HEK293 Cell Culture Research Articles

Characterization of the dynamic viscosity of cell cultures and its effect on mixing performance in a spinner flask bioreactor

Computational fluid dynamics (CFD) models have been developed to simulate cell culturing bioreactors but assume water-like viscosity properties due to significant data gaps. This study characterized the dynamic viscosity of HEK-293 cell cultures and evaluated its effect on mixing performance in a spinner flask bioreactor. Viscosity measurements indicated that the cell culture media, media with microcarriers, and cell cultures presented shear thinning behaviors within the measured shear rate range of 1–100 s−1. The viscosity also increased with the microcarrier concentrations and growth of cell culture. The CFD model, incorporating dynamic viscosity data, showed that shear stress and Kolmogorov length profiles are significantly influenced by microcarrier concentrations and cell culture growth. Higher microcarrier concentrations led to higher average shear stress and Kolmogorov values. The cultured HEK-293 cells after seven days of growth also had higher average shear stress and Kolmogorov values than at the day of seeding, indicating an impact caused by the cells’ metabolism and biomass. Overall, the results indicated that assuming water-like properties underestimates shear stress and Kolmogorov length scales, especially at zones of lower shear rates due to the observed shear thinning behavior. Thus, careful monitoring of dynamic viscosity of cell cultures and proper control of mixing parameters are critical to deliver the desired mixing conditions for optimized cell growth especially during scale-up production operations.

Human eRF1 Translation Regulation

Eukaryotic translation release factor eRF1 is an important cellular protein that plays a key role in translation termination, nonsense-mediated mRNA decay (NMD), and readthrough of stop codons. The amount of eRF1 in the cell influences all these processes. The mechanism of regulation of eRF1 translation through an autoregulatory NMD-dependent expression circuit has been described for plants and fungi, but the mechanisms of regulation of human eRF1 translation have not yet been studied. Using reporter constructs, we studied the effect of eRF1 mRNA elements on its translation in cell-free translation systems and HEK293 cell culture. Our data indicate the absence of an NMD-dependent autoregulatory circuit for human eRF1 expression. We found that the translation of the eRF1 coding sequence is most strongly influenced by the 5′ untranslated region of eRF1 mRNA and the start codon of the upstream open reading frame. According to the transcription start database, eRF1 mRNA is characterized by high heterogeneity of the transcription start and a variable 5' untranslated region in length. In addition, the start codon of the CDS in eRF1 mRNA is located within the known translational regulator of short 5' untranslated regions (TISU), which also stimulates mRNA transcription of genes with high transcription start heterogeneity. We hypothesize that regulation of human eRF1 synthesis occurs at both the transcriptional and translational levels. At the transcription level, the length of the eRF1 5' untranslated region and the number of the upstream open reading frames in it are regulated. This regulation in turn, regulates the production of eRF1 at the translation level.

Single-use chromatographic clarification to eliminate endonuclease treatment in production of recombinant adeno-associated viral vectors

In current manufacturing processes for recombinant adeno-associated viral vectors (rAAV), endonuclease treatment is used prior to depth filtration to break down host cell DNA that is released from cells post-lysis. Without this treatment, traditional depth filters exhibit negligible clearance of host cell DNA, a key process-related impurity. In this work, we evaluate single-use chromatographic clarification using Harvest RCTM filters (3 M) to process rAAV8 and rAAV9 HEK293 cell culture lysate with and without endonuclease pretreatment. We demonstrate endonuclease-free clarification for both serotypes with yield >90 % and up to 3 log reduction of host cell DNA, from >104 ng/mL to <50 ng/mL, matching or exceeding the filtrate quality in traditional depth filtration with 100 U/mL of endonuclease added during lysis. The addition of 100 mM of salt in the harvest buffer is also shown to improve filtration throughput, prevent filter clogging, and limit rAVV binding to the filter device. Finally, a mechanistic cake-fiber fouling model is shown to be a good representation of the Harvest RCTM filtration mechanism. The endonuclease-free clarification approach enables cost savings of USD 100,000 per 500 L batch, equivalent to the cost of cGMP-grade endonuclease at this scale. The benefits of endonuclease-free clarification further include easier raw material sourcing as well as eliminating the need for a residual endonuclease assay in rAAV manufacturing processes.

