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/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.
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D001940:Breast / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D001943:Breast Neoplasms / Q000473:pathology; D002118:Calcium / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002875:Chromosomes / Q000648:ultrastructure; D003470:Culture Media / Q000494:pharmacology; D046508:Culture Techniques / Q000379:methods*; D004848:Epithelium / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D005260:Female; D006801:Humans; D007633:Keratins / Q000096:biosynthesis
D003470:Culture Media; D007633:Keratins; D002118:Calcium