scholarly journals Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 797
Author(s):  
Aline Do Minh ◽  
Alexandra T. Star ◽  
Jacek Stupak ◽  
Kelly M. Fulton ◽  
Arsalan S. Haqqani ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Extracellular Vesicles ◽  
Downstream Processing ◽  
Hek293 Cells ◽  
Human Embryonic Kidney Cells ◽  
Transmission Electron ◽  
Before And After ◽  
Product Profile ◽  
Gene And Cell Therapy

Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered “impurities” in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.

scholarly journals Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures

2021 ◽  
Author(s):  
Aline Do Minh ◽  
Alexandra T. Star ◽  
Jacek Stupak ◽  
Jean-Francois Gélinas ◽  
Jianjun Li ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Extracellular Vesicles ◽  
Downstream Processing ◽  
Hek293 Cells ◽  
Enveloped Viruses ◽  
Human Embryonic Kidney Cells ◽  
Transmission Electron ◽  
Product Profile ◽  
Similar Enrichment

Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purify with LVs during downstream processing, becoming “impurities” in the context of cell therapy. To characterize EVs from an inducible lentivirus producing cell line, two conditions were studied: non-induced and induced. EVs’ identity was confirmed by transmission electron microscopy and western blot. Seven proteins were identified by mass spectrometry as potential EV markers. Lipid composition of EVs and LVs showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in genes involved in viral processes and binding functions. Flow virometry, GTA and ddPCR results also confirmed the heterogenic nature of EVs and LVs populations. These findings provide insights on the product profile of lentiviral preparation and could help develop separation strategies of co-produced EVs.

An Electron Microscope Study of Interdiffusion and Compound Formation in Ta-Au Thin Films

1973 ◽  
Vol 31 ◽  
pp. 32-33 ◽  
Author(s):  
T. C. Tisone ◽  
S. Lau
Keyword(s):  
Electron Microscope ◽  
Electron Microscope Study ◽  
Thermally Grown Oxide ◽  
Ion Milling ◽  
Compound Formation ◽  
Technology Application ◽  
Transmission Electron ◽  
Before And After ◽  
Au Thin Films

In a study of the properties of a Ta-Au metallization system for thin film technology application, the interdiffusion between Ta(bcc)-Au, βTa-Au and Ta2M-Au films was studied. Considered here is a discussion of the use of the transmission electron microscope(TEM) in the identification of phases formed and characterization of the film microstructures before and after annealing.The films were deposited by sputtering onto silicon wafers with 5000 Å of thermally grown oxide. The film thicknesses were 2000 Å of Ta and 2000 Å of Au. Samples for TEM observation were prepared by ultrasonically cutting 3mm disks from the wafers. The disks were first chemically etched from the silicon side using a HNO3 :HF(19:5) solution followed by ion milling to perforation of the Au side.

scholarly journals Identification and Characterization of Extracellular Vesicles and Its DNA Cargo Secreted During Murine Embryo Development

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 203 ◽  
Author(s):  
Blanca Simon ◽  
David Bolumar ◽  
Alicia Amadoz ◽  
Jorge Jimenez-Almazán ◽  
Diana Valbuena ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Preimplantation Embryos ◽  
Gene Enrichment ◽  
Transmission Electron ◽  
Genome Wide ◽  
Significant Gene ◽  
Identification And Characterization ◽  
Old Females ◽  
Total Concentrations

Extracellular vesicles (EVs) are known to transport DNA, but their implications in embryonic implantation are unknown. The aim of this study was to investigate EVs production and secretion by preimplantation embryos and assess their DNA cargo. Murine oocytes and embryos were obtained from six- to eight-week-old females, cultured until E4.5 and analyzed using transmission electron microscopy to examine EVs production. EVs were isolated from E4.5-day conditioned media and quantified by nanoparticle tracking analysis, characterized by immunogold, and their DNA cargo sequenced. Multivesicular bodies were observed in murine oocytes and preimplantation embryos together with the secretion of EVs to the blastocoel cavity and blastocyst spent medium. Embryo-derived EVs showed variable electron-densities and sizes (20–500 nm) and total concentrations of 1.74 × 107 ± 2.60 × 106 particles/mL. Embryo secreted EVs were positive for CD63 and ARF6. DNA cargo sequencing demonstrated no differences in DNA between apoptotic bodies or smaller EVs, although they showed significant gene enrichment compared to control medium. The analysis of sequences uniquely mapping the murine genome revealed that DNA contained in EVs showed higher representation of embryo genome than vesicle-free DNA. Murine blastocysts secrete EVs containing genome-wide sequences of DNA to the medium, reinforcing the relevance of studying these vesicles and their cargo in the preimplantation moment, where secreted DNA may help the assessment of the embryo previous to implantation.

