Effects of hormones and growth factors on human mammary epithelial cells in collagen gel culture.

Abstract

Studies on growth regulation were performed on normal human mammary epithelial cells obtained from a number of different sources: ( a ) reduction mammoplasties; ( b ) areas away from the primary lesion in mastectomies; and ( c ) colostrums. These cells were embedded inside collagen gels and cultured in Ham's F-12 medium containing serum, insulin, cortisol, cholera toxin, and urine extract or epidermal growth factor. Sustained growth leading to an increase of 10- to 30-fold in cell number over the initial value in 2 to 3 weeks has been accomplished in primary culture. Similar growth was also achieved in secondary culture after passaging the cultures with a split of one to eight. The relative contributions of the supplements to overall growth have been assessed by deletion experiments. The magnitude of effect of deleting any single additive was variable among the samples tested, but, in all cases, omission of any one additive resulted in reduction of the final cell number. This suggests that each of the additives must be involved in the stimulation of growth of normal mammary epithelial cells in culture. The three-dimensional outgrowths derived from single cells and small clumps were of different morphologies. In general, the outgrowth ranged in morphology from a duct-like appearance to a spherical mass. Colostrums gave rise to predominantly spherical mass-type outgrowths, whereas mammoplasties and mastectomies gave rise to more heterogeneous types. These differences in outgrowth morphology may be a reflection of the possibility that different cell types may give rise to different colony shapes. The collagen gel system provides a reproducible and consistent method for sustained three-dimensional growth of human normal mammary epithelial cells in primary culture and has been used to analyze growth regulation as well as morphogenesis in the present study.