HEK293 Cell Culture Research Articles

Engineering the Escherichia coli Nitroreductase NfsA to Create a Flexible Enzyme-Prodrug Activation System.

Bacterial nitroreductase enzymes that can efficiently convert nitroaromatic prodrugs to a cytotoxic form have numerous applications in targeted cellular ablation. For example, the generation of cytotoxic metabolites that have low bystander potential (i.e., are largely confined to the activating cell) has been exploited for precise ablation of specific cell types in animal and cell-culture models; while enzyme-prodrug combinations that generate high levels of bystander cell killing are useful for anti-cancer strategies such as gene-directed enzyme-prodrug therapy (GDEPT). Despite receiving substantial attention for such applications, the canonical nitroreductase NfsB from Escherichia coli has flaws that limit its utility, in particular a low efficiency of conversion of most prodrugs. Here, we sought to engineer a superior broad-range nitroreductase, E. coli NfsA, for improved activity with three therapeutically-relevant prodrugs: the duocarmycin analogue nitro-CBI-DEI, the dinitrobenzamide aziridine CB1954 and the 5-nitroimidazole metronidazole. The former two prodrugs have applications in GDEPT, while the latter has been employed for targeted ablation experiments and as a precise ‘off-switch’ in GDEPT models to eliminate nitroreductase-expressing cells. Our lead engineered NfsA (variant 11_78, with the residue substitutions S41Y, L103M, K222E and R225A) generated reduced metabolites of CB1954 and nitro-CBI-DEI that exhibited high bystander efficiencies in both bacterial and 2D HEK-293 cell culture models, while no cell-to-cell transfer was evident for the reduced metronidazole metabolite. We showed that the high bystander efficiency for CB1954 could be attributed to near-exclusive generation of the 2-hydroxylamine reduction product, which has been shown in 3D cell culture to cause significantly greater bystander killing than the 4-hydroxylamine species that is also produced by NfsB. We similarly observed a high bystander effect for nitro-CBI-DEI in HCT-116 tumor spheroids in which only a small proportion of cells were expressing variant 11_78. Collectively, our data identify variant 11_78 as a broadly improved prodrug-activating nitroreductase that offers advantages for both targeted cellular ablation and suicide gene therapy applications.

Effects of Collagen, Vitronectin and Gelatin on Three-Dimensional HEK293 Cell Culture and Cellular Collagen Release on ChitosanCoated SurfacesEffects of Collagen, Vitronectin and Gelatin on Three-Dimensional HEK293 Cell Culture and Cellular Collagen Release on ChitosanCoated Surfaces

Effects of Collagen, Vitronectin and Gelatin on Three-Dimensional HEK293 Cell Culture and Cellular Collagen Release on ChitosanCoated SurfacesEffects of Collagen, Vitronectin and Gelatin on Three-Dimensional HEK293 Cell Culture and Cellular Collagen Release on ChitosanCoated Surfaces

Production of antimicrobial metabolites against pathogenic bacteria and yeasts by Fusarium oxysporum in submerged culture processes.

The antimicrobial activity of the metabolites produced by Fusarium oxysporum PR-33 in submerged culture was evaluated against Gram-positive and Gram-negative bacteria and yeasts. Metabolites were determined by HPLC-DAD-MS/MS. An extract was obtained following the removal of mycelium by centrifugation and lyophilisation of the supernatant. The compounds in this extract demonstrated broad-spectrum antimicrobial action, with rates of inhibition between 60 and 80%, depending on the species and extract tested. The major compounds of the extracts were identified as fusarinolic acid and its isomer [56.9% flask extract (FE)] and 59.2% bioreactor extract (BE), dehydrofusaric acid (35.7% FE and 31.6% BE), and fusaric acid (6.5% FE and 1.1% BE). Fusaric acid has been shown to be responsible for antimicrobial activity. The cytotoxicity of the extracts was evaluated in culture of HEK-293 and SH-SY5Y animal cells and toxicity of these extracts was verified even in the lowest tested concentrations. Therefore, our results indicate that the compounds identified exhibit potential as antimicrobial agents.

