A simplified method for passage and long-term growth of human mammary epithelial cells |
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Authors: | Herbert D Soule Charles M McGrath |
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Institution: | (1) Department of Tumor Biology, Michigan Cancer Foundation, 110 E. Warren Avenue, 48201 Detroit, Michigan |
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Abstract: | Summary 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 105 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: (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. |
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Keywords: | mammary calcium cell culture |
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