scholarly journals Development of Novel High-Resolution Size-Guided Turbidimetry-Enabled Particle Purification Liquid Chromatography (PPLC): Extracellular Vesicles and Membraneless Condensates in Focus

2020 ◽  
Vol 21 (15) ◽  
pp. 5361 ◽  
Author(s):  
Hussein Kaddour ◽  
Yuan Lyu ◽  
Nadia Shouman ◽  
Mahesh Mohan ◽  
Chioma M. Okeoma
Keyword(s):  
Liquid Chromatography ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Therapeutic Implications ◽  
Fraction Collector ◽  
Transmission Electron ◽  
Compositional Properties ◽  
Culture Supernatants ◽  
Research Drug

Acellular particles (extracellular vesicles and membraneless condensates) have important research, drug discovery, and therapeutic implications. However, their isolation and retrieval have faced enormous challenges, impeding their use. Here, a novel size-guided particle purification liquid chromatography (PPLC) is integrated into a turbidimetry-enabled system for dye-free isolation, online characterization, and retrieval of intact acellular particles from biofluids. The chromatographic separation of particles from different biofluids—semen, blood, urine, milk, and cell culture supernatants—is achieved using a first-in-class gradient size exclusion column (gSEC). Purified particles are collected using a fraction collector. Online UV–Vis monitoring reveals biofluid-dependent particle spectral differences, with semen being the most complex. Turbidimetry provides the accurate physical characterization of seminal particle (Sp) lipid contents, sizes, and concentrations, validated by a nanoparticle tracking analysis, transmission electron microscopy, and naphthopyrene assay. Furthermore, different fractions of purified Sps contain distinct DNA, RNA species, and protein compositions. The integration of Sp physical and compositional properties identifies two archetypal membrane-encased seminal extracellular vesicles (SEV)—notably SEV large (SEVL), SEV small (SEVS), and a novel non-archetypal-membraneless Sps, herein named membraneless condensates (MCs). This study demonstrates a comprehensive yet affordable platform for isolating, collecting, and analyzing acellular particles to facilitate extracellular particle research and applications in drug delivery and therapeutics. Ongoing efforts focus on increased resolution by tailoring bead/column chemistry for each biofluid type.

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.

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.

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.

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