Synthesis and Anticancer Activity of Hybrid Molecules Based on Lithocholic and (5Z,9Z)-Tetradeca-5,9-dienedioic Acids Linked via Mono(di,tri,tetra)ethylene Glycol and α,ω-Diaminoalkane Units.

For the first time, hybrid molecules were synthesized on the basis of lithocholic and (5Z,9Z)-1,14-tetradeca-5,9-dienedicarboxylic acids, obtained in two stages using the homo-cyclomagnesiation reaction of 2-(hepta-5,6-diene-1-yloxy)tetrahydro-2H-pyran at the key stage. The resulting hybrid molecules containing 5Z,9Z-dienoic acids are of interest as novel synthetic biologically active precursors to create modern drugs for the treatment of human oncological diseases. The synthesized hybrid molecules were found to exhibit extremely high in vitro inhibitory activity against human topoisomerase I, which is 2–4 times higher than that of camptothecin, a known topoisomerase I inhibitor. Using flow cytometry and fluorescence microscopy, it was first shown that these new molecules are efficient apoptosis inducers in HeLa, U937, Jurkat, K562, and Hek293 cell cultures. In addition, the results of investigations into the effect of the synthesized acids on mitochondria and studies of possible DNA damage in Jurkat tumor cells are also presented.

Lipodendriplexes mediated enhanced gene delivery: a cellular to pre-clinical investigation

Clinical success of effective gene therapy is mainly hampered by the insufficiency of safe and efficient internalization of a transgene to the targeted cellular site. Therefore, the development of a safe and efficient nanocarrier system is one of the fundamental challenges to transfer the therapeutic genes to the diseased cells. Polyamidoamine (PAMAM) dendrimer has been used as an efficient non-viral gene vector (dendriplexes) but the toxicity and unusual biodistribution induced by the terminal amino groups (–NH2) limit its in vivo applications. Hence, a state of the art lipid modification with PAMAM based gene carrier (lipodendriplexes) was planned to investigate theirs in vitro (2D and 3D cell culture) and in vivo behaviour. In vitro pDNA transfection, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, cellular protein contents, live/dead staining and apoptosis were studied in 2D cell culture of HEK-293 cells while GFP transfection, 3D cell viability and live/dead staining of spheroids were performed in its 3D cell culture. Acute toxicity studies including organ to body index ratio, hematological parameters, serum biochemistry, histopathological profiles and in vivo transgene expression were assessed in female BALB/c mice. The results suggested that, in comparison to dendriplexes the lipodendriplexes exhibited significant improvement of pDNA transfection (p < 0.001) with lower LDH release (p < 0.01) and ROS generation (p < 0.05). A substantially higher cellular protein content (p < 0.01) and cell viability were also observed in 2D culture. A strong GFP expression with an improved cell viability profile (p < 0.05) was indicated in lipodendriplexes treated 3D spheroids. In vivo archives showed the superiority of lipid-modified nanocarrier system, depicted a significant increase in green fluorescent protein (GFP) expression in the lungs (p < 0.01), heart (p < 0.001), liver (p < 0.001) and kidneys (p < 0.001) with improved serum biochemistry and hematological profile as compared to unmodified dendriplexes. No tissue necrosis was evident in the animal groups treated with lipid-shielded molecules. Therefore, a non-covalent conjugation of lipids with PAMAM based carrier system could be considered as a promising approach for an efficient and biocompatible gene delivery system.

