Quantification Of Extracellular Vesicles Research Articles

Extracellular vesicles promote epithelial-to-mesenchymal transition of lens epithelial cells under oxidative stress

Posterior capsule opacification (PCO), resulting from residual lens epithelial cell (LEC) epithelial–mesenchymal transition (EMT), abnormal proliferation, and migration, is the most common complication of cataract surgery. A recent study determined that extracellular vesicles (EVs) and reactive oxygen species (ROS) regulate the EMT process during cutaneous wound healing and tumour metastasis. However, their underlying mechanism in PCO is unclear. In this study, we examined the secreted EVs from a scratch model in vitro. We found that the production of ROS was increased after mechanical injury, especially at the wound edge, and there was an increased viability of LECs, which can be blocked by diphenyleneiodonium, an NADPH oxidase inhibitor. Cell viability and migration were increased upon treatment with 1 μM H2O2, but significantly reduced when the concentration of H2O2 increased to 100 μM. Transwell assay showed that both post-surgery LECs and LECs treated with 1 μM H2O2 significantly induced the migration of normal LECs by EV secretion. Extraction and quantification of EVs derived from injured and H2O2-treated LECs showed a similar increase in production. Co-incubation of EVs from both injured and H2O2-treated LECs with normal LECs and organ-cultured mouse lenses activated EMT, which was attenuated by a ROS inhibitor. These results suggest that EVs participate in ROS-induced lens EMT, making EVs a potential target for treating PCO.

Extracellular vesicles on demand (EVOD) chip for screening and quantification of cancer-associated extracellular vesicles

While significant advancements have been made in cancer therapeutics and treatments, early disease detection and diagnosis remains critical to ensuring favorable outcomes for patients. To that end, we propose a microfluidic based approach to the sensitive detection of an intriguing cancer biomarker, extracellular vesicles (EVs). Our extracellular vesicles on demand (EVOD) chip utilizes a catalyst-free click chemistry to rapidly and specifically isolate EVs of interest. This specific isolation is followed by subsequent dithiothreitol release of the isolated EVs for downstream functional analysis. This joint isolation and release provide a powerful tool for the screening and quantification of EVs of interest. By incorporating antibodies against cancer associated surface proteins into the click-chemistry, we were able to selectively recover cancer-associated exosomes, allowing for important insights into patient disease. This platform was also tested using non-small cell lung cancer (NSCLC) patient samples, where anti-epidermal growth factor receptor (EGFR) assisted platform were able to selectively isolate and release 76% more exosomes from NSCLC patients than from healthy donors. This matches the previously reported higher EGFR expression commonly found in NSCLC EVs. Through its rapid isolation kinetics and adaptability in marker targeting, the EVOD device provides a highly versatile liquid biopsy platform for clinicians to use in the fight against cancer.

Carbon-Coated Superparamagnetic Nanoflowers for Biosensors Based on Lateral Flow Immunoassays.

Superparamagnetic iron oxide nanoflowers coated by a black carbon layer (Fe3O4@C) were studied as labels in lateral flow immunoassays. They were synthesized by a one-pot solvothermal route, and they were characterized (size, morphology, chemical composition, and magnetic properties). They consist of several superparamagnetic cores embedded in a carbon coating holding carboxylic groups adequate for bioconjugation. Their multi-core structure is especially efficient for magnetic separation while keeping suitable magnetic properties and appropriate size for immunoassay reporters. Their functionality was tested with a model system based on the biotin–neutravidin interaction. For this, the nanoparticles were conjugated to neutravidin using the carbodiimide chemistry, and the lateral flow immunoassay was carried out with a biotin test line. Quantification was achieved with both an inductive magnetic sensor and a reflectance reader. In order to further investigate the quantifying capacity of the Fe3O4@C nanoflowers, the magnetic lateral flow immunoassay was tested as a detection system for extracellular vesicles (EVs), a novel source of biomarkers with interest for liquid biopsy. A clear correlation between the extracellular vesicle concentration and the signal proved the potential of the nanoflowers as quantifying labels. The limit of detection in a rapid test for EVs was lower than the values reported before for other magnetic nanoparticle labels in the working range 0–3 × 107 EVs/μL. The method showed a reproducibility (RSD) of 3% (n = 3). The lateral flow immunoassay (LFIA) rapid test developed in this work yielded to satisfactory results for EVs quantification by using a precipitation kit and also directly in plasma samples. Besides, these Fe3O4@C nanoparticles are easy to concentrate by means of a magnet, and this feature makes them promising candidates to further reduce the limit of detection.

