THE BICARBONATE ION IS ESSENTIAL FOR EFFICIENT DNA-SYNTHESIS BY PRIMARY CULTURED RAT HEPATOCYTES

Bicarbonate in the culture medium is essential for DNA synthesis of primary cultured rat hepatocytes stimulated by epidermal growth factor (EGF). When primary cultured hepatocytes in supplemented Leibovitz L15 medium were placed in a 100% air incubator, no increase in DNA synthesis was observed even after stimulation by EGF. However, when these cells were cultured with NaHCO3 and EGF and placed in a 5% CO2:95% air incubator, a stimulus of DNA synthesis more than 10-fold greater than in cultures in air only was seen, and many mitotic figures could be identified. Furthermore, NaHCO3 added to supplemented DMEM/F12 medium enhanced the DNA synthesis of primary cultured rat hepatocytes in this medium. The ideal pH of the medium for DNA synthesis of cultured hepatocytes was in the range of 7.6 to 8.0. A dose response of NaHCO3 in several media showed that DNA synthesis of the cells increased as the concentration of NaHCO3 increased and that 25 to 30 mM NaHCO3 in the medium was optimal for the replication of DNA by primary cultured rat hepatocytes.