Elaboration and characterization of novel polyamide-12-layered titanoniobates nanocomposites

2009 ◽  
Vol 24 (11) ◽  
pp. 3358-3371 ◽  
Author(s):  
Sophie Chausson ◽  
Richard Retoux ◽  
Jean-Michel Rueff ◽  
Loïc LE Pluart ◽  
Pierre-Jean Madec ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
Scanning Transmission Electron Microscope ◽  
Thermomechanical Properties ◽  
Polyamide 12 ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Before And After ◽  
Basic Reaction

This article describes the synthesis and the characterization of a polyamide-12 filled with a nanostructured organic/inorganic titanoniobate hybrid material. The pristine oxide KTiNbO5 has been successfully organomodified by N-alkyl amines via an acido-basic reaction after a cationic exchange step as shown by x-ray diffraction. Transmission electron microscope study and scanning transmission electron microscope observations have been used to describe the change of morphology of the nanofillers before and after processing; the micronic aggregates were changed into single sheets and dispersed in the polymer. Thermomechanical properties of the composites have been determined, and their analyses with structure-properties models are consistent with the exfoliation of the organomodified titanoniobates.

scholarly journals Urinary Extracellular Vesicles for Renal Tubular Transporters Expression in Patients With Gitelman Syndrome

2021 ◽  
Vol 8 ◽  
Author(s):  
Chih-Chien Sung ◽  
Min-Hsiu Chen ◽  
Yi-Chang Lin ◽  
Yu-Chun Lin ◽  
Yi-Jia Lin ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Membrane Transporters ◽  
Gitelman Syndrome ◽  
Healthy Controls ◽  
Nanoparticle Tracking Analysis ◽  
Non Invasive ◽  
Transmission Electron ◽  
Renal Tubular ◽  
Inactivating Mutations

Background: The utility of urinary extracellular vesicles (uEVs) to faithfully represent the changes of renal tubular protein expression remains unclear. We aimed to evaluate renal tubular sodium (Na+) or potassium (K+) associated transporters expression from uEVs and kidney tissues in patients with Gitelman syndrome (GS) caused by inactivating mutations in SLC12A3.Methods: uEVs were isolated by ultracentrifugation from 10 genetically-confirmed GS patients. Membrane transporters including Na+-hydrogen exchanger 3 (NHE3), Na+/K+/2Cl− cotransporter (NKCC2), NaCl cotransporter (NCC), phosphorylated NCC (p-NCC), epithelial Na+ channel β (ENaCβ), pendrin, renal outer medullary K1 channel (ROMK), and large-conductance, voltage-activated and Ca2+-sensitive K+ channel (Maxi-K) were examined by immunoblotting of uEVs and immunofluorescence of biopsied kidney tissues. Healthy and disease (bulimic patients) controls were also enrolled.Results: Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Compared with healthy controls, uEVs from GS patients showed NCC and p-NCC abundance were markedly attenuated but NHE3, ENaCβ, and pendrin abundance significantly increased. ROMK and Maxi-K abundance were also significantly accentuated. Immunofluorescence of the representative kidney tissues from GS patients also demonstrated the similar findings to uEVs. uEVs from bulimic patients showed an increased abundance of NCC and p-NCC as well as NHE3, NKCC2, ENaCβ, pendrin, ROMK and Maxi-K, akin to that in immunofluorescence of their kidney tissues.Conclusion: uEVs could be a non-invasive tool to diagnose and evaluate renal tubular transporter adaptation in patients with GS and may be applied to other renal tubular diseases.

A Detailed Interface Characterization of the Explosively Welded Three-Layered Ti Gr 1/Alloy 400/1.4462 Steel Clads Before and After Heat Treatment

2021 ◽  
pp. 1-9
Author(s):  
Marta Janusz-Skuza ◽  
Agnieszka Bigos ◽  
Łukasz Maj ◽  
Jerzy Morgiel ◽  
Marek Faryna ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Heat Treatment ◽  
Hexagonal Phase ◽  
Cubic Phase ◽  
Interface Zone ◽  
Transmission Electron ◽  
Before And After ◽  
Diffusion Phenomena ◽  
Zone Microstructure

The presented research focused on the microstructural characteristics of explosively welded three-layered Ti Grade (Gr) 1/Alloy 400/1.4462 steel clads before and after heat treatment being of large practical potential. Scanning electron microscopy (SEM) analyses have shown that both interfaces formed between the plates are continuous and without defects. The in-depth examination was dedicated to the upper Ti Gr 1/Alloy 400 interface, located closer to the explosive material, therefore, subjected to more extreme welding conditions. The presence of cubic phase Ti2Ni, hexagonal phase Ni3Ti, and tetragonal phase (Cu x Ni1−x)2Ti were confirmed within the melted zones, which slightly widened due to annealing, being an essential step in the manufacturing of these modern materials. Transmission electron microscopy observations in the nano scale confirmed the preliminary chemical composition analyses collected with energy-dispersive X-ray spectroscopy in SEM. They additionally revealed the interface zone microstructure transformation due to the annealing. It was evidenced that initially mixed phases in the form of grains, after heat treatment formed irregular bands arranged in the following sequence: Alloy 400/Ni3Ti/(Cu x Ni1−x)2Ti/Ti2Ni/Ti Gr 1. A clear segregation of Cu and Ni forming two separate layers was also noticed. These diffusion phenomena may influence the strength of the final product, therefore need further studies regarding the prolonged annealing state.

