Insect cell lines are finding utility in many areas of biology, but their application as an in vitro tool for ecotoxicity testing has been given less attention. Our study aimed to demonstrate the utility and sensitivity of Sf21 cells to commonly used fungicides: Propiconazole and CuSO4, as well as dimethyl sulphoxide (DMSO) an industrial solvent. Sf21 cells were readily cultured from frozen stocks in 3-4 days and showed utility as an invertebrate in vitro acute toxicity test. The data showed the threshold levels of cell survivability against propiconazole and CuSO4. The EC50 values were 135.1 μM and 3.31 mM respectively. The LOAEL (lowest observed adverse effect level) was ≈ 1 μM for propiconazole and ≈ 10 μM for CuSO4. Culturing of Sf21 cells in media containing the solvent DMSO showed that 0.5% DMSO concentration did not effect cell viability. Sf21 cells are sensitive and useful as a robust ecologically relevant screening tool for acute toxicity testing.

Nonviral transfection has been used to express various recombinant proteins, therapeutics, and virus-like particles (VLP) in mammalian and insect cells. Virus-free methods for protein expression require fewer steps for obtaining protein expression by eliminating virus amplification and measuring the infectivity of the virus. The nonviral method uses a nonlytic plasmid to transfect the gene of interest into the insect cells instead of using baculovirus, a lytic system. In this chapter, we describe one of the transfection methods, which uses polyethyleneimine (PEI) as a DNA delivery material into the insect cells to express the recombinant protein in both adherent and suspension cells.

Insect cell expression has been successfully used for the production of viral antigens as part of commercial vaccine development. As expression host, insect cells offer advantage over bacterial system by presenting the ability of performing post-translational modifications (PTMs) such as glycosylation and phosphorylation thus preserving the native functionality of the proteins especially for viral antigens. Insect cells have limitation in exactly mimicking some proteins which require complex glycosylation pattern. The recent advancement in insect cell engineering strategies could overcome this limitation to some extent. Moreover, cost efficiency, timelines, safety, and process adoptability make insect cells a preferred platform for production of subunit antigens for human and animal vaccines. In this chapter, we describe the method for producing the SARS-CoV2 spike ectodomain subunit antigen for human vaccine development and the virus like particle (VLP), based on capsid protein of porcine circovirus virus 2 (PCV2d) antigen for animal vaccine development using two different insect cell lines, SF9 & Hi5, respectively. This methodology demonstrates the flexibility and broad applicability of insect cell as expression host.

The baculovirus expression vector system (BEVS) has now found acceptance in both research laboratories and industry, which can be attributed to many of its key features including the limited host range of the vectors, their non-pathogenicity to humans, and the mammalian-like post-translational modification (PTMs) that can be achieved in insect cells. In fact, this system acts as a middle ground between prokaryotes and higher eukaryotes to produce complex biologics. Still, industrial use of the BEVS lags compared to other platforms. We have postulated that one reason for this has been a lack of genetic tools that can complement the study of baculovirus vectors, while a second reason is the co-production of the baculovirus vector with the desired product. While some genetic enhancements have been made to improve the BEVS as a production platform, the genome remains under-scrutinized. This chapter outlines the methodology for a CRISPR-Cas9-based transfection-infection assay to probe the baculovirus genome for essential/nonessential genes that can potentially maximize foreign gene expression under a promoter of choice.

Adaptive laboratory evolution (ALE) is a powerful tool for enhancing the fitness of cell lines in specific applications, including recombinant protein production. Through adaptation to nonstandard culture conditions, cells can develop specific traits that make them high producers. Despite being widely used for microorganisms and, to lesser extent, for mammalian cells, ALE has been poorly leveraged for insect cells. Here, we describe a method for adapting insect High Five and Sf9 cells to nonstandard culture conditions via an ALE approach. Aiming to demonstrate the potential of ALE to improve productivity of insect cells, two case studies are demonstrated. In the first, we adapted insect High Five cells from their standard pH (6.2) to neutral pH (7.0); this adaptation allowed to improve production of influenza virus-like particles (VLPs) by threefold, using the transient baculovirus expression vector system. In the second, we adapted insect Sf9 cells from their standard culture temperature (27 °C) to hypothermic growth (22 °C); this adaptation allowed to improve production of influenza VLPs by sixfold, using stable cell lines. These examples demonstrate the potential of ALE for enhancing productivity within distinct insect cell hosts and expression systems by manipulating different culture conditions.

The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-β protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2 weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins.

The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.

Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and determining viral titers. This chapter describes cell maintenance in shake flasks using serum-free conditions and the expansion of virus stocks from a single plaque purified virus. Insect cells can be passaged over multiple generations, but as the cells may undergo changes over multiple passages, limiting the use of your cells to a defined number of passages such as 50 passages is recommendable. Baculovirus stocks once created using serum-free media are not very stable at 4-8 °C. This chapter also includes a simple method to store cells from an early cell passage and your virus stock in liquid nitrogen.

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests.