A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E1 and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three or four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelial morphology, distributiop of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells.Growth of human mammary epithelial cells (HMEC) in medium MM, which contains hormones, fetal bovine serum, and conditioning factors from human epithelial and myoepithelial cells, has been reported previously (1,2). This medium supports rapid monolayer growth of normal HMEC for three or four passages with 1:10 splits. However, good clonal growth in MM requires cholera toxin and a fibroblast feeder layer (3). Tumor-derived cells respond similarly, but their doubling potential is more variable.Although medium MM makes possible many types of experiments with cultured HMEC (4-7), its usefulness is limited by lack of definition and by early cellular senescence. Other culture systems described for HMEC are generally even more restrictive (reviewed in ref. 8).This report describes long-term growth of HMEC with pituitary extract as the only undefined supplement and shorter-term monolayer and clonal growth in a defined medium. These results were achieved by applying to HMEC methods previously used in similar studies with other cell types (9-11). MATERIALS AND METHODSMedia and Solutions. Medium MM was prepared as previously described (2), except that HS578 Bst conditioned medium and cholera toxin were omitted. Medium MCDB 170 (Table 1) is an optimized formulation for HMEC. Medium MCDB 202a differs from MCDB 170 in concentrations of four components: cysteine HCl, 2.0 x 10-4 M; glutamine, 1.0 X 10-3 M, sodium pyruvate, 5.0 x 10-4 M, and zinc sulfate, 1.0 x 10-7 M. MCDB 202a is a minor modification of previously published medium MCDB 202 (13). MCDB 170 and MCDB 202a were both prepared according to the protocol for MCDB 104 (12), with appropriate adjustment of concentrations. These media were prepared withou...

A new medium (MCDB 104) has been developed which will support clonal growth of WI-38 cells at concentrations of serum protein as low as 25 micrograms per ml (equivalent to 0.05% serum). The principal factors responsible for reduction of the protein requirement are: (a) adjustment of all nutrient concentrations in medium F12 to experimentally determined optimum values for WI-38 cells; (b) supplementation with trace elements; (c) replacement of hypoxanthine and folic acid with adenine and folinic acid; and (d) coating of the culture surface with polylysine. Individually, many of these modifications exert only a small effect on cellular growth at reduced protein concentrations, but collectively their effect has been very substantial. Other strains of fibroblast-like human diploid cells from amniotic fluid, fetal lung and newborn foreskin also will grow at reduced concentrations of serum protein in the new medium.

Clonal growth of WI-38 cells with a plating efficiency of 45% has been achieved in a synthetic nutrient mixture (MCDB 102) supplemented with either whole or dialyzed fetal bovine serum precisely. Deviation by a factor of three in either direction from the optimum concentration (9.0 x 10(-5) M) eliminates essentially all clonal growth. A high concentration of glutamine (2.5 x 10(-3) M) is also needed for optimum clonal growth.