Isolation and Therapeutic Application of Extracellular Vesicles Derived from Self-Assembled Mesenchymal Stem Cell Aggregates.

Extracellular vesicles (EVs) are vital mediators of intercellular communication and promising cell-free agents for regenerative medicine. Among EV subpopulations, large extracellular vesicles (LEVs) are particularly suited for tissue repair due to their enriched pro-regenerative cargo. However, standardized and reproducible methods to generate functional dental stem cell-derived LEVs for downstream characterization and therapeutic evaluation remain limited. Utilizing the exceptional osteogenic potential of stem cells from the apical papilla (SCAPs), we developed a 3D culture model under low-adhesion conditions to induce self-assembled SCAP aggregates. This model mimics physiological mesenchymal condensation, significantly enhancing cell-cell interactions and boosting LEV secretion. Key culture parameters and handling notes are provided to improve aggregate consistency and maximize LEV output while maintaining cell viability. Here, we present a standardized protocol for the efficient production of functional SCAP-derived LEVs, encompassing 3D aggregate generation, isolation via differential centrifugation, nanoparticle tracking analysis (NTA)-based particle size distribution and concentration analysis, and therapeutic delivery using a thermally crosslinked hydrogel for mandibular bone defect repair. This approach streamlines LEV production for downstream mechanistic studies and preclinical evaluation in craniofacial regeneration. This optimized workflow ensures the reproducible preparation of bioactive LEVs with consistent yields and enriched pro-osteogenic cargo, facilitating fundamental research and accelerating the clinical translation of EV-mediated craniofacial reconstruction. In representative applications, hydrogel-loaded LEVs provide a localized depot at defect sites and facilitate convenient handling during implantation. Collectively, this method provides a scalable platform to prepare SCAP-derived LEVs and highlights key considerations for standardized reporting and translational study design. This approach supports broader adoption across craniofacial EV laboratories.