Separation Of Extracellular Vesicles Research Articles

Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

Extracellular vesicles isolated from Arabidopsis thaliana leaves reveal characteristics of mammalian exosomes.

Plant-derived extracellular vesicles (EVs), containing a myriad of bioactive proteins, microRNAs, lipids, and secondary metabolites, have recently become the focus of rising interest due to their important roles in various applications. The widely accepted method for isolating plant EVs is differential ultracentrifugation plus density gradient centrifugation. However, the combination of differential ultracentrifugation and density gradient centrifugation for the isolation of plant EVs is time-consuming and labor-intensive. Hence, there is a need for more efficient methods to perform the separation of plant EVs. In this study, EVs were separated from Arabidopsis thaliana leaves by a cost-effective polyethylene glycol (PEG)-based precipitation approach. The mean size of purified Arabidopsis thaliana EVs determined by dynamic light scattering was 266nm, which is consistent with nanoparticle tracking analysis. The size was also confirmed via transmission electron microscopy with morphology of a cup-shaped appearance which is the typical mammalian exosome's morphology. Additionally, Western blotting of the purified Arabidopsis thaliana EVs, using commercially available mammalian exosomal kits, displayed surface marker tetraspanin proteins (CD9, CD63, and CD81), and endosomal sorting complexes required for transport (ESCRT)-associated proteins (TSG101 and ALIX). This demonstrates that the purified Arabidopsis thaliana EVs reveal the typical proteins reported in mammalian exosomes.

Efficient EVs separation and detection by an alumina-nanochannel-array-membrane integrated microfluidic chip and an antibody barcode biochip

BackgroundSmall endosome-derived lipid nanovesicles (30–200 nm) are actively secreted by living cells and serve as pivotal biomarkers for early cancer diagnosis. However, the study of extracellular vesicles (EVs) requires isolation and purification from various body fluids. Although traditional EVs isolation and detection technologies are mature, they usually require large amount of sample, consumes long-time, and have relatively low-throughput. How to efficiently isolate, purify and detect these structurally specific EVs from body fluids with high-throughput remains a great challenge in in vitro diagnostics and clinical research. ResultsHerein, we suggest a nanosized microfluidic device for efficient and economical EVs filtration based on an alumina nanochannel array membrane. We evaluated the filtration device performance of alumina membranes with different diameters and found that an optimized chamber array with a hydrophilic-treated channel diameter of 90 nm could realize a filtration efficiency of up to 82% without any assistance from chemical or physical separation methods. Importantly, by integrating meticulously designed multichannel microfluidic biochips, EVs can be captured in-situ and monitored by antibody barcode biochip. The proposed filtration chip together with the high-throughput detection chip were capable of filtration of a few tens of μL samples and recognition of different phonotypes. The practical filtration and detection of EVs from clinical samples demonstrated the high performance of the device. SignificantOverall, this work provides a cost-effective, highly efficient and automated EVs filtration chip and detection dual-function integrated chip platform, which can directly separate EVs from serum or cerebrospinal fluid with an efficiency of 82% and conduct in-situ detection. This small fluidic device can provide a powerful tool for highly efficient identifying and analyzing EVs, presenting great application potential in clinical detection.

Goat milk extracellular vesicles: Separation comparison of natural carriers for theragnostic application

Goat milk is a complex biological fluid, which in addition to having a high nutritional value, it is an interesting source of extracellular vesicles (EVs). Despite the countless potential applications that they offer in many biological fields, is not easy to compare the different proposed systems, and this is a major limitation for the real translatability of these natural nanoplatforms for theragnostic purposes. Thus, it is useful to further investigate reproducible methods to separate goat milk EVs. The choice of methods but also the preprocessing of milk has an immense impact on the separation, quality, and yield of EVs. Here, we tested four protocols to separate EVs from unpasteurised goat milk: two based on differential ultracentrifugation (DUC) and two on size-exclusion chromatography (SEC). Moreover, we assessed two different approaches of pre-treatment (acidification and precipitation) to facilitate milk protein discharge. To the best of our knowledge, a similar comparison of all performed protocols on raw goat milk has never been published before. Therefore, enriched EV samples were successfully obtained from goat milk using both DUC and SEC. Taken together, our results may be helpful to obtain natural carriers for future theragnostic applications in personalised medicine.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

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.

