Cytosol type electrolyte medium suitable for long term growth of human cells with very low membrane potential

Culture media for mammalian cells contain electrolytes at concentrations that approximate those of the extracellular fluid (ECF). However, we observed that HeLa strain cells can grow in a cytosol type, serum-free, high amino acid medium (KN13) in which the concentrations of K+, Mg2+ and Pi were increased to about 10 times those in ECF by replacing NaCl. The Ca2+ concentration of KN13 is 10% of that in ECF. The additions of Mg2+ and Pi are essential to prevent the harmful effect of a high concentration of K+ in the medium. Even an increase in the K/Na ratio to 7 and 9 permitted growth for at least 2 weeks and 4 days, respectively. HeLaP3 cells adapted to KN13 (HeLaK cells) have proliferated (4 fold/week) for 4 years in this medium. The Na,K-ATPase of HeLaK cells was the alpha 1 isoform. HeLaK cells in KN13 medium have a lower membrane potential (-3.0 +/- 6.1 mV) than that of the original HeLaP3 cells in DM160 (-26 +/- 10 mV).