Mag-Net Strong Anion Exchange Enables Isolation of Ovarian Cancer Ascites Extracellular Vesicles for Proteomic Biomarker Discovery

A novel immunoassay for ApoB-100, the main protein component of lipoproteins, enables the development of methods to enrich extracellular vesicles from human plasma while depleting both lipoproteins and free proteins.

Extracellular vesicles (EVs) are currently of tremendous interest in many research disciplines and EVs have potential for development of EV diagnostics or therapeutics. Most well-known single EV isolation methods have their particular advantages and disadvantages in terms of EV purity and EV yield. Combining EV isolation methods provides additional potential to improve the efficacy of both purity and yield.This review assesses the contribution and efficacy of using combined EV isolation methods by performing a two-step systematic literature analysis from all papers applying EV isolation in the year 2019. This resulted in an overview of the various methods being applied for EV isolations. A second database was generated for all studies within the first database that fairly compared multiple EV isolation methods by determining both EV purity and EV yield after isolation.From these databases it is shown that the most used EV isolation methods are not per definition the best methods based on EV purity or EV yield, indicating that more factors play a role in the choice which EV isolation method to choose than only the efficacy of the method. From the included studies it is shown that ~60% of all the included EV isolations were performed with combined EV isolation methods. The majority of EV isolations were performed with differential ultracentrifugation alone or in combination with differential ultrafiltration. When efficacy of EV isolation methods was determined in terms of EV purity and EV yield, combined EV isolation methods clearly outperformed single EV isolation methods, regardless of the type of starting material used. A recommended starting point would be the use of size-exclusion chromatography since this method, especially when combined with low-speed centrifugation, resulted in the highest EV purity, while still providing a reasonable EV yield.

Free flow electrophoresis allows preparation of extracellular vesicles with high purity

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

Cellular crosstalk between hematopoietic stem/progenitor cells and the bone marrow (BM) niche is vital for the development and maintenance of myeloid malignancies. These compartments can communicate via bidirectional transfer of extracellular vesicles (EVs). EV trafficking in acute myeloid leukemia (AML) plays a crucial role in shaping the BM microenvironment into a leukemia-permissive niche. Although several EV isolation methods have been developed, it remains a major challenge to define the most accurate and reliable procedure. Here, we tested the efficacy and functional assay compatibility of four different EV isolation methods in leukemia-derived EVs: (1) membrane affinity-based: exoEasy Kit alone and (2) in combination with Amicon filtration; (3) precipitation: ExoQuick-TC; and (4) ultracentrifugation (UC). Western blot analysis of EV fractions showed the highest enrichment of EV marker expression (e.g., CD63, HSP70, and TSG101) by precipitation with removal of overabundant soluble proteins [e.g., bovine serum albumin (BSA)], which were not discarded using UC. Besides the presence of damaged EVs after UC, intact EVs were successfully isolated with all methods as evidenced by highly maintained spherical- and cup-shaped vesicles in transmission electron microscopy. Nanoparticle tracking analysis of EV particle size and concentration revealed significant differences in EV isolation efficacy, with exoEasy Kit providing the highest EV yield recovery. Of note, functional assays with exoEasy Kit-isolated EVs showed significant toxicity towards treated target cells [e.g., mesenchymal stromal cells (MSCs)], which was abrogated when combining exoEasy Kit with Amicon filtration. Additionally, MSC treated with green fluorescent protein (GFP)-tagged exoEasy Kit-isolated EVs did not show any EV uptake, while EV isolation by precipitation demonstrated efficient EV internalization. Taken together, the choice of EV isolation procedure significantly impacts the yield and potential functionality of leukemia-derived EVs. The cheapest method (UC) resulted in contaminated and destructed EV fractions, while the isolation method with the highest EV yield (exoEasy Kit) appeared to be incompatible with functional assays. We identified two methods (precipitation-based ExoQuick-TC and membrane affinity-based exoEasy Kit combined with Amicon filtration) yielding pure and intact EVs, also suitable for application in functional assays. This study highlights the importance of selecting the right EV isolation method depending on the desired experimental design.

