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要旨:
Recently, the suitability of the avian suspension cell line AGE1.CR.pIX as a producer cell line for vaccinia virus in a chemically-defined process was demonstrated. Normally, one would expect that higher cell densities should lead to higher virus yields. However, for many viruses the so called cell density effect results in reduced yields at high cell densities and thus limits the production capacity. To understand whether a cell density effect occurs in this vaccinia production process, we monitored cell physiology of AGE1.CR.pIX cells before and after virus infection. A recombinant modified vaccinia virus with an enhanced green fluorescence protein (egfp) insert was used.
Cultures were infected after 3 or 5 days of batch growth at 4x106 cells/mL and 8x106 cells/mL, respectively, by adding an equal volume of production medium. We analyzed five parameters: i) cell viability, ii) cell cycle distribution by measuring DNA content, iii) apoptosis via annexin V staining, iv) infection status via detection of intracellular egfp and v) virus titer.
Experimental data did not indicate a cell density effect. No significant alterations of the cell population neither in terms of cell viability, nor in cell cycle proportions or fraction of apoptotic cells were observed in high cell density cultures. Cell-specific virus yields were also not impaired. In contrast, although cell concentration was doubled, virus yields increased by a factor of three and a maximum concentration of 5x108 viruses/mL was reached. As a cell concentration of 8x106 cells/mL can easily be achieved in a batch process, the use of fed-batch or perfusion cultivations could lead to even higher virus yields.
