A comparison of methods for the production of monodispersed cell suspensions from human primary breast carcinomas
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Production of monodispersed cell suspensions from primary human breast tumors is difficult due to the predominant stromal composition of most breast tumors. Our studies were designed to optimize dispersion of breast tumors of known stromal content and histopathology. In a first series of experiments three enzymatic protocols were compared to disperse minced tissue: (A) treatment with collagenase (2 mg/ml) in the presence of 5% serum for 24 hours; (B) treatment with collagenase (6 mg/ml) and DNase (0.002%) in 10% serum for 3 hours; (C) treatment with collagenase (2 mg/ml) for 3 hours followed by pronase (0.075%) for l hour. Protocol A produced better cell yields than B or C for all tumors tested. The monodispersed cells were suspended in a 0.3% semi-solid agar with alpha modified Eagles medium (αMEM), 10% serum, and selected hormones, then layered over similarly enriched 0.5% semi-solid agar. The cells prepared by protocol A had a higher plating efficiency and larger average colony size than B or C. In a second series of experiments, protocol A was repeated and compared to two additional procedures: (D) treatment with collagenase (2 mg/ml) and hyaluronidase (1 mg/ml) in the presence of 5% serum for 24 hours; and (E) mechanical disaggregation. Protocol D exhibited a small but significant negative difference from A, while E was the least efficient in producing viable monodispersed cells from the tumors. All enzymatically monodispersed cells produced clonal growth in our agar system. However, mechanically dispersed cells gave growth in only 4 of 7 tumors. Protocol A, in addition to yielding the highest number of viable cells per gram of tissue, gave the highest plating efficiency of all protocols tested.
Keywordscell culture cell monodispersion electron microscopy human breast carcinoma
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