High density mammalian cell growth in Leibovitz bicarbonate-free medium: effects of fructose and galactose on culture biochemistry

Abstract

The most commonly used buffering system for mammalian cell cultures is a bicarbonate/CO2 system, which requires CO2 regulators and incubators to supply a constant level of CO2. As a replacement, Leibovitz developed a bicarbonate-free medium, L15, with relatively high levels of certain amino acids in the free base form. We found that a modified form of L15, containing 10 mM-fructose instead of galactose, supported high density growth of Vero and MDCK cells, with maintenance of a stable pH and lactate/pyruvate ratio. We report here investigations of Vero and MDCK cell growth and culture biochemistry at different concentrations of the two carbohydrates. The initial fructose concentration in the medium affected the eventual pH of the medium, the rate of production of lactic acid and ammonia, and the fructose utilization rate. The initial galactose concentration affected the growth rate but did not affect eventual culture pH, the rates of lactate and ammonia production, or the rate of its own utilization. Thus, Leibovitz' formula, modified to contain 10 mM-fructose, appears to yield satisfactory stability of culture pH and the lactate/pyruvate ratio. At all concentrations of galactose tested, the lactate/pyruvate ratio drifted out of the physiological range.