A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation.

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening.

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction phase problem. Novel structures that lack a suitable homology model are often derivatized with heavy atoms to provide experimental phase information. The presented protocol efficiently generates derivatized protein crystals by combining random microseeding matrix screening with derivatization with a heavy atom molecule I3C (5-amino-2,4,6-triiodoisophthalic acid). By incorporating I3C into the crystal lattice, the diffraction phase problem can be efficiently solved using single wavelength anomalous dispersion (SAD) phasing. The equilateral triangle arrangement of iodine atoms in I3C allows for rapid validation of a correct anomalous substructure. This protocol will be useful to structural biologists who solve macromolecular structures using crystallography-based techniques with interest in experimental phasing.

Isolation and characterization of extracellular vesicle subpopulations from tissues.

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

Impact of various iodine concentrations of iohexol and iodixanol contrast media on image reconstruction techniques in a vascular-specific contrast media phantom: quantitative and qualitative image quality assessment.

PURPOSE: The aim of our study is to investigate the impact of iodine quantification on image reconstruction when employing a vascular-specific contrast media phantom with varying iodine concentrations. MATERIALS AND METHODS: A 30-cm phantom simulating arterial and venous blood vessel diameters was manufactured. Small (9 mm) and medium (12 mm) cylinders contained iodine concentrations from 10 to 100% while large (21 mm) cylinders were in quartiles from 25 to 100% diluted in blood equivalent medium. Each phantom was filled with either iohexol 350 mgI/mL (Group A) or iodixanol 320 mgI/mL (Group B) and then scanned separately. For each group, tube potential (80-140 kVp) and current (50-400 mAs) were changed and all image series were reconstructed with filtered back projection (FBP), hybrid-based iterative reconstruction (HBIR) and model-based iterative reconstruction (MBIR). Mean opacification was measured in all groups. All data were compared employing an independent t test and Pearson's correlation. Visual grading characteristic (VGC) and Cohens' kappa analyses were performed. RESULTS: At 80 kVp, mean opacification using HBIR was significantly higher in Group B (2165 ± 1108 HU) than in Group A (2040 ± 1036 HU) (p < 0.009). At 140 kVp, MBIR and HBIR were greater in Group A (1704 ± 1033 HU and 1685 ± 1023 HU) versus Group B (1567 ± 1036 HU and 1567 ± 1034 HU) (p < 0.022). CNR using FBP, HBIR and MBIR was higher in Group B (46 ± 42 HU, 70 ± 163 HU and 83 ± 74 HU, respectively) than in Group A (43 ± 39 HU, 174 ± 130 HU and 80 ± 65 HU, respectively) (p < 0.0001-0.035). Qualitative image analysis demonstrated no difference in Cohen's kappa analysis. VGC was higher in Group A at all image reconstruction groups. CONCLUSION: Iohexol outperforms iodixanol in observer performance when assessing image reconstruction techniques and iodine concentrations in a vascular-specific contrast media phantom.

A comparison of AAV-vector production methods for gene therapy and preclinical assessment.

Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.

Visualizing Coronavirus Entry into Cells.

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids.

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

From in vitro evaluation to human postmortem pre-validation of a radiopaque and resorbable internal biliary stent for liver transplantation applications.

The implantation of an internal biliary stent (IBS) during liver transplantation has recently been shown to reduce biliary complications. To avoid a potentially morbid ablation procedure, we developed a resorbable and radiopaque internal biliary stent (RIBS). We studied the mechanical and radiological properties of RIBS upon in vivo implantation in rats and we evaluated RIBS implantability in human anatomical specimens. For this purpose, a blend of PLA50-PEG-PLA50 triblock copolymer, used as a polymer matrix, and of X-ray-visible triiodobenzoate-poly(ε-caprolactone) copolymer (PCL-TIB), as a radiopaque additive, was used to design X-ray-visible RIBS. Samples were implanted in the peritoneal cavity of rats. The radiological, chemical, and biomechanical properties were evaluated during degradation. Further histological studies were carried out to evaluate the degradation and compatibility of the RIBS. A human cadaver implantability study was also performed. The in vivo results revealed a decline in the RIBS mechanical properties within 3 months, whereas clear and stable X-ray visualization of the RIBS was possible for up to 6 months. Histological analyses confirmed compatibility and resorption of the RIBS, with a limited inflammatory response. The RIBS could be successfully implanted in human anatomic specimens. The results reported in this study will allow the development of trackable and degradable IBS to reduce biliary complications after liver transplantation. STATEMENT OF SIGNIFICANCE: Biliary reconstruction during liver transplantation is an important source of postoperative morbidity and mortality although it is generally considered as an easy step of a difficult surgery. In this frame, internal biliary stent (IBS) implantation is beneficial to reduce biliary anastomosis complications (leakage, stricture). However, current IBS are made of non-degradable silicone elastomeric materials, which leads to an additional ablation procedure involving potential complications and additional costs. The present study provides in vitro and human postmortem implantation data related to the development and evaluation of a resorbable and radiopaque internal biliary stent (RIBS) that could tackle these drawbacks.

