Monolayer and suspension cultures of HEK 293 cells for recombinant adenovirus production for gene therapy

Abstract

In recent years, the need for the production of recombinant adenovirus for gene therapy is constantly increasing. To produce E1-deficient recombinant adenovirus in high titer, HEK (Human Embryo Kidney) 293 cells which support the propagation of E1-deficient adenovirus are required. Because the 293 cells transfected with the left 11% of the adenoviral genome express E1A and E1B viral gene products, they are permissive for growth of E1-deficient adenovirus. The 293 cells are obtained at the passage number of 31 from ATCC, and the cell of successive passages are required to produce adenovirus in large scale. Thus, we investigated the effect of passage number of 293 cells on cell growth and adenovirus production. In monolayer culture, the cell size of 293 cells became smaller and the specific growth rate (μ) became faster as the passage number increased. The μ at the passage number of 73 was increased approximately 2.5-fold, compared with that at the passage number of 43. In monolayer culture, the adenovirus production was also influenced by cell passage number. The virus titer obtained from 293 cells at the passage number of 66 was increased approximately 10-fold, compared with that at the passage number of 44. Furthermore, the optimal time for virus harvest post-infection became shorter for the cells at the later passage. In suspension culture, the cell size and μ of 293 cells did not change significantly as passage number increased up to 35. Taken together, the passage number of 293 cells influenced cell growth and adenovirus production in the monolayer culture. Furthermore, replication-competent adenovirus was not detected during cell passages tested in monolayer culture.