Insect cells–Baculovirus system: Factors affecting growth and low MOI infection (original) (raw)
Related papers
Continuous production of baculovirus in a cascade of insect-cell reactors
Applied Microbiology and Biotechnology, 1990
Insect cells (Spodopterafrugiperda) were cultured in a continuous stirred-tank reactor. The effluent was led to a cascade of another two reactors, each containing half the volume of the cell-growth reactor, where the cells were infected with Autographa californica nuclear polyhedrosis virus. For about 10 days production of 107 polyhedra (virus particles embedded in a protein capsule) per cm 3 was achieved. This short production time compared to previous experiments involving an analogous system with a single infection vessel of equal volume to the cell-growth vessel is ascribed to the accelerated occurrence of the so-called passage effect (a decrease of infectious virus with time). From the results of a computer model it was concluded that this passage effect was accelerated by the change in residence time distribution as compared to the earlier experiments.
Biotechnology and Bioengineering, 1992
An experimental study has been carried out to investigate the effectiveness of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cells were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2 % fetal bovine serum (FBS), SF-900, and a serumfree medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of 8. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown in 10% serum-supplemented medium. 0 1992 John Wiley & Sons, Inc.
Baculovirus and Insect Cell Expression
The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described throughout this book. In this chapter, the most widely techniques are summarized and the reader is directed to detailed information found elsewhere in this book. Furthermore, many useful tips and the author's personal preferences that are rarely published are discussed in this chapter along with quantitative methods to characterize cell growth, baculovirus infection, and metabolism.
A mathematical model of baculovirus infection on insect cells at low multiplicity of infection
Acta biochimica et biophysica Sinica, 2004
The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of infection (MOI), which is based on the multi-infection cycles of insect cell infection at low MOI described. A mathematical model for the amount of viruses released from primary infected cells and the amount of free viruses before secondary infected cells release viruses has been developed. Comparison of the simulation results with the experimental data confirms qualitatively that this model is highly reasonable before secondary infected cells release viruses. This model is considered as a base for further modeling the entire complicated infection process.
Production of selected baculoviruses in newly established lepidopteran cell lines
in Vitro Cellular & Developmental Biology-animal, 2001
One key to the in vitro mass production of baculoviruses is the development of insect cell lines capable of producing high levels of extracellular virus (ECV) and/or occlusion bodies (OBs). For this study, 34 newly established cell lines from 10 lepidopteran species were screened for their ability to produce ECV and OBs from a variety of baculoviruses. The selected baculoviruses included: the alfalfa looper virus (AcMNPV); the celery looper virus (AfMNPV); the velvetbean caterpillar virus (AgMNPV), the bollworm virus (HzSNPV), the diamondback moth virus (PxMNPV), and the beet armyworm virus (SeMNPV). ECV titers were determined using TCID50 assays (50% tissue culture infectivity dose), with the presence or absence of OBs being noted. For AcMNPV, 28 new cell lines were tested, with eight producing AcMNPV ECV titers of 1.1–47.3×106 TCID50/ml and 11 producing OBs. For AgMNPV, six new cell lines were tested, with all producing AgMNPV ECV titers of 3.5–62.3×106 TCID50/ml and generating OBs. For HzSNPV, four new cell lines were tested with three lines producing HzSNPV ECV titers of 1.4–5.0×106TCID50/ml, but none generating OBs. For PxMNPV, 10 new cell lines were tested with seven generating PxMNPV ECV titers of 4.7–232.6×106TCID50/ml and eight producing OBs. Lastly, using qualitative or semiquantitative methods, homologous cell lines were tested for AfMNPV and SeMNPV production, all of which produced OBs. Overall, many of the cell lines tested were found to produce OBs and generate moderate to high levels of ECVs of one or more baculoviruses.
Biotechnology and Bioengineering, 1991
Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL-41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.035, and 0.034 h-', respectively, at 33°C. The IPL-41 medium supported the highest maximum cell density (10.6 x lo6 cells/mL) compared to 3.5 x lo6 and 8.7 x lo6 cells/mL with the Grace's and serum-free media, respectively. In temperature shiftdown experiments with a temperature-sensitive baculovirus (ActslOYMlCAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL-41 (5.1 x lo7 PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x lo6 vs 4.1 x lo5 PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02. At MOI of 2.0 virtually no increase in virus or CAT titer was observed. This result is contrary to those obtained at constant-temperature (27°C) infection and cell culture, in which higher virus titers and recombinant protein expression are obtained at higher MOI.
Strong buffering capacity of insect cells. Implications for the baculovirus expression system
Cytotechnology, 1995
Insect cells are widely used for expression of a variety of different proteins by using the baculovirus expression system. The applicability of this system depends on production of proteins which have biological properties similar to their native counterparts. One application has been the expression of viral capsid proteins and their assembly into empty capsid structures to provide new viral immunogens which retain complex antigenic sites. An important parameter for efficient folding and assembly of proteins into viral procapsids may be the intracellular pH, particularly for acid-labile particles such as foot-and-mouth disease virus (FMDV). Benzoic acid was used as an effective indicator of intracellular pH in insect cells and 3-O-methyl glucose to measure cell volumes. We have determined the intracellular volume of the Spodopterafrugiperda IPLB-Sf21 insect cells 0.50 :t: 0.08 pL per cell. Using the distribution of [laC]-benzoic acid, we show that the intracellular pH remains constant at pH 7.0 when the cells are grown in media with pH values ranging from 6.2 to 6.8 and, moreover, is not affected by baculovirus infection. These results suggest that insect cells are suitable to express and produce acid-labile structures via the baculovirus expression system and that assembly of proteins and viral procapsids could occur.
Baculovirus and Insect Cell Expression Protocols
Methods in molecular biology, 2016
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