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 × 2 × 2 mm cubes) with matrices of relatively small pores (pore diameter 60 μm). 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 non-immobilized 293-F cells by maintaining favorable conditions in the culture environment.

Fig. 1

Relationship between the number of viable non-immobilized 293-F cells in the exponential growth phase in static cultures and difference in absorbance at 450 nm and at 650 nm (A₄₅₀–A₆₅₀) in the WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reduction assay

Fig. 2

Growth of 293-F cells immobilized within reticulated polyvinyl formal (PVF) resin biomass support particles (BSPs) without (a) and with (b) polyethyleneimine treatment in static (circle) and shake-flask (filled rectangle) cultures. Cell density in the untreated BSPs in shake-flask culture is not shown

Fig. 3

Adenovirus vector production by 293-F cells immobilized within polyethyleneimine-treated BSPs in static (a) and shake-flask (b) cultures. Immobilized cells at an initial density of 2.1 × 10⁷ cells cm⁻³-BSP in static culture and 1.1 × 10⁷ cells cm⁻³-BSP in shake-flask culture were respectively infected with Ad EGFP at a multiplicity of infection (MOI) of 10 transductional units (TU) cell⁻¹. The culture medium was replaced completely with 15 mL of fresh medium every 2 days. The time courses of Ad EGFP yield in the BSPs (open rectangle), cumulative vector yield in the culture broth (open triangle), and total yield (filled diamond) are presented. Bars represent the means ± SD of 3–5 different determinations