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 fetal bovine serum (FBS) as a supplement, which naturally contains EVs. For this reason, the purpose of the study was to evaluate novel tools to investigate mammary gland EVs in vitro and in a 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 EV 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. Transmission electron microscopy revealed that we obtained EV-like particles in five out of eight protocols. The cleanest samples with the highest number of particles and detectable amounts of RNA were obtained by using UF-SEC-UC.

Conclusion: Our chemically defined, FBS-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. In conclusion, we propose a new culture system and EVs isolation protocols for further research on mammary epithelial EVs.