This study attempted to establish a culture model to recreate the milk production pathway in bovine mammary epithelial cells (BMECs). BMECs were isolated from Holstein cows (nonlactating, nonpregnant, and parous) and were stored by cryopreservation. To separate the apical and basolateral compartments, BMECs were cultured on a cell culture insert with a collagen gel in the presence of bovine pituitary extract and dexamethasone to induce milk production and tight junction (TJ) formation. The culture model showed the secretion of the major milk components, such as β-casein, lactose, and triglyceride, and formed less-permeable TJs in BMECs. Moreover, the TJs were distinctly separated from the apical and basolateral membranes. Glucose transporter-1, which transports glucose into the cytoplasm through the basolateral membrane, localized in the lateral membrane of BMECs. Toll-like receptor-4, which binds to lipopolysaccharide in the alveolar lumen in mastitis, localized in the apical membrane. Beta-casein was mainly localized near the Golgi apparatus and the apical membrane. Moreover, milk components were almost secreted into the upper chamber of the cell culture insert. These findings indicate that this model has clear cell polarity as well as in vivo and is effective to study of milk production and the blood–milk barrier in lactating BMECs.
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