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Systematic Optimization of the Clonal Growth of Human Primary Breast Carcinoma Cells¹

Department of Human Oncology, Wisconsin Clinical Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin 53792

Human breast carcinomas have been one of the most difficult tumor types to culture in agar-based clonogenic assays. This fact has limited their clinical applicability. We have used statistically motivated experimental designs to systematically improve the clonal culture of enzymatically monodispersed primary human carcinoma cells in an anchorage-independent agar system. Based upon an initial comparison of two basal media, we selected one which gave the best colony growth and then sought to optimize the individual additives in the medium. Hydrocortisone, fetal bovine serum, and red blood cells all improved both plating efficiency and median size of colonies derived from breast carcinoma cells. Next, the concentrations of these three components were simultaneously idealized using response surface methodology. By these methods, it was found that the optimal concentration of hydrocortisone was 0.35 µg/ml, fetal bovine serum was 6.5%, and red blood cells was 2.1 x 10⁷ cells/ml. Using these culture conditions, we have achieved plating efficiencies of 0.39% and 0.19% for colonies with diameters greater than 50 (50 cells) or 70 (130 cells) µm, respectively.

¹ This work is supported in part by USPHS, NIH, National Cancer Institute Grants CA20432, CA30295, and CA35464. G. J. B. was a National Cancer Institute predoctoral trainee supported by Grant 5T32 CA09206.

² To whom requests for reprints should be addressed.

Received 2/ 5/85. Revised 10/22/85. Revised 1/22/86. Accepted 1/29/86.