Dwight E. Lynn

NOVEL TECHNIQUES TO ESTABLISH NEW INSECT CELL LINES

SummaryThe success of insect cell culture is demonstrated by reports of over 500 established cell lines. While established procedures that can be used for developing new cell lines exist, these usually require some fine-tuning for various tissue sources. This paper attempts to depict some of the variations that can be applied.

Available Lepidopteran Insect Cell Lines

This chapter lists the known cell lines from Lepidoptera, largely based on previous compilations of insect cell lines published by W. Fred Hink. More than 320 lines from 65 species are listed. The official designation is given for each cell line as well as the species, tissue source, and, when known, the susceptibilities to baculoviruses.

Fluorescent brightener inhibits apoptosis in baculovirus-infected gypsy moth larval midgut cells in vitro

Abstract Fluorescent brighteners significantly lower the LC50 and LT50 in a variety of nucleopolyhedrovirus–insect host systems. In larvae of the gypsy moth, Lymantria dispar (L.), a European NPV strain of virus (LdMNPV) does not normally replicate in the midgut, but addition of a fluorescent brightener (Calcofluor M2R) to the virus suspension results in productive infections. In the current study, we show that LdMNPV also does not replicate in a larval midgut primary cell culture system unless a fluorescent brightener (Blankophor P167) is added. Morphological and cellular changes characteristic of apoptotic cell death were noted in infected midgut cells in vitro. We used the TUNEL assay to measure apoptosis in virus-challenged midgut cell cultures at 24–48 h post-inoculation. A significant decrease in apoptotic midgut cells was noted in the presence of 0.01 M brightener. The inhibition of apoptosis and presumptive inhibition of shedding of infected midgut cells in the presence of fluorescent brightener in the insect midgut appeared to promote virus replication and are likely to be partly responsible for enhancement of LdMNPV activity that is observed in gypsy moth larvae.

Transfection of insect cell lines using polyethylenimine

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

Methods for the development of cell lines from insects

This paper describes techniques for the development of continuous cell lines from insect embryos and other tissues. Methods include how to obtain aseptic primary tissues, how to dissociate the tissue, and then how to maintain the primary culture until cells grow consistently. The relative advantages of different tissues (embryos, ovaries, etc.) in establishing insect cell lines are discussed and several insect tissue culture media are described.

Development of insect cell lines: Virus susceptibility and applicability to prawn cell culture

Insect cells have been successfully cultured in vitro as continuous cell lines for over 35 years. The media, culture methodology and conditions have been well resolved such that, for many insects, new cells lines can be routinely developed. Factors that are considered important for developing insect cell cultures are described as well as some of the history that led to the success. One of the major rationales for developing insect cell lines was for the study of insect viruses. This was particularly true for species of Lepidoptera from which over 900 viruses have been reported. Since many species of Lepidoptera are serious agricultural and forestry pests, effects have been made to utilize some of these pathogens as biological pesticides. Cell cultures are important in this endeavor since viruses require a living cell to reproduce. Of the known insect viruses, the most intensely studied have been the baculoviruses. In addition to their potential for controlling insect pests, they also have been used as expression vectors for producing recombinant proteins. Details of some of these experiments are described. Finally, experiences with insect cells are considered in relation to efforts to develop prawn cell cultures.

A new insect cell line from the Colorado potato beetle

Dear Editor: Insect cell lines have become increasingly useful in studies on insect pathogens, in developmental biology, and as a tool in the production of recombinant proteins through the use of the baculovirus expression vector. As of 1989, over 400 insect cell lines had been reported, yet only 3 of these were from beetles (Lynn, 1991). This despite the fact that it has been estimated that more species of beetles exists than all other animals combined. Since that review, several additional coleopteran cell lines have been reported (Barcenas et al., 1989; Mitsuhashi, 1989; Diibendorfer and Liebig, 1992; Stiles et al., 1992), but beetle cell lines are still greatly underrepresented relative to their numbers and importance. In this report, I describe a new cell line developed from eggs of the Colorado potato beetle, Leptinotarsa decimlineata (Coleoptera: 5

Effects of temperature on the susceptibility of insect cells to infection by baculoviruses.

Three insect cell lines were tested for susceptibility to baculovirus infection by use of a typical endpoint assay procedure. Cell lines from Spodoptera frugiperda (IPLB-Sf21AE), Lymantria dispar (IPLB-LdEIta), and Heliothis virescens (IPLB-HvE6s) in 96-well tissue culture plates were each infected with dilutions of extra cellular virus suspensions of the Autographa californica nucleopolyhedrovirus (AcMNPV). In addition, the L. dispar and H. virescens cells were also infected with L. dispar nucleopolyhedrovirus, and Helicoverpa zea nucleopolyhedrovirus, respectively. Each cell/virus combination was incubated at three temperatures: 22, 27 and 32 degrees C and wells were scored for positive infection (presence of occlusion bodies in cell nuclei) at 2 to 4 day intervals for up to 4 weeks. The resulting data were analyzed by the Spearman-Kärber method, providing virus titers for each combination of virus, cell line, and temperature. The results were categorized by accuracy (assuming the highest titer achieved was the most accurate) and by rapidity of maximum titer. AcMNPV reached the highest titer in each line at 22 degrees C although equivalent titers were reached with both AcMNPV and HzSNPV in the HvE6a line at all three temperatures. This line actually reported about 100-fold less AcMNPV than the other two lines with the same virus sample. Alternatively, the Sf21AE and LdEIta lines reached 10-fold higher titers at the lowest temperature as compared with the higher temperatures, although also at a slower rate.

