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Breast Cancer Research and Treatment

, Volume 3, Issue 1, pp 15–22 | Cite as

A comparison of methods for the production of monodispersed cell suspensions from human primary breast carcinomas

  • Greta J. Besch
  • William H. Wolberg
  • Kennedy W. Gilchrist
  • Joseph G. Voelkel
  • Michael N. Gould
Report

Summary

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.

Keywords

cell culture cell monodispersion electron microscopy human breast carcinoma 

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References

  1. 1.
    Hamburger AW, Salmon SE: Primary bioassay of human tumor stem cells. Science 197:461–463, 1977PubMedGoogle Scholar
  2. 2.
    Hamburger AW, Salmon SE, Kim MB, Trent JM, Soehnlen BJ, Alberts DS, Schmidt HJ: Direct cloning of human ovarian carcinoma cells in agar. Cancer Res 38:3438–3444, 1978PubMedGoogle Scholar
  3. 3.
    Von Hoff DD, Casper J, Bradley E, Trent JM, Hodack A, Reichert C, Makuch R, Altman A: Direct cloning of human neuroblastoma cells in soft agar culture. Cancer Res 40:3591–3597, 1980Google Scholar
  4. 4.
    Slocum HK, Pavelic ZP, Rustum YM, Creaven PJ, Karakousis C, Takita H, Greco WR: Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma, and lung tumor. Cancer Res 41:1428–1434, 1981PubMedGoogle Scholar
  5. 5.
    Ray DB, Horst IA, Jansen RW, Kowal J: Normal mammary cells in long-term culture. I. Development of hormonedependent functional monolayer culture and assay of α-lactalbumin production. Endocrinology 108:573–583, 1981PubMedGoogle Scholar
  6. 6.
    Gould MN: Mammary gland cell-mediated mutagenesis of mammalian cells by organ-specific carcinogens. Cancer Res 40:1836–1841, 1980PubMedGoogle Scholar
  7. 7.
    Yang J, Guzman R, Richards J, Jentoft V, DeVault MR, Welllings SR, Nandi S: Primary culture of human mammary epithelial cells embedded in collagen gels. J Natl Cancer Inst 65:337–341, 1980PubMedGoogle Scholar
  8. 8.
    McDivitt RW, Stewart FW, Berg JW: Atlas of Tumor Pathology. Second Series. Fascicle 2. Tumors of the breast. Armed Forces Institute of Pathology, Washington DC, 1968Google Scholar
  9. 9.
    Pavelic ZP, Slocum HK, Rustum YM, Creaven PJ, Nowak NJ, Karakousis C, Takita H, Mittelman A: Growth of cell colonies in soft agar from biopsies of different human solid tumors. Cancer Res 40:4151–4158, 1980Google Scholar
  10. 10.
    Gould MN, Cathers LE, Moore CJ: Human breast cellmediated mutagenesis of mammalian cells by polycyclic aromatic hydrocarbons. Cancer Res, in press, 1982Google Scholar

Copyright information

© Martinus Nijhoff Publishers 1983

Authors and Affiliations

  • Greta J. Besch
    • 1
  • William H. Wolberg
    • 1
    • 2
  • Kennedy W. Gilchrist
    • 1
    • 3
  • Joseph G. Voelkel
    • 1
  • Michael N. Gould
    • 1
    • 4
  1. 1.Department of Human OncologyUniversity of Wisconsin-Madison, Wisconsin Clinical Cancer CenterMadisonUSA
  2. 2.Department of SurgeryUniversity of Wisconsin-Madison, Wisconsin Clinical Cancer CenterMadisonUSA
  3. 3.Department of PathologyUniversity of Wisconsin-Madison, Wisconsin Clinical Cancer CenterMadisonUSA
  4. 4.Department of Human OncologyUniversity of WisconsinMadisonUSA

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