Three-Dimensional Culture Promoting Extracellular Vesicle Secretion From Dpscs

To investigate the mechanism by which three-dimensional culture enhances extracellular vesicle (EV) secretion from dental pulp stem cells (DPSCs). For 3D culture, DPSCs were seeded in the rotating cell culture system (RCCS) and cultured for 24 h, 48 h, and 72 h. The cell culture supernatant was collected, and cell counts at each time point were measured. The collected culture medium was centrifuged at 3000 g to remove cell debris, followed by ultracentrifugation at 120,000 g for 70 minutes to obtain the EV pellet, which was resuspended in PBS. The isolated EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis (NTA), and Western blotting. NTA was used to measure the number of EV particles produced by DPSCs under 2D and 3D culture conditions at different time points. Western blotting detected the expression levels of CD63, CD81, and CD9 in EVs. The concentration of EV RNA was measured by Nanodrop. Three-dimensional culture significantly increased EV production per cell compared to two-dimensional culture, with the highest yield observed at 48 hours. EVs secreted under 3D culture contained higher levels of CD63, CD81, and CD9 proteins. Additionally, RNA content in the EVs was significantly elevated under 3D culture conditions. Three-dimensional culture promotes the secretion of extracellular vesicles from DPSCs, with increased protein and nucleic acid content. The mechanism underlying this promotion may be related to the sustained low-shear stress provided by the RCCS.