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Zusammenfassung:
Every year epidemic influenza virus infections cause up to 500.000 deaths and 3 to 5 million cases of severe illness with enormous healthcare expenses and non-productive time. Because of a high demand of vaccines effective production of virus material is inevitable. Since several years there are increasing efforts to develop mammalian cell culture-based production systems to overcome the limitations and drawbacks of existing vaccine production in hen’s eggs. Typically, large scale influenza vaccine production consists of two distinct phases: the cultivation of cells in suspension or microcarrier systems and the subsequent virus replication phase. To optimize these processes and to identify bottlenecks we focus on analyzing cell physiology during both cell growth and virus infection. A suitable means to investigate cell populations on the single-cell level is flow cytometry. Using this technique we are able to measure cell cycle distributions and the degree of infection as well as necrosis and apoptosis. So far, these parameters were mainly determined qualitatively in fundamental biological research. In contrast, only a limited number of detailed investigations were done in bioprocess engineering applications. Here, we investigate the replication of influenza A virus in adherent Madin Darby canine kidney (MDCK) cells. The distribution of the cell cycle phases was determined by measuring the DNA content per cell; the degree of influenza infection in the cells was quantified via antibody binding. Necrosis was measured by propidium iodide exclusion while apoptosis could be quantified by reduced cellular DNA content and binding of a fluorescent pancaspase inhibitor (FLICA). Based on a better understanding of the complex biological mechanisms involved in cell growth and virus replication we eventually aim to improve vaccine production and to increase process productivity.
