A simplified method for passage and long-term growth of human mammary epithelial cells

Abstract

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.

Stem Cells in the Human Breast

“…A similar conclusion was reached in an independent study based on a completely different approach. Here, long-term growth of human breast epithelial cells was achieved for more than 50 generations (corresponding to a 1000-fold expansion of the original inoculum), based on a combination of serum supplementation and low Ca 2þ to overcome renewal inhibition (Soule and McGrath 1986). Intriguingly, under these conditions, clonal cells were released from the culture surface as free-floating cells in suspension, which could then be replated for further expansion.…”

“…Intriguingly, under these conditions, clonal cells were released from the culture surface as free-floating cells in suspension, which could then be replated for further expansion. The cells were characterized as breast epithelial cells because, under differentiating conditions (high Ca 2þ concentration), they formed domes in monolayer culture, a functional hallmark of luminal epithelial cells, and ductlike structures inside collagen gels (Soule and McGrath 1986).…”

Induction of Endothelial Cell Apoptosis by Solid Tumor Cells

“…Normal mammary epithelial cells were obtained from reduction mammoplasty and processed immediately after surgery as previously described [19]. Primary cultures were performed in Joklik-modified Eagle's medium supplemented with 10% heat-inactivated FCS, glutamine (2 mM) (GIBCO), nonessential amino acids (10 ml/L) (GIBCO), insulin (10 µg/ml) (GIBCO), cholera toxin (100 ng/ml) (Sigma), Cortisol (0.5 µM) (Sigma), epidermal growth factor (20 ng/ ml) (Sigma), and 100 IU/ml penicillin-streptomycin (GIBCO).…”

Application of floating cells for improved harvest in human chondrocyte culture

“…We postulate that the initially nonadherent cells represent the population of cells that did not have adequate space to adhere in primary cultures. Cells may also become less adherent during cell division (19). During this period, some cells detach from the culture dishes, possibly becoming floating cells.…”