Extracellular Vesicles Isolation Research Articles

Characterization of Extracellular Vesicles from Human Saliva: Effects of Age and Isolation Techniques.

Salivary extracellular vesicles (EVs) represent an attractive source of biomarkers due to the accessibility of saliva and its non-invasive sampling methods. However, the lack of comparative studies assessing the efficacy of different EV isolation techniques hampers the use of salivary EVs in clinical settings. Moreover, the effects of age on salivary EVs are largely unknown, hindering the identification of salivary EV-associated biomarkers across the lifespan. To address these questions, we compared salivary EV concentration, size mode, protein concentration, and purity using eight EV isolation techniques before and after magnetic bead immunocapture with antibodies against CD9, CD63, and CD81. The effects of age on salivary EVs obtained with each isolation technique were further investigated. Results showed higher expression of CD63 on isolated salivary EVs compared to the expression of CD81 and flotillin-1. Overall, magnetic bead immunocapture was more efficient in recovering salivary EVs with Norgen's Saliva Exosome Purification Kit and ExoQuick-TC ULTRA at the cost of EV yield. Regardless of age, Invitrogen Total Exosome Isolation Solution showed the highest level of protein concentration, whereas Izon qEVOriginal-70nm columns revealed the highest purity. This study provides the first comprehensive comparison of salivary EVs in younger and older adults using different EV isolation techniques, which represents a step forward for assessing salivary EVs as a source of potential biomarkers of tissue-specific diseases throughout the life cycle.

Isolation and Characterization of Extracellular Vesicles Derived from Ex Vivo Culture of Visceral Adipose Tissue.

Extracellular vesicles (EVs) are a heterogeneous group of nanoparticles possessing a lipid bilayer membrane that plays a significant role in intercellular communication by transferring their cargoes, consisting of peptides, proteins, fatty acids, DNA, and RNA, to receiver cells. Isolation of EVs is cumbersome and time-consuming due to their nano size and the co-isolation of small molecules along with EVs. This is why current protocols for the isolation of EVs are unable to provide high purity. So far, studies have focused on EVs derived from cell supernatants or body fluids but are associated with a number of limitations. Cell lines with a high passage number cannot be considered as representative of the original cell type, and EVs isolated from those can present distinct properties and characteristics. Additionally, cultured cells only have a single cell type and do not possess any cellular interactions with other types of cells, which normally exist in the tissue microenvironment. Therefore, studies involving the direct EVs isolation from whole tissues can provide a better understanding of intercellular communication in vivo. This underscores the critical need to standardize and optimize protocols for isolating and characterizing EVs from tissues. We have developed a differential centrifugation-based technique to isolate and characterize EVs from whole adipose tissue, which can be potentially applied to other types of tissues. This may help us to better understand the role of EVs in the tissue microenvironment in both diseased and normal conditions. Key features • Isolation of tissue-derived extracellular vesicles from ex vivo culture of visceral adipose tissue or any whole tissue. • Microscopic visualization of extracellular vesicles' morphology without dehydration steps, with minimum effect on their shape. • Flow cytometry approach to characterize the extracellular vesicles using specific protein markers, as an alternative to the time-consuming western blot.

Quality and efficiency assessment of five extracellular vesicle isolation methods using the resistive pulse sensing strategy.

Extracellular vesicles (EVs) have attracted great interest due to their great potential in disease diagnosis and therapy. The separation of EVs from complex biofluids with high purity is essential for the accurate analysis of EVs. Despite various methods, there is still no consensus on the best method for high-quality EV isolation and reliable mass production. Therefore, it is important to offer a standardized method for characterizing the properties (size distribution, particle concentration and purity) of EV preparations from different isolation methods. Herein, we employed a NanoCoulter Counter based on the resistive pulse sensing (RPS) strategy that enabled multi-parameter analysis of single EVs to compare the quality and efficiency of different EV isolation techniques including traditional differential ultracentrifugation, ultrafiltration, size exclusion chromatography, membrane affinity binding and polymer precipitation. The data revealed that the NanoCoulter Counter based on the RPS strategy was reliable and effective for the characterization of EVs. The results suggested that although higher particle concentrations were observed in three commercial isolation kits and ultrafiltration, traditional differential ultracentrifugation showed the highest purity. In conclusion, our results from the NanoCoulter Counter provided reliable evidence for the assessment of different EV isolation methods, which contributed to the development of EV-based disease biomarkers and treatments.