Experimental Validation of a Cascade Control Strategy for Continuously Perfused Animal Cell Cultures

This paper is dedicated to the experimental validation of a cascade control strategy for simultaneously regulating the glucose and biomass levels in continuously perfused HEK-293 cell cultures. The inner loop consists of a partial feedback linearization, which requires the estimation of the biomass specific growth rate and glucose uptake rate. This latter task is achieved by sliding mode observers, which do not require a priori process knowledge in the form of a process model. The linearized process is then regulated by the outer loop, including two classical PI controllers with autotuning. The four manipulated variables are a feed flow rate with low glucose concentration, another feed flow with a higher glucose content, a bleed flow, and a perfusion stream. The experimental results demonstrate the ability of the control strategy to reach and regulate the prescribed setpoints. The main advantage of the strategy is that it can be applied in a plug and play manner and shows satisfactory robustness. To the best of our knowledge, this is the first time that such a multivariable control strategy, together with sliding mode observers, is applied at the lab scale to an industrial process in the pharmaceutical sector.

Improvements of ER-Ca2+ Based High-Throughput Screening Method for Searching Novel RyR2 Inhibitors

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic reticulum (ER) and plays a central role in excitation-contraction coupling in the heart. When the RyR2 activity is abnormally enhanced by miss-sense mutations and/or excessive phosphorylation, spontaneous Ca2+ release occurs from the ER, which often results in fatal ventricular arrhythmias. Therefore, specific inhibitors of RyR2 are expected to have an antiarrhythmic effect on the RyR2-related arrhythmias, but currently such specific drugs have not been reported. We have recently established a screening method for isoform specific ryanodine receptor (RyR) inhibitors using ER Ca2+ indicator, R-CEPIA1er, in HEK293 cells (Murayama et al., Mol Pharmacol, 2018). Although this method is very useful, it requires 3 consecutive experimental days, i.e., culture of HEK293 cells expressing inducible RyR and R-CEPIA1er in 96-well plates on the first day, induction of protein expression by doxycycline on the next day, and assay using FlexStation3 on the third day. In order to screen a larger number of compounds more efficiently using a high-throughput screening device equipped with EM-CCD camera such as FDSS7000 (Hamamatsu), we made several improvements to this method. First, a novel bright green fluorescent indicator, G-CEPIA2er was used instead of R-CEPIA1er, which greatly improved signal-to-noise ratio. Second, measurements were done in 384 well plate with FDSS7000 just after seeding of cryopreserved cells which had been treated with doxycycline to induce RyR2 expression. These improvements resulted in a 48-fold increase in assay efficiency. This method, having allowed us to screen more than 20,000 compounds so far for both RyR1 and RyR2, proved to be a versatile and powerful tool for the development of specific RyR2-modifying reagents. This research was supported by JSPS Kakenhi grant (19K07105), AMED grant (19ek0109202s0203) and BINDS from AMED (0033).

Multiplexed Quantitative Proteomic Analysis of HEK293 Provides Insights into Molecular Changes Associated with the Cell Density Effect, Transient Transfection, and Virus-Like Particle Production.

The production of virus-like particles (VLPs) has gained importance over the last few years owing to the benefits they provide compared to conventional vaccines. The biopharmaceutical industry is currently searching for safer candidates based on VLPs for new and existing vaccines and implementing new methods of manufacturing, thus allowing a more sustainable, effective, and species-specific production. Despite achieving lower yields compared to traditional platforms, the use of mammalian cells provides the right post-translational modifications, and consequently, the intensification of bioprocesses using mammalian cell platforms has become a matter of pressing concern. One of the methods subjected to intensification is transient gene expression, which has been proven to be highly effective regarding VLP production for preclinical or even clinical trials. In this work, a multiplexed quantitative proteomic approach has been applied to study the molecular characteristics of HEK293 cell cultures when growing at cell densities higher than 4 × 106 cells/mL and to study the effects related to cell transfection and VLP production. The obtained results revealed a set of functional and metabolic profiles of HEK293 under these three different conditions that allowed the identification of physiological bottlenecks regarding VLP production. Regarding the cell density effect, molecular alterations in the cell biology were proposed to help explain the difficulty for the cells to be transfected at higher densities. In addition, an overall disruption of cellular homeostasis after transfection was observed based on altered biological processes, and after identifying potential pathways liable to be optimized via metabolic engineering, different solutions were proposed to improve VLP production.

