Insect Cell Lines Research Articles

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

In vitro infectivity of Spodoptera frugiperda multiple nucleopolyhedrovirus to different insect cell lines

Abstract: The objective of this work was to evaluate the response of an in vitro host range to Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), a pathogenic virus to the fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae), for the further development of a biopesticide based on cell culture systems. The cell lines from Bombyx mori (BM-5), Lymantria dispar (IPLB-LD-625Y), Trichoplusia ni (BTI-Tn-5B1-4), Anticarsia gemmatalis (UFL-AG-286), and S. frugiperda (IPLB-SF-21AE and Sf9) were tested for their susceptibility to a highly-virulent Brazilian isolate of SfMNPV. The cytopathic effects induced by the virus, the production of viral particles, and the synthesis of viral polypeptides were examined and compared. Both S. frugiperda cell lines showed hypertrophy of cell nuclei and production of many polyhedra. The SDS-Page of radiolabed proteins showed that the cell protein synthesis was shutoff, while an intense band of about 30 kDa, recognized as polyhedrin, was synthesized. The other cell lines did not show polyhedra production, although some of them underwent morphological changes and protein synthesis shutdown in response to virus infection. The SF-21 and Sf9 cell lines are recommended for further in vitro production of SfMNPV.

Cytotoxicity evaluation and apoptosis-inducing effects of furanone analogues in insect cell line SL2

ABSTRACT Insecticides containing furan ring have attracted more attention owing to their unique mechanism of action. 25 furanone analogues were used to conduct a preliminary screening on insect cell line SL2 and these compounds exhibited good cytoactivity. Particularly, compound 14 presented good inhibitory effect with an IC50 value of 28.14 μM. Subsequently, we investigated the selectivity against insect cells by testing the cytotoxicity of it on human cell line HEK293 and we noticed that compound 14 was less toxicity than the reference insect cells. Finally, we attempted to illustrate the act mechanism of compound 14 on SL2 cells at the cellular level and found it initiated apoptosis through a caspase-dependent mitochondrial pathway that down-regulated mitochondrial membrane potential level, increased the activity of caspase-3 and altered the cell cycle. The result showed that compound 14 could be considered as a new potent insecticide to be used for further optimization.

African and Asian Zika Virus Isolates Display Phenotypic Differences Both In Vitro and In Vivo.

Zika virus (ZIKV) is a mosquito-borne member of the genus Flavivirus that has emerged since 2007 to cause outbreaks in Africa, Asia, Oceania, and most recently, in the Americas. Here, we used an isolate history as well as genetic and phylogenetic analyses to characterize three low-passage isolates representing African (ArD 41525) and Asian (CPC-0740, SV0127-14) lineages to investigate the potential phenotypic differences in vitro and in vivo. The African isolate displayed a large plaque phenotype (∼3-4 mm) on Vero and HEK-293 cells, whereas the Asian isolates either exhibited a small plaque phenotype (∼1-2 mm) or did not produce any plaques. In multistep replication kinetics in nine different vertebrate and insect cell lines, the African isolate consistently displayed faster replication kinetics and yielded ∼10- to 10,000-fold higher peak virus titers (infectious or RNA copies) compared with the Asian isolates. Oral exposure of Aedes aegypti mosquitoes with the African isolate yielded higher infection and dissemination rates compared with the Asian isolates. Infection of Ifnar1-/- mice with the African isolate produced a uniformly fatal disease, whereas infection with the Asian isolates produced either a delay in time-to-death or a significantly lower mortality rate. Last, the African isolate was > 10,000-fold more virulent than the Asian isolates in an interferon type I antibody blockade mouse model. These data demonstrate substantial phenotypic differences between low-passage African and Asian isolates both in vitro and in vivo and warrant further investigation. They also highlight the need for basic characterization of ZIKV isolates, as the utilization of the uncharacterized isolates could have consequences for animal model and therapeutic/vaccine development.

Radioresistant Sf9 insect cells readily undergo an intrinsic mode of apoptosis in response to histone deacetylase (HDAC) inhibition.