Production of Recombinant Adeno-Associated Viruses (rAAVs) by Transient Transfection.

The most commonly used method for production of recombinant adeno-associated virus (rAAVs) in research laboratories is by transient triple transfection of 293 cells with AAV cis and trans plasmids and an adenovirus helper plasmid. This protocol describes the processes required to prepare the transfected cell suspension for virus purification.

Purification of Recombinant Adeno-Associated Viruses (rAAVs) by Iodixanol Gradient Centrifugation.

This is a simple method for rapid preparation of recombinant adeno-associated virus (rAAV) stocks, which can be used for in vivo gene delivery. The purity of these vectors is considerably lower than that obtained by either CsCl gradient centrifugation or by combination of iodixanol gradient ultracentrifugation followed by column chromatography.

Enrichment of Fully Packaged Virions in Column-Purified Recombinant Adeno-Associated Virus (rAAV) Preparations by Iodixanol Gradient Centrifugation Followed by Anion-Exchange Column Chromatography.

This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.

Effect of iodinated contrast media on the oxygen tension in the renal cortico-medullary region of pigs.

Repeated injections of iodinated contrast media (CM) can lead to a deterioration of the renal blood flow, can redistribute blood from the renal cortex to other parts of the kidney and can cause small decreases of the blood flow in cortical capillaries, a significant reduction in blood flow in peritubular capillaries and a significant reduction in blood flow in the vasa recta. Therefore, a study in pigs was designed, to show whether the repeated injection of CM boli, alone, can cause a reduction of oxygenation in the cortico-medullar renal tissue - the region with the highest oxygen demand in the kidney - of pigs.While the mean pO2-value had only decreased by 0.3 mmHg from 29.9±4.3 mmHg to 29.6±4.3 mmHg (p = 0.8799) after the tenth Iodixanol bolus, it decreased by 5.9 mmHg from 34.0±4.3 mmHg to 28.1±4.3 mmHg after the tenth Iopromide bolus (p = 0.044). This revealed a remarkable difference in the influence of these CM on the oxygen partial pressure in the kidney.Repeated applications of CM had a significant influence on the renal oxygen partial pressure. In line with earlier studies showing a redistribution of blood from the cortex to other renal areas, this study revealed that Iodixanol - in contrast to Iopromide - induced no changes in the pO2 in the cortico-medullar region which confirms that Iodixanol did not hinder the flow of blood through the renal micro-vessels. These results are in favor of a hypothesis from Brezis that a microcirculatory disorder might be the basis for the development of CI-AKI.

Combining random microseed matrix screening and the magic triangle for the efficient structure solution of a potential lysin from bacteriophage P68.

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

The Effects of Iodinated Radiographic Contrast Media on Multidrug-resistant K562/Dox Cells: Mitochondria Impairment and P-glycoprotein Inhibition.

Iodinated radiographic contrast media is used in cancer radiography for cancer diagnosis. The aim of this present study was to examine five iodinated radiographic contrast media (IRCM) (i.e., iohexol, iopamidol, iobitridol, ioxaglate, and iodixanol) in terms of their cytotoxicity, mitochondria membrane potential (ΔΨm), and P-glycoprotein function in multidrug resistant K562/Dox cancer cells and corresponding sensitive cancer cells. The cytotoxicity was determined by colorimetric resazurin reduction assay. The ΔΨm and P-glycoprotein function was measured using a noninvasive functional spectrofluorometry. Rhodamine B, fluorescence probe, was used to estimate ΔΨm. The kinetic of P-glycoprotein-mediated efflux pirarubicin was used to monitor P-glycoprotein function in multidrug resistant (MDR) cancer cells. The results showed that ioxaglate and iodixanol show similar efficacy in MDR cancer cells and for their corresponding sensitive cancer cells. Iopamidol, iohexol, and iobitridol showed higher efficacy in MDR cancer cells than for the corresponding sensitive cancer cells by approximately 2 fold. The results also showed no significant change in the |ΔΨm| values in treated K562 and K562/Dox cancer cells when compared to the non-treated K562 and K562/Dox cancer cells. However, there were notable changes detected for iobitridol and iodixanol at 50 mgI/mL. Similarly, the results showed significant differences in P-glycoprotein function of K562/Dox cancer cells after treatment with IRCM when compared to the non-treated K562/Dox cancer cells, with iohexol and iodixanol being the notable exceptions once again. In this present study, IRCM exhibited cytotoxicity on MDR cancer cells and their corresponding sensitive cancer cells. IRCM also showed potential as an anticancer agent in the future.

