Journal of Extracellular Biology

Extracellular vesicles (EVs), including exosomes, are widely believed to mediate intercellular communication by delivering molecular cargo to recipient cells. However, the efficiency and mechanisms of such cargo delivery remain unclear. In this study, we investigated whether EVs bearing the canonical exosomal marker CD63 are capable of fusing with recipient cell membranes to release their contents. Using the highly sensitive NanoBiT split luciferase system, we tracked potential fusion events between HiBiT-tagged CD63-EVs and HEK cells expressing cytosolic or endosome-localized LgBiT. While HiBiT and LgBiT reconstitution was readily detected when EVs carried the viral fusion protein VSV-G, no luminescence was observed with native CD63-EVs, despite their uptake or binding by recipient cells. Similarly, EVs carrying HiBiT-HSP70 failed to show evidence of cargo release. These findings demonstrate that unmodified CD63-EVs do not fuse with the plasma or endosomal membranes of recipient cells, suggesting that EV-mediated cargo delivery via membrane fusion is inefficient or absent under physiological conditions. Our results challenge the prevailing view of exosome function in intercellular communication and underscore the need for re-evaluation of EV cargo transfer mechanisms.

Serum-free synthetic media are frequently used as an alternative to extracellular vesicle-depleted serum containing media (EV-DEP) for EV production and collection. Here, we fully characterised the EVs released by MDA-MB-231 or HEK-293T cells cultured in 10% EV-DEP DMEM or a commonly used serum-free synthetic medium, Opti-MEM. Cells cultured in Opti-MEM released a significantly higher yield of CD9- and CD63-positive EVs as compared to cells grown in EV-DEP DMEM. In MDA-MB-231 cells the increased EV release was likely driven by nutrient deprivation (decreased phospho-S6) and increased cell stress (increased phospho-AKT). MDA-MB-231 cells grown in EV-DEP DMEM relied upon actin cytoskeleton dynamics for up to 50% of EVs released, while cells in Opti-MEM did not use ROCK kinases for EV biogenesis. Mass spectrometry analysis showed that the EV proteomes from both conditions were largely unchanged other than EV-DEP DMEM EVs contained more histones and bovine proteins. Addition of 2.5 - 10% serum or 2.5 mg/mL albumin to Opti-MEM medium partially rescued some cell stress phenotypes and returned EV release rates to that of cells in EV-DEP DMEM. The choice of media is an important consideration when designing EV studies and their downstream applications. While Opti-MEM increased cell stress, it achieved a high yield of EVs that were depleted of histones and contaminating bovine proteins - useful for therapeutic applications. Alternatively, EV-DEP DMEM produced a decreased yield of EVs in lower stress conditions, however, the EVs were histone and bovine protein rich which may have important implications for use for use in therapeutic or immunological experiments.

Mesenchymal stromal cells (MSCs) are a promising source of therapeutic extracellular vesicles (EVs), however it is not clear how heterogeneity within a non-clonal MSC population will affect the collective EV pool. Here we used immortalised clonal MSC lines, termed Y201 and Y202, to examine how MSC phenotype influences EV character and function. Although morphologically similar, Y201 EVs were more abundant in EV biomarkers versus Y202 EVs, with an enhanced miRNA and proteomic content, predicted to contribute to an elaborate EV corona particularly abundant in RGD-containing proteins fibronectin and MFG-E8. We demonstrated that Y201 EVs, but not Y202 EVs, significantly increased the proliferation of articular chondrocytes and that the proliferative effect was mediated at least in part via an RGD (integrin)-FAK-ERK1/2 axis. Both Y201 and Y202 EV subsets significantly reduced proliferative index scores of activated T cells. However, only Y201 EVs, not Y202 EVs, suppressed disease activity compared to controls in different in vivo models of inflammatory peritonitis and arthritis. EVs released by closely related MSC subtypes within the same heterogeneous population differ significantly in terms of cargo abundance, bioactivity, and pre-clinical in vivo efficacy. Analysis of defined EV subsets will aid mechanistic understanding and prioritisation for EV therapeutics.

Extracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on a cell line that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to this A549 cell line caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

The biological functions of extracellular vesicles (EVs) depend on their cellular source. Further, different subpopulations of EVs from the same cells carry different cargo, but differences in their biological functions are less understood. We here identify a very small EV subpopulation released by HEK293F cells (miniEVs). These EVs, in contrast to the larger EVs, were found to have anti-inflammatory properties. Quantitative proteomics identified a potential anti-inflammatory molecule, Syndecan-4 (SDC4), on the surface of the miniEVs, but not larger EVs. We engineered HEK293F cells to overexpress SDC4, which results in the molecule being highly expressed in all EV subpopulations. Expression of SDC4, a proteoglycan, also increased the presence of heparan sulfate on the EV surface. Furthermore, these EVs were found to have potent anti-inflammatory effects in vitro, which heparinase treatment could slightly reduce. Furthermore, the SDC4 EVs showed anti-inflammatory effects in vivo in a model of peritonitis. We conclude that HEK293F EVs can be engineered to become anti-inflammatory, and that SDC4-expressing HEK293F-EV potentially could become an anti-inflammatory therapeutic.

