Immobilization of 293 cells using porous support particles for adenovirus vector production

Abstract

Adenovirus vector production by anchorage-independent 293 cells immobilized using porous biomass support particles (BSPs) was investigated in static and shake-flask cultures for efficient large-scale production of adenovirus vectors for gene therapy applications. The density of cells immobilized within BSPs was evaluated by measuring their WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction activity. In shake-flask culture, 293-F cells, which were adapted to serum-free suspension culture, were not successfully retained within reticulated polyvinyl formal (PVF) resin BSPs (2 9 2 9 2 mm cubes) with matrices of relatively small pores (pore diameter 60 lm). When the BSPs were coated with a cationic polymer polyethyleneimine, a high cell density of more than 10 7 cells cm -3 -BSP was achieved in both static and shake-flask cultures with regular replacement of the culture medium. After infection with an adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP), the specific Ad EGFP productivity of the immobilized cells was comparable to the maximal productivity of nonimmobilized 293-F cells by maintaining favorable conditions in the culture environment.