Growth and continuous passage of COMMA-D mouse mammary epithelial cells in hormonally defined serum-free medium

Abstract

Growth of the mouse mammary epithelial cell line designated COMMA-D has been studied in serum-free medium (SFM) formulated with Ham's F12 and Dulbecco's modified Eagle's medium (1/1) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM glutamine, gentamicin (50 micrograms/ml; basal medium) and supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), selenous acid (10 ng/ml), epidermal growth factor (20 ng/ml; EGF), 10 nM 3,5,3'-triiodothyronine, 50 microM ethanolamine, 1.0 nM 17 beta-estradiol, 65 microM glutathione, and ovalbumin (100 micrograms/ml). COMMA-D cells were able to undergo serial passage and continued to exhibit dome formation after 20 passages in SFM. Cells seeded at low density in SFM underwent four population doublings at low passage number in 1 week compared to six doublings for cells grown in medium containing insulin, transferrin, selenium, EGF, and 1% fetal bovine serum. After many passages in SFM, the growth rates of cells were similar to those in serum-supplemented medium used for stock culture. Deletion of insulin or EGF from SFM resulted in cell growth similar to that of cells seeded in basal medium alone. When cells were seeded in basal medium without added supplements, addition of insulin or EGF resulted in 29 and 22%, respectively, of the number of cells grown in SFM for 5 days. However, when insulin and EGF were combined in basal medium, the cell number at 5 days was 83% of that in SFM. When insulin was deleted from SFM, COMMA-D cells became responsive to insulin-like growth factors I and II. The growth-promoting characteristics of EGF and transforming growth factor alpha were compared in SFM and were not distinguishable, showing identical dose-response curves. When incorporation of [3H]thymidine was used as an assay of cell growth, saturating levels of basic fibroblast growth factor (20 ng/ml) showed a stimulation 1.35 times greater than EGF (20 ng/ml). When EGF and fibroblast growth factor were combined, the stimulation was 1.75 times greater than EGF alone suggesting that COMMA-D cells are responsive to multiple classes of growth factors. COMMA-D cells seeded in basal medium supplemented with insulin, transferrin, and selenous acid have been used to detect mitogenic activity present in extracts of hypothalamus, uterus, and pituitary. The results show that COMMA-D cells can be grown long term in a hormonally defined serum-free medium and that maximal mitogenic effects were seen only with the addition of two or more growth factors.