Synthesis and Characterization of Superfine Nanostructure Manganese Dioxide Spontaneously Coated onto Carbon Nanotubes

2011 ◽  
Vol 364 ◽  
pp. 398-401
Author(s):  
Johari Md Salleh ◽  
Aziah Buang Nor ◽  
Muhammad Zamir Othman
Keyword(s):  
Electron Microscopy ◽  
Carbon Nanotubes ◽  
Manganese Dioxide ◽  
Synthesis And Characterization ◽  
X Ray ◽  
Transmission Electron ◽  
Before And After ◽  
Cnts Surface ◽  
Thermal Stability Of

Manganese dioxide nanostructered (MnO2) was coated onto carbon nanotubes (CNTs) by simple immersion of the CNTs into a KMnO4 aqueous solution. The morphology of the CNTs before and after MnO2 deposition was examined using field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM). The superfine coral-like MnO2 deposited and fully covered on the CNTs surface at pH 8. At pH 2 and 6 the MnO2 deposited as the superfine nanorod structure. The superfine MnO2 phase was identified as Birnessite-type MnO2 by X-ray powder diffraction and FESEM. The thermal stability of the superfine nanostructure MnO2 coated CNTs is increase based on the TGA with the weight loss of 4% at 400°C to 900°C.

Characteristics of Alumina Diffusion Barrier Films on Hastelloy

2004 ◽  
Vol 19 (4) ◽  
pp. 1175-1180 ◽  
Author(s):  
I. Usov ◽  
P. Arendt ◽  
L. Stan ◽  
DePaula R. ◽  
H. Wang ◽  
...  
Keyword(s):  
Ion Beam ◽  
Diffusion Mechanism ◽  
Barrier Films ◽  
Alumina Films ◽  
Ion Beam Assisted Deposition ◽  
Transmission Electron ◽  
Before And After ◽  
Wide Interface ◽  
Secondary Ion

The diffusion behavior of elements constituting Hastelloy C-276 (C, Si, Mn, Co, W, Fe, Cr, Mo, and Ni) in alumina films was investigated using secondary ion mass spectroscopy. The films were deposited by ion-beam-assisted deposition and annealed in vacuum over a temperature range of 500–1000 °C. Characterization of film microstructure was performed using transmission electron microscopy and selected area diffraction analyses. The films were predominantly amorphous with alumina nanocrystallites nonuniformly dispersed throughout the volume both before and after annealing. A relatively wide interface region between the Hastelloy substrate and alumina film was formed in the as-deposited sample due to ion beam mixing. No diffusion of any of the substrate elements was observed after annealing, except for Mn, Cr, and Ni. The impurity depth distributions consisted of two components, which differed by several orders of magnitude with respect to diffusion coefficient and solubility. Activation energies and temperature dependencies of the diffusion coefficients were determined, and a diffusion mechanism was discussed.

FE-SEM Study of Fine Nb Precipitates in Carbon Extraction Replicas

2005 ◽  
Vol 500-501 ◽  
pp. 663-668 ◽  
Author(s):  
Rocco Varano ◽  
A.M. Elwazri ◽  
Fulvio Siciliano ◽  
D.Q. Bai ◽  
Raynald Gauvin ◽  
...  
Keyword(s):  
High Strength ◽  
Volume Effect ◽  
Carbon Steels ◽  
Low Carbon ◽  
Thin Foils ◽  
Transmission Electron ◽  
Sem Study ◽  
Before And After ◽  
Linepipe Steel

Precipitation strengthening is an important parameter controlling the mechanical properties of low carbon steels. These precipitates are very fine and are normally analyzed using either thin foils or carbon extraction replicas under a transmission electron microscopy (TEM). In this work, field emission gun scanning electron microscope (FE-SEM) was applied successfully in the characterization of niobium (Nb) carbo-nitride (C,N) precipitates using carbon extraction replicas. FE-SEM observation of high strength linepipe steel replicas before and after aging at 400°C for 1 hr confirmed the presence of Nb(C,N) precipitates in ferrite. The FE-SEM could analyze small particles (below 50 nm) embedded in the steel but the analysis had to be carried out at low voltages to maximize spatial resolution resulting in a poor signal. However, carbon extraction replicas in the FE-SEM can be analyzed using high voltages, since the interaction volume effect is no longer a problem.

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