Extracellular Vesicles Isolation Research Articles

Isolation of goat milk small extracellular vesicles by novel combined bio-physical methodology.

Introduction: Goat milk is notable as a cost-effective source of exosomes, also known as small extracellular vesicles (sEVs). These nanoparticle-like structures are naturally secreted by cells and have emerged as potential diagnostic agents and drug delivery systems, also supported by their proven therapeutic effects. However, the complexity of goat milk and the lack of standardized protocols make it difficult to isolate pure sEVs. This work presents an optimized approach that combines well-established physical isolation methods with the biological treatment of milk with rennet. Methods: sEVs derived from goat milk were purified using a methodology that combines differential ultracentrifugation, rennet, and size-exclusion chromatography. This novel strategy was compared with two of the main methodologies developed for isolating extracellular vesicles from bovine and human milk by means of physico-chemical characterization of collected vesicles using Transmission Electron Microscopy, Western blot, Bradford Coomassie assay, Dynamic Light Scattering, Nanoparticle Tracking Analysis and Zeta Potential. Results: Vesicles isolated with the optimized protocol had sEV-like characteristics and high homogeneity, while samples obtained with the previous methods were highly aggregated, with significant residual protein content. Discussion: This work provides a novel biophysical methodology for isolating highly enriched goat milk sEVs samples with high stability and homogeneity, for their further evaluation in biomedical applications as diagnostic tools or drug delivery systems.

Extracellular vesicles as a potential source of tumor-derived DNA in advanced pancreatic cancer.

Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer.

One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles.

Extracellular vesicle (EV) proteomics emerges as an effective tool for discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, here we introduce a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. In comparison with the existing workflow, the one-pot pipeline was able to obtain better peptide yield and identify the equivalent number of unique EV proteins from 1 mL of urine. Finally, we applied the one-pot pipeline to profile proteomes in urinary EVs of bladder cancer patients. A total of 2774 unique proteins were identified in 53 urine samples using a 15 min gradient library-free data-independent acquisition method. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications.

One-week storage of refrigerated bovine milk does not affect the size, concentration, or molecular properties of extracellular vesicles

Milk extracellular vesicles (EVs) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EVs with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2-quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EVs' size, concentration, or morphology. We neither detected changes in the EV cargo regarding the amount of protein and RNA nor the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low somatic cell counts. Overall, we observed fewer CD81 and CD9 markers in quarters with a high SCC. Moreover, there was no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to one week is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EVs are highly stable in the raw milk matrix.

Extracellular Vesicles: Techniques and Biomedical Applications Related to Single Vesicle Analysis.

Extracellular vesicles (EVs) are extensively dispersed lipid bilayer membrane vesicles involved in the delivery and transportation of molecular payloads to certain cell types to facilitate intercellular interactions. Their significant roles in physiological and pathological processes make EVs outstanding biomarkers for disease diagnosis and treatment monitoring as well as ideal candidates for drug delivery. Nevertheless, differences in the biogenesis processes among EV subpopulations have led to a diversity of biophysical characteristics and molecular cargos. Additionally, the prevalent heterogeneity of EVs has been found to substantially hamper the sensitivity and accuracy of disease diagnosis and therapeutic monitoring, thus impeding the advancement of clinical applications. In recent years, the evolution of single EV (SEV) analysis has enabled an in-depth comprehension of the physical properties, molecular composition, and biological roles of EVs at the individual vesicle level. This review examines the sample acquisition tactics prior to SEV analysis, i.e., EV isolation techniques, and outlines the current state-of-the-art label-free and label-based technologies for SEV identification. Furthermore, the challenges and prospects of biomedical applications based on SEV analysis are systematically discussed.