Internalization of PEI-based complexes in transient transfection of HEK293 cells is triggered by coalescence of membrane heparan sulfate proteoglycans like Glypican-4

Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).

PLGA nanocapsules as a delivery system for a recombinant LRP-based therapeutic.

Telomerase activity is directly affected by the laminin receptor precursor (LRP) protein, a highly conserved nonintegrin transmembrane receptor, which has been shown to have therapeutic effects in ageing, and age-related diseases. Recently, it has been found that overexpression of LRP-FLAG, by plasmid transfection, leads to a significant increase in telomerase activity in cell culture models. This may indicate that upregulation of LRP can be used to treat various age-related diseases. However, transfection is not a viable treatment strategy for patients. Therefore, we present a nanoencapsulated protein-based drug synthesised using poly(lactic-co-glycolic acid) (PLGA) nanocapsules for delivery of the 37 kDa LRP protein therapeutic. PLGA nanocapsules were synthesised using the double emulsification-solvent evaporation technique. Different purification methods, including filtration and centrifugation, were tested to ensure that the nanocapsules were within the optimal size range, and the BCA assay was used to determine encapsulation efficiency. The completed drug was tested in a HEK-293 cell culture model, to investigate the effect on cell viability, LRP protein levels and telomerase activity. A significant increase in total LRP protein levels with a concomitant increase in cell viability and telomerase activity was observed. Due to the observed increase in telomerase activity, this approach could represent a safer alternative to plasmid transfection for the treatment of age-related diseases.

Bioactivity of &lt;i&gt;Lantana camara&lt;/i&gt; Flowers: Antilithiatic and Nephroprotective Implications in Male Wistar Rats

Lantana camara is a well-known medicinal plant with various reported therapeutic compounds. This study explores the antilithiatic and nephroprotective effects of Lantana camara flower hydroalcoholic extract. Using animal models and cell culture, we assessed its impact on ethylene glycol-induced nephrolithiasis and gentamicin-induced nephrotoxicity. The ethylene glycol-induced nephrolithiasis model revealed elevated levels of calcium, phosphate, and oxalate, indicating the formation of calculi. Treatment with the hydroalcoholic extract resulted in a significant decrease in uric acid levels, reducing the likelihood of calculi formation. Additionally, the animals treated with the extract showed reduced levels of urea, and creatinine, indicating improved kidney function. The results highlighted the notable difference between the preventive and curative treatments. In the HEK-293 cell culture, the hydroalcoholic extract demonstrated a significant inhibition of abnormal cell morphology induced by gentamicin, a nephrotoxic drug. The extract also exhibited a marked improvement in cell growth compared to the standard treatment. The findings of this study provide substantial evidence supporting the antilithiatic and nephroprotective effects of the hydroalcoholic extract of Lantana camara flowers. The extract effectively reduced the risk of calculi formation, improved renal parameters, and demonstrated potential in mitigating drug-induced nephrotoxicity. These results validate the traditional claims regarding the efficacy of Lantana camara as a therapeutic agent for kidney-related disorders. The hydroalcoholic extract of Lantana camara flowers could serve as a valuable alternative treatment option, offering potential benefits in the management of lithiasis and nephrotoxicity.

Sodium acetate increases the productivity of HEK293 cells expressing the ECD-Her1 protein in batch cultures: experimental results and metabolic flux analysis.

Human Embryonic Kidney cells (HEK293) are a popular host for recombinant protein expression and production in the biotechnological industry. This has driven within both, the scientific and the engineering communities, the search for strategies to increase their protein productivity. The present work is inserted into this search exploring the impact of adding sodium acetate (NaAc) into a batch culture of HEK293 cells. We monitored, as a function of time, the cell density, many external metabolites, and the supernatant concentration of the heterologous extra-cellular domain ECD-Her1 protein, a protein used to produce a candidate prostate cancer vaccine. We observed that by adding different concentrations of NaAc (0, 4, 6 and 8mM), the production of ECD-Her1 protein increases consistently with increasing concentration, whereas the carrying capacity of the medium decreases. To understand these results we exploited a combination of experimental and computational techniques. Metabolic Flux Analysis (MFA) was used to infer intracellular metabolic fluxes from the concentration of external metabolites. Moreover, we measured independently the extracellular acidification rate and oxygen consumption rate of the cells. Both approaches support the idea that the addition of NaAc to the culture has a significant impact on the metabolism of the HEK293 cells and that, if properly tuned, enhances the productivity of the heterologous ECD-Her1 protein.

