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Current Protocols in Cell Biology Sep 2020Exosomes are 50- to 150-nm-diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and...
Exosomes are 50- to 150-nm-diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation- and column-based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek ) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream ). High-performance liquid chromatography followed by nano-flow liquid chromatography-mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5-fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Pre-analytic fluid collection and processing Basic Protocol 2: Exosome isolation by ultracentrifugation Alternate Protocol 1: Exosome isolation by precipitation Basic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysis Alternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometry Basic Protocol 4: Downstream analysis of exosomes using high-performance liquid chromatography Basic Protocol 5: Downstream analysis of the exosome proteome using nano-flow liquid chromatography-mass spectrometry.
Topics: Animals; Chemical Precipitation; Exosomes; Nanoparticles; Proteomics; Ultracentrifugation
PubMed: 32633898
DOI: 10.1002/cpcb.110 -
Journal of Extracellular Vesicles Sep 2022Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo...
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles.
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.
Topics: Animals; Chromatography, Gel; Extracellular Vesicles; Filtration; Humans; Mice; Reproducibility of Results; Ultracentrifugation
PubMed: 36124834
DOI: 10.1002/jev2.12266 -
European Biophysics Journal : EBJ May 2021Analytical ultracentrifugation (AUC) is based on the concept of recording and analyzing macroscopic macromolecular redistribution that results from a centrifugal force...
Analytical ultracentrifugation (AUC) is based on the concept of recording and analyzing macroscopic macromolecular redistribution that results from a centrifugal force acting on the mass of suspended macromolecules in solution. Since AUC rests on first principles, it can provide an absolute measurement of macromolecular mass, sedimentation and diffusion coefficients, and many other quantities, provided that the solvent density and viscosity are known, and provided that the instrument is properly calibrated. Unfortunately, a large benchmark study revealed that many instruments exhibit very significant systematic errors. This includes the magnification of the optical detection system used to determine migration distance, the measurement of sedimentation time, and the measurement of the solution temperature governing viscosity. We have previously developed reference materials, tools, and protocols to detect and correct for systematic measurement errors in the AUC by comparison with independently calibrated standards. This 'external calibration' resulted in greatly improved precision and consistency of parameters across laboratories. Here we detail the steps required for calibration of the different data dimensions in the AUC. We demonstrate the calibration of three different instruments with absorbance and interference optical detection, and use measurements of the sedimentation coefficient of NISTmAb monomer as a test of consistency. Whereas the measured uncorrected sedimentation coefficients span a wide range from 6.22 to 6.61 S, proper calibration resulted in a tenfold reduced standard deviation of sedimentation coefficients. The calibrated relative standard deviation and mean error of 0.2% and 0.07%, respectively, is comparable with statistical errors and side-by-side repeatability in a single instrument.
Topics: Calibration; Macromolecular Substances; Solvents; Ultracentrifugation; Viscosity
PubMed: 33398460
DOI: 10.1007/s00249-020-01485-2 -
Analytical Biochemistry Sep 2022Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC extends the... (Review)
Review
Multi-wavelength analytical ultracentrifugation (MW-AUC) is a recent development made possible by new analytical ultracentrifuge optical systems. MW-AUC extends the basic hydrodynamic information content of AUC and provides access to a wide range of new applications for biopolymer characterization, and is poised to become an essential analytical tool to study macromolecular interactions. It adds an orthogonal spectral dimension to the traditional hydrodynamic characterization by exploiting unique chromophores in analyte mixtures that may or may not interact. Here we illustrate the utility of MW-AUC for experimental investigations where the benefit of the added spectral dimension provides critical information that is not accessible, and impossible to resolve with traditional AUC methods. We demonstrate the improvements in resolution and information content obtained by this technique compared to traditional single- or dual-wavelength approaches, and discuss experimental design considerations and limitations of the method. We further address the advantages and disadvantages of the two MW optical systems available today, and the differences in data analysis strategies between the two systems.
Topics: Biopolymers; Hydrodynamics; Ultracentrifugation
PubMed: 35609686
DOI: 10.1016/j.ab.2022.114728 -
Gene Therapy Aug 2023Adeno-associated virus (AAV) vector-based gene therapy is potentially curative for various genetic diseases; however, the development of a scalable purification method...
Adeno-associated virus (AAV) vector-based gene therapy is potentially curative for various genetic diseases; however, the development of a scalable purification method for full-genome AAV vectors remains crucial to increase productivity and reduce cost of GMP production. In this study, we developed a large-scale short-term purification method for functional full-genome AAV particles by using 2-step cesium chloride (CsCl) density-gradient ultracentrifugation with a zonal rotor. The 2-step CsCl method with a zonal rotor improves separation between empty and full-genome AAV particles, reducing the ultracentrifugation time (4-5 h) and increasing the AAV volume for purification. The highly purified full-genome AAV particles were confirmed by analytical ultracentrifugation (AUC), droplet digital PCR (ddPCR) in the whole region of the AAV vector genome, transduction efficiency in target cells, and transmission electronic microscopy (TEM). The high-purity AAV9 particles were obtained using culture supernatant during vector preparation rather than cell lysate. CsCl could be simply removed by a hydroxyapatite column. Interestingly, ddPCR analysis revealed that "empty" AAV particles contain small fragments of the inverted terminal repeat (ITR), probably due to unexpected packaging of Rep-mediated ITR fragments. This large-scale functional AAV vector purification with ultracentrifugation would be effective for gene therapy.