BackgroundExtracellular vesicles (EVs) are nano and macro-sized, lipid-bound particles, involved in cellular communication. Interestingly, cancer-derived EVs show a heterologous and cross-species tumour tropism which makes them a potential tool for efficient delivery of therapeutic small interfering RNA (siRNA) to the tumour cells. MethodsEVs derived from glioblastoma cells (U373P and U373vIII) were loaded with EGFRvIII siRNA to develop a targeted therapeutic strategy against glioblastoma. EV biodistribution was evaluated using fluorescent indocyanine green (ICG) staining followed by ex vivo imaging. Different loading strategies, including passive loading, sonication, saponin-mediated membrane permeabilization, electroporation, and transfection were assessed for their efficiency in loading siRNA into EVs. The efficiency of each method was evaluated by nano flowcytometry, in vitro uptake assay followed by immunoblot (western blot) analysis. Eventually, the most effective formulation was tested for the systemic siRNA administration and selective tumour delivery in vivo, followed by evaluation of tumour size and EGFRvIII expression. ResultsHere, we showed that siRNA transfection into EVs was the most effective loading strategy, as confirmed by nano-flow cytometry, uptake assays, and western blot analysis, achieving over 90% knockdown efficiency in vitro for EVs carrying EGFRvIII siRNA. In vivo, EGFRvIII siRNA-loaded EVs homed to the tumour site and downregulated EGFRvIII expression compared with the PBS-siRNA control group; however, no significant tumour shrinkage was observed. ConclusionEGFRvIII-targeting, glioblastoma cell-derived EVs can be used as siRNA delivery carriers for targeted gene therapy in glioblastoma. However, further optimization of siRNA delivery and treatment duration is required.

Extracellular vesicles (EVs) may contain a variety of molecular cargo including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated by small RNA sequencing that vault RNA (vtRNA) were highly abundant in EV pellets enriched by differential centrifugation. EVs were prepared by commonly used enrichment methods and biochemical assays used to determine whether vault particle components were bona fide EV cargo. EVs were isolated by differential centrifugation, size exclusion chromatography (SEC) and Dynabead immunocapture. RNase and proteinase treatment of EV preparations demonstrated that most vtRNA and major vault protein (MVP) were not enclosed and protected within the EV membrane. Vault-like particles were visualised in differential centrifugation pellets by cryo-transmission electron microscopy. EVs enriched by size exclusion chromatography and those isolated by immunocapture post-ultracentrifugation showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. Taken together, commonly used isolation techniques, such as differential centrifugation and SEC, can lead to contamination of EVs with vault particles. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.

Most approaches to extracellular vesicle (EV) characterization focus on EV size or density. However, such approaches provide few clues regarding EV origin, molecular composition, and function. New methods to characterize the EV surface proteins may aid our understanding of their origin, physiological roles, and biomarker potential. Recently developed immunoassays for intact EVs based on ELISA, NanoView, SIMOA and MesoScale platforms are highly sensitive, but have limited multiplexing capabilities, whereas MACSPlex FACS enables the detection of multiple EV surface proteins, but requires significant quantities of purified EVs, which limits its adoption. Here, we describe a novel Luminex-based immunoassay, which combines multiplexing capabilities with high sensitivity and, importantly, bypasses the enrichment and purification steps that require larger sample volumes. We demonstrate the methods specificity for detecting EV surface proteins using multiple EV depletion techniques, EVs of specific cellular origin isolated from culture media, and by co-localization with established EV surface markers. Using this novel approach, we elucidate differences in the tetraspanin profiles of the EVs carrying erythrocyte and neuron markers. Using size exclusion chromatography, we show that plasma EVs of putative neuronal and tissue macrophage origin are eluted in fractions distinct from those derived from erythrocytes, or from their respective cultured cells. In conclusion, our novel multiplexed assay differentiates between EVs from erythrocytes, macrophages, and neurons, and offers a new means for capture, classification, and profiling of EVs from diverse sources.

