Extracellular‐Vesicle Isolation from Different Biological Fluids by Size‐Exclusion Chromatography

2019 ◽  
Vol 49 (1) ◽  
pp. e82 ◽  
Author(s):  
Marta Monguió‐Tortajada ◽  
Miriam Morón‐Font ◽  
Ana Gámez‐Valero ◽  
Laura Carreras‐Planella ◽  
Francesc E. Borràs ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Biological Fluids ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Exclusion Chromatography

Extracellular vesicle isolation methods: rising impact of size-exclusion chromatography

2019 ◽  
Vol 76 (12) ◽  
pp. 2369-2382 ◽  
Author(s):  
Marta Monguió-Tortajada ◽  
Carolina Gálvez-Montón ◽  
Antoni Bayes-Genis ◽  
Santiago Roura ◽  
Francesc E. Borràs
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Isolation Methods ◽  
Exclusion Chromatography

scholarly journals Size-Exclusion Chromatography as a Technique for the Investigation of Novel Extracellular Vesicles in Cancer

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3156
Author(s):  
Daniel S. K. Liu ◽  
Flora M. Upton ◽  
Eleanor Rees ◽  
Christopher Limb ◽  
Long R. Jiao ◽  
...  
Keyword(s):  
Systematic Review ◽  
Cancer Cells ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Isolation Method ◽  
Isolation And Purification ◽  
Exclusion Chromatography

Cancer cells release extracellular vesicles, which are a rich target for biomarker discovery and provide a promising mechanism for liquid biopsy. Size-exclusion chromatography (SEC) is an increasingly popular technique, which has been rediscovered for the purposes of extracellular vesicle (EV) isolation and purification from diverse biofluids. A systematic review was undertaken to identify all papers that described size exclusion as their primary EV isolation method in cancer research. In all, 37 papers were identified and discussed, which showcases the breadth of applications in which EVs can be utilised, from proteomics, to RNA, and through to functionality. A range of different methods are highlighted, with Sepharose-based techniques predominating. EVs isolated using SEC are able to identify cancer cells, highlight active pathways in tumourigenesis, clinically distinguish cohorts, and remain functionally active for further experiments.

scholarly journals Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer‐based precipitation and size exclusion chromatography

2021 ◽  
Vol 10 (6) ◽  
Author(s):  
Juan A. Martínez‐Greene ◽  
Karina Hernández‐Ortega ◽  
Ricardo Quiroz‐Baez ◽  
Osbaldo Resendis‐Antonio ◽  
Israel Pichardo‐Casas ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Proteomic Analysis ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Quantitative Proteomic Analysis ◽  
Exclusion Chromatography

scholarly journals A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

2020 ◽  
Vol 21 (18) ◽  
pp. 6466 ◽  
Author(s):  
Karim Sidhom ◽  
Patience O. Obi ◽  
Ayesha Saleem
Keyword(s):  
Size Exclusion Chromatography ◽  
Culture Media ◽  
Biological Fluids ◽  
Vital Role ◽  
Size Exclusion ◽  
Isolation Techniques ◽  
Exclusion Chromatography ◽  
Specialized Equipment ◽  
Pros And Cons ◽  
Poly Ethylene

Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media, among others. While differential ultracentrifugation (dUC) has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics, and size-exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise. However, it must be noted that while SEC is a good candidate method to isolate exosomes, direct comparative studies are required to support this conclusion.

scholarly journals Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation

Biomedicines ◽  
2020 ◽  
Vol 8 (8) ◽  
pp. 246 ◽  
Author(s):  
Anders Askeland ◽  
Anne Borup ◽  
Ole Østergaard ◽  
Jesper V. Olsen ◽  
Sigrid M. Lund ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
High Speed ◽  
Proteome Analysis ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Label Free ◽  
Protein Biomarkers ◽  
Transmission Electron ◽  
Exclusion Chromatography

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.

scholarly journals A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

2020 ◽  
Author(s):  
Karim Sidhom ◽  
Patience O. Obi ◽  
Ayesha Saleem
Keyword(s):  
Size Exclusion Chromatography ◽  
Culture Media ◽  
Biological Fluids ◽  
Vital Role ◽  
Size Exclusion ◽  
Isolation Techniques ◽  
Exclusion Chromatography ◽  
Specialized Equipment ◽  
Pros And Cons ◽  
Poly Ethylene

Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media among others. While differential ultracentrifugation (dUC), has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics and size exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise.

scholarly journals Micro size exclusion chromatography combined with a multiplex protein profiling method for extracellular vesicle protein detection from small sample volumes

2020 ◽  
Author(s):  
Li Sun ◽  
David Meckes
Keyword(s):  
Size Exclusion Chromatography ◽  
Biological Fluids ◽  
Protein Detection ◽  
Cancer Diagnostics ◽  
Small Sample ◽  
Surface Proteins ◽  
Protein Profiling ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Pcr Method

Abstract Extracellular vesicles (EVs) are small nanometer-sized membrane sacs secreted into biological fluids by all cells. EVs encapsulate proteins, RNAs and metabolites from its origin cell and play important roles in intercellular communication events. Over the past decade, EVs have become a new emerging source for cancer diagnostics. One of the challenges in the study of EVs and there utility as diagnostic biomarkers is the amount of EVs needed for traditional protein analysis methods. Here, we present a new immuno-PCR method that takes advantage of commercially available TotalSeq™ antibodies containing DNA conjugated oligos to identify immobilized protein analysts using real-time qPCR. Using this method, we demonstrate that multiple EV surface proteins can be profiled simultaneously with high sensitivity and specificity. This approach was also successfully applied to similar protocol using live cell and serum samples. We further described the development of a micro-size exclusion chromatography method, where we were able to detect EV surface proteins with as little as 10 μL of human serum when combined with immuno-PCR. Overall, these results show that the immuno-PCR method results in rapid detection of multiple EV markers from small sample volumes in a single tube.

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