Detection and Quantification of Extracellular Vesicles with Cargo Protein and RNA Using the Amnis® CellStream® Flow Cytometer

Abstract Extracellular vesicles (EVs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. These EVs—released under normal physiological conditions as well as in the pathogenesis of neurological, vascular, hematological, and autoimmune diseases—have been shown to transfer biological molecules such as protein and RNA between cells, potentially transmitting signals. To understand more about these signaling mechanisms, there is a need for detecting and quantifying EVs with cargo protein and RNA in a reproducible and reliable manner. However, this has been challenging due to the small size of EVs (ranging from 30 to 100 nm in diameter), and the lack of specific staining reagents. Here, we showcase the Amnis® CellStream® Flow Cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. We demonstrate that a charge-coupled device (CCD)-based, time-delay-integration image capturing system can be used to detect and quantify EVs and their cargo labeled with ExoGlow™-Protein or ExoGlow™-RNA.

Characterization of Extracellular Vesicles Isolated From Human Milk Using a Precipitation-Based Method.

Extracellular vesicles (EV) function in intercellular communication, and those in human milk may confer immunologic benefits to infants. Methods of EV isolation such as ultracentrifugation (UC) may not be feasible for the study of EVs in human milk due to the need for large sample volume. A technique to isolate EVs from a small volume of human milk using a precipitation reagent is described herein. Electron microscopy, nanoparticle tracking analysis, and semi-quantitative antibody array were conducted to confirm isolation of human milk EVs. Count, size, protein content, and fatty acid quantification of EVs were determined. This isolation technique yielded 8.9 x 109 (± 1.1 × 109) EV particles/mL of human milk. The present method meets the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. An established EV isolation method suitable for a low volume of human milk will facilitate further research in this growing area.

Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.

A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses. EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2×107 cells were stained with AlexaFluor 488-conjugated specific antibodies or corresponding isotype controls. Amount and size of particles in normal scattering light mode (N mode) versus fluorescence mode (F mode, laser wavelength 488nm) was measured using ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Cryo electron microscopy (EM) was used to verify the presence of membrane bilayer surrounded nanoparticles. All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit. Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.

EV-origin: Enumerating the tissue-cellular origin of circulating extracellular vesicles using exLR profile

Extracellular vesicles (EVs) are complex ecosystems that can be derived from all body cells and circulated in the body fluids. Characterizing the tissue-cellular source contributing to circulating EVs provides biological information about the cell or tissue of origin and their functional states. However, the relative proportion of tissue-cellular origin of circulating EVs in body fluid has not been thoroughly characterized. Here, we developed an approach for digital EVs quantification, called EV-origin, that enables enumerating of EVs tissue-cellular source contribution from plasma extracellular vesicles long RNA sequencing profiles. EV-origin was constructed by the input matrix of gene expression signatures and robust deconvolution algorithm, collectively used to separate the relative proportions of each tissue or cell type of interest. EV-origin respectively predicted the relative enrichment of seven types of hemopoietic cells and sixteen solid tissue subsets from exLR-seq profile. Using the EV-origin approach, we depicted an integrated landscape of the traceability system of plasma EVs for healthy individuals. We also compared the heterogenous tissue-cellular source components from plasma EVs samples with diverse disease status. Notably, the aberrant liver fraction could reflect the development and progression of hepatic disease. The liver fraction could also serve as a diagnostic indicator and effectively separate HCC patients from normal individuals. The EV-origin provides an approach to decipher the complex heterogeneity of tissue-cellular origin in circulating EVs. Our approach could inform the development of exLR-based applications for liquid biopsy.

Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels.

Extracellular vesicles (EVs) are cell-derived membranous vesicles that exist in nearly all biological fluids, including blood and urine; and carry a great number of cargo molecules such as protein, nucleic acids, and lipid. They may play important roles in cell-cell communication and modulation of pathological processes, which, however, are not yet well understood, calling for highly sensitive, specific, and rapid methods for EV detection and quantification in biological samples. Here, we report the CuS-enclosed microgels that not only help enrich EVs carrying specific protein markers from complex biomatrices, but also produce strong chemiluminescence (CL) to realize sensitive detection of the target EVs. A detection limit of 104 EV particles/mL was achieved with these microgels by targeting EV proteins like CD63 and HER2, with a dynamic range up to 108 particles/mL. Direct detection of EVs in human serum and cell culture medium without tedious sample preparation was demonstrated, consuming much less sample compared to ELISA and Western Blot. We envision that our method will be valuable for quick quantification of EVs in biological samples, benefiting disease monitoring and functional study.

Conductance-Based Biophysical Distinction and Microfluidic Enrichment of Nanovesicles Derived from Pancreatic Tumor Cells of Varying Invasiveness.