Extracellular vesicle depletion and UGCG overexpression mitigate the cell density effect in HEK293 cell culture transfection

The hitherto unexplained reduction of cell-specific productivity in transient gene expression (TGE) at high cell density (HCD) is known as the cell density effect (CDE). It currently represents a major challenge in TGE-based bioprocess intensification. This phenomenon has been largely reported but the molecular principles governing it are still unclear. The CDE is currently understood to be caused by the combination of an unknown inhibitory compounds in the extracellular medium and an uncharacterized cellular change at HCD. This study investigates the role of extracellular vesicles (EVs) as extracellular inhibitors for transfection through the production of HIV-1 Gag virus-like particles (VLPs) via transient transfection in HEK293 cells. EV-depletion from the extracellular medium restored transfection efficiency in conditions suffering from the CDE, also enhancing VLP budding and improving production by 60%. Moreover, an alteration in endosomal formation was observed at HCD, sequestering polyplexes and preventing transfection. Overexpression of UGCG enzyme removed intracellular polyplex sequestration, improving transfection efficiency. Combining EV-depletion and UGCG overexpression improved transfection efficiency by ∼45% at 12x106 cells/mL. These results suggest that the interaction between polyplexes and extracelluar and intracellular vesicles plays a crutial role in the CDE, providing insights for the development of strategies to mitigate its impact.

A methodological primer of extracellular vesicles isolation and characterization via different techniques.

We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the "best method" for their work under their individual conditions.

Shiga-Toxin-Producing Strains of Escherichia coli O104:H4 and a Strain of O157:H7, Which Can Cause Human Hemolytic Uremic Syndrome, Differ in Biofilm Formation in the Presence of CO2 and in Their Ability to Grow in a Novel Cell Culture Medium.

One pathogen that commonly causes gastrointestinal illnesses from the consumption of contaminated food is Escherichia coli O157:H7. In 2011 in Germany, however, there was a prominent outbreak of bloody diarrhea with a high incidence of hemolytic uremic syndrome (HUS) caused by an atypical, more virulent E. coli O104:H4 strain. To facilitate the identification of this lesser-known, atypical E. coli O104:H4 strain, we wanted to identify phenotypic differences between it and a strain of O157:H7 in different media and culture conditions. We found that E. coli O104:H4 strains produced considerably more biofilm than the strain of O157:H7 at 37 °C (p = 0.0470-0.0182) Biofilm production was significantly enhanced by the presence of 5% CO2 (p = 0.0348-0.0320). In our study on the innate immune response to the E. coli strains, we used HEK293 cells that express Toll-like receptors (TLRs) 2 or 4. We found that E. coli O104:H4 strains had the ability to grow in a novel HEK293 cell culture medium, while the E. coli O157:H7 strain could not. Thus, we uncovered previously unknown phenotypic properties of E. coli O104:H4 to further differentiate this pathogen from E. coli O157:H7.

Evaluation of virus-neutralizing antibody level after novel coronavirus infection COVID-19: development of an instant assay assessing protective antibodies using a pseudovirus-based reaction

The continued emergence of SARS-CoV-2 variants with immune evasion properties of concern, such as Delta (B.1.617.2) and Omicron (B.1.1.529), calls into question the extent of the antibody-mediated immune response from the virus. The presence of virus-neutralizing antibodies against SARS-CoV-2 in the blood serum of recovered and immunized volunteers is the most accurate indicator of the level of protective activity. Methods for reliable, sensitive and rapid detection of anti-SARS-CoV-2 nAbs are needed for preclinical and clinical vaccine research. In addition, quantification of virus-neutralizing antibodies in recovered COVID-19 subjects may be useful in identifying potential donors for passive immunization and therapeutic use of class G immunoglobulins. Pseudoviruses are actively used as an alternative to infectious viral isolates of pathogenicity groups III in serological studies to determine the titers of neutralizing antibodies formed in vaccinated or infected volunteers. In addition, using several pseudotypes with different reporter genes, it is possible to simultaneously detect antibodies to different types of viruses in one biological sample. Currently, the pseudolentiviral system is widely used, in which pseudoviral particles are obtained by transfection of producer cells with vectors of a multiplasmid system of 45 plasmids: one for the vector genome, the second for Gag-Pol, the third for Rev, and one or two for protein(s) envelope, or for the co-expression of a labeled viral protein such as GAG-GFP or VPR-GFP, the main advantage of which is safety due to the minimal risk of generating a replication-competent virus. The article discusses the development of a technique that allows to determine the presence of virus-specific neutralizing antibodies to the SARS-CoV-2 antigen in the blood serum of volunteers who have had a new coronavirus infection COVID-19 and/or immunized with specific prophylaxis drugs, healthy volunteers in a neutralization reaction on a HEK 293-cell culture. T-hAce2 using pseudotyped viral constructs based on human immunodeficiency virus. The results of the development and validation of the method, as well as its subsequent prospects for use, are shown.

