Impact of chemically defined culture media formulations on extracellular vesicle production by amniotic epithelial cells

<jats:title>Abstract</jats:title><jats:p>The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell‐free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP‐compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry‐based proteomic profiling revealed proteome composition of hAEC‐EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical‐grade scalable EV manufacture for generation of therapeutic EVs.</jats:p>