We wished to determine methods that could reveal how the cell cycle is regulated in nonimmortalised, very early MEC cultures, and therefore examined ways of extending this brief window of proliferation that characterised the first 3�C5 days of primary cell culture, using cells from pregnant mammary glands. A key cell cycle determinant of breast epithelia is growth factors. Despite previous studies indicating that EGF and insulin are sufficient for the growth of normal MECs, these factors together with serum were not able to maintain more than 15% cells in S-phase after day-4 of culture. Moreover, adding fresh growth factors did not reactivate cell cycle. In the mammary gland in vivo, the growth factors that stimulate proliferation during puberty and pregnancy include IGFs, Amphiregulin, Fibroblast Growth Factor-2, Receptor Activator for Nuclear Factor k B Ligand and Wnt. To determine if these factors promote cell cycle in MECs, cells were cultured using amounts of bFGF, RANKL, or Wnt3a known to have a physiological effect. None of the growth factors showed any significant effect on the percentage of cells in S-phase compared to control cells. In addition to growth factors, ECM proteins can alter the proliferative response of luminal MECs. MECs were cultured on different ECM proteins and proliferation was assessed 4 d after Bortezomib supply plating cells on collagen I, laminin I, vitronectin, fibronectin or directly on the plastic of the culture dish. The proportion of proliferating cells on collagen I was approximately 8%, but less than 5% on the other substrata. Thus, the proliferation potential of MECs cannot be extended or enhanced by manipulating the culture medium by addition of growth factors, or by altering the 2D ECM protein substratum. Contact inhibition and spatial restriction are negative regulators of epithelial cell cycle. During cell-cell contact, the ligation of Ecadherin up-regulates the cell cycle inhibitor p27, blocking proliferation. Since MECs on collagen I were nearly confluent at day 4 when the proliferation levels were very low we reasoned that releasing contact inhibition by replating the cells might reactivate cell cycle. MECs were PR-171 Proteasome inhibitor replated at a density of approximately 2.56104 cells per cm2, either when the peak of cells were in S-phase, i.e.,45% at 2-days after isolation, or once proliferation had decreased, i.e.,10%, after 4 days. Proliferation was analysed each day for 4 days following replating, but at no point were more than 6% of cells in S-phase. Replating onto different ECM also did not promote proliferation ; similarly the addition of HGF, bFGF, RANKL, or Wnt3a to complete media either alone or in combination, to replated cells failed to promote proliferation. MECs harvested from different pregnancy time points also failed to proliferate following replating.