One-pot preparation of bi-functional POSS-based hybrid monolith via photo-initiated polymerization for isolation of extracellular vesicles

Extracellular vesicles (EVs) are important participants in numerous pathophysiological processes, and could be used as valuable biomarkers to detect and monitor various diseases. However, facile EV isolation methods are the essential and preliminary issue for their downstream analysis and function investigation. In this work, a polyhedral oligomeric silsesquioxanes (POSS) based hybrid monolith combined metal affinity chromatography (MAC) and distearoyl phospholipid ethanolamine (DSPE) function was developed via photo-initiated thiol-ene polymerization. This synthesis process was facile, simple and convenient, and the obtained hybrid monolith could be applied to efficiently isolate EVs from bio-samples by taking advantages of the specific bond of Ti4+ and phosphate groups on the phospholipid membrane of EVs and the synergistic effect of DSPE insertion. Meanwhile, the eluted EVs could maintain their structural integrity and biological activity, suggesting they could be used for downstream application. Furthermore, 75 up-regulated proteins and 56 down-regulated proteins were identified by comparing the urinary EVs of colorectal cancer (CRC) patients and healthy donors, and these proteins might be used as potential biomarkers for early screening of CRC. These results demonstrated that this hybrid monolith could be used as a simple and convenient tool for isolating EVs from bio-samples and for wider applications in biomarker discovery.

Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization.

Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.

Therapeutic potential in rheumatic diseases of extracellular vesicles derived from mesenchymal stromal cells.

The incidence of rheumatic diseases such as rheumatoid arthritis and osteoarthritis and injuries to articular cartilage that lead to osteochondral defects is predicted to rise as a result of population ageing and the increase in high-intensity physical activities among young and middle-aged people. Current treatments focus on the management of pain and joint functionality to improve the patient's quality of life, but curative strategies are greatly desired. In the past two decades, the therapeutic value of mesenchymal stromal cells (MSCs) has been evaluated because of their regenerative potential, which is mainly attributed to the secretion of paracrine factors. Many of these factors are enclosed in extracellular vesicles (EVs) that reproduce the main functions of parental cells. MSC-derived EVs have anti-inflammatory, anti-apoptotic as well as pro-regenerative activities. Research on EVs has gained considerable attention as they are a potential cell-free therapy with lower immunogenicity and easier management than whole cells. MSC-derived EVs can rescue the pathogenetic phenotypes of chondrocytes and exert a protective effect in animal models of rheumatic disease. To facilitate the therapeutic use of EVs, appropriate cell sources for the production of EVs with the desired biological effects in each disease should be identified. Production and isolation of EVs should be optimized, and pre-isolation and post-isolation modifications should be considered to maximize the disease-modifying potential of the EVs.

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs. This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg. Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Isolation of plasma small extracellular vesicles by an optimized size-exclusion chromatography-based method for clinical applications

Isolation of plasma small extracellular vesicles by an optimized size-exclusion chromatography-based method for clinical applications

Integrating extracellular vesicle and circulating cell-free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients.

Multi-analyte liquid biopsies represent an emerging opportunity for non-invasive cancer assessment. We developed ONCE (One Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell-free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining EV-associated RNA (EV-RNA) and cfDNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we isolated EV-enriched samples by a charge-based (CB) method and investigated EV-RNA and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV-RNA for HER2 cancer assessment and supports ONCE as a valuable tool for multi-analytes liquid biopsies' clinical implementation.

EVs from primary neurons: Characterization of extracellular vesicles derived from mouse primary neurons

It is now clear that the specific composition of extracellular vesicles (EV) is largely determined by the type and developmental or biological state of their donor cells. Careful characterization of EV secretion is therefore important for understanding their functions in specific contexts. Here, we assess neuronal EV secretion, using mouse primary cortical and hippocampal neurons and at two developmental stages. Our proteomics data further shows the presence of widely reported EV proteins such as CD81, Alix and TSG101, and the absence of the commonly used EV marker CD63. Moreover, neuron-derived EVs were significantly enriched in neuronal-enriched proteins, including specific synaptic proteins. Overall, this data provides an initial framework for the isolation of EVs from primary neurons that could be beneficial for specific studies of neuron-derived EVs.

Critical considerations in determining the surface charge of small extracellular vesicles.

