Isolation and Characterization of Dental Pulp Stem Cell-Derived Exosomes Across Cell Passages

Dental pulp stem cells (DPSCs) represent an accessible and clinically relevant source of mesenchymal stem cells, and their derived exosomes have emerged as promising bioactive vesicles for cell-free applications in tissue engineering and regenerative medicine. However, reproducible and standardized methods for isolating and functionally validating DPSC-derived exosomes remain limited, particularly with respect to variability introduced during cell expansion across passages. Here, we describe a comprehensive, standardized, kit-based workflow for the isolation and purification of exosomes from DPSC-conditioned medium that is compatible with routine laboratory practice. This method enables consistent recovery of intact vesicles suitable for morphological, molecular, and functional characterization using commonly available techniques. Exosome identity is assessed based on morphology, size distribution, and marker expression, while biological activity is evaluated using a defined in vitro anti-inflammatory assay. To examine passage-related effects, exosomes derived from different DPSC passages are compared using this functional readout, providing a practical framework for assessing consistency during cell expansion. By integrating isolation, purification, characterization, and functional validation into a single workflow, this protocol offers a reproducible and scalable approach for generating functionally validated DPSC-derived exosomes for biomaterials research and regenerative applications.