Optimization of Isolation Method for Extracellular Vesicles from Pancreatic Juice and Impact of Protease Activity

2022 ◽  
Author(s):  
Koichiro Tsutsumi ◽  
Eijiro Ueta ◽  
Hironari Kato ◽  
Kazuyuki Matsumoto ◽  
Shigeru Horiguchi ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Pancreatic Juice ◽  
Protease Activity ◽  
Isolation Method

scholarly journals A multiparametric extraction method for Vn96-isolated plasma extracellular vesicles and cell-free DNA that enables multi-omic profiling

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jeremy W. Roy ◽  
Catherine A. Taylor ◽  
Annie P. Beauregard ◽  
Surendar R. Dhadi ◽  
D. Craig Ayre ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Small Rna Sequencing ◽  
Isolation Method ◽  
Protein Biomarkers ◽  
Cell Free Dna ◽  
Extraction Protocol ◽  
Free Dna ◽  
Rich Material ◽  
Short Timeframe

AbstractExtracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and ‘omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.

scholarly journals Tetraspanin immunocapture phenotypes extracellular vesicles according to biofluid source but may limit identification of multiplexed cancer biomarkers

2021 ◽  
Author(s):  
Rachel R. Mizenko ◽  
Terza Brostoff ◽  
Tatu Rojalin ◽  
Hanna J. Koster ◽  
Hila S. Swindell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Fluorescence Detection ◽  
Single Particle ◽  
Cancer Biomarkers ◽  
Surface Markers ◽  
Isolation Method ◽  
Standard Methods

AbstractTetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their general detection and classification from background contaminants. This common practice typically assumes a consistent expression of tetraspanins across EV sources, thus obscuring subpopulations of variable or limited tetraspanin expression. While some recent studies indicate differential expression of tetraspanins across bulk isolated EVs, here we present analysis of single EVs isolated using various field-standard methods from a variety of in vitro and in vivo sources to identify distinct patterns in colocalization of tetraspanin expression. We report an optimized method for the use of antibodycapture single particle interferometric reflectance imaging sensing (SP-IRIS) and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. Using SP-IRIS and immunofluorescence, we report that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles as measured by flow cytometry do not share similar trends, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. Our findings may reveal key insights into the complexities of the EV biogenesis and signaling pathways and better inform EV capture and detection platforms for diagnostic or other downstream use.

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 Tetraspanins are unevenly distributed across single extracellular vesicles and bias sensitivity to multiplexed cancer biomarkers

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Rachel R. Mizenko ◽  
Terza Brostoff ◽  
Tatu Rojalin ◽  
Hanna J. Koster ◽  
Hila S. Swindell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Extracellular Vesicles ◽  
Fluorescence Detection ◽  
Cancer Biomarkers ◽  
Surface Markers ◽  
Isolation Method ◽  
Antibody Capture

Abstract Background Tetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their detection and classification, a practice that typically assumes their consistent expression across EV sources. Results Here we demonstrate that there are distinct patterns in colocalization of tetraspanin expression of EVs enriched from a variety of in vitro and in vivo sources. We report an optimized method for the use of single particle antibody-capture and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. We found that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles measured by flow cytometry do not totally agree, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. Conclusion Our findings may reveal key insights into protein expression heterogeneity of EVs that better inform EV capture and detection platforms for diagnostic or other downstream use. Graphical abstract

67 Optimizing a protocol for isolating extracellular vesicles from medium conditioned by bovine embryos in vitro

2019 ◽  
Vol 31 (1) ◽  
pp. 159
Author(s):  
K. C. Pavani ◽  
A. Hendrix ◽  
B. Leemans ◽  
A. Van Soom
Keyword(s):  
Conditioned Medium ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Downstream Processing ◽  
Size Exclusion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Isolation Method ◽  
Exclusion Chromatography

