A method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue are plated in medium containing 1.05 mM Ca++ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca++ to overcome "renewal inhibition" and to stimulate growth. In low Ca++ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca++, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca++, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of (linear) growth. Cells released from monolayer were greater than 90% viable and yielded 10(5) cells/cm2 of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin for dissociation. Long-term free-floating cells were typical mammary epithelium: they formed domes and exhibited renewal inhibition, they produced ductlike formations in collagen gels, they contained epithelium-specific keratin filaments, and they were diploid.
The concentration of Ca++ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca++ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of alpha-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca++ to less than 0.08 mM. The reduction of Ca++ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca++ on growth and differentiation were specific (Mg++ and Mn++ variations were without effect) and reversible and in many respects resembled Ca++ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca++-induced coupling and uncoupling of proliferation and the program of differentiation, we proposed that Ca++ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu.
A stable cell line (MCF-7), derived from a pleural effusion of a patient with metastatic breast carcinoma, was maintained in these laboratories for more than 3 years in conventional monolayer culture. To further characterize the tumor origin of the MCF-7 line, cells were grown on collagen-coated cellulos sponges. On the three-dimensional sponge matrix, the cells formed clusters, ductlike structures, and lumina similar to the patterns observed in the antecedent primary tumor and in the pleural metastasis. The similarity between the original tumor and the cells grown in sponge suggested that the MCF-7 cells did in fact retain the potential to express the histologic patterns of tumor, even in the absence of stroma support. This study confirmed the utility of sponge culture for the investigation of the retention of tumor characteristics by cultured cells of neoplastic origin.
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