Derivation of conditionally immortal cell lines
Enrichment procedures have been described for partially purifying epithelial cells from the other types of cells that are present in the mammary gland [28]. These methods use enzymatic treatment and differential centrifugation to obtain a clean, fibroblast-free population of primary epithelial cells, and did not work well in our hands with the transgenic mice. We therefore developed a novel, simpler method that is based on the unpublished observation of one of us (BB) that, on a collagen-coated surface, fibroblast outgrowth from mammary explants requires the presence of serum, whereas epithelial cells grow out easily in a chemically defined medium. Initially, explant cultures were established from mammary glands excised from several lines of mice, with different transgene copy numbers. This procedure was most successful with tissue from the lowest copy number line SV40-2, and subsequent work was carried out with mammary tissue from this line. Mammary explant cultures were prepared by slicing the glands into 1 mm square pieces, seeding into collagen type 1 coated flasks and culturing for 2 weeks at the permissive (33°C) and semipermissive temperature (37°C) until outgrowths were well established (Fig. 3a). The explants were removed and the cells cultured until the islands stopped expanding in size. During this time period dome-like structures began to appear only in the cultures grown at 37°C (Fig. 3b).
The primary cultures were routinely passaged as clumps of five to 10 cells, because single cell passaging results in the appearance of cells with a different morphology that was reminiscent of a fibroblastic phenotype. These cells are now routinely passaged every 3-4 days as clumps and divided 1:3 or 1:4. These cells, which we have designated KIM-2, have been maintained in culture at 37°C for over 60 passages and have a stable phenotype both on plastic and collagen as assessed by immunocytochemistry.