Small extracellular vesicles (EVs) have emerged as a focal point of EV research due to their significant role in a wide range of physiological and pathological processes within living systems. However, uncertainties about the nature of these vesicles have added considerable complexity to the already difficult task of developing EV-based diagnostics and therapeutics. Whereas small EVs have been shown to be negatively charged, their surface charge has not yet been properly quantified. This gap in knowledge has made it challenging to fully understand the nature of these particles and the way they interact with one another, and with other biological structures like cells. Most published studies have evaluated EV charge by focusing on zeta potential calculated using classical theoretical approaches. However, these approaches tend to underestimate zeta potential at the nanoscale. Moreover, zeta potential alone cannot provide a complete picture of the electrical properties of small EVs since it ignores the effect of ions that bind tightly to the surface of these particles. The absence of validated methods to accurately estimate the actual surface charge (electrical valence) and determine the zeta potential of EVs is a significant knowledge gap, as it limits the development of effective label-free methods for EV isolation and detection. In this study, for the first time, we show how the electrical charge of small EVs can be more accurately determined by accounting for the impact of tightly bound ions. This was accomplished by measuring the electrophoretic mobility of EVs, and then analytically correlating the measured values to their charge in the form of zeta potential and electrical valence. In contrast to the currently used theoretical expressions, the employed analytical method in this study enabled a more accurate estimation of EV surface charge, which will facilitate the development of EV-based diagnostic and therapeutic applications.

An enzyme-based system for extraction of small extracellular vesicles from plants

Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are the next generation of nanocarrier platforms for biotherapeutics and drug delivery. EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitations of existing isolation methods, the EVs extraction efficiency is low, and a large amount of plant material is wasted, which is of concern for rare and expensive medicinal plants. We proposed and validated a novel method for isolation of plant EVs by enzyme degradation of the plant cell wall to release the EVs. The released EVs can easily be collected. The new method was used for extraction of EVs from the roots of Morinda officinalis (MOEVs). For comparison, nanoparticles from the roots (MONVs) were extracted using the grinding method. The new method yielded a greater amount of MOEVs, and the vesicles had a smaller diameter compared to MONVs. Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxic effect and promoted the expression of miR-155. The promotion of miR-155 by MOEVs was dose-dependent. More importantly, we found that MOEVs and MONVs were enriched toward bone tissue. These results support our hypothesis that EVs in plants could be efficiently extracted by enzymatic cell wall digestion and confirm the potential of MOEVs as therapeutic agents and drug carriers.

The Adaptation of Botrytis cinerea Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process.

Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several sets of macromolecules implicated in cell communication. EVs have become a relevant topic in the study of pathogenic fungi due to their relationship with fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EVs since plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, EVs of Botrytis cinerea are little known, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the characterization of B. cinerea EVs proteome changes under two pathogenic conditions and the description of their potential role during the infective process. All the experimental procedure was conducted in B. cinerea growing in a minimal salt medium supplemented with glucose as a constitutive stage and deproteinized tomato cell walls (TCW) as a virulence inductor. The isolation of EVs was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EVs fractions were visualised by TEM using negative staining. Proteomic analysis of EVs cargo was addressed by LC-MS/MS. The methodology used allowed the correct isolation of B. cinerea EVs and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in morphology under both assayed conditions. GO analysis of EV proteins showed enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed the difference in EVs function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, among others. This work describes the first evidence of EVs protein cargo adaptation in B. cinerea, which seems to play an essential role in its infection process, sharing crucial functions with the conventional secretion pathways.

Modeling and optimization of parallelized immunomagnetic nanopore sorting for surface marker specific isolation of extracellular vesicles from complex media

The isolation of specific subpopulations of extracellular vesicles (EVs) based on their expression of surface markers poses a significant challenge due to their nanoscale size (< 800 nm), their heterogeneous surface marker expression, and the vast number of background EVs present in clinical specimens (1010–1012 EVs/mL in blood). Highly parallelized nanomagnetic sorting using track etched magnetic nanopore (TENPO) chips has achieved precise immunospecific sorting with high throughput and resilience to clogging. However, there has not yet been a systematic study of the design parameters that control the trade-offs in throughput, target EV recovery, and ability to discard background EVs in this approach. We combine finite-element simulation and experimental characterization of TENPO chips to elucidate design rules to isolate EV subpopulations from blood. We demonstrate the utility of this approach by reducing device background > 10× relative to prior published designs without sacrificing recovery of the target EVs by selecting pore diameter, number of membranes placed in series, and flow rate. We compare TENPO-isolated EVs to those of gold-standard methods of EV isolation and demonstrate its utility for wide application and modularity by targeting subpopulations of EVs from multiple models of disease including lung cancer, pancreatic cancer, and liver cancer.