Method for obtaining recombinant antibodies produced by a cell line transduced with recombinant adenoviruses

Objectives. To develop a technology for obtaining recombinant antibodies in a suspension culture of human HEK293 cells using transduction with recombinant adenovirus serotype 5 (rAd5) carrying genes expressing heavy and light chains of antibodies on the example of two broadspectrum anti-influenza antibodies 27F3 and CR9114.Methods. Ad5-27F3-H, Ad5-CR9114-H, and Ad5-27F3-L recombinant adenoviruses carrying the 27F3 antibody heavy chain gene, CR9114 antibody heavy chain gene, and 27F3 light chain gene, respectively, were generated using the AdEasy™ Adenoviral vector system. To accumulate preparative amounts of recombinant r27F3 and rCR9114 antibodies, the HEK293 suspension cell line was transduced with recombinant adenoviruses carrying genes for heavy and light chains of antibodies. The cells were cultured in a wave-type bioreactor. Chromatography was used to purify recombinant antibodies from the culture medium. After analyzing the molecular weights of purified antibodies using protein electrophoresis, their ability to interact with influenza A and B viruses was analyzed using the Western blot technique, while their ability to neutralize influenza A and B viruses was evaluated using the virus neutralization assay.Results. A method for the accumulation and purification of recombinant r27F3 and CR9114 antibodies from the culture medium of a suspension culture of human cells following transduction with its recombinant adenoviruses carrying the genes for heavy and light chains of these antibodies was developed. The ability of the r27F3 antibody to interact with and neutralize influenza A viruses of group 1 (except influenza A virus subtype H2) and group 2 was shown. The ability of the rCR9114 antibody to interact with influenza A viruses of group 1 and influenza B viruses, as well as to neutralize influenza A viruses of group 1, was demonstrated.Conclusions. A technology for obtaining recombinant antibodies in a suspension culture of HEK293 cells using transduction with recombinant adenoviruses carrying genes expressing heavy and light chains of antibodies was developed along with a confirmation of their specificity.

Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier.

Biotherapeutic soluble proteins that are recombinantly expressed in mammalian cells can pose a challenge when biomanufacturing in three-dimensional (3D) suspension culture systems. Herein, we tested a 3D hydrogel microcarrier for a suspension culture of HEK293 cells overexpressing recombinant Cripto-1 protein. Cripto-1 is an extracellular protein that is involved in developmental processes and has recently been reported to have therapeutic effects in alleviating muscle injury and diseases by regulating muscle regeneration through satellite cell progression toward the myogenic lineage. Cripto-overexpressing HEK293 cell lines were cultured in microcarriers made from poly (ethylene glycol)-fibrinogen (PF) hydrogels, which provided the 3D substrate for cell growth and protein production in stirred bioreactors. The PF microcarriers were designed with sufficient strength to resist hydrodynamic deterioration and biodegradation associated with suspension culture in stirred bioreactors for up to 21 days. The yield of purified Cripto-1 obtained using the 3D PF microcarriers was significantly higher than that obtained with a two-dimensional (2D) culture system. The bioactivity of the 3D-produced Cripto-1 was equivalent to commercially available Cripto-1 in terms of an ELISA binding assay, a muscle cell proliferation assay, and a myogenic differentiation assay. Taken together, these data indicate that 3D microcarriers made from PF can be combined with mammalian cell expression systems to improve the biomanufacturing of protein-based therapeutics for muscle injuries.

Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1–42 in Alzheimer’s disease

Reduced insulin-like growth factor 2 (IGF2) levels in Alzheimer’s disease (AD) may be the mechanism relating age-related metabolic disorders to dementia. Since Igf2 is an imprinted gene, we examined age and sex differences in the relationship between amyloid-beta 1–42 (Aβ42) accumulation and epigenetic regulation of the Igf2/H19 gene cluster in cerebrum, liver, and plasma of young and old male and female 5xFAD mice, in frontal cortex of male and female AD and non-AD patients, and in HEK293 cell cultures. We show IGF2 levels, Igf2 expression, histone acetylation, and H19 ICR methylation are lower in females than males. However, elevated Aβ42 levels are associated with Aβ42 binding to Igf2 DMR2, increased DNA and histone methylation, and a reduction in Igf2 expression and IGF2 levels in 5xFAD mice and AD patients, independent of H19 ICR methylation. Cell culture results confirmed the binding of Aβ42 to Igf2 DMR2 increased DNA and histone methylation, and reduced Igf2 expression. These results indicate an age- and sex-related causal relationship among Aβ42 levels, epigenomic state, and Igf2 expression in AD and provide a potential mechanism for Igf2 regulation in normal and pathological conditions, suggesting IGF2 levels may be a useful diagnostic biomarker for Aβ42 targeted AD therapies.

Characterization and Analysis of HEK293/Adenovirus Type 5 Cell Cultures under Simulated Microgravity Using Differential Neural Network Modeling

HEK293 cells are the common system to produce adenoviral vectors for vectorized vaccines and gene therapy products. Therefore, the improvement of this type of cell culture is highly relevant nowadays. Furthermore, the high aspect ratio vessel (HARV) bioreactor provides a simulated microgravity condition (SMGC) which allows the establishment of in vitro models of mammalian cells. The present work is aimed at showing the characterization of HEK293 cell cultures in serum‐free medium under SMGC and to identify a set of adaptable time differential equations from experimental data through a nonparametric mathematical modeling using differential neural networks (DNN). Results showed that the HARV bioreactor enables to obtain a gravity percentage equal to 〖%g〗_maximum = −0.053% (where minus sign implies against the gravity direction). In addition, the characterization of HEK293 culture under SMGC revealed that such condition favored the production of infective viral particles as well as the uptake of basic nutrients such as glucose and glutamine, but the cell concentration was not significantly affected. As shown, these results contribute to improve the adenoviral vector production, and the DNN analysis is useful to model the kinetic changes and to build the foundations for control strategies and optimization of adenoviral production in further studies.

Towards advanced cardiac miRNA therapies through DNA-based nanostructures

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Proyecto MINECO-Ministerio de Ciencia e Innovación (Spain)- PID2019-105674RB-I00 Proyecto DGA- Diputación General de Aragón (Spain)- LMP128_21 Introduction Cardiac microRNAs (miRNAs) have been found to be dysregulated in cardiac disease and aging both in animal models and humans. Thanks to their pleiotropic effects, miRNAs are spotlighted as promising therapeutic targets to treat cardiac conditions. However, their low stability and potential off-target effects in vivo difficult the development of effective and safe miRNA therapies. Nanotechnology enables the fabrication of biocompatible carriers to perform targeted delivery of both conventional and advanced therapies. In particular, nanostructures built with DNA (DNS) have been successfully developed for miRNA therapies in cancer, but they have not been applied in cardiac therapy yet [1]. Purpose The main goal of this study is to demonstrate the capacity of DNS to deliver miRNA therapies in human cardiomyocytes. Methods Our group has identified MIR24-2 upregulated with age in the human left ventricle and miR24-2-5p to interact with genes related with the cardiac function, such as SERCA2 [2]. Different versions of DNS were designed and generated by self-assembly of DNA strands containing anti-miR24-2-5p capture segments. Physicochemical characterization of DNS was then conducted to determine their auto-assembling capacity, size, morphology and thermal stability. The characterization of the biological effect of the DNS was also conducted in vitro and in cell culture of HEK293 and human induced pluripotent stem cells (iPSC)-derived cardiomyocytes (iCM) to assess their cytotoxicity and internalization capacity. Results DNS were assembled correctly and reproducibly into carriers of around 30-50 nm. In vitro, the DNS showed thermal stability at physiological temperature, proper stability in serum and they disassembled specifically in the presence of the target miR24-2-5p sequence. In HEK293 and iCM cultures, the DNS showed lack of cytotoxicity demonstrating in vitro biocompatibility. DNS were efficiently internalized by HEK293, but the uptake capacity of DNS by iCM was remarkably lower. Conclusions We created and characterized biocompatible anti-miR-loaded DNS that effectively captured the target miR-24-2-5p in vitro. DNS internalized in iCM, but with lower efficiency than in HEK293 cells. Our results indicate that DNS are suitable candidates to carry miRNA therapies to human cardiac cells in vitro, but the observed cell-type specific differences suggest that future efforts need to be directed towards developing DNS functionalization capable of promoting efficient and specific carrier uptake in vivo by primary cardiac cells in vivo.

