Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro.

Abstract

An epithelial cell line, designated COMMA-1D, was derived from mammary tissue of BALB/c mice in the middle of pregnancy. This Considerable effort has been directed toward the characterization of normal, preneoplastic, and neoplastic mammary cells and tissues in vivo and in vitro with respect to growth regulation, hormonal responsiveness, and biochemical properties (for a review, see ref.2). As part of this effort, a significant amount of work has been directed toward the development of cell culture methodology for establishing cell lines of mammary epithelial origin. Although numerous tumorigenic mouse mammary cell lines of epithelial morphology have been described (3-6), only two nontumorigenic mouse tnammary cell lines have been successfully established from nonneoplastic tissue (7-9). Neither of these lines produces any type of cellular outgrowth when transplanted in the mammary fat pads of syngeneic mice. Difficulties in maintaining the growth potential of cultured epithelial cells, coupled with complications due to overgrowth of fibroblastic cells, have dictated that studies on normal mouse mammary epithelial cells in vitro be confined to short-term primary cultures.To date, no established epithelial cell line retaining mammary gland-specific morphological and functional differentiation in vivo has been reported. We describe herein the isolation and characterization of an epithelial cell line, COMMA-1D, established from normal mouse mammary gland tissue. This cell line exhibits several properties specific for normal mammary gland function. MATERIALS AND METHODSIsolation of the COMMA-1D Cell Line. Mammary glands were collected from BALB/c mice in the middle of pregnancy. The tissues were minced finely and the cells were dispersed by collagenase treatment as described (10). The cells were plated in two 75-cm2 cell culture flasks in the presence of Dulbecco's modified Eagle's medium (DME medium), supplemented with 10% fetal bovine serum and insulin (5 /.g/ml). Cell cultures were incubated at 370C in a water-saturated atmosphere containing 7.5% CO2 in air. The cells were not passaged for several weeks. Thereafter, at irregular intervals over the following 5 months, overgrowing fibroblasts were removed from the epithelial monolayer by treatment with trypsin/EDTA (GIBCO) in phosphate-buffered saline. After 6 months, to facilitate the preferential growth of epithelial-like cells over fibroblastic cells, the culture was incubated in the presence of DME medium with 1% fetal bovine serum and and the following components (designated as "growth supplement"): insulin (5 pug/ml), transferrin (5 Ag/ml), fibronectin (1 pug/ml), epidermal growth factor (10 ng/ml), endothelial cell growth factor (10 ,ug/ml), and sodium selenite (50 nM) (11). After several passages (subculturing the cells 1:2 at 2-wk intervals), a sufficient number of cells was produced for use in the experiments described below. Presently, the COMMA-1D cell line is subcultured weekly with 1:2 splits and is maintained in DME medium containing 1% fet...

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