Expansion and Storage of Insect Cells and Virus Stocks.

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.

A Time and Cost-Effective Pipeline for Expression Screening and Protein Production in Insect Cells Based on the HR-Bac Toolbox to Generate Recombinant Baculoviruses.

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-ß 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.

Optimized Workflow from Gene to Product.

This chapter outlines the workflow using the ExpiSf™ Expression System designed for high-density infection of suspension ExpiSf9™ cells. The system utilizes a chemically defined, serum-free, protein-free, and animal origin free medium, making it suitable for recombinant protein expression experiments. The ExpiSf™ chemically defined medium allows efficient transfection and baculovirus production directly within the same culture medium. The ExpiSf™ Expression System Starter Kit provides all necessary components, including cells, culture medium, and reagents needed to infect one (1) liter of cell culture. The system's versatility and animal origin free nature make it a valuable tool for various protein expression studies and biotechnological applications.

Construction of Recombinant Baculoviruses.

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.

RNAi-Mediated Silencing in the Insect Cell-Baculovirus Expression System.

RNA interference (RNAi) serves as an indispensable tool for gene function studies and has been substantiated through extensive research for its practical applications in the baculovirus expression vector system (BEVS). This chapter expands the RNAi toolkit in insect cell culture by including small interfering RNA (siRNA) in the protocol, in addition to the conventional use of double-stranded RNA (dsRNA). This chapter also brings attention to key design and reporting considerations, based on Minimum Information About an RNAi Experiment (MIARE) guidelines. Recommendations regarding online tools for dsRNA and siRNA design are provided, along with guidance on choosing suitable methods for measuring silencing outcomes.

Generation of Complex Protein Structures by Coinfection with High-Quality Recombinant Baculovirus.

The baculovirus expression vector system (BEVS) is a powerful platform for protein expression in insect cells. A prevalent application is the expression of complex protein structures consisting of multiple, interacting proteins. Coinfection with multiple baculoviruses allows for production of complex structures, facilitating structure-function studies, allowing augmentation of insect cell functionality, and production of clinically relevant products such as virus-like particles (VLPs) and adeno-associated viral vectors (AAV). Successful coinfections require the generation of robust and well-quantified recombinant baculovirus stocks. Virus production through homologous recombination, combined with rigorous quantification of viral titers, allows for synchronous coinfections producing high end-product titers. In this chapter, we describe the streamlined workflow for generation and quantification of high-quality recombinant baculovirus stocks and successful coinfection as defined by a preponderance of dually infected cells in the insect cell culture.

Adaptive Laboratory Evolution to Improve Recombinant Protein Production Using Insect Cells.

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.

Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System.

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.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Extraction of Modified Adeno-Associated Virus-Like Particles Displaying Peptides on Their Surface.

Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.

Purification Process for a Secreted Protein Produced in Insect Cells.

The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).

A Microsphere Immunoassay for the Quantitative Detection of Antigens in Cell Culture Supernatant.

Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.

Nonviral Platform for Expression of Recombinant Protein in Insect Cells.

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.

TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Preventing loss of sirt1 lowers mitochondrial oxidative stress and preserves C2C12 myotube diameter in an in vitro model of cancer cachexia.

Cancer cachexia is a multifactorial syndrome associated with advanced cancer that contributes to mortality. Cachexia is characterized by loss of body weight and muscle atrophy. Increased skeletal muscle mitochondrial reactive oxygen species (ROS) is a contributing factor to loss of muscle mass in cachectic patients. Mice inoculated with Lewis lung carcinoma (LLC) cells lose weight, muscle mass, and have lower muscle sirtuin-1 (sirt1) expression. Nicotinic acid (NA) is a precursor to nicotinamide dinucleotide (NAD+) which is exhausted in cachectic muscle and is a direct activator of sirt1. Mice lost body and muscle weight and exhibited reduced skeletal muscle sirt1 expression after inoculation with LLC cells. C2C12 myotubes treated with LLC-conditioned media (LCM) had lower myotube diameter. We treated C2C12 myotubes with LCM for 24 h with or without NA for 24 h. C2C12 myotubes treated with NA maintained myotube diameter, sirt1 expression, and had lower mitochondrial superoxide. We then used a sirt1-specific small molecule activator SRT1720 to increase sirt1 activity. C2C12 myotubes treated with SRT1720 maintained myotube diameter, prevented loss of sirt1 expression, and attenuated mitochondrial superoxide production. Our data provides evidence that NA may be beneficial in combating cancer cachexia by maintaining sirt1 expression and decreasing mitochondrial superoxide production.

In vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing.

Electrospun (ES) fibrous nanomaterials have been widely investigated as novel biomaterials. These biomaterials have to be safe and biocompatible; hence, they need to be tested for cytotoxicity before being administered to patients. The aim of this study was to develop a suitable and biorelevant in vitro cytotoxicity assay for ES biomaterials (e.g. wound dressings). We compared different in vitro cytotoxicity assays, and our model wound dressing was made from polycaprolactone and polyethylene oxide and contained chloramphenicol as the active pharmaceutical ingredient. Baby Hamster Kidney cells (BHK-21), human primary fibroblasts and MTS assays together with real-time cell analysis were selected. The extract exposure and direct contact safety evaluation setups were tested together with microscopic techniques. We found that while extract exposure assays are suitable for the initial testing, the biocompatibility of the biomaterial is revealed in in vitro direct contact assays where cell interactions with the ES wound dressing are evaluated. We observed significant differences in the experimental outcome, caused by the experimental set up modification such as cell line choice, cell medium and controls used, conducting the phosphate buffer washing step or not. A more detailed technical protocol for the in vitro cytotoxicity assessment of ES wound dressings was developed.