BackgroundP-glycoprotein (P-gp/ABCB1) is a key efflux transporter that maintains barrier integrity by clearing xenobiotics and toxic metabolites. At the feto-maternal interface, trophoblast-derived extracellular vesicles (CTC-EVs) naturally and transiently transfer functional P-gp to maternal decidual cells, restoring lost and or reduced P-gp function (exofection) to sustain pregnancy homeostasis. A similar loss of P-gp at the blood brain barrier (BBB) contributes to impaired amyloid-{beta} (A{beta}) clearance and neuroinflammation in Alzheimers disease. We investigated whether CTC-EV-mediated exofection could restore P-gp function in human brain endothelial cells (hBECs) and enhance A{beta} clearance under inflammatory and neurodegenerative conditions. MethodsCTC-EVs were isolated and characterized by nanoparticle tracking analysis and western blotting for P-gp and EV markers. Transcriptomic profiling of CTC-EVs identified enrichment of transporter-related genes, including solute carriers and ABC transporters, along with inflammatory mediators. Network analysis revealed coordinated modules linking EV cargo to transporter regulation, endocytosis/trafficking pathways, and inflammatory remodeling processes converging on BBB efflux activity. hBECs were exposed to LPS (500 ng/mL, 48 h) with or without CTC-EVs. P-gp expression was assessed by immunofluorescence (mean fluorescence intensity, MFI) and western blotting, while functional efflux was measured using Calcein-AM assays. A{beta} oligomer transport was evaluated using a transwell hBEC model. In vivo, 3xTg-AD mice received intravenous CTC-EVs (1x10L/day for 5 days), followed by assessment of P-gp expression, A{beta} burden, and neuroinflammatory markers. Pharmacokinetic studies in P-gp knockout mice were conducted to confirm functional transporter recovery. ResultsLPS exposure significantly reduced P-gp expression in hBECs (41.3% decrease in MFI, p=0.0084), which was restored by CTC-EVs (46.7% increase vs. LPS, p=0.0121). Exofection increased P-gp by a 2.1-fold following EV treatment as determined by western blot. Functional assays demonstrated enhanced efflux, with a 38.5% reduction in intracellular Calcein fluorescence (p<0.001). Network-informed mechanisms supported coordinated regulation of transporter and trafficking pathways. CTC-EVs improved A{beta} transport across inflamed hBEC monolayers. In vivo, EV-treated 3xTg-AD mice exhibited increased P-gp expression in the frontal cortex (38.6%) and hippocampus (42.1%), reduced A{beta} plaque burden (27.9%), and decreased inflammatory markers (IL-1{beta} and TNF-, p<0.05). In P-gp knockout mice, EVs reduced brain drug accumulation by 22.4% (p=0.032), confirming restoration of transporter function. ConclusionCTC derived EVs are natural carriers of functional transporter proteins and restore efflux capacity in compromised endothelial barriers. Integration of transcriptomic and network analyses highlights coordinated regulation of transporter, trafficking, and inflammatory pathways underlying exofection. This reproductive biology inspired strategy offers a promising therapeutic approach for enhancing A{beta} clearance and mitigating neuroinflammation in Alzheimers disease.

Separating and enriching specific extracellular vesicle (EV) subpopulations from the broader EV pool present in tissues and blood is crucial for understanding their role in physiological and pathological conditions. However, high-purity enrichment of specific EV-subpopulations remains challenging due to the lack of suitable techniques. Initial studies have shown that Fluorescence-Activated Cell Sorting (FACS) has great potential for enriching EV subpopulations, despite the technical challenges posed by their small size. Yet, existing protocols have been inconsistent, and proper validation using state-of-the-art sorters has been inadequate. Here, we introduce an EV sorting workflow that overcomes technical challenges and allows for the analysis of EVs from various species, tissue sources and cell culture. We used two fluorescence cell sorters, the BD FACSAria Fusion and the BD FACSDiscover S8, to sort EVs with different fluorescent labels. The successful sorting of EVs was validated using high-sensitivity imaging flow cytometry, transmission electron microscopy, and liquid chromatography tandem mass spectrometry. We defined the optimal parameters for nozzle sizes, flow rates, sample dilutions, and sorting modes, enabling the enrichment of EV populations of interest to nearby 100% purity, including low-frequency EV populations of under 10%, while preserving compatibility with downstream analyses. The workflow presented here provides a powerful tool for both, basic science and translational applications.

