OBJECTIVES: Objectives of this research were to determine if initial spatial arrangement of cells determined spheroid structure and observe the movement and self-organization of cells in the spheroid depending on the order of the layers. STUDY DESIGN: The research was conducted as an in vitro study - a controlled experiment. MATERIALS AND METHODS: Two commercially available cell lines (BJ normal human fibroblasts and D54 human glioblastoma) were used for the research. Spheroids were formed using the magnetic levitation method, cut in a cryostat and stained using the Masson trichrome staining method. Preparations from all three weeks were examined using a light microscope and photographed with an Olympus DP70 camera. Images were analyzed in Image J, and statistical analysis was done with Statistica 12 program. RESULTS: No significant differences in the morphology of the spheroids can be observed in the images. The difference between the layers of spheroids is visible only in the first week of cultivation, and in the second and third weeks no demarcation between individual layers of cells is visible. The size of the spheroids decreases over time due to the formation of necrotic centers. The t test showed that there was a statistically significant difference in size between spheroids with different cell arrangements in the third week of cultivation. CONCLUSION: The initial spatial arrangement of the cells does not determine the structure of the spheroid and during the three weeks of cultivation the cells migrate, there is no clear boundary between the two layers. There is statistically significant difference in the size of spheroids between groups of spheroids with different cellular arrangement in the last week of growth.