Cultivo in vitro tridimensional de tecido ovariano bovino associado à nanopartículas e levitação magnética

Abstract

The two-dimensional (2D) system still widely used for cultivation of preantral follicles and ovarian tissue cannot be considered optimal because it prevents the preservation of spatial arrangements of cells in vivo, impairing cellular integrity. The use of the three-dimensional in vitro culture system (3D) is an attempt to represent the in vivo system to recreate the environment similar to the extracellular matrix of the ovary. The objective of this work was to evaluate the viability of bovine ovarian tissue fragments in in vitro culture in 3D system using nanoparticles composed of gold, iron oxide and poly-L-lysine, associated with magnetic levitation at different concentrations. For this, the cell viability of the fragments was evaluated by checking the production of reactive oxygen species (ROS), apoptosis, cell degeneration and the cell viability and follicular activation. The ovaries were collected from a refrigerator. Three replicates were made, in each replica the 3x3x0.5 mm size fragments were distributed in 24- well plates with the following treatments: conventional (2D), three-dimensional (3D) 300µL / mL and three-dimensional (3D) 400µL/mL. Confocal and histological analyzes were performed immediately after collection (D0), 24 hours after cultivation (D1) and after seven days of cultivation (D7). At the end of cultivation, the 2D group had a lower follicular viability (P <0.05) than the fresh control and the 3D cultivation groups. In addition, the 3D 400 µL / ml group had superior follicular viability indices (P <0.05) compared to the control group and the 300 µL / ml 3D group. In general, regardless of the culture day, the 3D 400 µL / ml group presented the highest (P <0.05) proportion of viable follicles. Follicular activation was higher in all cultivation groups compared to control and the 3D 400 µL / ml group had a higher activation rate compared to the other groups. Overall, the three-dimensional culture treatment with 400 µL / ml nanoparticles showed the lowest (P <0.05) rate of cell degeneration. At the end of the cultivation period all treatments presented similar apoptosis rates (P> 0.05) and lower than the fresh control (P <0.05). On the first day of in vitro culture, the emission of reactive oxygen species (ROS) was lower (P <0.05) in the three-dimensional culture groups compared to fresh control and two-dimensional culture. In general, three-dimensional (3D) cultivation presented better results for the coverings evaluated in this work when compared to the conventional methodology.