Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production.

Insect cells are largely used for industrial production of vaccines, viral vectors and recombinant proteins as well as in research and development as an important tool for biology and bioprocess studies. They grow in suspension and are semi-adherent cells. Among the cell culture systems enabling scalable bioprocess the single-use fixed-bed iCELLis(®) bioreactors offer great advantages. We have established the conditions for Drosophila melanogaster Schneider 2 (S2) and Spodoptera frugiperda (Sf9) cells entrapment into the fixed-bed, cell growth and recover from the fixed-bed once high cell densities were attained. Our established protocol allowed these cells, at a cell seeding of 2×1E5 cells/microfiber carriers (MC) (3.5×1E6cells/mL; 1.7×1E4cells/cm(2)), to grow inside a 4m(2)/200mL fixed-bed attaining a concentration of 5.3×1E6 cells/MC (9.5×1E7cells/mL; 4.7×1E5 cells/cm(2)) for S2 cells or 4.6×1E6 cells/MC (8×1E7cells/mL; 4.1×1E5cells/cm(2)) for Sf9 cells. By washing the fixed-bed, entrapped cells could then be recovered from the fixed-bed at a high rate (>85%) with high viability (>95%) by increasing the agitation to 1200/1500rpm. Although the cell yields in the fixed-bed bioreactor were comparable to those obtained in a stirred tank (respectively, 1.3×1E10 and 2.5×1E10 total cells), S2 cells stably transfected with a cDNA coding for the rabies virus glycoprotein (RVGP) showed a 30% higher preserved rRVGP production (2.5±0.1 and 1.9±0.1µg/1E7 cells), as evidenced by a conformational ELISA evaluation. These findings demonstrate not only the possibility to entrap, cultivate to high densities and recover insect cells using a single-use fixed-bed bioreactor, but also that this system provides suitable physiological conditions for the entrapped cells to produce a cell membrane associated recombinant protein with higher specific biological activity as compared to classical suspension cell cultures.

Kinetic studies of recombinant rabies virus glycoprotein (RVGP) cDNA transcription and mRNA translation in Drosophila melanogaster S2 cell populations.

Recombinant rabies virus glycoprotein (RVGP) was expressed in cell membranes of stably transfected Drosophila S2 cells using constitutive and inducible promoters. Although with quantitative differences of RVGP expression in both systems, the cDNA transcription, as evaluated by relative RVGP mRNA levels measured by qRT-PCR, sustained the amount of RVGP producing cells and the RVGP volumetric (ΠRVGP) productivity. At the transition to the stationary cell growth phase, once the cell culture slowed down its rate of multiplication, an accumulation of RVGP mRNA and RVGP was clearly observed in both cell populations. Nevertheless, cell cultures performed under sub-optimal temperatures indicated that an envisaged increase in the RVGP production is not only dependent on cell growth rate, but essentially on optimal cell metabolic state.