Glycosylation sterically inhibits platelet adhesion to von Willebrand factor without altering intrinsic conformational dynamics

BackgroundA molecular basis for von Willebrand factor (VWF) self‐inhibition has been proposed by which the N‐terminal and C‐terminal flanking sequences of the globular A1 domain disulfide loop bind to and suppress the conformational dynamics of A1. These flanking sequences are rich in O‐linked glycosylation (OLG), which is known to suppress platelet adhesion to VWF, presumably by steric hindrance. The inhibitory mechanism remains unresolved as to whether inhibition is due to steric exclusion by OLGs or a direct self‐association interaction that stabilizes the domain. ObjectivesThe platelet adhesive function, thermodynamic stability, and conformational dynamics of the wild‐type and type 2M G1324S A1 domain lacking glycosylation (Escherichia coli) are compared with the wild‐type glycosylated A1 domain (HEK293 cell culture) to decipher the self‐inhibitory mechanism. MethodsSurface plasmon resonance and analytical rheology are utilized to assess Glycoprotein Ibα (GPIbα) binding at equilibrium and platelet adhesion under shear flow. The conformational stability is assessed through a combination of protein unfolding thermodynamics and hydrogen‐deuterium exchange mass spectrometry (HXMS). ResultsA1 glycosylation inhibits both GPIbα binding and platelet adhesion. Glycosylation increases the hydrodynamic size of A1 and stabilizes the thermal unfolding of A1 without changing its equilibrium stability. Glycosylation does not alter the intrinsic conformational dynamics of the A1 domain. ConclusionsThese studies invalidate the proposed inhibition through conformational suppression since glycosylation within these flanking sequences does not alter the native state stability or the conformational dynamics of A1. Rather, they confirm a mechanism by which glycosylation sterically hinders platelet adhesion to the A1 domain at equilibrium and under rheological shear stress.

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurodegenerative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson’s disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of death-associated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

Effective Stabilization of Viral Vectors in Liquid Using an Algorithm-Based Development Approach

Background In recent years approvals of both CAR-T cell therapies and promising clinical data of gene therapy trials such as in hemophilia or sickle cell disease, prove that significant progress of gene and cell therapy in the field of hematological malignancies as well as rare hematologic genetic disorders has been made. Replication-deficient recombinant viral vectors such as adenovirus serotype 5 (Ad5), adeno-associated virus (AAV) and members of the poxvirus family represent a rapidly growing field of vaccine development and gene therapy. Viral vectors are known as complex supra-molecular ensembles of macromolecules produced by living organisms (nucleic acids, proteins, polysaccharides and in the case of lipid enveloped viruses of phospholipids) which are prone to a variety of complex chemical and physical degradation pathways, in particular due to stress induced by manufacturing, storage and distribution; hence representing a significant hurdle for the development and administration of stable vector-based pharmaceuticals such as vaccines or gene therapeutics. AIM To effectively develop stabilizing formulations for replication-deficient recombinant viral vectors, we applied a database and algorithm-based development strategy including Design of Experiment (DoE). Methods As a representative example of viral vectors we used Ad5. DoE was applied to select the most effectively stabilizing formulations. Ad5 was stored at 37 °C and 25 °C as accelerated aging temperature to identify the most effective stabilizing excipients and combinations thereof. Based on these results, the best selected formulations were further iteratively optimized and used for real-time storage at 5 °C in both liquid and lyophilized formulation. Infectivity was measured by titration assay and immunostaining of the Ad5 specific Hexon protein in HEK293 cell culture. ResultS By analysis of the infectious virus titers and mathematical combination of these results with the DoE matrix, the linear influence of each amino acid used in the DoE matrix was determined. The accelerated aging conditions were shown to be predictive for real-time aging. Several of the excipients indicated a neutral influence, whereby a well-balanced combination of the components was important regarding concentration and stabilizing interactions. The predictive power of this approach was confirmed by by long-term storage of two iteratively improved formulations tailored for Ad5. Conclusion The highly efficient pre-selection strategy of effectively stabilizing excipients by an algorithm-based development approach and an accelerated aging model enables the generation of best-in-class stable liquid formulations for viral vectors in short development time. Stable titres in liquid formulations can avoid lyophilization, loss of infectivity via freeze/thaw procedures or high overage and thus significantly reduce manufacturing costs. This approach is transferred to AAV and could have beneficial impact when applied early in downstream processing. A transfer to the frequently applied gene therapy vector system AAV is currently being implemented and an update will be presented at the conference. Disclosures No relevant conflicts of interest to declare.

