STUDY OF ADENOVIRUS PRODUCTION IN SERUM-FREE 293SF SUSPENSION-CULTUREBY GFP-EXPRESSION MONITORING

The red-shifted S65T mutant green fluorescent protein (GFP) was used t o compare the adenovirus (Ad) production and post-infection survival o f 293SF and 293S cells in serum-free and serum-containing flask cultur es, respectively. The GFP-expressing vector permitted the quantificati on of both the level of GFP expressed by infected cells and the infect ious viral content of the cultures by flow cytometry in a simple, fast , sensitive, and reliable way. The GFP has the main advantage of fluor escing without any substrate addition. Infected cultures showed the co existence of two populations of fluorescent cells, high-fluorescence c ells (HFCs) and low-fluorescence cells (LFCs), in proportions that var ied between 20 and 75 hpi. The gradual increase in the number of LFCs at the expense of HFCs correlated well with the increase in the number of dead cells. This relationship could be used for the continuous mea sure of a culture's viability with the appropriate on-line instrumenta tion. The post-infection death rate of infected 293SF cells was higher than that of infected 293S cells, but the level of GFP fluorescence i n viable, highly fluorescent cells was similar in the two infected cel l lines. The number of infectious viral particles (IVPs) was quantifie d in less than 24 h by an infection assay of 293S cells in wells with viral particles extracted from the culture samples, and the results we re more reproducible (+/-10% variation) than those generally reported for conventional plaque assay titrations or end-point dilutions. The v iable cell-specific IVP concentrations were for most experiments simil ar, indicating again that the difference between the two cell lines wa s their unequal post-infection viabilities, not the virus production b y the infected living cells.