Extracellular vesicles (EVs) are increasingly recognized as key players in fungal biology, mediating intercellular communication, modulating virulence, and shaping host-pathogen interactions. Despite their growing importance, the lack of standardized protocols for EV isolation in fungi has hindered reproducibility and the accurate interpretation of functional data. This chapter presents a comprehensive, optimized protocol for the isolation and characterization of EVs produced by Aspergillus fumigatus under both solid and liquid culture conditions. Culture parameters, including medium composition and growth format, are critically evaluated for their impact on EV yield, molecular composition, and biological significance. The protocol encompasses detailed procedures for fungal cultivation, EV isolation via differential ultracentrifugation, and multiparametric characterization through nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and molecular marker profiling, all aligned with MISEV2023 guidelines. Designed to support both exploratory and high-resolution investigations, this standardized approach robustly analyzes EV-associated biomarkers. The isolated EVs are suitable for a range of downstream applications, including studies of immune modulation, molecular composition, fungal pathogenesis, and intercellular signaling dynamics.

Background/Aims: Extracellular vesicles (EVs), including microvesicles and exosomes, deliver bioactive cargo mediating intercellular communication in physiological and pathological conditions. EVs are increasingly investigated as therapeutic agents and targets, but also as disease biomarkers. However, a definite consensus regarding EV isolation methods is lacking, which makes it intricate to standardize research practices and eventually reach a desirable level of data comparability. In our study, we performed an inter-laboratory comparison of EV isolation based on a differential ultracentrifugation protocol carried out in 4 laboratories in 2 independent rounds of isolation. Methods: Conditioned medium of colorectal cancer cells was prepared and pooled by 1 person and distributed to each of the participating laboratories for isolation according to a pre-defined protocol. After EV isolation in each laboratory, quantification and characterization of isolated EVs was collectively done by 1 person having the highest expertise in the respective test method: Western blot, flow cytometry (fluorescence-activated cell sorting [FACS], nanoparticle tracking analysis (NTA), and transmission electron microscopy (TEM). Results: EVs were visualized with TEM, presenting similar cup-shaped and spherical morphology and sizes ranging from 30 to 150 nm. NTA results showed similar size ranges of particles in both isolation rounds. EV preparations showed high purity by the expression of EV marker proteins CD9, CD63, CD81, Alix, and TSG101, and the lack of calnexin. FACS analysis of EVs revealed intense staining for CD63 and CD81 but lower levels for CD9 and TSG101. Preparations from 1 laboratory presented significantly lower particle numbers (p < 0.0001), most probably related to increased processing time. However, even when standardizing processing time, particle yields still differed significantly between groups, indicating inter-laboratory differences in the efficiency of EV isolation. Importantly, no relation was observed between centrifugation speed/k-factor and EV yield. Conclusions: Our findings demonstrate that quantitative differences in EV yield might be due to equipment- and operator-dependent technical variability in ultracentrifugation-based EV isolation. Furthermore, our study emphasizes the need to standardize technical parameters such as the exact run speed and k-factor in order to transfer protocols between different laboratories. This hints at substantial inter-laboratory biases that should be assessed in multi-centric studies.

Extracellular vesicles (EV) are enriched with proteins and RNA cargo, promoting cell-to-cell communication. Biofluid derived EV cargo is used for discovering disease specific markers for diagnosis and disease monitoring. Blood is a complex fluid with an abundance of protiens and thus isolation of EVs is challenging. Therefore, methods for EV isolation, including commercial kits use thromboplastin D (TP-D) for pretreatment of plasma to increase EV purity and yield. This pretreatment can introduce contaminants. We performed a comparative study to evaluate the effect ofEV isolation methods focusing on (a) pretreatment of plasma with additives, which include: rabbit TP (rTP) versus human recombinant thromboplastin (huTP), to increase purity and yield (b) an additional centrifugation step prior to freezing plasma and (c) comparison of frozen versus fresh plasma EV isolations. Pretreatment with rTP generated a dynamic range of proteins, however, most of these proteins were contaminants, introduced from the rTP (99.1% purity). As an alternative, huTP was used, which did not introduce any significant contaminants, however, this did not increase yield or purity. Additionally, an extra 10,000g centrifugation did not improve either EV yield or purity. Finally, comparison of fresh or frozen plasma showed no significant difference, an important factor when sourcing plasma from biobanks. Appropriate controlsare required when adding any additives during EV isolationas even a small percentage of contaminants can have a major effect on results. Furthermore, biobanked plasma can be used with no major changes to processing.

