Increased Virus Production in Suspension Culture by a Trichoplusia ni Cell Line in Serum-Free Media

Abstract

Trichoplusia ni-derived BTI-Tn5B1-4 (Tn-5B1-4) cells were grown in 50 mL spinner flasks in four different formulations of serum-free medium (SFM). The objectives of this work were to determine the capacity of this cell line to grow and support virus replication in suspension culture in different SFM. Maximum cell densities exceeding 3.0 × 10 6 cells/mL were attained for all cell/medium combinations. Likewise, cell doubling times were all within 20-22 h. Infection studies with a wild-type isolate of Autographa californica nuclear polyhedrosis virus (AcMNPV) yielded between 156 and 187 occlusion bodies (OBs)/cell. The lethal effect of AcMNPV-OBs produced in this cell line in the various types of media was demonstrated in vivo using T. ni neonate larvae. The percent mortality at a single dose (1.0 × 10 6 OBs/mL) ranged from 40 to 60% at 4 days post-inoculation (p.i.). These results compared favorably with mortality rates found for AcMNPV-OBs produced in vivo. DNA quantification analysis, comparing polyhedra-derived viral DNA concentrations from an equal number of cell culture-and larval-derived OBs, was also determined. The larval-derived OB DNA concentration averaged 0.514 µg/10 8 OBs, whereas the cell culture-derived viral DNA concentrations ranged from 0.347 to 0.710 µg/10 8 OBs. These results suggest that no significant differences exist in DNA content between larvae-and cell culture-derived samples. Expression of the recombinant protein, secreted alkaline phosphatase (SEAP), ranged from 2.7 to 5.5 IU/mL for each cell/medium combination, which was consistent with previous static culture results for this same cell line in serumcontaining TNM-FH medium. Electron microscopic analysis of OBs produced in vivo or in vitro revealed no obvious differences in packaging or number of enveloped nucleocapsids per OB between samples. However, AcMNPV-OBs from infected larvae appeared physically smaller (1.28 ( 0.164 µm) than those produced in cell culture (1.72 ( 0.120). The Tn-5B1-4 cell line is adaptable to suspension culture in serumfree medium and is suitable for further development in the production of viruses and recombinant proteins on an industrial scale.

Screening of Transformed Insect Cell Lines for Recombinant Protein Production

“…By means of three stages of screening, it was demonstrated that the High Five insect cell line is a superior host system for the production of recombinant proteins using our new lepidopteran insect cell protein expression system. This result is in agreement with studies conducted by Farrell et al (1998; with several transformed insect cell lines and more comprehensive studies employing the baculovirus/insect cell system (Wickham et al, 1992;McKenna et al, 1997). A transformed clonal High Five cell line produced a maximum GM-CSF concentration of 46 and 27 mg/L in static and suspension culture, respectively.…”

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

“…Chakraborty et al (1999) assayed the production of PIBs of the Helicoverpa armigera singly enveloped nucleopolyhedrovirus in serum-free cultures of the insect cell line BCIRL-HZ-AM1, obtaining specific yields varying from 27 PIBs per cell in a serum-supplemented medium to 112 PIBs per cell in a serum-free medium. Higher specific yields of PIBs of Autographa californica multicapsid nucleopolyhedrovirus were obtained by McKenna et al (1997) in suspension cultures of Tn-5B1-4 cells in several serum-free media, where they attained up to 185 PIBs per cell.…”

Production of Insecticidal Baculoviruses in Insect Cell Cultures: Potential and Limitations

“…More recently, Nguyen et al (2011) have argued that the minimum volumetric yield for an economically feasible process of production of HaSNPV in insect cell cultures should be 40 times higher. The OBs yields effectively reached for several baculoviruses in serum-free cultures of different cell lines, according to the available information, ranged from 1 x 10 9 to 3 x 10 11 OBsL -1 , with cell specific yields ranging from 3 to 700 OBs per cell (Chakraborty et al, 1999;Gioria et al, 2006;McKenna et al, 1997;Micheloud et al, 2009;Rodas et al, 2005). The insecticidal potency of baculovirus occlusion bodies that are produced in insect cell cultures is a controversial topic.…”

“…The insecticidal potency of baculovirus occlusion bodies that are produced in insect cell cultures is a controversial topic. Some papers have shown that occlusion bodies produced in infected insect cell cultures are less potent than polyhedra produced by infection of insect larvae (Chakraborty et al, 1999;McKenna et al, 1997). However, this reduced activity may not be an intrinsic characteristic of the occlusion bodies produced in cell cultures, but related to the extraction method used.…”