In Vitro Cell. Dev. Biol. 27A:585-594. July 1991 © 1991 Tissue Culture Association 0883-8364/91 $01.50+0.00 ESTABLISHMENT AND CHARACTERIZATION OF BOVINE MAMMARY EPITHELIAL CELL LINES C. ANNE GIBSON, JUAN R. VEGA, CRAIG R. BAUMRUCKER 1, CORI S. OAKLEY, ~ u CLIFFORD W. WELSCH Department of Dairy and Animal Science, The Pennsylvania State University, 302 Henniag Building, University Park, Pennsylvania 16802 (C. A. G., J. R. V., C. R. B.) and Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824 (C. S. 0., C. W. W.) (Received 28 September 1990; accepted 8 March 1991) SUMMARY One bovine mammary epithelial cell clone, designated PS-BME-C1, and two bovine mammary epithehal cell lines, designated PS-BME-L6 and PS-BME-L7, were derived from mammary tissue of a pregnant (270 day) Holstein cow. The cells exhibit the distinctive morphologic characteristics of mammary epithelial cells and express the milk fat globule membrane protein, PAS-III. They form domes when cultured on plastic substrata and acinilike aggregates when cultured on a collagen matrix. These cells are capable of synthesizing and secreting o~-lactalbumin and a-sl-casein when cultured on a collagen matrix in the presence of insulin, cortisol, and prolactin. The cells have a near-normal diploid number and do not grow in suspension culture. When transplanted to the cleared mammary fat pads of female athymic nude mice, the cells readily proliferate forming noninvasive palpable spherical cellular masses within 8 wk after inoculation. The cells may become a useful tool to study the regulation of ruminant mammary epithelial cell growth and differentation. Key words: bovine; mammary; epithelial cell line; cell growth; differentiation. INTRODUCTION Many in vitro studies of growth, differentiation, and lactation have used murine mammary tissue as a model. These studies have used explants (Nicholas et al., 1988), primary cell culture (Ethier, 1986; Barcellos-Hoff et at., 1989), and estabhshed epithelial cell lines such as the COMMA-D cell line (Danielson et al., 1984). Although the rodent has proved to be a good model, it is not always possible to make assumptions about the behavior of bovine mam- mary epithelial cells based on a rodent model. Researchers using tissue culture to study the bovine mammary gland have used explants (Collier et al., 1977; Goodman et al., 1983), organoid culture (Baumrucker et al., 1988; O'Brien et al., 1981; Park et al., 1978), and primary cell culture (Mackenzie et al., 1982; Shamay and Gertler, 1986). McGrath (1987) has advo- cated the preparation of large numbers of primary ceils from a single donor, enriching the preparation for epithelial cells followed by cryopreservation. The lack of a well-characterized bovine mammary epithelial cell line has limited the study of growth and differentiation in these secretory cells. Schmid et al. (1982) have used an immortalized bovine mammary epithelial cell line to study various cytoskeleton proteins, and recently Turner and Hung (1989) have established a bovine mammary epithelial cell line that has been transformed by transfection with SV 40, but a manuscript has not appeared. Our objectives in this study were to isolate and grow a bovine mammary epithelial cell line and to initially characterize the cells to establish 1 To whom correspondence should be addressed. 585 them clearly as bovine mammary epithelial cells. In this report, we describe the isolation and characterization of three bovine mam- mary epithelial cell lines. These cell lines designated as Penn State Bovine Mammary Epithelial Clone 1 and Penn State Bovine Mam- mary Epithelial Cell Lines 6 and 7 (PS-BME-C1, PS-BME-L6, and PS-BME-L7) exhibit properties specific for normal mammary epi- thelial cell physiology. MATERIALS AND METHODS Isolation of the PS-BME cell lines. Mammary tissue was ob- tained at slaughter from a nonlactating, pregnant (270 day) Hol- stein cow from the Pennsylvania State University dairy herd. The tissue was transported to the laboratory, minced, and digested for 2 h at 37 ° C with 400 IU/ml collagenase (Worthington, Freehold, NJ), 0.04% DNAse (Sigma, St. Louis, MO), and 0.005% hyaluroni- dase (Sigma) in Hanks' buffered salt solution (HBSS; Sigma) con- taining 5 U-5 gg/ml penicillin-streptomycin(Sigma) and 50 ttg/ml gentamycin (Sigma). The final volume of HBSS was 5 ml/g of tissue. The digested tissue was filtered through nylon mesh and washed with HBSS. Bovine mammary epithelial cells were isolated using a preformed percoll gradient (Imagawa et al., 1982). The isolated cells were cultured in RPMI 1640 (Sigma) containing 2% fetal bovine serum (FBS), 5 mM sodium acetate, 10/.tg/ml trans- ferfin (Sigma), 5 U-5 #g/ml penicillin-streptomycin, and 50 #g/ml gentamicin. A mixed population of cells was observed. Selective trypsinization and the use of serum-free medium (SFM: RPMI 1640 with 0.5 mg/ml bovine serum albumin (BSA) 10 #g/ml transferrin, and 5 mM sodium acetate) prevented overgrowth of the