Introduction: Extracellular vesicles (EVs) contain protein and nucleic cargo that has been shown to be reflective of physiological and pathological states, or their originating cells, in many diseases including cancer. Specifically, these EV-associated biomarkers have been shown to fluctuate with the change in pathological processes. As a result, characterization of blood-based circulating EVs have been widely investigated for diagnostic applications such as disease monitoring and treatment selection. The heterogeneity of blood-based EVs present significant challenges to current rapid isolation methods due to enrichment with unwanted sub-populations, e.g. apoptotic bodies, or contamination with lipids, e.g. APOB, for both capture-based chemistry-based isolation methods. We have developed a novel lab-on-a-chip technology for isolation and on-chip characterization of EVs from blood-based matrices using an AC electrokinetics (ACE) methodology. Methods: Blood samples were collected from 10 healthy volunteers and 10 donors with known cancer diagnosis into K2EDTA tubes under IRB approved protocols. Plasma was processed from the blood and stored at -80C. 120 µL of thawed plasma was applied to the microelectrode array flow cell (ExoVerita Flex, Biological Dynamics, San Diego, CA) and the EVs were isolated on the microelectrodes. Following isolation, the flow cell was washed, the isolated material was released from the array and eluted from the flow cell. For comparison, isolation of EVs from the same amount of plasma was performed using a chemistry-based method (ExoQuick Ultra, System Biosciences, Palo Alto, CA) according to manufacturer's instructions. Following isolation, the eluates were evaluated for the presence of EV-associated biomarkers using Western blotting capillary electrophoresis and mass spectrometry (Orbitrap Fusion Lumos, TFS). Nanoparticle tracking analysis (qNano Gold, Izon Science, New Zealand) was performed to determine EV concentration and size distribution. Quantitative qRT-PCR analysis of the purified plasma Evs was performed to confirm presence of EV-bound mRNA. Results: Western Blotting demonstrated that eluates from both ACE-based methodology and chemistry-based isolation contain the expected CD9 and CD63. The ACE-isolated EVs showed a single prominent band for CD63, whereas chemistry-based isolated EVs display multiple smaller CD63-reactive species, which was suggestive of proteolysis. Mass spectrometry analysis confirmed presence of CD9 and CD81 in EVs isolated by ACE method; but not in EVs purified by chemistry-based method. APOB contamination was present in chemistry-based EVs, but not in ACE-isolated EVs. For RT-PCR experiments, EVs were purified from 5 subjects with lung cancer and 3 subjects with melanoma patient plasma using ACE method. Quantitative PCR confirmed presence of mRNA, via successful amplification of the housekeeping genes PGK1 and β-actin, in all ACE-isolated EVs. Conclusions: The novel ACE-based platform successfully demonstrated direct isolation of EVs from plasma while preserving the integrity of protein and mRNA biomarkers. The compatibility of the eluted EVs with multiple downstream technologies, such as mass spectrometry, Western blotting and qRT-PCR, may enable novel biomarker discovery and use of EV-associated biomarkers in diagnostic assays. Citation Format: Rajaram Krishnan, Jean Lewis, David Searson, Orlando Perrera, Alfred Kinana, Heath Balcer, Iryna Clark, Juan Pablo Hinestrosa. Characterization of circulating extracellular vesicles isolated from plasma of cancer subjects using novel AC electrokinetics platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2850.