Assessment of the Antigen-Binding Capacity and Separation of Extracellular Vesicles Coated with Antigen-Specific Antibody Light Chains.

Many researchers are interested in the possibility of manipulating the targeting specificity of extracellular vesicles (EVs) for their use as physiological delivery vehicles for drugs and bioactive molecules. Our studies demonstrated the possibility of directing EVs toward the desired acceptor cell by coating them with antigen-specific antibody light chains. Here, we describe the methods for detection of the presence of antibody light chains on the EV surface, proving their ability to specifically bind the antigen and for separating the antigen-binding EV subpopulation.

Exploring Circular RNA Profile and Expression in Extracellular Vesicles.

Extracellular vesicles (EVs) are small vesicles secreted by various cell types and are enriched in multiple body fluids. EVs containing RNA have the potential to modulate biological processes and are being investigated for their diagnostic and therapeutic applications. Circular RNAs (circRNAs), generated through back-splicing of exons, are enriched in EVs. Given their unique characteristics and diverse functions, EV-circRNAs are important players in disease pathology. This chapter describes a workflow for investigating the expression profile of EV-circRNAs, which includes EVs separation, library preparation, and bioinformatics analysis. This workflow can aid the investigation of EV-circRNAs and their potential role in disease pathology.

Exosome Precipitation by Ionic Strength Modulation: ExoPRISM.

Extracellular vesicles (EVs) are emerging as crucial materials for precision theragnostic applications. However, current separation methods are time-consuming, costly, and not scalable and deliver limited yields or purity. Here, we present EV precipitation by ionic strength modulation (ExoPRISM), a simple, low-cost, user-friendly, and readily adaptable approach for separating EVs in high yields without compromising their biological functions. Adding an electrolyte solution to blood plasma in small increments generates the sequential precipitation of proteins and EVs, allowing for fractional separation of EVs using low-speed centrifugation. The coprecipitated electrolytes are easily washed away, and the entire EV separation and washing process takes less than an hour. This approach successfully separates EVs from a broad range of volumes and types of biological fluids, including culture medium, urine, plasma, and serum, showing promise as a robust tool for next-generation liquid biopsies and regenerative medicine.

Nanostructure enabled extracellular vesicles separation and detection

Extracellular vesicles (EVs) have recently attracted significant research attention owing to their important biological functions, including cell-to-cell communication. EVs are a type of membrane vesicles that are secreted into the extracellular space by most types of cells. Several biological biomolecules found in EVs, such as proteins, microRNA, and DNA, are closely related to the pathogenesis of human malignancies, making EVs valuable biomarkers for disease diagnosis, treatment, and prognosis. Therefore, EV separation and detection are prerequisites for providing important information for clinical research. Conventional separation methods suffer from low levels of purity, as well as the need for cumbersome and prolonged operations. Moreover, detection methods require trained operators and present challenges such as high operational expenses and low sensitivity and specificity. In the past decade, platforms for EV separation and detection based on nanostructures have emerged. This article reviews recent advances in nanostructure-based EV separation and detection techniques. First, nanostructures based on membranes, nanowires, nanoscale deterministic lateral displacement, and surface modification are presented. Second, high-throughput separation of EVs based on nanostructures combined with acoustic and electric fields is described. Third, techniques combining nanostructures with immunofluorescence, surface plasmon resonance, surface-enhanced Raman scattering, electrochemical detection, or piezoelectric sensors for high-precision EV analysis are summarized. Finally, the potential of nanostructures to detect individual EVs is explored, with the aim of providing insights into the further development of nanostructure-based EV separation and detection techniques.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.