Extracellular Vesicles as Surrogates for Drug Metabolism and Clearance: Promise vs. Reality.

Drug-metabolizing enzymes (DMEs) and transporters play a major role in drug efficacy and safety. They are regulated at multiple levels and by multiple factors. Estimating their expression and activity could contribute to predicting drug pharmacokinetics and their regulation by drugs or pathophysiological situations. Determining the expression of these proteins in the liver, intestine, and kidney requires the collection of biopsy specimens. Instead, the isolation of extracellular vesicles (EVs), which are nanovesicles released by most cells and present in biological fluids, could deliver this information in a less invasive way. In this article, we review the use of EVs as surrogates for the expression and activity of DMEs, uptake, and efflux transporters. Preliminary evidence has been provided for a correlation between the expression of some enzymes and transporters in EVs and the tissue of origin. In some cases, data obtained in EVs reflect the induction of phase I-DMEs in the tissues. Further studies are required to elucidate to what extent the regulation of other DMEs and transporters in the tissues reflects in the EV cargo. If an association between tissues and their EVs is firmly established, EVs may represent a significant advancement toward precision therapy based on the biotransformation and excretion capacity of each individual.

Isolation of Hepatic and Adipose-Tissue-Derived Extracellular Vesicles Using Density Gradient Separation and Size Exclusion Chromatography.

In recent years, the study of extracellular vesicles (EVs) in the context of various diseases has dramatically increased due to their diagnostic and therapeutic potential. Typically, EVs are isolated in vitro from the cell culture of primary cells or cell lines or from bodily fluids. However, these cell culture methods do not represent the whole complexity of an in vivo microenvironment, and bodily fluids contain a high heterogeneous population of vesicles since they originate from different tissues. This highlights the need to develop new methods to isolate EVs directly from tissue samples. In the present study, we established a protocol for isolating EVs from hepatic and adipose tissue of mice, using a combination of ultracentrifugation and iodixanol-sucrose density gradient separation. EV isolation was confirmed with EV protein marker enrichment in Western blot assays, total protein quantification, and transmission electron microscopy. Regarding the liver tissue, we additionally implemented size exclusion chromatography (SEC) to further increase the purity grade of the EVs. The successful isolation of EVs from tissue samples will allow us to uncover a more precise molecular composition and functions, as well as their role in intercellular communication in an in vivo microenvironment.

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.

Polyethylene glycol-based isolation of urinary extracellular vesicles, an easily adoptable protocol

Urine is a highly advantageous biological specimen for biomarker research and is a non-invasive source. Most of the urinary biomarkers are non-specific, volatile and need extensive validation before clinical adoption. Extracellular vesicles are secreted by almost all cells and are involved in homoeostasis, intercellular communication, and cellular processes in healthy and pathophysiological states. Urinary extracellular vesicles (UEVs) are released from the urogenital system and mirror the molecular processes of physiological and pathological states of their source cells. Therefore, UEVs serve as a valuable source of biomarkers for the non-invasive diagnosis of various pathologies. They hold a promising source of multiplex biomarkers suitable for prognosis, diagnosis, and therapy monitoring. UEVs are easily accessible, non-invasive, and suited for longitudinal sampling. Although various techniques are available for isolating UEVs, there is yet to be a consensus on a standard and ideal protocol. We have optimized an efficient, reliable, and easily adoptable polyethylene glycol (PEG) based UEV isolation technique following MISEV guidelines. The method is suitable for various downstream applications of UEVs. This could be a cost-effective, consistent, and accessible procedure for many clinical labs and is most suited for longitudinal analysis. Adopting the protocol will pave the way for establishing UEVs as the ideal biomarker source. •Urine can be collected non-invasively and repeatedly, hence a very useful specimen for biomarker discovery. Urinary EVs (UEVs), derived from urine, offer a stable diagnostic tool, but standardised isolation and analysis approaches are warranted.•To have enough UEVs for any study, large volumes of urine sample are necessary, which limits different isolation methods by cost, yield, and time.•The protocol developed could help researchers by offering a cost-effective and dependable UEV isolation method and may lay the foundation for UEVs adoption in clinical space.