Extracellular vesicles (EVs) are enclosed by phospholipid bilayers and carry a variety of biological molecules including proteins, nucleic acids, lipids, and metabolites. They are released by almost all cell types into extracellular space. The role of these vesicles in intercellular communications is very important for the influence on many physiological and pathological processes, placing them among emerging research for diagnostic and therapeutic purposes. Despite the great potential, the field of EV research is highly challenging due to the complicated nature of EVs in terms of their size, composition, cellular origin, and their source matrix. This complexity highlights the need of developing standardized methodologies to address the issue of diversity in EVs and the variability in both pre-analytical and analytical phase of EV research.Chapter 1 of this dissertation reviews the advancements in EV isolation and characterization technologies, indicating a need for a collective effort on the standardization of EV research. Although significant advances have been made in EV research recently, our knowledge of the functional and biological mechanisms of EVs and their significance to certain pathophysiological states is not sufficiently developed. The detailed characterization of the molecular composition of EVs and EV subpopulations still presents a major challenge. The inconsistency of experimental results across different labs, even though they used similar techniques, underscores the importance of universally standard protocols. Covering the entire research spectrum, these protocols should address specimen collection, EV isolation, analytical characterization, and data interpretation to improve consistency and reproducibility. Through standardizing, optimizing and verifying these protocols, the field can move towards a more comprehensive view of EV biology and development of new EV-centric diagnostic and therapeutic approaches. Chapter 2 focuses on particular challenges and innovations in the realm of discovery proteomics for EVs, segueing from discussion in Chapter 1 that stresses the importance of standardization in EV research. This chapter introduces a new hypothesis, which exploits the electrostatic characteristics of EVs to ameliorate the sensitivity and dynamic range limitations in liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomic analysis. Plasma-derived EVs and EV subpopulations demonstrate the potential as a minimally invasive approach for biomarker discovery. However, the isolation and detailed molecular characterization of blood-derived EVs remain challenging due to technical limitations in separating them from high-abundance free plasma proteins and in fractionating EV subpopulations. Addressing these issues, we introduced a novel hypothesis centered on the surface charge properties of EVs. We proposed a charge-based fractionation method to enhance the purity and subpopulation specificity of plasma-derived EVs, leveraging their inherent negative charge and variations in charge density under different biological and pathological conditions. This study started with a thorough evaluation for method development and optimization on samples from healthy donors, including transmission electron microscopy, nanoparticle tracking analysis, western blotting, and LC-MS/MS proteomics. Subsequently, a pilot testing on clinical samples from prostate cancer patients and age-matched controls was performed to assess the applicability of the developed method in the real-world challenges. This research paves the way for advanced EV-based diagnostics, offering a promising window for the development of precise biomarker discovery and enhancing our understanding of EVs' role in cancer and other pathological conditions. --Author's abstract

Extracellular vesicles (EVs) are nano-sized vesicles containing nucleic acid and protein cargo that are released from a multitude of cell types and have gained significant interest as potential diagnostic biomarkers. Human serum is a rich source of readily accessible EVs; however, the separation of EVs from serum proteins and non-EV lipid particles represents a considerable challenge. In this study, we compared the most commonly used isolation techniques, either alone or in combination, for the isolation of EVs from 200 µl of human serum and their separation from non-EV protein and lipid particles present in serum. The size and yield of particles isolated by each method was determined by nanoparticle tracking analysis, with the variation in particle size distribution being used to determine the relative impact of lipoproteins and protein aggregates on the isolated EV population. Purification of EVs from soluble protein was determined by calculating the ratio of EV particle count to protein concentration. Finally, lipoprotein particles co-isolated with EVs was determined by Western blot analysis of lipoprotein markers APOB and APOE. Overall, this study reveals that the choice of EV isolation procedure significantly impacts EV yield from human serum, together with the presence of lipoprotein and protein contaminants.

