Effects of Ammonia on Cell Growth

Although ammonia inhibited cell growth in all these studies, the sensitivity of the growth rate to ammonia concentration varied among the cell lines used. The extent of ammonia inhibition seems to be more severe for primary cells, whereas transformed cells tolerate higher ammonia levels. For 3T3 cells, ammonia levels as low as 0.6 mM reduced cell growth by 60%. However, when the cells are transformed, the reduction was reduced to 15% (64). Addition of 2 mM ammonia reduced growth by 30% for BSC-1 monkey epithelial cells (65). For established cell lines, inhibitory effects of ammonia were observed at higher ammonia concentrations. Hassell et al. (66) reported reduction of cell growth at 2 mM for a variety of cell lines. Several reports indicate ammonia inhibition for hybridoma cells with varying severity (6,41,50,52,67-82). Inhibitory concentrations of ammonia were observed to be in the range of 2 to 10 mM in these studies. For BHK cells, even lower concentrations of ammonia resulted in growth inhibition. Wentz and SchUgerl (83) reported a 80% reduction at 1 mM ammonia, and Butler and Spier (84) reported a 75% decrease in growth at 3 mM of ammonia in BHK cells. For CHO cells, the inhibitory ammonia levels seems to be higher. Kurano et al. (85) obtained a 50% reduction in growth at 8 mM ammonia. Some cell lines are reported to be more resistant to ammonia. For instance, Schneider (81) observed only a 20% reduction in growth at 6 mM ammonia for HeLa cells, and Hansen and Emborg (86) did not observe any inhibition at up to 8 mM for a CHO clone. Other factors that can influence the inhibitory effects of ammonia on growth are the serum level and cell adaptation. Holley et al. (65) for instance, observed that increasing the level of serum from 0.1 to 10% reduced the ammonia inhibition. Miller et al. (52) observed adaptation of hybridoma cells to 8 mM ammonia. Maiorella et al. (54) used serial passaging in the presence of 5 mM ammonia and demonstrated that the resulting cells survived better in 10 mM ammonia. Adaptation to high ammonia concentrations can be used for selection of ammonia-resistant cell lines.

The effects of ammonia on cell growth in most of these studies reported were quantified as the reduction in cell density, and only a few studies evaluated the specific growth rates as a function of ammonia concentration. Ammonia inhibition on the specific growth rate can be described by a second-order inhibition model:

where i and i0 are the growth rates in the presence and absence of ammonia, respectively; [NH/j] is the initial ammonia concentration; and Ka is the inhibition constant. The square root of Ka corresponds to the ammonia concentration at which the specific growth rate decreases by 50%. Figure 2 presents the effect of ammonia on cell growth rate and cell density for 163.4.G5.3 murine hybridoma cells. Equation 2 describes the cell growth rate accurately with parameters i0 = 0.037/h and Ka = 24 mM2. For comparison, Ka value of 26 mM2 was reported for the CRL-1606 hybridoma cell line (87), however, the value reported for the SB-4082 hybridoma cell line was almost one order of magnitude lower (Ka = 3.2 mM2) (65).

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