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.
Instituto Butantan, Laboratório de Imunologia Viral, Av. Vital Brasil 1500, 05503-900 São Paulo, Brazil; Universidade de São Paulo, Escola Politécnica, Departamento de Engenharia Química, Laboratório de Células Animais, Av. Prof. Luciano Gualberto 380, 05508-900 São Paulo, Brazil.
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Ventini-Monteiro
Pall Life Science/ATMI/Artelis, rue de Ransbeek 310, 1120 Brussels, Belgium.
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Dubois
Instituto Butantan, Laboratório de Imunologia Viral, Av. Vital Brasil 1500, 05503-900 São Paulo, Brazil; Universidade de São Paulo, Escola Politécnica, Departamento de Engenharia Química, Laboratório de Células Animais, Av. Prof. Luciano Gualberto 380, 05508-900 São Paulo, Brazil.
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Astray
Pall Life Science/ATMI/Artelis, rue de Ransbeek 310, 1120 Brussels, Belgium.
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Castillo
Instituto Butantan, Laboratório de Imunologia Viral, Av. Vital Brasil 1500, 05503-900 São Paulo, Brazil; Universidade de São Paulo, Escola Politécnica, Departamento de Engenharia Química, Laboratório de Células Animais, Av. Prof. Luciano Gualberto 380, 05508-900 São Paulo, Brazil. Electronic address: carlos.pereira@butantan.gov.br.
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Pereira
D000818:Animals; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D018929:Cell Culture Techniques; D018914:Cells, Immobilized / Q000166:cytology*; D004331:Drosophila melanogaster / Q000166:cytology*; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera / Q000166:cytology*
D011994:Recombinant Proteins
Cell associated recombinant rabies vírus glycoprotein-rRVGP; Cell recovering; Insect cells; Scaling-up; Single-use fixed-bed bioreactor