Comparative study on the effects of 2D and 3D cell culture on the functions of stem cells from the apical papilla

Dental-derived stem cells (DSCs), especially stem cells from the apical papilla (SCAP), are vital for regenerative medicine. Three-dimensional (3D) cell culture is superior to two-dimensional (2D) culture in mimicking in vivo environments. A dynamic 3D culture system consisting of microcarriers and a spinner flask further improves efficiency via mechanical stimulation, but key parameters have not been standardized and require further investigation. Key 3D culture parameters, including initial seeding cell density (cell number), SCAP passage number, and culture duration, were investigated to establish and optimize a 3D cell culture system. SCAP cultured in both 2D and 3D systems were evaluated for proliferation, migration, adipogenic and osteogenic differentiation potential, and exosome secretion ability. Proteomic analysis was performed on SCAP. Levels of reactive oxygen species (ROS), mitochondrial membrane potential, and adenosine triphosphate (ATP) were measured to assess changes in cellular energy metabolism between 3D- and 2D-cultured SCAP. Initial seeding density and culture duration significantly affected SCAP proliferation, whereas SCAP passage number did not significantly affect proliferation. The optimized 3D culture system significantly enhanced SCAP proliferation and migration capabilities and promoted adipogenic differentiation compared to the 2D system, while osteogenic differentiation showed no significant difference. Under 3D culture conditions, exosome secretion was significantly increased, accompanied by elevated ROS levels, decreased mitochondrial membrane potential, and reduced ATP level in SCAP. By optimizing key 3D culture parameters for SCAP, this study significantly improved their proliferation capacity and exosome secretion efficiency. The findings demonstrate the effects of the 3D dynamic environment on cellular functions and energy metabolism compared to 2D culturing.