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Abstract

The potential of Madine Darby Canine Kidney (MDCK) cells for the production of influenza vaccines have been greatly explored in the past decades. Recently, a new MDCK cell line (MDCKsus) that was able to grow in suspension in a fully defined system was established (Lohr, V., et al., 2010. Vaccine. 28:6256-64). This new cell line is suitable for the development of robust industrial manufacture of viral based products. In our laboratory we are interested in the production of canine adenoviruses (CAV) for gene therapy. In fact, we have validated the use of MDCK cells for the amplification of CAV-2 vectors in monolayer cultures (Santiago et al., 2010 ASGCT). In this work, we investigated whether the MDCKsus cell line was suitable for the amplification of CAV-2 vectors in single-cell/small aggregate suspension cultures. We tested four different serum-free media: two formulations of SMIF8, AEM, ExCELL MDCK and OptiPRO SFM. The maximal cell densities achieved varied from 2 x 106 cells/ml (SMIF8 and OptiPRO SFM) to 5 x 106 cells/ml (AEM and ExCELL MDCK). A first screening of CAV 2 production in the four media resulted in low amplification of the vector (below 30) but the reduction of the multiplicity of infection led to an increase of the amplification ratio to values up to 250 (and cell specific productivities up to 750 IP/cell). Although further optimization experiences are in progress, our data shows that canine adenovirus vectors can be effectively produced in serum-free suspension cultures of MDCKsus cells. It is anticipated that the CAV-2 production system described here can be successfully used to obtain the high titer vector stocks required for gene therapy trials.