Features of spheroidogenesis of L929 cell line during prolonged three-dimensional cultivation

Cellular 3D cultures, in particular spheroids, are widely used to study intercellular interactions and test biocompatibility and toxicity, as they more accurately reproduce physiological conditions compared to 2D cultures. The aim of the study was to investigate the features of spheroid formation in L929 cell line in static suspension culture during long-term cultivation. To obtain spheroids, a suspension with a concentration of 2 × 106 cells/ml was placed in a 24-well plate coated with a 2% agar solution. Cultivation was carried out for 34 days. Every three days, half of the medium volume was replaced. The average diameter and volume of the spheroids were determined. The presence of necrotic cells in the spheroids was assessed by double staining of the spheroids with fluorescein diacetate and ethidium bromide. It was found that maintaining L929 cell line in a static suspension culture allowed spheroids with an average diameter of 118 μm to be obtained without the presence of a necrotic nucleus. Spheroid formation was a multi-step process that included initial cell aggregation, followed by compaction of the spheroids, which were kept in a floating state for one month of cultivation. During the study, it was found that L929 cell line spheroids were formed in several stages, including aggregation, compaction, and stabilisation of cells. All spheroids retained their spherical shape and remained viable, showing no signs of necrotic changes even on the 34th day of cultivation. The results of the study can be used to improve methods for testing biocompatibility and cytotoxicity, particularly in the development of new drugs and biomaterials using 3D cultures of L929 cell line