Extracellular vesicles (EVs) are lipid membrane-bound structures that are secreted by almost all cell types into the extracellular space. They are involved in different cell communication processes; particularly, it has been identified that EVs derived from cancer cells play an important role in modifying the tumor microenvironment since they have the capacity to transport different types of biomolecules, such as proteins and types of RNA, which act as regulators in the cell cycle. These characteristics allow the use of EVs as biomarkers for the diagnosis and monitoring of different 4 pathologies, including breast cancer, as well as their use in targeted therapies. The principal aim of this study was to standardize a methodology for the isolation of EVs present in the culture supernatant of two breast cancer cell lines, as well as their characterization using antibody-based techniques. The results show that the vesicles produced by the cells, both in luminal A cancer and in triple negative cancer, have micro-RNA (miRNA), for which they effectively constitute a tool for diagnosis and monitoring of the disease. Finally, this study allowed the isolation of EVs from cell culture, proving the efficiency of IZON qEV2 SEC columns in laboratories located above 2000 m.a.s.l. and eliminating EV contamination from fetal bovine serum (FBS) without significant effect on the cell culture viability using unsupplemented culture medium
