Tissue engineering is primarily associated with medical disciplines, and research has thus focused on mammalian cells. For applications where clinical relevance is not a constraint, it is useful to evaluate the potential of alternative cell sources to form tissues in vitro. Specifically, skeletal muscle tissue engineering for bioactuation and cultured foods could benefit from the incorporation of invertebrate cells because of their less stringent growth requirements and other versatile features. Here, we used a Drosophila muscle cell line to demonstrate the benefits of insect cells relative to those derived from vertebrates. The cells were adapted to serum-free media, transitioned between adherent and suspension cultures, and manipulated with hormones. Furthermore, we analyzed edible scaffolds to support cell adhesion and assayed cellular protein and minerals to evaluate nutrition potential. The insect muscle cells exhibited advantageous growth patterns and hold unique functionality for tissue engineering applications beyond the medical realm.

Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.

Novel neuroactive insecticides are discovered/registered differently, have a lower value in use, and exert their physiological actions in manners distinct from neuroactive pharmaceuticals, but there are clear similarities in their biochemical modes of action. Insecticides are generally discovered using whole pest insect screens, and this eases difficulties in \'translational science\' from laboratory to field, as opposed to pharmaceutical translation from biochemical or cell-based targets to animal models to human clinical trials to registered drug. This paper examines recent trends in pharmaceutical science and identifies some technologies which may represent complementary approaches to insecticide discovery screening and mode of action determination beyond the sound processes in common practice today. Examples will be drawn from nanoparticle delivery of neuroactives, unique ligand-polymer conjugates, proposed advances in insect cell culture following from pharmaceutical cell biology, and laboratory or organ-on-a-chip approaches. It is hoped that these concepts will stimulate novel thinking which may enable discovery of efficacious new neuroactive insecticides. © 2020 Society of Chemical Industry.

Virus-like particles (VLPs) are hollow nanoparticles composed of recombinant viral surface proteins without a virus genome. In the present study, we investigated the production of influenza VLPs using recombinant insect cells. DNA fragments encoding influenza A virus hemagglutinin (HA) and matrix protein 1 (M1) were cloned with the Drosophila BiP signal sequence in plasmid vectors containing a blasticidin and a neomycin resistance gene, respectively. After Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with a pair of constructed plasmid vectors, stably transformed cells were established via incubation with blasticidin and G418. Western blot analyses showed that recombinant High Five cells secreted HA and M1 proteins into the culture supernatant. Immunoprecipitation of the culture supernatant with an anti-HA antibody and transmission electron microscopy suggested that secreted HA and M1 proteins were in a particulate structure with a morphology similar to that of an influenza virus. Hemagglutination assay indicated that expressed HA molecules retained hemagglutination activity. In a shake-flask culture, recombinant cells achieved a high HA yield (≈ 10 μg/ml) comparable to the yields obtained using the baculovirus-insect cell system. Recombinant insect cells may serve as excellent platforms for the efficient production of influenza VLPs for use as safe and effective vaccines and diagnostic antigens.

With ongoing colony losses driven in part by the Varroa mite and the associated exacerbation of the virus load, there is an urgent need to protect honey bees (Apis mellifera) from fatal levels of virus infection and from the non-target effects of insecticides used in agricultural settings. A continuously replicating cell line derived from the honey bee would provide a valuable tool for the study of molecular mechanisms of virus-host interaction, for the screening of antiviral agents for potential use within the hive, and for the assessment of the risk of current and candidate insecticides to the honey bee. However, the establishment of a continuously replicating honey bee cell line has proved challenging. Here, we provide an overview of attempts to establish primary and continuously replicating hymenopteran cell lines, methods (including recent results) of establishing honey bee cell lines, challenges associated with the presence of latent viruses (especially Deformed wing virus) in established cell lines and methods to establish virus-free cell lines. We also describe the potential use of honey bee cell lines in conjunction with infectious clones of honey bee viruses for examination of fundamental virology.

Monoclonal antibodies and antibody fragments are widely used in therapeutics and diagnoses. While mammalian cells serve as the host cells for antibody production, insect cells can produce large quantities of secretory antibodies in serum-free suspension cultures. The effects of lithium on the processes of autophagy and apoptosis in mammalian cells are well chronicled. In the present study, stably transformed insect cells, which produce an engineered antibody molecule, were cultured with lithium chloride in a serum-free medium. Treatment with lithium chloride induced autophagy and apoptosis in recombinant insect cells and led to increases in the yields of secreted antibodies.

The generation of monoclonal cell lines is an important early process development step for recombinant protein production. Although single-cell cloning is an established method in mammalian cell lines, straightforward protocols are not yet available for insect cells. We describe a new method for the generation of monoclonal insect cells without using fetal bovine serum and/or feeder cells pretreated by irradiation or exposure to mitomycin. Highly productive clones of Drosophila melanogaster S2 cells were prepared in a two-step procedure, comprising the establishment of a polyclonal population and subsequent single cell isolation by limiting dilution. Necessary growth factors were provided by co-cultivation of single transformants with untransfected feeder cells, which were later removed by antibiotic selection. Enhanced expression of EGFP and two target peptides was confirmed by flow cytometry and dot/western blotting. Highly productive clones were stable, showed a uniform expression profile and typically a sixfold to tenfold increase in cell-specific productivity.

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.

Since its inception more than 30 years ago, the baculovirus expression vector system (BEVS) has been used prolifically to produce heterologous proteins for research and development. In the cell, a cornerstone of biological activity are multiprotein complexes, catalyzing essential functions. BEVS has been uniquely successful to unlock such complex assemblies for high-resolution structural and functional analysis. Synthetic biology approaches have been implemented to optimize multigene assembly methods, accelerating upstream processes. Specialized baculoviral genomes are being created with functions tailored to enhance production of particular target protein classes. Here we comment on current and emerging developments in the field and their potential to accelerate protein complex research.

Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10(7) cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10(8) insect cells at -80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were \"expression ready\" immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at -80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.