Isolation and Characterization of Extracellular Vesicles from Cell Culture Media.

Extracellular vesicles (EVs) were once believed to serve as a means of disposing of cellular waste. However, recent discoveries have identified their crucial roles in intercellular communication between neighboring and distant cells. Almost all cell types have now been identified to produce EVs, which play a vital role in transporting cellular cargo. The functional roles of EVs, along with their implications in (patho)physiology of various diseases, are still being explored. In the last decade, the identification of EV roles in pathophysiology, pharmacology, and diagnostics has gained significant interest, albeit the development of universal methods for the isolation and characterization of EVs has been the limiting factor. A further challenge is ensuring that EVs of various size categories, which are thought to be produced via independent cellular mechanisms and often differ in their cargo and physiological purpose, can be separated and studied in isolation.This protocol provides an efficient and accessible method for isolating and characterizing EV samples from conditioned cell culture media. The combination of differential centrifugation and the use of a commercial EV-precipitation kit allows for the rapid isolation of a highly pure sample of EVs separated by size. A microfluidic resistive pulse sensing (MRPS)-based method is then used to quantify the particles, as well as to assess the size distribution of the EV sample. As a result, this protocol provides a reproducible means to isolate and characterize EVs of a variety of sizes from nearly any cultured cells.

Analysis of Cryptococcus Extracellular Vesicles.

Extracellular vesicles (EVs) are produced by all domains of life. In fungal pathogens, they participate in virulence mechanisms and/or induce protective immunity, depending on the pathogenic species. EVs produced by pathogenic members of the Cryptococcus genus mediate virulence, antifungal resistance, as well as humoral and cell-mediated immunity. The isolation of cryptococcal EVs has been laborious and time-consuming for years. In this chapter, we detail a fast protocol for the isolation and analysis of EVs produced by members of the Cryptococcus genus.

Isolation of Extracellular Vesicles Using Formulas to Adapt Centrifugation to Different Centrifuges.

Extracellular vesicles (EVs) are small lipid bilayer vesicles released by cells to facilitate cell-to-cell communication. To study their biological roles and functions, they need to be isolated and purified, which can be achieved through a variety of methods. Here, we describe different methods for isolating and purifying EVs, with a focus on calculating the required g-force and centrifugation time with different centrifuges and rotors. We have compiled key formulas and tested predicted parameters for EV acquisitions to provide a comprehensive guide for EV isolation.

Isolation of Extracellular Vesicles from Cell Culture and Blood Through Nano-Targeted DLD Microfluidic Device.

Extracellular vesicles (EVs) are secreted by cells and found in biological fluids such as blood, with concentration correlated with oncogenic signals, making them attractive biomarkers for liquid biopsy. The current gold-standard method for EVs isolation requires an ultracentrifugation (UC) step among others. The cost and complexity of this technique are forbiddingly high for many researchers, as well as for routine use in biological laboratories and hospitals. This chapter reports on a simple microfluidic method for EVs isolation, based on a microfluidic size sorting technique named Deterministic Lateral Displacement (DLD). With the design of micrometric DLD array, we demonstrated the potential of our DLD devices for the isolation of nano-biological objects such as EVs, with main population size distribution consistent with UC technique.

Isolation of Extracellular Vesicles Using Titanium Dioxide Microspheres.

