Proteomic study of secretomes from cellular agriculture for milk production

Cultivation of bovine mammary epithelial cells (bMECs) is regarded as a potential future source of milk constituents; however, there is a research gap concerning the investigation of compositional similarity between such secretomes and milk, and furthermore, how culture conditions can be optimized to make it a feasible strategy for potential production of milk constituents. To perform such an initial, detailed quantitative proteomic study of secretomes from in vitro grown bMECs, these cells were isolated from the mammary tissue of two lactating cows and cultured on Matrigel®-coated inserts in a trans-well system. Cells were treated with or without prolactin at the basolateral sides, and the secretomes together with media from the apical side were collected and subsequently compositionally characterized for their protein contents and label-free quantitative proteomics. Using sensitive nLC-timsTOF Pro MS/MS, more than 500 proteins were identified, though in very low total concentrations, across proteomes from the four secretomes collected from prolactin- or non-prolactin-treated cells. The proteins included many cellular proteins and characteristic milk proteins, but with caseins accounting for less than 1% of total protein abundance. Secretomes from prolactin-treated bMECs showed significant (P < 0.05) up-regulation of 351 different proteins, including milk proteins such as αS1-casein, κ-casein, β-lactoglobulin, lactotransferrin, and osteopontin, indicating a positive regulation of lactation-associated proteins with prolactin treatment. These findings elucidate a potential for future cellular agriculture using in vitro cultured bMECs for the production of milk constituents, together with challenges, such as upscale and sufficient lactogenesis.