Primary Culture of Human Mammary Epithelial Cells Embedded in Collagen Gels

Abstract

Human mammary epithelial cells were dissociated from mastectomy tissues. The contaminating fibroblasts were removed by the use of Percoll density-gradient centrifugation, which utilizes the difference in buoyant densities between epithelial cells and fibroblasts. A preparation highly enriched for mammary epithelial cells was then embedded in collagen gel and cultured in Ham's F12 medium containing 12.5% horse serum, 2.5% fetal calf serum, 0.1 μg cholera toxin/ml, an extract prepared from human male urine (6 μg protein/ml), and a hormone combination of 10 μg insulin/ml, 10 μg human placental lactogen/ml, 1 μg aldosterone/ml, and 0.5 μg hydrocortisone/ml. Sustained growth leading to an increase of tenfold to thirtyfold in cell number over the initial value was accomplished in primary culture, and this growth was maintained even after passage to secondary culture. Deletion of either the urine extract or the hormone combination resulted in less than optimal growth. Subsequent studies showed that hydrocortisone alone could replace the hormone combination. In addition, urine extract could be replaced by extracts prepared from human kidneys or brains. The collagen gel system provides a reproducible and consistent method for sustained three-dimensional growth of mammary epithelial cells from human breast tissue in primary as well as passaged cultures.