Adeno-Associated Virus Production, Purification, and Titering.

Adeno-associated virus (AAV) vectors are exemplary tools for studying gene function in vivo and are particularly favorable for transferring genes of interest into brain tissues. They have shown great promise as a gene therapy vector for preclinical and clinical applications. However, the ability to use this tool is often hampered because the viruses themselves are not readily available. Many methods have been developed for AAV production. Here, we describe a simple method for small- to medium-scale (1012 -1013 viral particles) production of AAV based on Polyethylenimine Max (PEI Max)-mediated triple transfection of HEK 293 cells and purification with iodixanol gradient ultracentrifugation. These methods will provide users with ample material of sufficient quality for performing in vivo gene transfer. © 2018 by John Wiley & Sons, Inc.

Magnetic iodixanol - a novel contrast agent and its early characterization.

AIMS: Contrast-induced nephropathy is a commonly encountered problem in clinical practice. The purpose of the study was to design and develop a novel contrast agent, which could be used to prevent contrast-induced nephropathy in the future. METHODS: In total, 20-220nm magnetic nanoparticles were conjugated with iodixanol, and their radio-opacity and magnetic properties were assessed thereafter. Scanning electron microscopy pictures were acquired. Thereafter, the nanoparticles conjugate was tested in cell culture (HUVEC cells), and Quantibody® assay was studied after cell treatment in 1:5 dilutions for 48h, compared with control. RESULTS: The conjugate preparation had an adequate radio-opacity. A 4mm magnetic bubble was attached to a bar magnet and the properties were studied. The magnetic bubble maintained its structural integrity in all angles including antigravity position. Scanning electron microscopy showed magnetic nanoparticles in all pictures and the particles are of 100-400nm agglomerates with primary particle sizes of roughly 20nm. 1:5 diluted particles had no effect on secretion of IL-1a, IL-1b, IL-4, IL-10, IL-13 and TNFa. Particles increased secretion of IL-8 from 24h and 48h. Secretion of IFNg was also increased when particles were added to the cells as early as 1h. Likewise, IL-6 was strongly secreted by HUVEC treated with particles from 24h incubation time. In contrast, the secretion of MCP-1 was slightly reduced on HUVEC treated with particles. CONCLUSION: There is potential for a novel iodixanol-magnetic nanoparticle conjugate to be used in cineradiography. Further investigations need to be performed to study its performance in vitro and in vivo.

A new formulation of poly(MAOTIB) nanoparticles as an efficient contrast agent for in vivo X-ray imaging.

Polymeric nanoparticles (PNPs) are gaining increasing importance as nanocarriers or contrasting material for preclinical diagnosis by micro-CT scanner. Here, we investigated a straightforward approach to produce a biocompatible, radiopaque, and stable polymer-based nanoparticle contrast agent, which was evaluated on mice. To this end, we used a nanoprecipitation dropping technique to obtain PEGylated PNPs from a preformed iodinated homopolymer, poly(MAOTIB), synthesized by radical polymerization of 2-methacryloyloxyethyl(2,3,5-triiodobenzoate) monomer (MAOTIB). The process developed allows an accurate control of the nanoparticle properties (mean size can range from 140 nm to 200 nm, tuned according to the formulation parameters) along with unprecedented important X-ray attenuation properties (concentration of iodine around 59 mg I/mL) compatible with a follow-up in vivo study. Routine characterizations such as FTIR, DSC, GPC, TGA, 1H and 13C NMR, and finally SEM were accomplished to obtain the main properties of the optimal contrast agent. Owing to excellent colloidal stability against physiological conditions evaluated in the presence of fetal bovine serum, the selected PNPs suspension was administered to mice. Monitoring and quantification by micro-CT showed that iodinated PNPs are endowed strong X-ray attenuation capacity toward blood pool and underwent a rapid and passive accumulation in the liver and spleen. STATEMENT OF SIGNIFICANCE: The design of X-ray contrast agents for preclinical imaging is still highly challenging. To date, the best contrast agents reported are based on iodinated lipids or inorganic materials such as gold. In literature, several attempts were undertaken to create polymer-based X-ray contrast agents, but their applicability in vivo was limited to their low contrasting properties. Polymer-based contrast agents present the advantages of an easy surface modification for future application in targeting. Herein, we develop a novel approach to design polymer-based nanoparticle X-ray contrast agent (polymerization of a highly iodine-loaded monomer (MAOTIB)), leading to an iodine concentration of 59 mg/mL. We showed their high efficiency in vivo in mice, in terms of providing a strong signal in blood and then accumulating in the liver and spleen.