Establishment of bovine mammary epithelial cells, an in vitro model for milk protein synthesis.

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We isolated two kinds of mammary epithelial cells by a collagenase method from Holstein cows in late and early pregnancy, respectively.(1) Mammary epithelial cells isolated from a cow in late pregnancy (270-d).Primary cultured cells increased 4-5 times within 5days in 10 % FCS DME on collagen coated dishes. The cellular contaminants, especially fibroblasts, could be removed because fibroblasts detached earlier from collagen-coated dishes than mammary epithelial cells following 0.1 % trypsin treatment Mammary epithelial cells formed an alveolar-like structure on Matrigel, while only flat shaped cells were observed on type-I collagen. IGF-I stimulated the proliferation of the cells at a dose of 62 ng/ml in low FCS condition. This effect was inhibited by concomitant addition of lactogenic hormones to the culture.(2) Mammary epithelial cells from a cow in early pregnancy (26-d)To overcome the problem of fibroblast outgrowth, cells were cloned by limiting dilution. One colony arising from single cell with epithelial morphology and showing positive cytokeratin staining was obtained. The clone showed a population doubling time of approximately 24 h and have been cultured more than 20 passages without showing any sign of senescence. The effect of lactogenic hormones, insulin, prolactin, and dexamethasone, on the proliferation of these cells was not observed when cultured on collagen type-I coated dishes. The mammary cells were differentiated when cultured on Matrigel with lactogenic hormones.