Plant extracellular vesicles (EVs) play critical roles in the cross-kingdom trafficking of molecules from hosts to interacting microbes, most notably in plant defense responses. However, the isolation of pure, intact EVs from plants remains challenging. A variety of methods have been utilized to isolate plant EVs from apoplastic washing fluid (AWF). Here, we compare published plant EV isolation methods, and provide our recommended method for the isolation and purification of plant EVs. This method includes a detailed protocol for clean AWF collection from Arabidopsis thaliana leaves, followed by EV isolation via differential centrifugation. To further separate and purify specific subclasses of EVs from heterogeneous vesicle populations, density gradient ultracentrifugation and immunoaffinity capture are then utilized. We found that immunoaffinity capture is the most precise method for specific EV subclass isolation when suitable specific EV biomarkers and their corresponding antibodies are available. Overall, this study provides a guide for the selection and optimization of EV isolation methods for desired downstream applications.

In this study, we evaluated the ability to detect novel proteins on extracellular vesicles (EVs) following isolation using an alternating current electrokinetic (ACE) microarray. EVs circulating in blood contain a wealth of biomarkers that are thought to reflect the tumor’s proteome; however, detecting the full diversity of EV proteins may depend on choice of method to purify EVs for analysis. We compared an ACE microarray isolation method with a conventional chemical precipitation method to isolate EVs from blood plasma cancer and control samples. EVs were isolated from 1 mL each of control or pancreatic cancer patient plasma using the ExoVerita platform (Biological Dynamics) or ExoQuick Ultra kit (System Biosciences). Purified EVs were processed using tandem mass tag (TMT)-based multiplexing mass spectrometry proteomics (ThermoFisher Scientific) and the resulting peptide spectra were analyzed and quantified to determine the proteins present. Additional analyses, such as bioanalyzer measurements of contaminating plasma proteins and nanoparticle tracking analysis were performed in parallel to further characterize the isolated EVs. Cancer EVs isolated by ACE were enriched in proteins related to cancer functions when compared to non-cancer control EVs. They also showed enrichment in ontology clusters such as NABA core matrisome proteins, with depressed expression of wound healing family proteins. In contrast, EVs isolated using chemical purification demonstrated enrichment chiefly in proteins functionally grouped into immune system or blood regulation functions. After analyzing EVs isolated by ACE, a total of fifty-one (51) proteins were observed to be upregulated in the cancer donor samples. EV isolation method choice significantly impacted the proteomic profile observed. EV isolation using ACE-based technology can provide more insights into understanding the proteome by possibly reducing or removing non-specific proteins from the EVs. With better separation of EVs from blood proteins, the EV proteins identified may have greater biological significance. Citation Format: Juan P. Hinestrosa, Jean M. Lewis, David J. Searson, Heath I. Balcer, David J. Gonzalez, Rajaram Krishnan. Proteomic analysis of extracellular vesicles from oncology donor samples purified using an alternating current electrokinetic microelectrode chip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5082.

Increasing yield of msc-evs in scalable xeno-free manufacturing

Reliable isolation and quantitation of extracellular vesicles (EVs), which function as natural bioactive nanocarriers in biological processes, are essential to uncovering their underlying mechanisms and applications. To meet these requirements, we present here a peptide-engineered biomimetic nanoplatform featuring cell-membrane camouflage. This biomimetic nanoplatform utilizes specific peptide ligands to facilitate the "capture-release" isolation of EVs while enhancing performance by harnessing the antifouling and fluidity advantages afforded by the camouflage of red blood cell membranes. Furthermore, this nanoplatform streamlines the electrochemical quantitation of EVs via a nondestructive labeling and delabeling process, showing accuracy comparable to that of widely used nanoparticle tracking analysis. Validating with EVs from breast cancer cells and human embryonic stem cells, this nanoplatform is also proved to effectively maintain the biological activities of the isolated EVs, thereby enabling precise regulation of cell migration and antiapoptotic response. As such, this biomimetic nanoplatform stands as a highly effective solution for isolating and quantitatively assessing EVs from diverse sources, thus propelling the potential applications of EVs in biomedical and clinical research.