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  • Title: Growth limitations in high density microcarrier cultures.
    Author: Butler M.
    Journal: Dev Biol Stand; 1985; 60():269-80. PubMed ID: 3899789.
    Abstract:
    The growth of anchorage-dependent animal cells on microcarriers has enabled treatment of these cell lines as quasi-suspension cultures allowing the production of high cell densities. Analysis of the parameters affecting the final cell yield shows that if an optimal microcarrier/cell seeding ratio is provided, the surface area for cell growth is unlikely to be limiting. The culture medium could become limiting to cell growth by nutrient depletion or through the accumulation of growth inhibitors. In batch cultures of MDCK cells the analysis of amino acid utilization showed that some amino acids are nearly completely depleted from the medium during the growth period. Glutamine in particular was rapidly consumed reflecting its likely role as a major energy source. The use of a novel perfusion system for such cultures produces much higher cell densities. In 500 ml cultures perfused at 1 ml/min most of the amino acids maintained a steady concentration. The glutamine concentration was reduced not completely depleted. Under these conditions the ammonia concentration of the medium increased to a value of 2.3 mM when cell growth ceased. This level of ammonia accumulation occurred in both perfused and unperfused cultures. Investigations of the parameters affecting the growth of BHK cells in microcarrier cultures showed the rapid utilization of glutamine which was optimal at an initial concentration of 4 mM. Glucose showed rapid but not complete utilization in these experiments. The ammonia accumulation in the medium was shown to be directly related to the initial glutamine concentration and under optimal conditions rose to a level of 2.5 mM. This level of ammonia was shown to be growth inhibitory when added to cultures during inoculation. Growth experiments using culture medium diluted with isotonic salt solutions showed that the final cell yield was not directly related to the medium dilution in either perfused or unperfused cultures. This suggests that the accumulation of a growth inhibitor is responsible for growth limitation rather than nutrient depletion. The measurements of ammonia accumulation suggest that this could be the growth inhibitor which limits the final cell yield.
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