Diagnostics based on exosomes and other extracellular vesicles (EVs) are emerging as strategies for informing cancer progression and therapies, since the lipid content and macromolecular cargo of EVs can provide key phenotypic and genotypic information on the parent tumor cell and its microenvironment. We show that EVs derived from more invasive pancreatic tumor cells that express high levels of tumor-specific surface proteins and are composed of highly unsaturated lipids that increase membrane fluidity, exhibit significantly higher conductance versus those derived from less invasive tumor cells, based on dielectrophoresis measurements. Furthermore, through specific binding of the EVs to gold nanoparticle-conjugated antibodies, we show that these conductance differences can be modulated in proportion to the type as well as level of expressed tumor-specific antigens, thereby presenting methods for selective microfluidic enrichment and cytometry-based quantification of EVs based on invasiveness of their parent cell.

Rapid Lipid-Based Approach for Normalization of Quantum-Dot-Detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples.

Extracellular vesicles (EVs) are of considerable interest as tumor biomarkers because tumor-derived EVs contain a broad array of information about tumor pathophysiology. However, current EV assays cannot distinguish between EV biomarker differences resulting from altered abundance of a target EV population with stable biomarker expression, altered biomarker expression in a stable target EV population, or effects arising from changes in both parameters. We now describe a rapid nanoparticle- and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker levels to EV abundance. In this approach, EVs are captured from complex samples (e.g., serum), stained with a lipophilic dye, and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant and nonmalignant pancreatic cell lines exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its modular design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple expedient of swapping the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker.

A simple approach for rapid and cost-effective quantification of extracellular vesicles using a fluorescence polarization technique

Extracellular vesicles (EVs) are membrane-bound phospholipid vesicles actively secreted by all cells. As they carry specific markers expressed by their parental cells, EVs are utilized to identify specific cells via liquid biopsy. To facilitate EV-based clinical diagnosis, a fast and reliable method to count EVs is critical. We developed a method for rapid and cost-effective quantification of EVs which relies on the fluorescence polarization (FP) detection of lipophilic fluorescein probe, 5-dodecanoylamino fluorescein (C12-FAM). The alkyl tail of C12-FAM is specifically incorporated into the EVs, producing high FP values due to a slow diffusional motion. We quantified EVs derived from two cell lines, HT29 and TCMK1 using the new strategy, with good sensitivity that was at par with the commercial method. The new method involves minimal complexity and hands-on time. In addition, FP signaling is inherently ratiometric and is robust against environmental noise.

Simple and rapid extracellular vesicles quantification via membrane biotinylation strategy coupled with fluorescent nanospheres-based lateral flow assay

Extracellular vesicles (EVs) are cell-excreted membrane particles existing in a variety of biological fluids. As potential noninvasive biomarkers, EVs have received wide attention in recent years. However, usual EVs assays are complex, time-consuming and costly, thus limiting their clinical utility. Simple and rapid EVs quantification within biological fluids remains challenging. Here, we developed a simple, rapid strategy for EVs quantification, which combined with lateral flow assay and membrane biotinylation strategy. By utilizing biotin-functionalized phosphatidylethanolamine (DSPE-PEG-Biotin), the membrane of EVs could be successfully modified with biotin under strong hydrophobic interactions. Subsequently, based on the high affinity between streptavidin and biotin, quantification assay was achieved by lateral flow assay with fluorescent nanospheres (FNs) as a reporter. Biotinylation of biogenic EVs could be reached to 85%. This proposed method enables sensitive detection of 2.0 × 103 particles/μL. The whole procedure time was within 1 h. Furthermore, this approach was used to detect EVs in biological samples, demonstrating potential clinical applications.

Extracellular Vesicle Quantification and Characterization: Common Methods and Emerging Approaches

Extracellular vesicles (EVs) are a family of small membrane vesicles that carry information about cells by which they are secreted. Growing interest in the role of EVs in intercellular communication, but also in using their diagnostic, prognostic and therapeutic potential in (bio) medical applications, demands for accurate assessment of their biochemical and physical properties. In this review, we provide an overview of available technologies for EV analysis by describing their working principles, assessing their utility in EV research and summarising their potential and limitations. To emphasise the innovations in EV analysis, we also highlight the unique possibilities of emerging technologies with high potential for further development.

Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging.

Characterization of the tumor microenvironment, including extracellular vesicles (EVs), is important for understanding cancer progression. EV studies have traditionally been performed on dissociated cells, lacking spatial information. Since the distribution of EVs in the tumor microenvironment is associated with cellular function, there is a strong need for visualizing EVs in freshly resected tissues. We intraoperatively imaged untreated human breast tissues using a custom nonlinear imaging system. Label-free optical contrasts of the tissue, correlated with histological findings, enabled point-of-procedure characterization of the tumor microenvironment. EV densities from 29 patients with breast cancer were found to increase with higher histologic grade and shorter tumor-to-margin distance and were significantly higher than those from 7 cancer-free patients undergoing breast reduction surgery. Acquisition and interpretation of these intraoperative images not only provide real-time visualization of the tumor microenvironment but also offer the potential to use EVs as a label-free biomarker for cancer diagnosis and prognosis.