Extracellular vesicles (EVs) released by resting endothelial cells support vascular homeostasis. To better understand endothelial cell EV biogenesis, we examined cultured human umbilical vein endothelial cells (HUVECs) prepared by rapid freezing, freeze-substitution, and serial thin section electron microscopy. Thin sections of HUVECs revealed clusters of membrane protrusions on the otherwise smooth cell surface. The protrusions contained membrane-bound organelles, including multivesicular bodies (MVBs), and appeared to be on the verge of pinching off to form microvesicles. Beyond cell peripheries, membrane-bound vesicles with internal MVBs were observed, and serial sections confirmed that they were not connected to cells. These observations are consistent with the notion that these multi-compartmented microvesicles (MCMVs) pinch-off from protrusions. Remarkably, omega figures formed by fusion of MVBs with the MCMV limiting membrane were directly observed, apparently caught in the act of releasing exosomes from the MCMV. In summary, MCMVs are a novel form of EV that bud from membrane protrusions on the HUVEC surface, contain MVBs and release exosomes. These observations suggest that exosomes can be harbored within and released from transiting microvesicles after departure from the parent cell, constituting a new site of exosome biogenesis occurring from endothelial and potentially additional cell types.

Extracellular vesicles (EVs) like exosomes are functional nanoparticles trafficked between cells and found in every biofluid. An incomplete understanding of which cells, from which tissues, are trafficking EVs in vivo has limited our ability to use EVs as biomarkers and therapeutics. However, recent discoveries have linked EV secretion to expression of genes and proteins responsible for EV biogenesis and found as cargo, which suggests that emerging "cell atlas" datasets could be used to begin understanding EV biology at the level of the organism and possibly in rare cell populations. To explore this possibility, here we analyzed 67 genes that are directly implicated in EV biogenesis and secretion, or carried as cargo, in [~]44,000 cells obtained from 117 cell populations of the Tabula Muris. We found that the most abundant proteins found as EV cargo (tetraspanins and syndecans) were also the most abundant EV genes expressed across all cell populations, but the expression of these genes varied greatly among cell populations. Expression variance analysis also identified dynamic and constitutively expressed genes with implications for EV secretion. Finally, we used EV gene co-expression analysis to define cell population-specific transcriptional networks. Our analysis is the first, to our knowledge, to predict tissue- and cell type-specific EV biology at the level of the organism and in rare cell populations. As such, we expect this resource to be the first of many valuable tools for predicting the endogenous impact of specific cell populations on EV function in health and disease.

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The study of EV secretion and EV characterization remains challenging due to the small size of these particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV separation techniques. We developed a method for in-cell EV labeling with fluorescent lipids (DiI), followed by DiI-labelled EV characterization in the conditioned medium by imaging flow cytometry (IFC). Direct IFC analysis of EVs in the conditioned medium, after removal of apoptotic bodies and cellular debris, significantly reduces sample processing and loss compared to established methods for EV separation, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by ultracentrifugation. In conclusion, our optimized protocol for EV labeling and analysis reduces EV sample processing and loss, and is well suited for cell biology studies that focus on modulation of EV secretion by cells in culture.

In some cell systems, small extracellular vesicles bearing PD-L1 (PD-L1+ sEVs) have been shown to be able to suppress T-cell immunity. We have herein investigated whether a distinct profile of PD-L1+ sEVs exists in human follicular fluid (FF). Single-particle interferometric reflectance imaging sensing combined with a single-particle antibody capture and immunofluorescence labelling were used to determine the expression and colocalization of CD63, CD81, CD9, and PD-L1 in sEVs derived from FF of women undergoing fertility treatments (n=10). In addition, the size distribution of sEVs was investigated via atomic force microscopy. Our data indicate that the bulk of tetraspanin-expressing EVs in human FF are less than 50 nm in size. Tetraspanins and PD-L1 exhibit distinct expression and colocalization profiles at sEV level across all cohort samples. A total of 42%, 46%, and 50% of all the particles captured by anti-CD63, anti-CD81, and anti-CD9 antibodies, respectively, were positive for CD81. PD-L1 was expressed at the highest level on CD9+ sEVs, with an average value of 5% within the cohort. The presence of distinct PD-L1+ sEV subpopulations suggests that they may play a role in regulating the immune response in the follicular microenvironment. Further research is needed to fully understand the functional significance of PD-L1+ sEVs in this context and their potential as biomarkers for predicting fertility outcomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/628903v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@77e67dorg.highwire.dtl.DTLVardef@1bd1ce2org.highwire.dtl.DTLVardef@b376d8org.highwire.dtl.DTLVardef@3f6fad_HPS_FORMAT_FIGEXP M_FIG C_FIG

