Assessing extracellular vesicles from bovine mammary gland epithelial cells cultured in FBS-free medium

Aim Mammary gland extracellular vesicles (EVs) are found in both human and livestock milk. Our knowledge of the role of EVs in the mammary gland development, breast cancer and mastitis derives mainly from in vitro cell culture models. However, a commonly shared limitation is the use of foetal bovine serum (FBS) as a supplement, which naturally contains EVs. For this reason, the purpose of the study is to establish a novel tool to investigate mammary gland EVs in vitro and in an FBS-free system. Methods Primary bovine mammary epithelial cells (pbMECs) and a mammary gland alveolar epithelial cell line (MAC-T) were cultured in a chemically defined EV-free medium. To find a reliable EVs isolation protocol from a starting cell conditioned medium (10 mL), we compared eight different methodologies by combining ultracentrifugation (UC), chemical precipitation (CP), size exclusion chromatography (SEC), and ultrafiltration (UF). Results The medium formula sustained both pbMECs and MAC-T cell growth and did not alter MAC-T cell identity. Transmission electron microscopy revealed that we obtained EV-like particles in five out of eight protocols. The cleanest samples with the highest particles amount and detectable amounts of RNA were obtained by using UF-SEC-UC and UC-SEC-UC. Conclusion Our chemically defined, EV-free medium sustains the growth of both pbMECs and MAC-T and allows the isolation of EVs that are free from any contamination by UF-SEC-UC and UC-SEC-UC. In conclusion, we propose a new culture system and EVs isolation protocols for further research on mammary epithelial EVs.