G. A. King

Engineering aspects of insect cell suspension culture: a review

SummaryThe development of insect cell suspension culture techniques for the production of insect pathogenic viruses and recombinant proteins has been reviewed, with an emphasis on process scale-up and reactor design considerations. The problems of culture media cost and insect cell shear sensitivity have also been addressed.

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

SummaryThe performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, Vg, in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, kLa, increased from 2.5 to 18.1 h-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 μm diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×107 cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 μg/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 μg/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×108 cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 μg antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.

Adaptation of insect cells to suspension culture

Abstract An assessment was made of the effects of successive passages, the concentration of fetal calf serum, and the presence of methyl cellulose and pluronic F-68 on the adaptation of Spodoptera frugiperda and Trichoplusia ni cells from stationary to suspension growth in shake flasks and an external loop airlift bioreactor. The studies showed that it takes about six successive passages to adapt Spodoptera frugiperda cells to suspension growth in shake flasks. The population doubling time was about 35 h with a maximum cell density of 5 × 106 cells/ml. In comparison, pour cell growth was observed for Trichoplusia ni cells in shake flasks. The presence of a higher serum concentration, methyl cellulose, and pluronic, while beneficial for cell growth, was not as important as the passage number. Preliminary experiments with an external loop airlift bioreactor suggest that Spodoptera frugiperda insect cells, previously adapted to shake-flasks, cannot survive air-sparging if the bubble diameter is less than 500 μm.

Growth of baculovirus-infected insect cells in microcapsules to a high cell and virus density

SummaryInsect cells (Spodoptera Frugiperda), infected with a temperature-sensitive mutant (TS10) of theAutographa Californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate-polylysine (PLL) microcapsules. It was possible to obtain intracapsular cell densities of 8× 107 cells/mL of capsules and virus concentrations of up to 109 IFU/mL of capsules. This was higher by a factor of 10 than that which could be achieved by conventional cell suspension culture.

Toxicity analysis of encapsulation solutions and polymers in the cultivation of insect cells

Microencapsulation of insect cells, hosts for baculovirus expression systems, requires that the encapsulation reagents and membrane-forming materials be non-toxic to the cells. Various encapsulation polymers (poly-l-lysine, chitosan and alginate) and solutions (KCl, CaCl2, CHES and sodium citrate) were tested for their toxicity toSpodopterafrugiperda cells. The effects of varying polymer molecular weight and concentration on cell viability were also investigated.

Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus

Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Graces). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×107 cells.ml−1, and a β-gal concentration of up to 104,000 unit.ml−1 were achieved.