Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Transfection and protein expression in mammalian cells

摘要

This is the first report of transient transfection of suspended cells with purified plasmid DNA in bioreactors or spinner flasks. DNA/calcium phosphate complexes were pumped or injected directly into stirred cultures of the immortalized human embryo kidney cell line 293 (HEK-293) which had been adapted to growth in suspension. We identified culture conditions suitable for this approach and modified the protocol for the generation of precipitate complexes, based on our earlier work. In order to stabilize the ‘DNA-vehicle’-complex in the culture medium, we identified pH ranges and ion-concentrations which prevent dissolution or aggregation of the precipitate particles. Such conditions maintained suspended fine particles in spinners or bioreactors for up to 6 hr. During that period, cells and precipitate complexes interacted sufficiently to allow DNA transfer and subsequent expression of recombinant protein. In a simple 5 day batch process, with a starting density of 0.3 × 106 cells mL-1, about 0.5 mg L-1 of a recombinant tissue plasminogen activator variant was observed.