ИСТИНА

Introduction. Three-dimensional cell culture systems are considered as a link between two ways of anticancer drug testing: in vitro screening models and in vivo drug testing on laboratory animals. The aim of this work was to create 3D cell spheroids on microspheres made from poly(3-hydroxybutyrate)(PHB). Materials & Methods. To obtain porous microspheres with various diameters, the water phase/oil phase/water phase (W/O/W) method was used, followed by washing out the porogen. An aqueous solution of ammonium carbonate was used as a porogen because of its ability of thermal decomposition to ammonia and carbon dioxide. To obtain stable spheroids, HEp-2 cells (human laryngeal cancer) were cultured together with porous PHB microspheres in wells coated with 1% agarose gel under constant stirring at a temperature of 37°C and 5% CO2 content. The dynamics of cell division and growth in spheroids was monitored by MTT analysis, the morphology of spheroids was assessed visually by histological methods, as well as by scanning electron (SEM) and confocal microscopy. Results. The growth of spheroids peaked on the 7th day of cultivation, after which the number of living cells decreased both in the control (cell spheroids) and in the experiment (cell spheroids with microspheres). Moreover, during cultivation with microspheres with a diameter of 50-100 μm, more active proliferation of cells was observed compared to the control and spheroids with microspheres over 100 μm. SEM studies also showed better cell attachment and aggregation with microspheres with smaller diameters. Conclusion. The described approach to creating 3D cell models from tumor cells using PHB is promising for obtaining systems for testing antitumor drugs. A more complete understanding of the mechanisms will be achieved through analysis of the expression of various transcription factors and during the scaling of the process via cultivation in a larger volume with a constant circulation of the growth media. The work was supported by Russian Foundation of Basic Research, project #18-29-09099. The equipment of the User Facilities Centers of MSU and RCB RAS was used in the work.