Extracellular Vesicles Isolated from Menstrual Blood-derived Mesenchymal Stem Cells in Regenerative Medicine

: Extracellular vesicles (EVs) are small particles ranging from 30 - 1000 nm released by different cells to mediate cell-cell communication. The content of these particles mirrors the cellular properties of their producing cells. The extracellular vesicles, including exosomes and microvesicles, could exert the same therapeutic effects as stem cell therapy. However, these stem cell-derived particles (cell-free derivatives) do not have concerns associated with stem cell-based therapies. Among various mesenchymal stem/stromal cells, Menstrual Blood Stem Cells (MenSCs) are known for their high proliferative activity, ease of harvesting, lack of moral dilemma, and high differentiation potential. Extracellular vesicles derived from MenSCs have widely been employed in various preclinical studies for regenerative purposes. This paper aims to provide an overview of the therapeutic utility of MenSCs-derived EVs in regenerative medicine. We also reviewed the current knowledge on the cellular profile, biogenesis, separation, and action mechanism of MenSCs-derived EVs in organ regeneration.

Electrostatic interactions control the adsorption of extracellular vesicles onto supported lipid bilayers

Communication between cells located in different parts of an organism is often mediated by membrane-enveloped nanoparticles, such as extracellular vesicles (EVs). EV binding and cell uptake mechanisms depend on the heterogeneous composition of the EV membrane. From a colloidal perspective, the EV membrane interacts with other biological interfaces via both specific and non-specific interactions, where the latter include long-ranged electrostatic and van der Waals forces, and short-ranged repulsive “steric-hydration” forces. While electrostatic forces are generally exploited in most EV immobilization protocols, the roles played by various colloidal forces in controlling EV adsorption on surfaces have not yet been thoroughly addressed. In the present work, we study the adsorption of EVs onto supported lipid bilayers (SLBs) carrying different surface charge densities using a combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and confocal laser scanning microscopy (CLSM). We demonstrate that EV adsorption onto lipid membranes can be controlled by varying the strength of electrostatic forces and we theoretically describe the observed phenomena within the framework of nonlinear Poisson-Boltzmann theory. Our modelling results confirm the experimental observations and highlight the crucial role played by attractive electrostatics in EV adsorption onto lipid membranes. They furthermore show that simplified theories developed for model lipid systems can be successfully applied to the study of their biological analogues and provide new fundamental insights into EV-membrane interactions with potential use in developing novel EV separation and immobilization strategies.

Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins.

Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.

Extracellular Vesicles Isolation from Large Volume Samples Using a Polydimethylsiloxane-Free Microfluidic Device.

Extracellular vesicles (EV) have many attributes important for biomedicine; however, current EV isolation methods require long multi-step protocols that generally involve bulky equipment that cannot be easily translated to clinics. Our aim was to design a new cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) asymmetric flow field fractionation microfluidic device that could isolate EV from high-volume samples in a simple and efficient manner. We tested the device with large volumes of urine and conditioned cell media samples, and compared it with the two most commonly used EV isolation methods. Our device was able to separate particles by size and buoyancy, and the attained size distribution was significantly smaller than other methods. This would allow for targeting EV size fractions of interest in the future. However, the results were sample dependent, with some samples showing significant improvement over the current EV separation methods. We present a novel design for a COC-OSTE microfluidic device, based on bifurcating asymmetric flow field-flow fractionation (A4F) technology, which is able to isolate EV from large volume samples in a simple, continuous-flow manner. Its potential to be mass-manufactured increases the chances of implementing EV isolation in a clinical or industry-friendly setting, which requires high repeatability and throughput.

