Optimization of production of vesicular stomatitis virus (VSV) in suspension serum-free culture medium at high cell density

Abstract During the last decade, oncolytic viruses such as vesicular stomatitis virus (VSV) have gained tremendous popularity as efficient vaccines for infectious diseases as well as for the treatment of cancer. Our laboratory has developed two stable cell lines, 293SF-3F6 (derived from HEK293A cells) and SF-BMAdR cells (a variant of A549 that expresses the E1 region of human adenovirus). These two cell lines were adapted to grow efficiently in suspension culture and in serum-free medium. In this report we evaluated the production of a recombinant VSV expressing the green fluorescent protein (VSV-GFP) in these two stable cell lines. At a relatively low cell density of 500,000 cells per ml, 293SF-3F6 produced 4.6 times more infectious particles than SF-BMAdR cells. There was a positive correlation between volumetric virus titer and cell density up to 2.E + 07 cells/ml. A fed-batch process using an in-house medium and feed was developed to support the growth of 293SF-3F6 cells up to a concentration of 1.E + 07 cells/ml for infection at higher cell density and VSV production at high titer. Shifting the temperature from 37 °C to 34 °C at infection time improved VSV titer up to 3.3 fold. After scaling up the optimal condition from small scale (3 ml) to larger volumes (50 & 200 ml), the maximal volumetric titer obtained using the 293SF-3F6 cells was in average 2.9E + 10 extracellular infectious particles/ml. In conclusion, our data demonstrated that 293SF-3F6 cells, for which a cGMP master cell bank is available, is a performant cell line to scale up VSV production in suspension culture using serum-free medium.