Extracellular vesicles (EVs) are bilayer membrane particles released from several cell types to the extracellular environment. EVs have a crucial role in cell-cell communication, involving different biological processes in health and diseases. Due to the potential of biomarkers for several diseases as diagnostic and therapeutic tools, it is relevant to understand the biology of the EVs and their content. One of the current challenges involving EVs is regarding the purification method, which is a critical step for EV's functional and characterization studies. Ultracentrifugation is the most used method for EV isolation, where the nanoparticles are separated in sequential centrifugation to isolate the EVs based on their size. However, for viscous biofluids such as plasma, there is a co-isolation of the most abundant proteins, which can impair the EV's protein identification due to the low abundance of these proteins and signal suppression by the most abundant plasma proteins. Emerging techniques have gained attention in recent years. Titanium dioxide (TiO2) is one of the most promising techniques due to its property for selective isolation based on the interaction with phospholipids in the EV membrane. Using a small amount of TiO2 beads and a low volume of plasma, it is possible to isolate EVs with reduced plasma protein co-isolation. This study describes a comprehensive workflow for the isolation and characterization of plasma extracellular vesicles (EVs) using mass spectrometry-based proteomics techniques. The aim of this chapter is describe the EV isolation using TiO2 beads enrichment and high-throughput mass spectrometry techniques to efficiently identify the protein composition of EVs in a fast and straightforward manner.

Extracellular Vesicle Isolation and Mass Spectrometry-Based Proteomic Analysis in Arabidopsis thaliana.

Extracellular vesicles (EVs) can transport various biological materials, including proteins, lipids, nucleic acids, and metabolites, through the unconventional protein secretion (UPS) pathway. Plant EVs can be classified into at least three major types: tetraspanin 8 (TET8)-positive EVs, penetration 1 (PEN1)-positive EVs, and exocyst-positive organelle (EXPO)-derived EVs. However, the research progress of plant EVs has been hindered due to the limitations inherent in EV isolation techniques. Moreover, since previous research on plant EVs has primarily focused on the interaction between plants and microbes, the biogenesis, transport, and secretion of plant EVs remain unexplored. Recent advances in centrifugation methods for extraction of apoplastic wash fluids, combined with mass spectrometry-based proteomic analysis, provide approaches to identify regulators and cargoes of plant EVs and thus serve as an important step for future studies on the biogenesis and function of plant EVs. Here, we illustrate detailed methods of EV isolation and mass spectrometry-based proteomic analysis in Arabidopsis.

Isolation, Quantification, and Visualization of Extracellular Membrane Vesicles in Rhizobia Under Free-Living Conditions.

Rhizobia are a group of soil proteobacteria that are able to establish a symbiotic interaction with legumes. These bacteria are capable to fix atmospheric nitrogen into ammonia within specific plant root organs called nodules. The rhizobia-legume interaction is established by a complex molecular dialogue that starts with flavonoids exudated by the plant roots. In response, signaling molecules known as Nod factors (NFs) are secreted by the bacteria. These factors are sensed by specific plant receptors that trigger a downstream signaling cascade leading to rhizobium-specific intracellular colonization of the root hair via the formation of infection threads and the eventual development of nodules on roots. In these organs, rhizobia can fix nitrogen from the atmosphere for the plant in exchange for photosynthates and the appropriate environment for nitrogen fixation. Recently, it has been demonstrated that extracellular membrane vesicles (EMVs) produced by some rhizobia carry NFs. EMVs are proteolipidic structures that are secreted to the milieu from the bacterial membranes and are involved in several important biological processes, including intercellular communication. Thus far, little is known about rhizobia vesicles, and further studies are needed to understand their functions, including their role as transporting vessels of signaling molecules during the process of symbiosis. Here, we present a detailed protocol to isolate high-purity EMVs from free-living cultured rhizobia, test their integrity, and quantify their abundance.

Studying the small extracellular vesicle capture efficiency of magnetic beads coated with tannic acid.

The isolation of small extracellular vesicles (sEVs), including those secreted by pathological cells, with high efficiency and purity is highly demanded for research studies and practical applications. Conventional sEV isolation methods suffer from low yield, presence of contaminants, long-term operation and high costs. Bead-assisted platforms are considered to be effective for trapping sEVs with high recovery yield and sufficient purity for further molecular profiling. In this study, magnetically responsive beads made of calcium carbonate (CaCO3) particles impregnated with iron oxide (Fe3O4) nanoparticles are fabricated using a freezing-induced loading (FIL) method. The developed magnetic beads demonstrate sufficient magnetization and can be collected by a permanent magnet, ensuring their rapid and gentle capture from an aqueous solution. The tannic acid on the surface of magnetic beads is formed by a layer-by-layer (LbL) method and is used to induce coupling of sEVs with the surface of magnetic beads. These tannic acid coated magnetic beads (TAMB) were applied to capture sEVs derived from MCF7 and HCT116 cell lines. Quantitative data derived from nanoparticle tracking analysis (NTA) and BCA methods revealed the capture efficiency and recovery yield of about 60%. High-resolution transmission electron microscopy (HRTEM) imaging of sEVs on the surface of TAMBs indicated their structural integrity. Compared with the size exclusion chromatography (SEC) method, the proposed approach demonstrated comparable efficiency in terms of recovery yield and purity, while offering a relatively short operation time. These results highlight the high potential of the TAMB approach for the enrichment of sEVs from biological fluids, such as cell culture media.