U6/miR-211 expression ratio as a purity parameter for HEK293 cell-derived exosomes.

Small extracellular vesicles (sEVs) including exosomes are produced by all cell types and can be isolated from biological fluids and cell culture supernatants. The separation of exosomes with high purity from protein-rich media remains challenging. Besides contaminating proteins, small microvesicles (MVs) and apoptotic bodies are usually co-isolated with exosomes. The optimization of exosome separation and purification depends on reliable methods for the determination of the purity of the preparation, but no standard measurement has been defined so far. We tried to advance purity assessment. sEVs were isolated from HEK293 cell culture supernatants by various combinations of centrifugation, precipitation and size exclusion chromatography. sEVs with a diameter within the size range of 30-150 nm, typical for exosomes, were obtained with all tested isolation methods as shown by electron microscopy. To estimate the levels of protein contamination, flow cytometric analysis of the obtained vesicles was used. Based on the controlled preferential loading and enrichment of miR-211 into exosomes, a novel approach for the estimation of the fraction of HEK293 derived exosomes as opposed to MVs and apoptotic bodies in sEV mixtures was developed. This novel approach represents a simple qRT-PCR-based approach to improve the precise characterization of sEV isolates that is necessary for the usage of exosomes as carriers for therapeutic nucleic acids. Compared to the precipitation and size exclusion chromatography, the differential ultracentrifugation turned out to give sEVs with fairly intact shape and the highest purity according to the novel qRT-PCR-based approach, as well as to other established methods for purification assessment.

Transduction of HEK293 Cells with BacMam Baculovirus Is an Efficient System for the Production of HIV-1 Virus-like Particles.

Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 ± 1.1%, and a VLP titer of 2.8 ± 0.1 × 109 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100–200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production.

Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.

Potential Antiaging Effects of DLBS1649, a Centella asiatica Bioactive Extract.

PurposeCentella asiatica is a traditional medicinal plant, especially for wound healing and as a neuroprotective agent. DLBS1649 is a bioactive extract from C. asiatica, and was studied to investigate its benefits as an antiaging agent.MethodsDLBS1649 was administered to HEK293 and 3T3L1 mammalian cells cultured in a time- or dose-dependent manner. Telomere length analysis was performed. TERT, CMYC, SIRT1, SIRT2, and KL expression were observed using reverse-transcription qPCR. Telomerase protein was studied with ELISA, while calorie restriction was observed using Oil Red O. In vivo study was conducted using Drosophila melanogaster with restricted mean survival time as the statistical method of analysis.ResultsDLBS1649 50 µg/mL showed an effect in the prevention of telomere shortening by 50% and decrease in telomerase activity by 28% compared to the controls (70% and 40%, respectively) in the HEK293 cell cultures. TERT-, CMYC-, SIRT1-, SIRT2-, and KL-expression degression was also reduced (29%, 9%, 18%, 25%, 9%, and 30%, respectively) compared to the controls (46%, 40%, 56%, 44%, and 46%, respectively) after ten serial passages. Calorie-restriction activity from DLBS1649 50 µg/mL was seen, with lower fat droplet counts being detected in the treated samples (37%) than the controls (28%) in 3T3L1 cells. DLBS1649 2 mg/mL increased restricted mean survival time in male and female D. melanogaster (23.87% [p<0.05] and 12.58%, respectively).ConclusionThe results revealed DLBS1649’s potential as an antiaging agent based on telomere-length preservation, decreased expression of aging-related genes, increased calorie restriction in vitro, and mortality reduction in D. melanogaster in vivo.