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Abstract: Cells of the human embryonic kidney cell line (HEK 293) were grown as suspended aggregates in stirred vessels and infected with a recombinant adenovirus vector (Ad-TH-GFP). Regular spherical aggregates with the mean diameter less than 300 μm and a viable cell density greater than 5×106 cells·ml−1 were readily achieved after 9 day culture in spinner flasks. The HEK 293 cells growing as suspended aggregates could be efficiently infected by Ad-TH-GFP at an MOI of 10 with a prolonging infection time up to 144 hour post-infection (hpi). The time profile of Ad-TH-GFP production was strongly corresponding to the infection process with a virus concentration peak occurred consistently at 144 h after infection. And the infected aggregates essentially maintained spherical in shape, the portion of dissociated cells from the infected aggregates was less than 5% at 144 hpi. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5 L stirred tank bioreactor and infected with Ad-TH-GFP at a density higher than 1×107 cells·ml−1 resulted in a similar Ad-TH-GFP production kinetics, but a much higher virus yield approximately at 5.7×1011 GTU ml−1 at 144 hpi to that of the infected spinner flask cultures. These results demonstrate the feasibility for using suspended cell aggregates as an immobilization system to facilitate perfusion in stirred tank bioreactors, and improve volumetric productivities by eliminating the cell density effect. [Copyright &y& Elsevier]