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キーワード:
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要旨:
In the standard process for production of influenza vaccines MDCK cells are cultivated on microcarriers (Cytodex 1) in GMEM, supplemented with fetal bovine serum (FBS) and peptone. However, drawbacks of serum are high costs, variable quality and the potential introduction of prions and microbial contaminants into the process. For infection with influenza virus trypsin is added to increase virus-infectivity. Serum contains trypsin-inhibitory substances and therefore is removed by media exchange before infection. A cultivation in serumfree medium could avoid this media change and reduce costs and contamination risks. The aim of this work was to cultivate MDCK cells in serum-free medium EX-CELLTM MDCK on Cytodex 1 microcarriers for production of influenza virus and optimization of cultivation conditions to increase cell and virus yield. The protocol for MDCK cell-cultivation in GMEM had to be changed for cultivation in EX-CELLTM MDCK medium as the cells did not attach to the microcarriers. Overcoming problems concerning adhesion and growth of the cells in the serum-free medium turned out to be most problematic and time consuming. The trypsinization-procedure used for passaging the cells seemed to be the key factor and was varied by two ways, resulting in a decrease of trypsin-concentration for attachment of the cells on the microcarriers. Results obtained were also influenced by the lot-to-lot-quality of the EX-CELLTM MDCK medium.Cultivation was performed in spinner flasks, Sixfors multi-fermenter system and a 5 L bioreactor. For infection with influenza virus two different influenza strains, equine (A/Equi 2 Newmarket 1/93 H3N8) and human (A/PR/38 H1N1), were used. Finally, cultivation of MDCK cells on microcarriers in EX-CELLTM medium in a 5 L-bioreactor and infection with human influenza virus resulted in a high virus-titer of 2.9 log HA-units/100 L, which was determined by a haemagglutination (HA)-assay. Analysing media-composition during cultivation showed sufficient nutrient concentration and low concentration of inhibitory lactate and ammonia allowing a process without medium exchange.