Topics: Genetic Vectors; Ultracentrifugation; Dependovirus
PubMed: 36977769
DOI: 10.1038/s41434-023-00398-x -
BioMed Research International 2023Exosomes were extracted from a variety of biological samples using several different purification processes, and our goal was to determine which method and sample were...
OBJECTIVE
Exosomes were extracted from a variety of biological samples using several different purification processes, and our goal was to determine which method and sample were the most effective for exosome extraction.
METHODS
We used ExoQuick-TC combined with ultrafiltration to separate and purify exosomes from the supernatant of gastric cancer cells, while we used the ExoQuick kit and ultracentrifugation to purify exosomes from human serum samples. Furthermore, exosomes were isolated and purified from human urine samples by diafiltration and from postparturition human breast milk samples by the filtration-polyethylene glycol precipitation method. The isolated exosomes were morphologically analyzed using a transmission electron microscope, the particle size was measured by NanoSight, and the protein content was analyzed by western blotting.
RESULTS
The isolated exosomes showed an obvious cup holder shape, with a clear outline and typical exosome morphological characteristics. The sizes of exosomes derived from gastric cancer cell supernatant, serum, urine, and milk were 65.8 ± 26.9 nm, 87.6 ± 50.9 nm, 197.5 ± 55.2 nm, and 184.1 ± 68.7 nm, respectively. Western blot results showed that CD9 and TSG101 on the exosomes were expressed to varying degrees based on the exosome source. Exosome abundance was higher in the serum, urine, and breast milk than in the supernatant. It is suggested that its exosomes can be extracted to obtain an excellent potential biological source of exosomes.
CONCLUSION
In this study, the extraction and separation methods of foreign bodies from different biological samples were obtained, and it was found that human breast milk was a potential excellent material for administration because of its high abundance.
Topics: Female; Humans; Exosomes; Stomach Neoplasms; Ultracentrifugation; Body Fluids; Milk, Human
PubMed: 37124929
DOI: 10.1155/2023/2336536 -
Journal of Pharmaceutical Sciences Jul 2022Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay... (Review)
Review
Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique.
Topics: Antibodies, Monoclonal; Chromatography, Gel; Ultracentrifugation
PubMed: 34986360
DOI: 10.1016/j.xphs.2021.12.023 -
Biomolecules Jun 2021Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs,... (Review)
Review
Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.
Topics: Computational Biology; Exosomes; Extracellular Vesicles; Humans; Mass Spectrometry; Milk Proteins; Milk, Human; Proteomics; Reproducibility of Results; Ultracentrifugation
PubMed: 34204944
DOI: 10.3390/biom11060833 -
Methods in Molecular Biology (Clifton,... 2018Understanding the role of extracellular RNA (exRNA) has emerged as an exciting avenue for biomarker, therapeutic, as well as basic cell-cell communication applications... (Review)
Review
Understanding the role of extracellular RNA (exRNA) has emerged as an exciting avenue for biomarker, therapeutic, as well as basic cell-cell communication applications and discoveries. Multiple protocols, kits, and procedures have been developed in the last decade to allow fractionation as well as isolation of subpopulations of macromolecules of interest found in biofluids. Here, we introduce the protocols decision tree developed by the Extracellular RNA Communication Consortium and available on their website (exRNA portal), and compare all methods currently available to the exRNA field and report pros and cons for each platform.
Topics: Animals; Chemical Precipitation; Extracellular Space; Extracellular Vesicles; Filtration; Humans; Molecular Biology; RNA; Ultracentrifugation
PubMed: 29388132
DOI: 10.1007/978-1-4939-7652-2_2 -
STAR Protocols Dec 2021Separation of highly enriched extracellular vesicles (EVs) fractions from milk is desirable for quantification, cargo analysis, functional characterization, and...
Separation of highly enriched extracellular vesicles (EVs) fractions from milk is desirable for quantification, cargo analysis, functional characterization, and investigation as delivery vehicles for nutrients and/or therapeutics. However, a rigorous, reproducible protocol is lacking. This protocol considers a crucial aspect typically overlooked, i.e., that caseins are of similar size to, but more abundant than, EVs in milk. Our protocol combines acid pre-treatment and gradient ultracentrifugation, producing EV-enriched fractions suitable for downstream orthogonal characterization approaches. For complete details on the use and execution of this protocol, please refer to Mukhopadhya et al. (2021).
Topics: Animals; Caseins; Extracellular Vesicles; Humans; Infant; Infant Formula; Milk; Ultracentrifugation
PubMed: 34568843
DOI: 10.1016/j.xpro.2021.100821