Open Access

Alec Geßner, Benjamin Florian Koch, Kevin Klann, Dominik Christian Fuhrmann, Samira Farmand, Ralf Schubert, Christian Münch, Helmut Geiger, Patrick C. Baer

• Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.