Growth of human mammary epithelial cells on collagen gel surfaces

We have developed a method for sustained growth of human mammary epithelial cells in monolayer cultures. Epithelial organoids derived from solid breast tissues were grown on the surface of thin (approximately 1 mm) collagen gel layers in an enriched growth medium supplemented with hormones, growth factors, fetal calf serum, and horse serum. To transfer the cultures, the collagen layers were dislodged and digested with collagenase. The monolayers of cells released into suspension were then dissociated into single cells using trypsin-ethylene-diaminetetraacetate. Dissociated single cells were repleted with 75 to 95% efficiency onto collagen layers or tissue culture plastic surfaces. The dissociated cells could also be cryopreserved and reactivated with greater than 80% plating efficiency on collagen layers. Normal human mammary epithelial cells grown under these conditions progressed through 12 to 15 population doublings. The population-doubling times for normal and malignant mammary cells on collagen layers were 34 and 65 hr, respectively. After reaching confluence, cells in some cultures, derived from either normal or malignant tissues, penetrated the gel surface and grew into the collagen. Within the gels, the cells became organized into three-dimensional tubular structures. The use of collagen layers eliminates a major problem in growth of human mammary epithelial cells in culture, difficulty in efficient dissociation, and cell transfer from monolayers.