In the absence of the maternal tract, pre-implantation bovine embryos cultured in group are able to promote their own development in vitro by releasing autocrine embryotropins. Recently we have identified extracellular vesicles (EV) among these embryotropins as one of the communication mechanisms among embryos. Extracellular vesicles are nano-sized (25-250nm), with a lipid bilayer, and are functionally active, since they contain proteins, lipids, and nucleic acids, including RNA and miRNA. However, one of the major challenges in isolating EV is an inadequate volume of medium conditioned by bovine embryo. As it requires larger volumes of conditioned medium to isolate EV, our study mainly focused on isolating high yields of functional EV from a minimal volume. There are 3 known isolation methods for EV: differential ultracentrifugation (DU), OptiPrep™ density gradient ultracentrifugation (ODGU), and size-exclusion chromatography (SEC). We have used these 3 protocols to determine the method that yielded the highest number of EV. We used routine in vitro maturation and fertilization methods, but for in vitro culture presumed zygotes were cultured until 8 days post-insemination (dpi) in medium (synthetic oviducal fluid supplemented with insulin, transferrin, selenium, and bovine serum albumin) that was ultracentrifuged to remove any possible contaminating EV. In vitro embryo culture took place in groups of 25 presumed zygotes in 50-mL drops, covered with mineral oil and incubated at 38°C in 5% CO2, 5% O2, and 90% N2. On 8 dpi, medium conditioned by bovine embryo was collected and pooled until 3mL. For each isolation method, 1mL of conditioned medium was used, and next, EV isolated from each isolation method were analysed with nanoparticle tracking, electron microscopy, and Western blot (CD9, Flotillin 1, and AGO 2). We observed higher concentrations (1.03×109 particles mL−1) of EV were isolated from the SEC compared with the other 2 methods (301.5×108 particles mL−1 and 64.5×108 particles mL−1 for DU and ODGU, respectively; P<0.05), whereas smaller size EV (20-50nm) were lost during the ultracentrifugation methods. Besides, it takes only 2h of time to perform size-exclusion chromatography for isolating EV, whereas it takes more than 1 day to perform ultracentrifugation methods. Therefore, we propose to use SEC for further downstream processing and sequencing of miRNA in isolated EV. We are currently focusing on optimizing an EV isolation protocol to extract EV from very low volumes of conditioned medium (less than 500 µL).

scholarly journals Detection and proteomic characterization of extracellular vesicles in human pancreatic juice

2018 ◽  
Vol 499 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Xabier Osteikoetxea ◽  
Márton Benke ◽  
Marta Rodriguez ◽  
Krisztina Pálóczi ◽  
Barbara W. Sódar ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Pancreatic Juice

scholarly journals Metabolomics Applied to the Study of Extracellular Vesicles

Metabolites ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 276 ◽  
Author(s):  
Charles Williams ◽  
Mari Palviainen ◽  
Niels-Christian Reichardt ◽  
Pia R.-M. Siljander ◽  
Juan M. Falcón-Pérez
Keyword(s):  
Cell Culture ◽  
Extracellular Vesicles ◽  
Pulmonary Arteries ◽  
Mechanisms Of Action ◽  
Clinical Applications ◽  
Metabolomic Analysis ◽  
Isolation Method ◽  
Non Invasive ◽  
Cell Culture Systems ◽  
Protein Nucleic Acid

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities—particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies.

scholarly journals Isolation of Extracellular Vesicles: General Methodologies and Latest Trends

2018 ◽  
Vol 2018 ◽  
pp. 1-27 ◽  
Author(s):  
Maria Yu. Konoshenko ◽  
Evgeniy A. Lekchnov ◽  
Alexander V. Vlassov ◽  
Pavel P. Laktionov
Keyword(s):  
Extracellular Vesicles ◽  
State Of The Art ◽  
Data Interpretation ◽  
Biochemical Properties ◽  
Mixed Population ◽  
Isolation Method ◽  
Modern Biology ◽  
Advantages And Disadvantages ◽  
Isolation Methods ◽  
Labor Consumption

Background. Extracellular vesicles (EVs) play an essential role in the communication between cells and transport of diagnostically significant molecules. A wide diversity of approaches utilizing different biochemical properties of EVs and a lack of accepted protocols make data interpretation very challenging. Scope of Review. This review consolidates the data on the classical and state-of-the-art methods for isolation of EVs, including exosomes, highlighting the advantages and disadvantages of each method. Various characteristics of individual methods, including isolation efficiency, EV yield, properties of isolated EVs, and labor consumption are compared. Major Conclusions. A mixed population of vesicles is obtained in most studies of EVs for all used isolation methods. The properties of an analyzed sample should be taken into account when planning an experiment aimed at studying and using these vesicles. The problem of adequate EVs isolation methods still remains; it might not be possible to develop a universal EV isolation method but the available protocols can be used towards solving particular types of problems. General Significance. With the wide use of EVs for diagnosis and therapy of various diseases the evaluation of existing methods for EV isolation is one of the key problems in modern biology and medicine.

Clinical significance of cathepsin E in pancreatic juice in the diagnosis of pancreatic ductal adenocarcinoma

2000 ◽  
Vol 15 (11) ◽  
pp. 1333-1338 ◽  
Author(s):  
Koji Uno ◽  
Takeshi Azuma ◽  
Masatsugu Nakajima ◽  
Kenjiro Yasuda ◽  
Takanobu Hayakumo ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Clinical Significance ◽  
Pancreatic Juice ◽  
Ductal Adenocarcinoma ◽  
Cathepsin E

Pancreatic stones shockwave lithotripsy induces oxidative stress. A study of lipoperoxidation products in pure pancreatic juice

2001 ◽  
Vol 120 (5) ◽  
pp. A217-A217
Author(s):  
C SPADA ◽  
S SANTINI ◽  
F FOSCHIA ◽  
M PANDOLFI ◽  
V PERRI ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pancreatic Juice ◽  
Shockwave Lithotripsy ◽  
Pure Pancreatic Juice ◽  
Pancreatic Stones
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