Isolation of Rhizobial Extracellular Membrane Vesicles from Bacteroids.

Extracellular-membrane vesicles (EMVs) are spherical buds of the extracellular membrane, commonly produced by Gram-negative bacteria, known to mediate intricate inter-kingdom communication. In this context, comprehensive research dissecting the role of EMVs in one of the most complex nature-occurring molecular dialogues, rhizobium-legume symbiosis, has been so far neglected. During the different stages of the symbiotic process, rhizobia and their host plants establish a very specific and controlled intercellular trafficking of signal molecules. Thus, as conveyors of a broad range of molecules into the target cell, EMVs are gaining weight in the field. Here, we describe a detailed protocol to isolate EMVs from bacteroids of legume nodules, opening a new door for discovering new authors of the symbiotic process.

First isolation and characterization of caprine oviduct fluid extracellular vesicles

First isolation and characterization of caprine oviduct fluid extracellular vesicles

A simplified and efficient extracellular vesicle-based proteomics strategy for early diagnosis of colorectal cancer.

Colorectal cancer (CRC) is a major cause of cancer-related death worldwide and an effective screening strategy for diagnosis of early-stage CRC is highly desired. Although extracellular vesicles (EVs) are expected to become some of the most promising tools for liquid biopsy of early disease diagnosis, the existing EV-based proteomics methods for practical application in clinical samples are limited by technical challenges in high-throughput isolation and detection of EVs. In the current study, we have developed a simplified and efficient EV-based proteomics strategy for early diagnosis of CRC. DSPE-functionalized beads were specifically designed that enabled direct capture of EVs from plasma samples in 10 minutes with good reproducibility and comprehensive proteome coverage. The single-pot, solid-phase-enhanced sample-preparation (SP3) technology was then combined with data-independent acquisition mass spectrometry (DIA-MS) for in-depth analysis and quantification of EV proteomes. From a cohort with 30 individuals including 11 healthy controls, 8 patients with adenomatous polyp and 11 patients with early-stage CRC, our streamlined workflow reproducibly quantified over 800 proteins from their plasma-derived EV samples, from which dysregulated protein signatures for molecular diagnosis of CRC were revealed. We selected a panel of 10 protein markers to train a machine learning (ML) model, which resulted in accurate prediction of polyp and early-stage CRC in an independent and single-blind validation cohort with excellent diagnostic ability of 89.3% accuracy. Our simplified and efficient clinical proteomic strategy will serve as a valuable tool for fast, accurate, and cost-effective diagnosis of CRC that can be easily extended to other disease samples for discovery of unique EV-based biomarkers.

From biogenesis to aptasensors: advancements in analysis for tumor-derived extracellular vesicles research.

Extracellular vesicles (EVs) are enclosed by a nanoscale phospholipid bilayer membrane and typically range in size from 30 to 200 nm. They contain a high concentration of specific proteins, nucleic acids, and lipids, reflecting but not identical to the composition of the parent cell. The inherent characteristics and variety of EVs give them extensive and unique advantages in the field of cancer identification and treatment. Recently, EVs have been recognized as potential tumor markers for the detection of cancer. Aptamers, which are molecules of single-stranded DNA or RNA, demonstrate remarkable specificity and affinity for their targets by adopting distinct tertiary structures. Aptamers offer various advantages over their protein counterparts, such as reduced immunogenicity, the ability for convenient large-scale synthesis, and straightforward chemical modification. In this review, we summarized EVs biogenesis, sample collection, isolation, storage and characterization, and finally provided a comprehensive survey of analysis techniques for EVs detection that are based on aptamers.

