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

J. Cell Set. D. Barngrover 0 J. Thomas 0 W. G. Thilly 0 0 Present address: Integrated Genetics , 31 New York Ave, Framingham, MA 01701 , U.S.A. fPresent address: Genentech, Inc. , 460 Point San Bruno Blvd, South San Francisco, California 94080 , U.S.A OF FRUCTOSE AND 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 L1S, containing 10 mMfructose 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 lOmM-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. bicarbonate-free Leibovitz; media; mammalian; cell culture; Vero cells; MDCK cells; fructose HIGH DENSITY MAMMALIAN CELL GROWTH IN MEDIUM: EFFECTS BIOCHEMISTRY D. BARNGROVER*, J. THOMASf AND W. G. THILLY The most common method in use today for buffering mammalian cell cultures is the bicarbonate/CO2 system, which is based on the following equilibrium: The use of bicarbonate is an attempt to mimic the buffering system of blood, but it has at least two major drawbacks. (1) The p/Q of bicarbonate is 6*1, which is far removed from the desired pH range of cell culture media (7-0-7-4). (2) Although bicarbonate is cheap, supplying a constant level of CO2 to cell cultures is definitely not, as it requires expensive CO2 incubators or fermentors. In high density cell culture, the production of CO2 during metabolism complicates the control of CO2 levels in the cell environment. A simpler solution to this buffering problem was developed by Leibovitz (1963). His L15 medium and a subsequent modification (Waymouth, 1981) were designed for use in cultures in free exchange with room air and were based on the following Fig. 1. Cell number (A) and pH of the medium (B) in microcarrier cultures of Vero cells. Cultures were grown in 100 ml total volume with Sg microcarrier/1 and the indicated medium with 10% horse serum. Cultures with DMEM or HiGEM were maintained in an atmosphere of 10 % CO2 :90 % air, while cultures with L15 were maintained in 100 % air. ( ) DMEM-glucose; ( ) HiGEM-fructose; (A) LIS-galactose; ( T ) L15-fructose. principles. (1) The use of high levels of the free base forms of amino acids (arginine, cysteine and histidine) to set the desired pH. (2) The use of galactose instead of glucose as a carbohydrate source, which results in a lower production of lactic acid and hence less need for buffering (Eagle, Barban, Levy & Schulze, 1958). (3) The use of pyruvate and alanine to generate the required bicarbonate for synthesis reactions. Earlier research from this laboratory has shown that the use of fructose instead of Fig. 2. Cell number (A) and pH of the medium (B) in microcarrier cultures of MDCK cells. Conditions (and symbols) were the same as in Fig. 1. glucose in cell culture media buffered with bicarbonate/CCh results in a lower lactic acid production and a more stable pH and lactate/pyruvate ratio (Imamura, Crespi, Thilly & Brunengraber, 1982). Therefore, it seemed reasonable to examine Leibovitz medium with the original galactose-containing formula or with fructose as carbohydrate source. Fig. 3. Cell number (A) and pH of the medium (B) in microcarrier cultures of Vero cells. Initial conditions (and symbols) were as in Fig. 1. After day 6, the medium was replaced with medium containing 2 % horse serum, and with fresh medium every 3 days thereafter (indicated by arrowheads). Another consideration with regard to analysing the effects of type and concentration of carbohydrate arises from the nature of energy sources in mammalian cell culture. Reitzer, Wice & Kennell (1979) have reported that for cultured HeLa cells, glutamine is the major energy source, not sugar. From the distribution of label from [U-14C]glutamine, Reitzer et al. concluded that a majority of the glutamine was completely oxidized, though a significant fraction of the label (13 %) did accumulate in lactate. They also concluded that when 2mM-fructose was used, 90% of it was metabolized via the oxidative pathway of the pentose phosphate cycle, with very little going through glycolysis. The utilization of glucose and glutamine is reciprocally regulated in human diploid fibroblasts (Zielke et al. 1978); that is, high levels of each reduce the utilization of the other. We were interested in determining if fructose and galactose had the same effect in Vero cells. One of the major byproducts of glutamine metabolism is ammonia, and earlier studies have shown that the accumulation of ammonia matches the decrease in glutamine (Imamura et al. 1982). We therefore measured ammonia production, as well as lactate, pyruvate and fructose levels in L15 microcarrier cultures with different kinds and concentrations of carbohydrates. Madin Darby canine kidney cells (MDCK) and Vero African green monkey cells were obtained from Flow Laboratories, Inc. (McLean, VA). Stocks were maintained in 490cm2 roller bottles in Fig. 4. Outline of amino acid and carbohydrate utilization for energy production. 10 Fig. 5. Cell number (A) and pH of the medium (B) in microcarrier cultures of Vero cells in L15 plus 10% horse serum with the indicated initial concentrations of fructose. Cultures (100 ml total volume) were maintained in free exchange with room air ( # ) 5 mM, ( ) 10mM, ( A ) 15mM, ( ) 20mM-fructose. 100 ml HiGEM (Flow Laboratories) with 10% horse serum (GIBCO, Grand Island, NY). MDCK cells were used between passages 64 and 66 and Vero between 139 and 145. All cells were routinely tested for mycoplasma by DNA fluorescence testing (Russell, Newman & Williamson, 1975) (Cell Culture Centre, Massachusetts Institute of Technology) and found to be negative. Fig. 6. Lactate levels in the medium of the Vero microcarrier cultures shown in Fig. 5. Symbols as in Fig. 5 for Figs 6-10. Dulbecco's modified Eagle's medium (DMEM), HiGEM (a modification of DMEM containing fructose instead of glucose), L15 and L15 without galactose were obtained as liquid media from Flow Laboratories. Media were supplemented with 4 mM (...truncated)