial organization of solid tumours, including the formation of proliferative and necrotic zone. Spheroids were generated using different initial cell densities (1000, 2000, 3000, 4000 and 10,000 cells) and cultured for one or two weeks. Histological preparations were used to determine the size of the necrotic and proliferative zones. The results confirmed the characteristic spatial organization of spheroids and showed that higher initial cell numbers and longer culture durations produced larger spheroids with more pronounced necrotic regions. Based on these findings, spheroids with moderately sized necrotic zones were selected for further analyses, in which metabolic assays were performed and spheroid volume was calculated. Metabolic activity assessment showed that the PrestoBlue™ assay detected an increasing trend in signal intensity with spheroid growth, although no statistically significant correlation with volume was observed. The ATPlite 3D assay did not prove reliable under the tested experimental conditions, as it did not yield reproducible results. We found that metabolic activity is not linearly associated with spheroid size, likely reflecting diffusion limitations and metabolic heterogeneity. The combined use of morphological and metabolic analyses therefore enables a more comprehensive understanding of spheroid status and provides a relevant foundation for further investigation of therapeutic approaches, such as electrochemotherapy.
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