Temporal in vitro expansion of the luminal lineage of human mammary epithelial cells achieved with the 3T3 feeder layer technique

Abstract

Human mammary epithelial cells from reduction mammoplasties were serially propagated in vitro from single cells and/or cell clusters using the NIH 3T3 cell feeder layer technique. In seven passages 46 cell population doublings, corrected for plating efficiency were achieved. The plating efficiency of epithelial cells in the primary culture was 0.2%. During subsequent passages it rose to 10–12% and decreased sharply towards the end of the culture life. In the third and fourth passages temporal prevalence of luminal cells was observed. The critical conditions for prevalence of the 1uminal phenotype were found to be the initial dissociation and optimum seeding density during subculturing. In primary cultures, after optimum dissociation of 0.15 cm³ mammary tissue with 0.05% collagenase A (Boehringher‐Mannheim) in Eagle's MEM for 16 h at 37 °C, the yield on day 13 was 20 large colonies of 8–10 mm diameter. About 30% of the epithelial cells, which stained positively for the luminal cell marker cytokeratin 19, occupied colony centres. The remaining 70% were actin positive myoepithelial cells at the periphery. In subsequent passages, when using the optimum seeding density of 2×10⁵ cells per 60 mm culture dish, the proportion of luminal cells gradually increased to 90% on day 35 in the fourth passage. A sudden rise in the proportion of rapidly growing myoepithelial cells to 65% was observed in the fifth passage. In the sixth and seventh passage small colonies were formed, most of which contained at least one keratin‐19‐positive (luminal) cell. Cells of human breast carcinomas are considered to be of luminal origin. Therefore, the described approach can be useful in studies of cell and molecular biology of mammary carcinomas.