Insect cell lines have been utilized as an important higher eukaryotic model system to decipher stress responses and cell death mechanisms. Lepidopteran Sf9 cells (derived from the ovaries of Spodoptera frugiperda) display nearly 100 times higher resistance to ionizing radiation in contrast to mammalian cells, which is partly contributed by an unusually high HDAC activity. However, their response to HDAC inhibition remains to be evaluated. In the present study, the effects of HDAC inhibitor (NaBt) on Sf9 cellular/nuclear morphology, cell cycle progression, DNA damage/repair, redox status, and mitochondrial perturbations were evaluated. NaBt-induced apoptosis was evident at 18h in Sf9 cells at 2mM concentration, primarily through mitochondrial induction of oxidative stress and subsequent DNA damage. Cell cycle analysis revealed appearance of sub-G1 DNA content at 12h onwards and DNA fragmentation by 18h. Initial few hours of treatment caused significant loss in MMP through oxidation of mitochondrial inner membrane protein, i.e., cardiolipin. HDAC inhibition-mediated apoptosis was associated with increased Bax/Bcl2 ratio, mitochondrial cytochrome-c release, and caspase-3 activation. The study thus infers that Sf9 cells, which can withstand very high radiation doses, are quite sensitive to the increase in the chromatin acetylation levels. In addition, HDAC inhibition also sensitized Sf9 cells to radiation-induced DNA damage, further corroborating our recent finding that chromatin compactness contributes significantly to their radioresistance. Therefore, the study demonstrates prominence of prevailing DNA/chromatin protective mechanisms in Lepidopteran insect cells.

Nutritional demands and metabolic characteristics of the DSIR-HA-1179 insect cell line during growth and infection with the Oryctes nudivirus.

The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8×105 cells ml-1) with a population doubling time of 4.2d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38×107 TCID50 ml-1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25cm2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.

CFD-aided optimization of a laboratory-scale centrifugation for a shear-sensitive insect cell line

A study has been conducted to assess cell damage during centrifugation and to optimize this operation for the insect cell line Se301. Experiments were carried out in a discontinuous centrifuge using Falcon tubes of different sizes as containers. The cells were easily recovered from the cell suspension and the time required for complete sedimentation was found to be independent of tube size or design. Cell damage was observed once the cells were sedimented, therefore this process depends on both the residence time of the cells in the pellet and on the acceleration applied. The sensitivity of the cells to this operation was higher than for other naked microalgae or for very sensitive red blood cells. Indeed, excessive treatment produced cell damage that reduced the productivity of subsequent cultures. CFD simulations were carried out in 2D and a good agreement was found between the CFD and experimental values in terms of the time required for full sedimentation. In addition, this technique allowed the calculation of shear stress, a key variable in the study of cell sensitivity to flow.

FUNCTIONAL ANALYSIS OF BRACON HEBETOR VENOM ON TARGET AND NON-TARGET INSECT CELL LINES

Insect cell cultures are widely used in studies of insect cell physiology, toxicology, developmental biology and microbial pathology. The lethal effects of crude venom extracted from the ectoparasitic wasp Bracon hebetor were examined with three cultured insect cell lines; Spodoptera frugiperda (Sf9), Tribolium castaneum (TcA) and Aedes aegypti (Aag-2). Venom caused cells to round-up, swell and eventually die. Despite similar sensitivities and overlapping LC 50 values [(0.00125-0.00695) venom reservoir equivalents (VRE)/µl], significant differences were noted at the onset of cytotoxicity among the three insect cell lines. Cells from the Tribolium castaneum (TcA) and Aedes aegypti (Aag-2) showed little sensitivity to the venom: 0.0046 VRE were needed to induce 50% mortality in TcA [50% lethal concentration (LC 50 ) = 0.0046 VRE/µl], and 0.0069 VRE were needed to induce 50% mortality in Aag-2 [50% lethal concentration (LC 50 ) = 0.0069 VRE/µl). Over 80% of the Sf9 cells were nonviable within 1 h after the addition of an LC 99 dose of venom, whereas the other cells required a 5-10-fold longer incubation period to produce mortality above 50%.

RMCE-based insect cell platform to produce membrane proteins captured on HIV-1 Gag virus-like particles.

Conformationally complex membrane proteins (MPs) are therapeutic targets in many diseases, but drug discovery has been slowed down by the lack of efficient production tools. Co-expression of MPs with matrix proteins from enveloped viruses is a promising approach to obtain correctly folded proteins at the surface of virus-like particles (VLPs), preserving their native lipidic environment. Here, we implemented a site-specific recombinase-mediated cassette exchange (RMCE) strategy to establish a reusable HIV-1 Gag-expressing insect cell line for fast production of target MPs on the surface of Gag-VLPs. The Sf9 cell line was initially tagged with a Gag-GFP-expressing cassette incorporating two flipase recognition target sites (FRTs), one within the fusion linker of Gag-GFP. The GFP cassette was afterwards replaced by a Cherry cassette via flipase (Flp) recombination. The fusion of Gag to fluorescent proteins enabled high-throughput screening of cells with higher Gag expression and Flp-mediated cassette exchange ability, while keeping the functionality of the VLP scaffold unaltered. The best cell clone was then Flp-recombinated to produce Gag-VLPs decorated with a human β2-adrenergic receptor (β2AR). Release of a fluorescently labeled β2AR into the culture supernatant was confirmed by immunoblotting, and its co-localization with Gag-VLPs was visualized by confocal microscopy. Furthermore, the differential avidity of β2AR-dsplaying Gag-VLPs versus "naked" Gag-VLPs to an anti-β2AR antibody measured by ELISA corroborated the presence of β2AR at the surface of the Gag-VLPs. In conclusion, this novel insect cell line represents a valuable platform for fast production of MPs in their native conformation, which can accelerate small-molecule and antibody drug discovery programs.

