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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...

Sustained growth of normal mouse mammary epithelial cells in primary culture, leading to an increase in cell number, in response to growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] or cholera toxin has been achieved by embedding the cells inside collagen cells. Inclusion of agents known to increase the level of cellular cAMP have been found to be favorable for mammary epithelial cell proliferation. Cholera toxin is by far the best of all of the agents tested (prostaglandins E1 and E2, isoproterenol, theophylline, and dibutyryl cAMP). When growth factors (EGF or FGF) are added with cholera toxin, a synergistic effect resulting in a response much greater than with either of them alone is seen. This synergism was best seen in normal mammary epithelial cells from nonpregnant mice. The extent of this synergistic effect was found to be less in normal cells from pregnant mice, suggesting that these cells may be less responsive to EGF during pregnancy. Tumor cells were found to be rather inconsistent in their responses to EGF and cholera toxin, ranging from a minimal response, similar to that of normal cells from pregnant animals, to a maximal response, similar to that of normal cells from nonpregnant animals.