Free flow electrophoresis allows quick and reproducible preparation of extracellular vesicles from conditioned cell culture media

Aim: Despite intensive research during the last decade, it remains challenging to prepare extracellular vesicles (EVs) of high purity, especially from primary body liquids or protein-rich conditioned media. For now, timeconsuming combinations of at least two orthogonal methods, e.g., density and size separation, are required to enrich EVs to high purity, often at the expense of processing time. Therefore, novel technologies are required that allow EV preparation in acceptable time intervals and to fair purities. Free-flow electrophoresis (FFE) constitutes a well-established semi-preparative method to separate and prepare analytes, e.g., by inherent differences in their electric charges. FFE combines a flow-driven longitudinal transport of sample material with vertical electrophoresis and allows the separation of sample components into up to 96 different fractions. It was our aim to evaluate the potential of FFE for the separation of EVs from other sample components of EV-containing protein-rich conditioned cell culture media. Methods: Exemplarily, conditioned media of mesenchymal stem/stromal cells raised in the presence of EV- containing 10% human platelet lysate were processed. We analyzed the obtained fractions by different technologies, including imaging flow cytometry, western blot and nanoparticle tracking analysis. Results: We demonstrate that FFE quickly and reproducibly separates EVs from a huge proportion of molecules included in the original sample. Conclusion: Our results qualify FFE as a feasible, quick and reproducible technology for the preparation of bona fide EVs.