ATP-loaded biomimetic nanoparticles as controlled release system for extracellular drugs in cancer applications.

PurposeThe antitumoral effect of ATP requires its accumulation in the extracellular space to interact with membrane receptors in target cells. We propose the use of albumin nanoparticles (ANPs) coated with erythrocyte membranes (EMs) to load, deliver, release, and enhance the extracellular anticancer activity of ATP.Materials and methodsANPs were synthesized by desolvation method and optimal values of pH, albumin concentration, and ethanol volume were determined. EMs were derived from erythrocyte lysates and were coated on to ANPs using an extruder. Size was determined by transmission electron microscopy (TEM) and hydrodynamic size and zeta potential were determined by dynamic light scattering. Coating of the ANPs with the EMs was verified by TEM and confocal microscopy. Nanoparticle cell uptake was analyzed by confocal microscopy using HeLa and HEK-293 cell cultures treated with nanoparticles stained with 1,1′-diocta-decyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) for EM-ANPs and Alexa 488 for ANPs. Cell viability was analyzed by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and Annexin V/propidium iodide assays.ResultsOptimal values of ANP preparation were as follows: pH=9, 10 mg/mL albumin concentration, and 2.33±0.04 mL ethanol volume. Size distributions as analyzed by TEM were as follows: ANPs =91.9±4.3 nm and EM-ANPs =98.3±5.1 nm; hydrodynamic sizes: ANPs =180.5±6.8 nm and EM-ANPs =197.8±3.2 nm; and zeta potentials: ANPs =17.8±3.5 mV, ANPs+ATP =−13.60±0.48 and EM-ANPs =−13.7±2.9 mV. The EMs coating the ANPs were observed by TEM and confocal microscopy. A fewer number of internalized EM-ANPs+ATP compared to non-coated ANPs+ATP was observed in HeLa and HEK-293 cells. Cell viability decreased up to 48.6%±2.0% with a concentration of 400 µM ATP after 72 hours of treatment and cell death is caused mainly via apoptosis.ConclusionOur current results show that it is possible to obtain nanoparticles from highly biocompatible, biodegradable materials and that their coating with EMs allows the regulation of the internalization process in order to promote extracellular activity of ATP.

Compact UV LED Lamp with Low Heat Emissions for Biological Research Applications

Much biomedical research focuses on the effects of UV light on human cells. UV light sources are a prerequisite for such research. This paper presents the design and achieved performance of a UVA (Ultraviolet A: 320–400 nm) and a UVB (Ultraviolet B: 290–320 nm) LED-based lamp suitable for use in bioassays, as well as inside an incubator. Numerical simulations were used to optimise the number, layout and output power of LEDs to achieve good irradiance homogeneity while maintaining low costs. Design was optimised for the efficient transfer of generated heat away from the irradiated samples through the heatsink at the back of the lamps. The average irradiance of the target surface by the UVA lamp was 70.1 W/m2 with a maximum deviation of 4.9%, and the average irradiance by the UVB lamp was 3.1 W/m2 with a maximum deviation of 4.8%. With the UVA and UVB lamps, the temperature of samples undergoing irradiation in the incubator rises from 37 to 42 °C within 40 and 67 min, respectively. This by far exceeds the required UV irradiation time in most cases. Tests on Jurkat and HEK-293 cell cultures confirmed the suitability of our lamps for biomedical research.

