Impact of Mechanical and Enzymatic Isolation Protocols on Extracellular Vesicle Release from Osteoarthritic Chondroprogenitors

INTRODUCTION: The therapeutic potential of extracellular vesicles (EVs) in cartilage regeneration is a rapidly evolving field. Beyond Mesenchymal Stem Cell (MSC)-derived EVs, there is increasing interest in utilizing EVs from primary chondrocytes isolated directly from patients with Osteoarthritis (OA). These patient-specific vesicles reflect the local joint environment, serving as key mediators for autotransplant and regenerative purposes. However, a major hurdle in utilizing primary cells for EV production is the lack of standardized isolation protocols. Since isolation methods, particularly prolonged enzymatic digestion, can profoundly alter the resulting secretome, establishing methods that preserve the biological integrity of released vesicles is crucial. This work aims to evaluate how different isolation protocols, specifically comparing traditional enzymatic digestion with mechanical-hybrid methods, influence the profile and yield of EVs released from OA-derived cartilaginous samples. MATERIAL AND METHODS: Human knee OA cartilage samples were obtained from surgical discards (Ethics Committee, University of Urbino, n.84, 25 July 2024). Explanted tissues were finely minced and pre-treated with Pronase E for one hour. Samples were processed via: 1. Traditional long-term collagenase digestion 2. A hybrid protocol using mechanical dissociators (Medicons white, CTSV) with significantly reduced collagenase exposure. Released EVs (harvested from media centrifuged at 2500g were characterized by CD63 and CD44 expression. For the heterogeneous, non-ultracentrifuged EV population, a lipophilic cationic dye (LCD) was employed in conjunction with Rosetta Calibration beads for standardized flow cytometric quantification. RESULTS: Preliminary data indicate that the duration of collagenase exposure significantly influences the EV release. Notable differences in EV counts were observed specifically for CD44-positive vesicles (the primary receptor for hyaluronic acid) depending on the isolation method used. CONCLUSION: Further validation via Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM) through a classic orthogonal approach is required to confirm the performance of the hybrid isolation method.