Sustained growth in primary culture of normal mammary epithelial cells embedded in collagen gels

Abstract

Normal mammary epithelial cells from BALB/cfC3H middpregnant mice were freed from stromal cell types by Percoll density gradient centrifugation after collagenase digestion and were then embedded within collagen gels. Sustained growth leading to an increase in cell number was accomplished in response to cholera toxin and high concentrations of horse serum. The extent of growth was found to be dependent on the horse serum concentration, the maximum growth being attained at 50%. A serum concentration of 12.5% horse serum and 2.5% fetal calf serum, along with cholera toxin at 0.01 Lg/ml, allowed maintenance but failed to cause any significant increase in cell number during the experimental period of 2 weeks. This same maintenance medium was used to determine the effects of various exogenously added steroids, protein hormones, and organ extracts on the proliferation of mammary epithelial cells in culture. Hormones failed to elicit any proliferative response, but extracts of kidney, brain, uterus, and spleen produced proliferative responses equal to or greater than the response obtained with 50% horse serum and cholera toxin. Kidney extracts prepared from midpregnant mice, virgin mice, and virgin mice given pituitary isografts all showed comparable activities, suggesting that the concentration of stimulatory factor(s) was not influenced by the hormonal status of the donor. Normal mammary epithelial cells that had undergone a 10-to 15-fold increase in cell number over initial values during 2-3 weeks in culture were passaged to secondary gel cultures. Outgrowths similar to those seen in primary culture were seen again in secondary culture. The present system provides a method for sustaining growth in culture of primary mammary epithelial cells from normal tissues.Classical endocrinology involving organ ablation and hormone therapy has developed the concept that pituitary, ovarian, and adrenocortical hormones are involved in mammary gland development (1, 2). Detailed analysis, however, requires an in vitro system in which the direct mitogenic effect of hormones can be assessed on appropriate target cells for prolonged periods. Early studies using organ and fragment cultures have shown insulin and growth factors to be the major factors responsible for mammary cell proliferation, but not the in vivo mammogenic hormones, an observation presenting a paradox with the in vivo situation (1, 2). Even insulin and the growth factors have been shown to cause only one or two rounds of DNA synthesis in vitro (3-6) and never a sustained growth.Recent studies indicate that, while estrogens have a direct mitogenic effect on a human mammary tumor cell line (MCF-7) (7) as assessed by an increase in cell number, estrogen mitogenicity cannot be demonstrated on the MTW9/PL mammary cell line (8). It has been proposed for the latter case that estrogen acts indirectly on the target cells through the production of intermediary growth factors that in turn promote growth of the mammary cell line (18). An inherent limitation of the use of e...

GAS6 is an estrogen-inducible gene in mammary epithelial cells

“…Mammary epithelial cells were separated from stromal cells by collagenase digestion and Percoll gradient centrifugation method (22). Total RNAs were isolated by TRizol (Invitrogen) method.…”

Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones.

“…To simulate the 3D condition of the normal mammary gland for culture experiments, several ECM-like biomatrices have been used, including collagen I matrices (rat tail collagen) (Emerman & Pitelka 1977, Hallowes et al 1980, Yang et al 1980, and reconstituted basement membrane (rBM; Englebreth-Holm-Swarm tumor or EHS; Matrigel) (Barcellos-Hoff et al 1989, Petersen et al 1992. Using these biomatrices, we asked whether normal human breast epithelial cells could respond and recapitulate certain aspects of their normal growth and differentiation program.…”

Linoleic acid, but not cortisol, stimulates accumulation of casein by mouse mammary epithelial cells in serum-free collagen gel culture.

“…Collagen Gel Culture. Collagen gel culture of mammary epithelial cells has been extensively described and reviewed elsewhere (24,30). The serum-free medium, which has also been described (27,28), was a 1:1 (vol/vol) mixture of Ham's F12 medium/Dulbecco's modified Eagle's medium (both from GIBCO) containing NaHCO3 at 1.2 g/liter and buffered with Hepes at 20 mM.…”

“…Toward this end we have attempted to determine the minimal requirement for casein accumulation by mammary epithelial cells from adrenalectomized and ovariectomized virgin mice using the serum-free collagen gel culture system developed in this laboratory (24)(25)(26)(27)(28). Since DNA synthesis has been shown to be a prerequisite to hormone-induced casein accumulation by mammary epithelial cells from virgin mice (29), we have used a two-step system in our studies.…”

“…Epithelial Cell Isolation. The cell dissociation was essentially as described (24,30 (24,30). The serum-free medium, which has also been described (27,28) 160 ,g/ml in all cultures, and, when used, the concentration of fatty acid was 10 ttg/ml.…”