Production and Purification of Filovirus Glycoproteins in Insect and Mammalian Cell Lines

Filoviruses are highly virulent pathogens capable of causing severe disease. The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding. As such, they are the main candidate vaccine antigen. Despite their virulence, most filoviruses are not comprehensively characterized, and relatively few commercially produced reagents are available for their study. Here, we describe two methods for production and purification of filovirus glycoproteins in insect and mammalian cell lines. Considerations of expression vector choice, modifications to sequence, troubleshooting of purification method, and glycosylation differences are all important for successful expression of filovirus glycoproteins in cell lines. Given the scarcity of commercially available filovirus glycoproteins, we hope our experiences with possible difficulties in purification of the proteins will facilitate other researchers to produce and purify filovirus glycoproteins rapidly.

Molecular analysis of genome segment-3 of bluetongue virus serotype 12 isolates from Haryana.

Aim:The present study was designed to characterize the genome segment 3 (Seg-3) of bluetongue virus (BTV) serotype 12 isolates from different outbreaks of Bluetongue disease in Haryana, India.Materials and Methods:Blood and swab samples were collected from goat and sheep suspected to be suffering of BT from different outbreaks from Gurugram, Sirsa, Hisar, and Karnal districts of Haryana. The samples were grown in insect and mammalian cell lines. After preliminary identification, serotyping was done using BTV type-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Sequencing was performed using terminal and walking internal primers specific for Seg-3 on ABI Capillary Sequencer 3130 using a “BigDye cycle sequencing kit.” The obtained sequence data were analyzed with various bioinformatic tools.Results:Real-time PCR results confirmed the samples to be positive for BTV-12. The Seg-3 of Indian isolates was most closely related to that of a south Indian isolate of BTV-12 from Andhra Pradesh (KC662614) with 97% nucleotide identity.Conclusions:The study confirmed the circulation of BTV-12 in Haryana, India. The variations shown in genome Seg-3 of BTV-12 isolates may have some significance and need to be further explored.

Agrotis segetum nucleopolyhedrovirus but not Agrotis segetum granulovirus replicate in AiE1611T cell line of Agrotisipsilon.

Both Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV) belong to a cluster of four baculoviruses that are infective for different Agrotis species. Belonging further to different baculovirus genera, namely Alphabaculovirus and Betabaculovirus, respectively, AgseNPV-B and AgseGV are candidates to investigate virus interactions in co-infections. However, for the investigation of virus interactions on a cellular level, permissive insect cell-lines are needed. The cell line AiE1611T deriving from Agrotisipsilon eggs has been shown to be permissive for several Alphabaculovirus isolates. In this study, virus replication was followed based on microscopic analysis of infected and transfected cells, as well as on a molecular level by PCR of DNA and cDNA of selected baculovirus transcripts. While the permissivity was not verified for AgseGV, AgseNPV-B produced occlusion bodies in both infection with hemolymph of infected larvae and Lipofectamin transfection with AgseNPV-B genomic DNA. In addition to the possibility to investigate virus interaction of AgseNPV-B with other alphabaculoviruses, the permissivity of AiE1611T for AgseNPV-B further offers the possibility a biological selection to separate AgseNPV-B from AgseGV.

Infectivity of Sf-rhabdovirus variants in insect and mammalian cell lines

Sf-rhabdovirus was only recently identified as an adventitious agent of Spodoptera frugiperda (Sf) cell lines used as hosts for baculovirus vectors. As such, we still know little about its genetic variation, infectivity, and the potential impact of variation on the Sf-rhabdovirus-host interaction. Here, we characterized Sf-rhabdoviruses from two widely used Sf cell lines to confirm and extend information on Sf-rhabdovirus variation. We then used our novel Sf-rhabdovirus-negative (Sf-RVN) Sf cell line to assess the infectivity of variants with and without a 320bp X/L deletion and found both established productive persistent infections in Sf-RVN cells. We also assessed their infectivity using heterologous insect and mammalian cell lines and found neither established productive persistent infections in these cells. These results are the first to directly demonstrate Sf-rhabdoviruses are infectious for Sf cells, irrespective of the X/L deletion. They also confirm and extend previous results indicating Sf-rhabdoviruses have a narrow host range.