Miniaturized Biomedical Sensors for Enumeration of Extracellular Vesicles.

In this research, we have realized a rapid extracellular vesicle (EV) quantification methodology using a high field modulated AlGaN/GaN high electron mobility (HEMT) biosensor. The unique sensing structure facilitated the detection of the sub-cellular components in physiological salt environment without requiring extensive sample pre-treatments. The high field operation of GaN HEMT biosensor provides high sensitivity and wide dynamic range of detection of EVs (107–1010 EVs/mL). An antibody specific to the known surface marker on the EV was used to capture them for quantification using an HEMT biosensor. Fluorescence microscopy images confirm the successful capture of EVs from the test solution. The present method can detect EVs in high ionic strength solution, with a short sample incubation period of 5 min, and does not require labels or additional reagents or wash/block steps. This methodology has the potential to be used in clinical applications for rapid EV quantification from blood or serum for the development of diagnostic and prognostic tools.

High Purity Isolation and Sensitive Quantification of Extracellular Vesicles Using Affinity to TIM4.

Almost all types of cells secrete extracellular vesicles (EVs), including exosomes and microvesicles. EVs carry various proteins, lipids, mRNAs, and microRNAs, and may participate in many aspects of physiological and pathophysiological processes. Various studies are currently being conducted to develop therapeutic and diagnostic methods targeting or utilizing EVs. Therefore, developing ideal methods for isolating and quantifying EVs is an active area of research. EVs express phosphatidylserine on their outer lipid bilayer. This unit describes an affinity-based method for isolating EVs using TIM4, which binds phosphatidylserine in a specific and calcium-dependent manner. EVs captured by TIM4 can be easily released by addition of a chelating agent, or can be retained for quantification by ELISA or flow cytometry. These methods enable the isolation of highly purified EVs and the sensitive quantification of EVs, which will accelerate EV research beyond what has been achievable with conventional methods. © 2017 by John Wiley & Sons, Inc.

Assessment of Extracellular Vesicles Purity Using Proteomic Standards.

The increasing interest in extracellular vesicles (EVs) research is fueled by reports indicating their unique role in intercellular communication and potential connection to the development of common human diseases. The unique role assumes unique protein and nucleic acid cargo. Unfortunately, accurate analysis of EVs cargo faces a challenge of EVs isolation. Generally used isolation techniques do not separate different subtypes of EVs and even more, poorly separate EVs from non-EVs contaminants. Further development of EVs isolation protocols urgently needs a quantitative method of EVs purity assessment. We report here that multiple reaction monitoring assay using internal standards carrying peptides for quantification of EVs and non-EVs proteins is a suitable approach to assess purity of EVs preparations. As a first step in potential standardization of EVs isolation, we have evaluated polymer-based precipitation techniques and compared them to traditional ultracentrifugation protocol.

A Novel Capturing Method for Quantification of Extra-Cellular Nanovesicles.

Extracellular vesicles (EVs), secreted by cells and found in body fluids play important roles in intercellular communication. Therefore, EVs are receiving increasing attention as potential biomarkers in the diagnosis and prognosis of various diseases. However, the detection and the quantification of EVs are hampered by the nanometer scale of these particles and by the lack of optimized quantification methods. Atomic force microscopy (AFM) is a powerful technology that can detect small particles. Here we report a 3D capture method for sample preparation of AFM which improves the accuracy, sensitivity and reproducibility for EVs’ detection, compared to conventional sample preparation methods. By shaking a mica plate in EV solution, all the EVs were captured onto the 2D surface. The majority of the captured particles have a size ranging from 10 to 120 nm, which correlates with size data obtained from transmission electron microscopy studies. This novel sample preparation method has high adaptability potential and can also be applied to other organic and inorganic nanoparticles.

An Integrated Double-Filtration Microfluidic Device for Detection of Extracellular Vesicles from Urine for Bladder Cancer Diagnosis.

Extracellular vesicles (EVs) are present in a variety of bodily fluids and they play an important role in cellular communications and signal transduction mechanisms. Studies have shown that the number of EVs and EV-associated biomarkers (i.e., proteins, nucleic acids and lipids) can be used to aid clinical diagnosis. Although ultracentrifugation is commonly used for EV isolation, it is not practical for clinical settings. Here, we developed an integrated double-filtration device that isolated and enriched EVs from urine, and subsequently detected/quantified EVs from urine via microchip ELISA. Results showed that the concentration of EVs was significantly elevated compared to healthy controls. Receiver operating characteristic analysis demonstrated that this integrated EV quantification device had a sensitivity of 81.3% at a specificity of 90% (16 bladder cancer patients and eight healthy controls). Thus, this integrated device shows great potential to supplement urine cytology for diagnosis of bladder cancer in point-of-care (POC) settings.