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Recent studies from our laboratory have demonstrated that clonal cell proliferation of early passage Syrian hamster embryo (SHE) cells is optimal at a bicarbonate concentration in the culture medium of 8.9 mM (pH 6.65-6.75) under the experimental conditions reported. The purpose of the studies reported here was to examine whether morphological transformation induced by benzo[a]pyrene (BP) was enhanced under optimal culture conditions for SHE cell proliferation. Culture media of pH 6.70, 7.11 and 7.34 under incubator conditions of 10% CO2 in air were obtained by the addition of 0.75 (8.9 mM), 2.25 (26.8 mM) and 3.75 g/l (44.6 mM) of NaHCO3 respectively to a modified formulation of Dulbecco's modified Eagles medium. The frequency of morphological transformation of SHE cells was increased at 8.9 mM bicarbonate (pH 6.70) relative to media containing 26.8 or 44.6 mM bicarbonate (pH 7.11 and 7.34 respectively). Additionally, the isolate of embryo cells and lot of fetal bovine serum used supported transformation induced by BP at 8.9 mM bicarbonate (pH 6.70), but did not with media of higher bicarbonate concentration and pH. The duration of cell culture and the no. of colonies per plate influenced the amount of increase of morphological transformation observed at 8.9 mM bicarbonate relative to media of higher bicarbonate concentration. Initial studies have shown that a fraction of morphologically transformed colonies generated at reduced bicarbonate concentration were tumorigenic in newborn hamsters. These results are discussed in terms of the potential utility of low bicarbonate concentration cultured SHE cells for transformation studies.

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The effect of the pH, Na+ concentration and osmolality of the culture medium on early passage Syrian hamster embryo (SHE) clonal cell proliferation was examined. The pH of the medium was adjusted from 6.49 to 7.45 by addition of different amounts of NaHCO3 to the medium and incubating the cell cultures in a fixed atmosphere of 10% CO2/90% air. Our results indicate that clonal SHE cell proliferation is optimal at pH 6.65-6.80 while plating efficiency is independent of pH between 6.65 and 7.45. Adjustment of Na+ to that concentration in the medium (3450 p.p.m., 0.15 M) of the greatest NaHCO3 addition caused a moderate depression of cell proliferation over the entire pH series. Adjusting the osmolality of the culture medium to a constant value of 338 mOsm/kg did not alter the pH effect on cell proliferation. The pH of the medium also affected cellular and colony morphology. Below pH 6.90 there was an increase in the number of colonies which exhibited a transformed-like morphology ('altered' colonies). The 'altered' phenotype was characterized by a multilayered, criss-cross pattern of growth throughout the colony. This phenotype was stable upon sub-cloning into pH 6.65 medium but was reversible if sub-cloned into pH 7.36 medium. The induction of 'altered' colonies at low pH could be partially suppressed by Na+ or osmolality adjustment. These results are discussed in terms of optimizing growth conditions for SHE cells in order to enhance their usefulness for cell transformation studies. The induction of 'altered' colonies by low pH is also discussed relative to the involvement of pH regulation in tumor-promoter and growth-factor action on cells in culture.