The Effect of Endoplasmic Reticulum Stress Activator-Tunicamycin on HEK293 Cells

DOI : 10.26650/experimed.2018. 462982 Objective : Among the functions of the Endoplasmic Reticulum (ER) are many different reactions that include the regulation of protein folding and modifications in the lumen, as well as the use of Ca+2 and glycogen storage, the biogenesis of cell membrane lipids. ER homeostasis becomes unbalanced and is recognized as ER stress by the cell. It triggers Unfolded Protein Responce (UPR) systems. Hypoxia, viral infections, unfolded protein accumulation, and some chemicals cause ER stress. Among the chemicals, tunicamycin is an antibiotic known to induce ER stress in the cell by inhibiting the enzyme GlcNAc phosphotransferase (GPT), which is involved in the first step of synthesis of asparagine-linked glycoproteins. The aim of this study is to determine the optimal experimental conditions for investigating ER stress in vitro. Material and Method : The HEK293 cells were cultured at the appropriate culture conditions and treated with five different doses (0.5, 1, 2, 5, and 10 μ g/mL) of tunicamycin for 12 hours. The morphology of the cells was evaluated with a light microscope for 1, 2, 4, 8, and 12 hours. The total RNA isolation and cDNA synthesis were performed from normal and treated HEK293 cells with 0.5 and 1 μ g/mL of tunicamycin. Expressions of CHOP, BiP, GADD34, ATF4, EDEM1, ASK1, XBP1 (spliced, unspliced and total), TRAF2, HERP, and PDIA4 selected from the UPR system were compared to normal cells for the evaluation of ER stress via tunicamycin application by qRT-PCR Results : After 1, 2, 4, 8, and 12 hour observations using light microscopy after treatment of tunicamycin, the morphology of HEK293 cells were preserved at concentrations of only 0.5 and 1 μ g/mL. In all other doses, the morphological structures of the cells were observed to be impaired within the first hour. Relative quantitation values of BiP, ASK1, PDIA4, s-xBP1, us-XBP1, t-XBP1, HERP, and CHOP were increased significantly on HEK293 cells treated with tunicamycin as commpared to normal cells. Despite ASK1 being in the same pathway, a decrease of TRAF2 relative quantitation was observed. Conclusion : It was found that the appropriate doses to induce ER stress with tunicamycin utilizing an HEK293 cell culture were 0.5 and 1 μ g/mL. Higher doses are thought to lead to cell apoptosis.

In vitro evaluation of copper oxide nanoparticle-induced cytotoxicity and oxidative stress using human embryonic kidney cells.

This study aimed to evaluate the in vitro cytotoxicity and oxidative stress induced by the copper oxide nanoparticles (CuO NPs) in human embryonic kidney cell line (HEK-293) cells following exposure. CuO NPs size <50 nm were used in this study. HEK-293 cell cultures were exposed to different concentrations of CuO NPs between 3 µg/ml and 300 µg/ml and quartz (known as cytotoxic agent) and assessed for cell viability-mitochondrial function (MTT assay), cell membrane damage (lactate dehydrogenase (LDH) assay), reduced glutathione (GSH), interleukin-8 (IL-8), and lipid peroxidation levels. The IC50 value of NPs was found to be 65.5 µg/ml. Exposure of HEK cells to CuO NPs (10-300 µg/ml) resulted in concentration-dependent cell membrane damage, increased production of IL-8, increased thiobarbituric acid reactive substance (TBARS), and decreased intracellular GSH levels. The significant increases in IL-8, TBARS, and LDH levels along with decreased GSH levels indicated induction of oxidative stress in cells. Our preliminary data suggest that oxidative stress might contribute to CuO NPs-induced cytotoxicity in HEK-293 cells.

Multimodal Production of Adeno-Associated Virus.

Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer.

