A Simplified Method for Passage and Long-Term Growth of Human Mammary Epithelial Cells

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$10^{5} cells/cm^2$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: (a) they formed domes and exhibited renewal inhibition, (b) they produced ductlike formations in collagen gels, (c) they contained epithelium-specific keratin filaments, and (d) they were diploid.