Slow cell infection, inefficient primary infection and inability to replicate in the fat body determine the host range of Thysanoplusia orichalcea nucleopolyhedrovirus

Thysanoplusia orichacea multicapsid nucleopolyhedrovirus (ThorMNPV) carrying an enhanced green fluorescent protein (EGFP) gene expression cassette (vThGFP) was used to study host-range mechanisms. Infection kinetics showed that vThGFP replication in Sf21 cells was too slow to suppress cell growth. Wide-host-range Autographa californica MNPV (AcMNPV) could speed up vThGFP infection and enhance the vThGFP infection rate in Sf21 cells. The enhancement was not due to recombination, as no recombinant virus was isolated from co-infection by plaque assay. No improvement of vThGFP infection in Sf21 was found by AcMNPV cosmid transactivation assay. However, culture medium from Sf21 cells infected with AcMNPV did enhance vThGFP replication in Sf21. Third-instar larvae of Spodoptera frugiperda, S. exigua and Helicoverpa zea were not killed by feeding with vThGFP polyhedra but were killed by intrahaemocoelic injection using budded viruses (BVs). This suggested that insufficient BVs were generated during the primary infection in the midgut. vThGFP infected haemocytes, tracheae and Malpighian tubules but not fat bodies of larvae of S. frugiperda, S. exigua and H. zea. Third-instar S. frugiperda larvae co-infected by injection with vThGFP and vAcDsRed2, an AcMNPV expressing a red fluorescent protein gene, showed EGFP expression in the fat body. This result suggests that vAcDsRed2 could help vThGFP to replicate in the fat body or trans-activate EGFP expression in the fat body. All these results suggested that slow cell infection, insufficient primary infection and inability to replicate in the fat body control the host range of ThorMNPV.

Comparative susceptibilities of twelve insect cell lines to infection by three baculoviruses.

Insect cell lines are commonly used to study insect viruses (Blissard, 1996) and have often been considered for production of certain virus species as biopesticides or for recombinant proteins. In the case of proteins, the Spodoptera frugiperda line IPLB-Sf21AE and the Sf-9 clone derived from it have been most frequently used with the baculovirus expression vector derived from Autographa californica nucleopolyhedrovirus (AcMNPV). However, many other lepidopteran cell lines are permissive to infection by this virus. In the present study, twelve insect cell lines (Table 1) were tested for susceptibility to baculovirus infection by use of a typical endpoint assay procedure (Lynn, 2002) using extracellular virus (ecv) suspensions of three nucleopolyhedroviruses (NPVs), including A. californica NPV (AcMNPV), Anagrapha falcifera NPV (AfMNPV), and Anticarsia gemmatalis NPV (AgMNPV). Each of these viruses was originally isolated from a noctuid moth species and infect many species from this family as well as some other Lepidoptera. By using the same virus dilutions on each line and scoring for infection, the relative susceptibility of the cells can be determined for each virus. Cultures were maintained in 25cm Greiner Cellstar tissue culture flasks (Frickenhausen, Germany) at 26 C on a weekly subculture interval. The specific media used for maintenance was the one that provided the best growth for each line and was used in the assays since this provides the most consistent results (Lynn, 2000). Cell identity was confirmed by isozyme analysis. The resulting titers obtained with the various cell/virus combinations are presented in Fig. 1. Because of its wide use in baculovirus research, the Sf21 cell line was used as a benchmark in Fig. 1 by dividing the TCID50/ ml (titer) obtained on each line by the value obtained with this line for each virus. This method eliminates the differences in actual titers for each virus suspension, making comparisons between viruses easier. Sensitivity to infection is one aspect of virus replication that can vary between cell lines. I have previously shown the TN-R line was more than 100-fold more susceptible to AcMNPV than the more commonly used Sf21 cell line (Lynn, 1992a). This was confirmed in the present study. The TN-R line was also most susceptible to AfMNPV which is not surprising since the AfMNPV virus has a great deal of genetic homology to AcMNPV (Harrison and Bonning, 1999). Ag286 from the velvetbean caterpillar also was considerably more susceptible to these viruses than Sf21 and was most susceptible to its homologous virus, AgMNPV, by 600-fold over Sf21. Journal of Invertebrate Pathology 82 (2003) 129–131 Journal of INVERTEBRATE PATHOLOGY