The Fast-Growing Strain of Hepatitis A Virus MB-7/293 (HepA-293) Adapted to HEK293 Cell Culture: Properties of Virus Production and Analysis of Genomic RNA

Hepatitis A virus (HAV) continues to cause outbreaks of viral hepatitis, which remains a socially significant disease despite existing vaccines. During the production of vaccines, HAV strains that require prolonged incubation of infected cell cultures, up to 4 weeks, are used. Several HAV strains, which are fast-growing on cell cultures, up to 7–10 days, are known. This paper presents the results of virological characterization and analysis of the genome of the HAV strain MB-7/293 (HepA-293) derived from MB-7/4647, one of the fast-growing strains, which was obtained by adapting it to HEK293 cell culture (human embryonic kidney cells). It was shown that the MB-7/293 strain significantly exceeds the parent MB-7/4647 strain in productivity when cultured on HEK293 cell culture, i.e., in the yield of the infectious virus and the virus-specific antigen. Analysis of the nucleotide sequence of full-length genomic RNA of the MB-7/293 strain showed that it differs from the genome sequence of MB-7/4647 in three nucleotide substitutions in the 5'-untranslated region and 20 nucleotide substitutions in the coding region, which lead to substitutions of ten amino acid residues in the proteins VP3, VP1, 2B and 2C. Comparative analysis of the genomic sequences of the strains MB-7/293, MB-7/4647, and other HAV strains, including the previously described fast-growing strains, allowed to reveal mutations that affect the reproductive properties of the virus.

Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures.

At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.

Low Ca2+ levels in the culture media support the heterologous expression of insect odorant receptor proteins in HEK cells

BackgroundHeterologous expression of insect odorant receptors (ORs) in mammalian or insect cells is challenging due to the insufficient intracellular trafficking of ORs and their ability to form leak ion channels. New MethodWe tested whether reducing the Ca2+ levels in the cell culture medium after electroporation by means of a Dulbecco's Modified Eagle Medium (DMEM) without calcium, in a 1:1 ratio with Ham's F12 nutrient mixture, together with 10% fetal calf serum, can improve the success rate of insect OR expression in HEK293 cells. ResultsWe show that a reduced extracellular Ca2+ level supports functional expression of insect ORs by increasing the fraction of cells responding to the co-receptor agonist VUAA1 and by reducing the intracellular Ca2+ base level of transfected cells. Comparison with existing method(s)A DMEM formula without calcium outperforms standard DMEM in a 1:1 ratio with Ham's F12 mix and 10% serum, when culturing HEK293 cells transiently expressing insect OR proteins. ConclusionsReducing the extracellular Ca2+ level of HEK293 cell culture media after transfection increases the success of functional insect OR expression.

Protective effects of isatin and its synthetic derivatives against iron, copper and lead toxicity

IntroductionWhile some metals are required for physiological functions in the form of essential trace elements, they can cause toxicity in the excessive concentrations. Chelation therapy was used to reduce the adverse effects of acute and chronic poisoning by metals. Isatin derivatives form complexes with copper ions indicating that they may have protective activity against metal overload. MethodIn this study, four compounds (isatin and three isatin-derivatives Mj1, TR and Mk1) were evaluated for drug-likeliness. Then their potency inhibiting cell proliferation was determined in HEK293 cell culture assay. Finally, IC50 values for lead, copper, and iron was evaluated in the absence and also the presence of isatin and its derivatives. ResultsIsatin and its derivatives used in this study complied with the Lipinski criteria for drug-likeliness. The greatest difference between the IC50 values and the non-toxic dose was obtained for TR and Mj1, respectively. Pretreatment with the Mj1 increased the IC50 values for lead, iron, and copper, by 2.1, 1.7 and 1.7 times, respectively. At non-toxic dose, TR has only increased the IC50 values for lead and copper by 1.4 and 1.3 times without affecting iron cytotoxicity. Mk1 increased the IC50 values for lead, copper, and iron by 1.3, 1.8 and 1.7 times, respectively. ConclusionsMj1 is suggested as a lead compound for developing therapeutic agents for lead (Pb) toxicity and Mk1 for copper and iron.