Two Different Serum-free Media and Osmolality Effect Upon Human 293 Cell Growth and Adenovirus Production

Abstract

Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called 'cell density effect', i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection. Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus production at cell densities higher than 1x10(6) cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when the infection was performed at 1x10(6) cells/ml, at 3x10(6) cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies, which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus productivity was only affected for osmolalities higher than 430 mOsm.

Responses of CHO-DHFR cells to ratio of asparagine to glutamine in feed media: cell growth, antibody production, metabolic waste, glutamate, and energy metabolism

“…The respiratory quotient of cells was constant in metabolic flux analysis, and the value was one (Sengupta et al 2011;Ferreira et al 2005;Duarte et al 2014;Ahn and Antoniewicz 2012). So, the specific rate of CO 2 production was equal to that of O 2 consumption.…”

Histone deacetylase inhibitor trichostatin A enhances the antitumor effect of the oncolytic adenovirus H101 on esophageal squamous cell carcinoma in vitro and in vivo

“…This leads to an inconsistency between the efficacy obtained from cell culture experiments in vitro and in clinical trials. Therefore, their efficacy of viral oncolysis need to be improved (42)(43)(44)(45). The present study examined whether the enhanced in vitro tumor cytotoxicity, mediated by TSA and H101 in combination, was also exhibited during EC1 cell tumor growth in vivo.…”

293 cell cycle synchronisation adenovirus vector production

“…It is important to notice that although infections were performed at different cell densities, namely, 3.0 Â 10 6 cell/mL in the control and 1.6 Â 10 6 cell/mL in the S phase synchronised cells, the specific productivity obtained in the synchronised cells was significantly higher when compared against the optimal cell concentration for infection (1.0 Â 10 6 cell/mL). 4 In addition, the time taken (139 h) to synchronise the cells led to poorer medium conditions (lower nutrients and higher by-products concentration at the time of infection). Therefore, the significant increase on the specific productivity observed can be correlated, albeit not uniquely, with the increase on the percentage of cells at the S phase of the cell cycle.…”

“…The cell density at infection is a very important parameter as it impacts volumetric titer. However, despite the fact that 293 cells can grow up to 8 Â 10 6 cell/ mL in simple batch mode operations, a maximum infection cell density of 1 Â 10 6 cell/mL has been reported as optimal for infection of 293 [4][5][6] and PER.C6 cells. 7,8 An approach to maintain AdV cell specific titer at increased cell density consists of infecting the cells after medium exchange.…”