Exosome free serum

Extracellular vehicles were isolated from incisor epithelium and mesenchyme cells of 5/6-day-old Sprague–Dawley rats. Briefly, the epithelium with intact cervical loop was carefully separated from mesenchyme tissue under a dissection microscope per our prior methods.^{14 (link)} Epithelium and mesenchyme stem/progenitor cells were digested and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% exosome-free serum (System Biosciences, Mountain View, CA, USA) and 1% PS (Gibco). Supernatants were collected after 5–7 days. Upon removing nonadherent cells and debris, extracellular vesicles were further purified by ultracentrifugation or a total exosome isolation kit (Invitrogen, Carlsbad, CA, USA). Ultracentrifugation was performed as the following step.^{37 (link)} Cell culture supernatant was centrifuged at 300g for 10 min. Supernatant was collected and centrifuged at 2000g for 10 min, followed by centrifugeation at 10000g for 60 min. The final supernatant is then ultracentrifuged (Beckman Coulter, USA) at 100000g for 70 min. The pellet was washed in a large volume of phosphate-buffered saline (PBS) to eliminate contamination of proteins and centrifuged at 100000g for 70 min. The collected dental epithelium and mesenchyme vesicles were resuspended in PBS and characterized by NanoSight LM10 (Particle Characterization Laboratories, Novato, CA, USA).