"Development of a protocol to isolate extracellular vesicles from 3D cultures" in ISEV2022 Abstract Book

Introduction: Even if the 3D cultures reflect the tumor’s architecture more realistically, there are currently no standardized protocols to isolate extracellular vesicles (EVs) from tumor cell lines cultured in 3D models. Thus, the goal of our study was to develop a simple, practical, reproducible, and economical protocol to isolate EVs from 3D cell cultures. Methods: Human ovarian cancer CABA I cells were cultured in RPMI+5% FBS (40 nm-filtered) by hanging drop to form tumor spheroids (30000 cells/spheroid). Spheroids were moved to and cultured (1mlmedium/10 spheroids) in Petri dishes coated with an anti-adhesion solution, commercially available. The supernatant was collected twice daily for 5 days, centrifuged at 4◦C at 600xg (15 min), 1,500xg (30 min), and 100,000xg (Rotor 70Ti, Quick-Seal Ultra-Clear tubes, kadj 221, brake 9, Optima XPN-110 Beckman Ultracentrifuge) for 90 min. Isolated EVs were resuspended in PBS, quantified by measuring the EV-associated proteins level (by Bradford method), and analyzed by Transmission Electron Microscopy (TEM), Nanosight (NTA), Western Blots. Scanning Electron Microscopy (SEM) imaged EVs’ release from spheroids. Cell viability in spheroids was evaluated by XTT. Results: CABA I cultured by hanging drop form compact and proliferating spheroids (334% viability at 48h vs 100% attributed to 24h); SEM revealed the release of many EVs from their surface. Indeed, the supernatant of cultured spheroids contained EVs; the TEM morphological analysis showed spherical and intact particles, which for the most part have a size attributable to sEVs (size range 55-120 nm), as confirmed by NTA. The identity of EVs was further confirmed by positivity for specific EV markers (CD63, CD9, CD81). The amount of released EVs was 0,17-0,53 μg/spheroid. Summary/Conclusion: Taking advantage of an anti-adhesion solution, compatible with EVs release and collection, we have set a simple method to isolate EVs from 3D spheroids in an effective and reproducible way.