Abstract 697: Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses

Abstract Virus-based therapies show promise in inducing systemic anti-tumor immunity. Over the past years, modified poxviruses were developed to induce a sustained antitumoral immune response. Several studies confirm this hypothesis and report induction of T cell response against the antigen targeted by these vaccines. We previously reported MVA induced T-cell response against HPV oncoviral antigens E6 and E7 in HPV anogenital cancers (NCT03260023) and against private tumor neoantigen in ovarian and head and neck cancer. Despite these promising results the molecular and cellular mechanisms involved in vaccine response remains to be elucidated. In this work, we inquired whether EVs, well known for mediating intercellular communication, play a role in these therapeutic vaccination process. We first asked whether poxviral infection of human primary cells modifies their EV secretion properties. To this end, peripheral blood mononuclear cells (PBMCs) from healthy donors were infected with poxviral vectors, cell culture supernatants were harvested, and different isolation methods were compared for their ability to separate EVs from viral particles: ultracentrifugation, immunocapture, density gradient, velocity gradient, size exclusion chromatography and filtration. EVs size distribution and concentration were determined by nanoparticle tracking analysis (NTA) and the presence of infectious viral particles was titrated by plaque assays. Our data showed that 0.1µm filtration allowed the complete separation of EVs from infectious viral particles. All other methods showed co-isolation of EVs and infectious viral particles. Using this method, we demonstrate that infection of PBMCs with the poxviruses VACV, MVA and PCPV significantly increased the amount of secreted EVs. Next, we investigated the molecular content of EVs isolated from MVA-infected PBMCs by mass spectrometry. We observed differences in the abundance of at least 57 human proteins between EVs from infected and non-infected cells. In addition, several viral proteins, including virus-encoded payloads were detected in EVs from infected cells. We are now confirming those data with flow cytometry to detect virus-encoded payloads like tumor antigens, antigen-presenting molecules, and EV markers such as tetraspanins. The RNA content of EVs from infected and non-infected cells will be compared. Finally, EVs were isolated from murine dendritic cells infected with therapeutic pox vaccines. We are now evaluating the effects of local and systemic application of EVs in murine tumor models. Citation Format: Lucas Walther, Caroline Tosch, Jules Deforges, Christine Carapito, Magali Rompais, Marie-Christine Claudepierre, Nathalie Silvestre, Kaïdre Bendjama, Eric Quemeneur, Jacky Goetz, Vincent Hyenne, Karola Rittner. Extracellular vesicles (EV) - mediators of therapeutic vaccination? In vivo and in vitro characterization of EVs generated after infection of human and murine cells with therapeutic poxviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 697.

From Conventional to Microfluidic: Progress in Extracellular Vesicle Separation and Individual Characterization (Adv. Healthcare Mater. 8/2023)

Single Extracellular Vesicles Physical and biochemical characterization for single extracellular vesicles (EVs) has been gaining more attention for disease diagnosis. With the development of micromachining technologies, microfluidic chips can analyze EVs with precise manipulation, low cost and high portability. In the foreseeable future, the combination of EVs characterization with microfluidics and gradually individualizing them will be a meaningful direction. These considerations are reviewed by Daxiang Cui, Hossam Haick, Ning Tang, and co-workers, in article 2202437.

Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

Alterations in Abundance and Compartmentalization of miRNAs in Blood Plasma Extracellular Vesicles and Extracellular Condensates during HIV/SIV Infection and Its Modulation by Antiretroviral Therapy (ART) and Delta-9-Tetrahydrocannabinol (Δ9-THC).

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

SIV Infection Regulates Compartmentalization of Circulating Blood Plasma miRNAs within Extracellular Vesicles (EVs) and Extracellular Condensates (ECs) and Decreases EV-Associated miRNA-128.

Bifurcated Asymmetric Field Flow Fractionation of Nanoparticles in PDMS-Free Microfluidic Devices for Applications in Label-Free Extracellular Vesicle Separation.

Extracellular vesicles are small membrane-bound structures that are released by cells and play important roles in intercellular communication garnering significant attention in scientific society recently due to their potential as diagnostic and therapeutic tools. However, separating EVs from large-volume samples remains a challenge due to their small size and low concentration. In this manuscript, we presented a novel method for separating polystyrene beads as control and extracellular vesicles from large sample volumes using bifurcated asymmetric field flow fractionation in PDMS-free microfluidic devices. Separation characteristics were evaluated using the control system of polystyrene bead mix, which offers up to 3.7X enrichment of EV-sized beads. Furthermore, in the EV-sample from bioreactor culture media, we observed a notable population distribution shift of extracellular vesicles. Herein presented novel PDMS-free microfluidic device fabrication protocol resulted in devices with reduced EV-loss compared to size-exclusion columns. This method represented an improvement over the current state of the art in terms of EV separation from large sample volumes through the use of novel field flow fractionation design.