Characterization of Site-Specific Glycosylation in Influenza A Virus Hemagglutinin Produced by Spodoptera frugiperda Insect Cell Line.

Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion was used to determine the glycosites and glycan occupancy. Three enzymes, trypsin, AspN, and pepsin, were used separately to generate suitable glycopeptides for online LC tandem MS analysis. The glycan structure of a given glycopeptide was determined by collision-induced dissociation MS/MS fragmentation, and the peptide backbone information was provided by collision-induced dissociation (CID)-MS3 fragmentation. With this approach, 100% sequence coverage of the hemagglutinin sample was obtained. Six glycosylation sites fitting the sequon N-X-S/T were successfully confirmed, and the glycan heterogeneity as well as the ratios of glycoforms were determined at each site.

Developing global regression models for metabolite concentration prediction regardless of cell line.

Following the Process Analytical Technology (PAT) of the Food and Drug Administration (FDA), drug manufacturers are encouraged to develop innovative techniques in order to monitor and understand their processes in a better way. Within this framework, it has been demonstrated that Raman spectroscopy coupled with chemometric tools allow to predict critical parameters of mammalian cell cultures in-line and in real time. However, the development of robust and predictive regression models clearly requires many batches in order to take into account inter-batch variability and enhance models accuracy. Nevertheless, this heavy procedure has to be repeated for every new line of cell culture involving many resources. This is why we propose in this paper to develop global regression models taking into account different cell lines. Such models are finally transferred to any culture of the cells involved. This article first demonstrates the feasibility of developing regression models, not only for mammalian cell lines (CHO and HeLa cell cultures), but also for insect cell lines (Sf9 cell cultures). Then global regression models are generated, based on CHO cells, HeLa cells, and Sf9 cells. Finally, these models are evaluated considering a fourth cell line(HEK cells). In addition to suitable predictions of glucose and lactate concentration of HEK cell cultures, we expose that by adding a single HEK-cell culture to the calibration set, the predictive ability of the regression models are substantially increased. In this way, we demonstrate that using global models, it is not necessary to consider many cultures of a new cell line in order to obtain accurate models. Biotechnol. Bioeng. 2017;114: 2550-2559. © 2017 Wiley Periodicals, Inc.

Whole-Genome Sequence of the Spodoptera frugiperda Sf9 Insect Cell Line.

ABSTRACTThe draft whole-genome sequence of the Spodoptera frugiperda Sf9 insect cell line was obtained using long-read PacBio sequence technology and Canu assembly. The final assembled genome consisted of 451 Mbp in 4,577 contigs, with 12,716× mean coverage and a G+C content of 36.53%.

Molecular Cloning and Expression of Osmotin in a Baculovirus-Insect System: Purified Osmotin Mitigates Amyloid-beta Deposition in Neuronal Cells

Osmotin is a pathogenesis-related plant protein, have gained focus of research because of its homology with mammalian adiponectin. The therapeutic properties of osmotin have been explored in recent years as it exhibits neuroprotective effects against amyloid beta-, glutamate- and ethanol-induced synaptic dysfunction and neurodegeneration. In the present study, the full-length gene of the tobacco plant osmotin was cloned and expressed in the Sf9 insect cell line using the baculovirus expression system. In vitro analysis of purified Osmotin protein showed excellent cell viability, p-AMPK activation and a reduction in amyloid-beta deposition. Immunofluorescent analysis showed significant reduction in amyloid beta deposition in APP over expressing neuronal cells. Osmotin inhibited amyloid beta deposition by influencing expression of APP processing genes including APP, ADAM 10 and BACE 1. Purified Osmotin showed reduction in amyloid beta deposition in different in vitro models as well. Osmotin showed similar mechanism when compared with mammalian adiponectin in different in vitro models. The present method will be an excellent approach for the efficient and cost-effective production of the functional protein to be utilized for therapeutic purposes. Reduction in amyloid beta deposition by activation of p-AMPK influencing APP processing genes makes osmotin a potent therapeutic candidate for neurodegenerative diseases.

CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

Erratum to: Prostaglandin actions in established insect cell lines.

Erratum to: Prostaglandin actions in established insect cell lines.

Toxicity, membrane binding and uptake of the Sclerotinia sclerotiorum agglutinin (SSA) in different insect cell lines.

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC50) ranging between 9 and 42μg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.