We are analyzing https://link.springer.com/article/10.1007/bf02623435.

Title:
A simplified method for passage and long-term growth of human mammary epithelial cells | In Vitro Cellular & Developmental Biology - Plant
Description:
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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28 years and 1 months (reg. 1997-05-29).

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Keywords {🔍}

cells, article, growth, mammary, google, scholar, culture, cell, human, pubmed, cas, vitro, epithelial, soule, medium, calcium, privacy, cookies, content, mcgrath, primary, publish, search, method, longterm, access, stampfer, normal, cancer, data, information, log, journal, research, cellular, biology, passage, tissue, monolayer, renewal, inhibition, cultures, freefloating, epithelium, chapter, discover, proliferation, proc, natl, usa,

Topics {✒️}

month download article/chapter long-term free-floating cells human cell line tissue culture medium human mammary epithelium cell culture related subjects typical mammary epithelium mouse mammary epithelium privacy choices/manage cookies full article pdf tumor-specific properties yielded 105 cells/cm2 serum-free growth maintaining calcium levels cell cycle cell physiol long-term growth european economic area time determined partly produced ductlike formations government printing office epithelial cells pleural effusion derived cholera toxin stimulation conditions privacy policy breast tissue rapid clonal growth overcome “renewal inhibition” exhibited renewal inhibition accepting optional cookies terminal differentiation occurs lymphoblast proliferation mcgrath rights michigan cancer foundation low ca++ media tumor biology primary culture main content log hormonally defined medium monolayer cultures suitable journal finder publish extended serial passage article soule organoids dissociated culture medium article log check access instant access cells continue

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