Abstract: Cell therapy is getting a growing interest in a wide range of indications in human. In many cases, a substantial part of the therapeutic effects relies on cell-secreted factors and the extracellular vesicles (EV) are proposed as a cell-free surrogate for cell therapy. Currently, during the EV production phase, human cells are placed in serum-free media to produce EV, with limited cell survival. Here, the inventors describe a new procedure for GMP-compatible human cells-derived EV wherein Human Platelet Lysate (HPL) is produced from which the EV are removed by tangential-flow-filtration resulting in an EV-depleted HPL. Said EV-depleted HPL may be then uses as a culture medium for the production of EV by cells of interest.