Genetically engineered cell-derived nanovesicles for cancer immunotherapy.

The emergence of immunotherapy has marked a new epoch in cancer treatment, presenting substantial clinical benefits. Extracellular vesicles (EVs), as natural nanocarriers, can deliver biologically active agents in cancer therapy with their inherent biocompatibility and negligible immunogenicity. However, natural EVs have limitations such as inadequate targeting capability, low loading efficacy, and unpredictable side effects. Through progress in genetic engineering, EVs have been modified for enhanced delivery of immunomodulatory agents and antigen presentation with specific cancer targeting ability, deepening the role of EVs in cancer immunotherapy. This review briefly describes typical EV sources, isolation methods, and adjustable targeting of EVs. Furthermore, this review highlights the genetic engineering strategies developed for delivering immunomodulatory agents and antigen presentation in EV-based systems. The prospects and challenges of genetically engineered EVs as cancer immunotherapy in clinical translation are also discussed.

RNA Extraction from Gram-Positive Bacteria Membrane Vesicles Using a Polymer-Based Precipitation Method.

Investigations into the biological role and composition of bacterial extracellular vesicles have grown in popularity in recent years. Vesicles perform a variety of functions during interactions with eukaryotic host cells, ranging from antibiotic resistance to immune modulation. It is necessary to isolate vesicles in order to understand their biological functions. Here we describe a polymer-based precipitation method allowing high-yield isolation of extracellular vesicles and their cargo RNA from the Gram-positive bacterium Staphylococcus aureus.

Recent research on material-based methods for isolation of extracellular vesicles.

Extracellular vesicles (EVs) are nanoparticles secreted by cells with a closed phospholipid bilayer structure, which can participate in various physiological and pathological processes and have significant clinical value in disease diagnosis, targeted therapy and prognosis assessment. EV isolation methods currently include differential ultracentrifugation, ultrafiltration, size exclusion chromatography, immunoaffinity, polymer co-precipitation and microfluidics. In addition, material-based biochemical or biophysical approaches relying on intrinsic properties of the material or its surface-modified functionalized monomers, demonstrated unique advantages in the efficient isolation of EVs. In order to provide new ideas for the subsequent development of material-based EV isolation methods, this review will focus on the principle, research status and application prospects of material-based EV isolation methods based on different material carriers and functional monomers.

Enhanced enrichment of extracellular vesicles for laboratory and clinical research from drop-sized blood samples.

In the realm of biomedical advancement, extracellular vesicles (EVs) are revolutionizing our capacity to diagnose, monitor, and predict disease progression. However, the comprehensive exploration and clinical application of EVs face significant limitations due to the current isolation techniques. The size exclusion chromatography, commercial precipitation reagents, and ultracentrifugation are frequently employed, necessitating skilled operators and entailing challenges related to consistency, reproducibility, quality, and yields. Notably, the formidable challenge of extracellular vesicle isolation persists when dealing with clinical samples of limited availability. This study addresses these challenges by aiming to devise a rapid, user-friendly, and high-recovery EVs isolation technique tailored for blood samples. The NTI-EXO precipitation method demonstrated a 5-fold increase in the recovery of serum EVs compared to current methodologies. Importantly, we illustrate that a mere two drops of blood (∼100 µL) suffice for the recovery of enriched EVs. The integrity and quality of these isolated EVs were rigorously assessed for the size, purity, and contaminants. This method was validated through the successful isolation of EVs from organ transplant recipients to detect disease-specific exosomal markers, including LKB1, SARS-CoV-2 spike protein, and PD-L1. In conclusion, NTI-EXO method can be used for small clinical samples, thereby advancing discoveries in the EV-centric domain and propelling the frontiers of biomedical research and clinical applications.

Extracellular vesicles: Methods for purification and characterization.

Extracellular vesicles (EVs) are membrane-bound particles released by cells that play a significant role in intercellular communication. They can be obtained from a variety of sources, including conditioned culture medium, blood and urine. In this chapter we detail the methods for EV isolation and characterization. Isolating and characterizing EVs is essential for understanding their functions in physiological and pathological processes. Advances in isolation and characterization techniques provide opportunities for deeper research into EV biology and its potential applications in diagnostics and therapeutics.