Study on the Antibacterial Activity and Bone Inductivity of Nanosilver/PLGA-Coated TI-CU Implants.

Background: Implants are widely used in the field of orthopedics and dental sciences. Titanium (TI) and its alloys have become the most widely used implant materials, but implant-associated infection remains a common and serious complication after implant surgery. In addition, titanium exhibits biological inertness, which prevents implants and bone tissue from binding strongly and may cause implants to loosen and fall out. Therefore, preventing implant infection and improving their bone induction ability are important goals. Purpose: To study the antibacterial activity and bone induction ability of titanium-copper alloy implants coated with nanosilver/poly (lactic-co-glycolic acid) (NSPTICU) and provide a new approach for inhibiting implant-associated infection and promoting bone integration. Methods: We first examined the in vitro osteogenic ability of NSPTICU implants by studying the proliferation and differentiation of MC3T3-E1 cells. Furthermore, the ability of NSPTICU implants to induce osteogenic activity in SD rats was studied by micro-computed tomography (micro-CT), hematoxylin-eosin (HE) staining, masson staining, immunohistochemistry and van gieson (VG) staining. The antibacterial activity of NSPTICU in vitro was studied with gram-positive Staphylococcus aureus (Sa) and gram-negative Escherichia coli (E. coli) bacteria. Sa was used as the test bacterium, and the antibacterial ability of NSPTICU implanted in rats was studied by gross view specimen collection, bacterial colony counting, HE staining and Giemsa staining. Results: Alizarin red staining, alkaline phosphatase (ALP) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis showed that NSPTICU promoted the osteogenic differentiation of MC3T3-E1 cells. The in vitro antimicrobial results showed that the NSPTICU implants exhibited better antibacterial properties. Animal experiments showed that NSPTICU can inhibit inflammation and promote the repair of bone defects. Conclusion: NSPTICU has excellent antibacterial and bone induction ability, and has broad application prospects in the treatment of bone defects related to orthopedics and dental sciences.

Sulfur dioxide inhibits mast cell degranulation by sulphenylation of galectin-9 at cysteine 74.

Objectives: Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation. Methods: HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC ß-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation. Results: Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions. Conclusion: These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.

Mitochondrial Targeted Cerium Oxide Nanoclusters for Radiation Protection and Promoting Hematopoiesis.

Purpose: Mitochondrial oxidative stress is an important factor in cell apoptosis. Cerium oxide nanomaterials show great potential for scavenging free radicals and simulating superoxide dismutase (SOD) and catalase (CAT) activities. To solve the problem of poor targeting of cerium oxide nanomaterials, we designed albumin-cerium oxide nanoclusters (TPP-PCNLs) that target the modification of mitochondria with triphenyl phosphate (TPP). TPP-PCNLs are expected to simulate the activity of superoxide dismutase, continuously remove reactive oxygen species, and play a lasting role in radiation protection. Methods: First, cerium dioxide nanoclusters (CNLs), polyethylene glycol cerium dioxide nanoclusters (PCNLs), and TPP-PCNLs were characterized in terms of their morphology and size, ultraviolet spectrum, dispersion stability and cellular uptake, and colocalization Subsequently, the anti-radiation effects of TPP-PCNLs were investigated using in vitro and in vivo experiments including cell viability, apoptosis, comet assays, histopathology, and dose reduction factor (DRF). Results: TPP-PCNLs exhibited good stability and biocompatibility. In vitro experiments indicated that TPP-PCNLs could not only target mitochondria excellently but also regulate reactive oxygen species (ROS)levels in whole cells. More importantly, TPP-PCNLs improved the integrity and functionality of mitochondria in irradiated L-02 cells, thereby indirectly eliminating the continuous damage to nuclear DNA caused by mitochondrial oxidative stress. TPP-PCNLs are mainly targeted to the liver, spleen, and other extramedullary hematopoietic organs with a radiation dose reduction factor of 1.30. In vivo experiments showed that TPP-PCNLs effectively improved the survival rate, weight change, hematopoietic function of irradiated animals. Western blot experiments have confirmed that TPP-PCNLs play a role in radiation protection by regulating the mitochondrial apoptotic pathway. Conclusion: TPP-PCNLs play a radiologically protective role by targeting extramedullary hematopoietic organ-liver cells and mitochondria to continuously clear ROS.

Unexpected regulatory functions of cyprinid Viperin on inflammation and metabolism.

BACKGROUND: Viperin, also known as radical S-adenosyl-methionine domain containing protein 2 (RSAD2), is an interferon-inducible protein that is involved in the innate immune response against a wide array of viruses. In mammals, Viperin exerts its antiviral function through enzymatic conversion of cytidine triphosphate (CTP) into its antiviral analog ddhCTP as well as through interactions with host proteins involved in innate immune signaling and in metabolic pathways exploited by viruses during their life cycle. However, how Viperin modulates the antiviral response in fish remains largely unknown. RESULTS: For this purpose, we developed a fathead minnow (Pimephales promelas) clonal cell line in which the unique viperin gene has been knocked out by CRISPR/Cas9 genome-editing. In order to decipher the contribution of fish Viperin to the antiviral response and its regulatory role beyond the scope of the innate immune response, we performed a comparative RNA-seq analysis of viperin-/- and wildtype cell lines upon stimulation with recombinant fathead minnow type I interferon. CONCLUSIONS: Our results revealed that Viperin does not exert positive feedback on the canonical type I IFN but acts as a negative regulator of the inflammatory response by downregulating specific pro-inflammatory genes and upregulating repressors of the NF-κB pathway. It also appeared to play a role in regulating metabolic processes, including one carbon metabolism, bone formation, extracellular matrix organization and cell adhesion.