Circulating extracellular vesicles (EVs) have gained significant attention for discovering tumor biomarkers. However, isolating EVs with well-defined homogeneous populations from complex biological samples is challenging. Different isolation methods have been found to derive different EV populations carrying different molecular contents, which confounds current investigations and hinders subsequent clinical translation. Therefore, standardizing and building a rigorous assessment of isolated EV quality associated with downstream molecular analysis is essential. To address this need, we introduce a statistical algorithm (ExoQuality Index, EQI) by integrating multiple EV characterizations (size, particle concentration, zeta potential, total protein, and RNA), enabling direct EV quality assessment and comparisons between different isolation methods. We also introduced a novel capture-release isolation approach using a pH-responsive peptide conjugated with NanoPom magnetic beads (ExCy) for simple, fast, and homogeneous EV isolation from various biological fluids. Bioinformatic analysis of next-generation sequencing (NGS) data of EV total RNAs from pancreatic cancer patient plasma samples using our novel EV isolation approach and quality index strategy illuminates how this approach improves the identification of tumor associated molecular markers. Results showed higher human mRNA coverage compared to existing isolation approaches in terms of both pancreatic cancer pathways and EV cellular component pathways using gProfiler pathway analysis. This study provides a valuable resource for researchers, establishing a workflow to prepare and analyze EV samples carefully and contributing to the advancement of reliable and rigorous EV quality assessment and clinical translation.

Cells communicate via extracellular vesicles (EVs) containing functional RNAs, proteins and lipids. Knowledge on the fate of internalized EVs, especially their capacity to fuse with target cell membranes and deliver luminal cargo, is limited. Currently available EV-cargo delivery assays are indirect and thus unlikely to uncover molecular players and conditions that specifically control the EV-fusion step. Here, we present a novel live-cell imaging assay for detection of EV-binding, -uptake, and -fusion in time and space. We employed the SunTag system for exceptional signal amplification. EV-donor cells were engineered to tag the luminal EV-membrane with a fluorescent label coupled to SunTag peptides. Recipient cells express fluorescent single-chain anti-SunTag antibody (STAb), which binds EV-enclosed SunTag upon its cytosolic exposure. Using SunTagged EVs carrying fusogen VSV-G, we visualize the EV-fusion process, quantify fusion kinetics and efficiency, and determine subcellular localization of fusion events. We term this methodology EV-FUSIM (Extracellular Vesicle Fusion Spatiotemporal Imaging Method). In the future, this technology can support identification of fusogenic EV-subsets, as well as molecular players and drugs that modulate EV-fusion, without confounding effects of post-fusion processes. This will extend knowledge on EV-biology and can aid in the engineering of EVs that efficiently deliver intraluminal therapeutic payloads.

The promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluid-specific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, which were used to model the potential biogenesis route of different EV subsets and their cellular origin.

Obesity is a growing global health concern, contributing to diseases such as cancer, autoimmune disorders, and neurodegenerative conditions. Adipose tissue dysfunction, characterized by abnormal adipokine secretion and chronic inflammation, plays a key role in these conditions. Adipose-derived extracellular vesicles (ADEVs) have emerged as critical mediators in obesity-related diseases. However, the study of mature adipocyte-derived EVs (mAdipo-EVs) is limited due to the short lifespan of mature adipocytes in culture, low EV yields, and the low abundance of these EV subpopulations in the circulation. Additionally, most studies rely on rodent models, which have differences in adipose tissue biology compared to humans. To overcome these challenges, we developed a standardized approach for differentiating human preadipocytes (preAdipos) into mature differentiated adipocytes (difAdipos), which produce high-yield, human adipocyte EVs (Adipo-EVs). Using visceral adipose tissue from bariatric surgical patients, we isolated the stromal vascular fraction (SVF) and differentiated preAdipos into difAdipos. Brightfield microscopy revealed that difAdipos exhibited morphological characteristics comparable to mature adipocytes (mAdipos) directly isolated from visceral adipose tissue, confirming their structural similarity. Additionally, qPCR analysis demonstrated decreased preadipocyte markers and increased mature adipocyte markers, further validating successful differentiation. Functionally, difAdipos exhibited lipolytic activity comparable to mAdipos, supporting their functional resemblance to native adipocytes. We then isolated preAdipo-EVs and difAdipo-EVs using tangential flow filtration and characterized them using bulk and single EV analysis. DifAdipo-EVs displayed classical EV and adipocyte-specific markers, with significant differences in biomarker expression compared to preAdipo-EVs. These findings demonstrate that difAdipos serve as a reliable surrogate for mature adipocytes, offering a consistent and scalable source of adipocyte-derived EVs for studying obesity and its associated disorders.

Extracellular vesicles (EVs) are nanometric lipid vesicles that shuttle cargo between cells. Their analysis could shed light on health and disease conditions, but EVs must first be preserved, extracted and often pre-concentrated. Here we firstly compare plasma preservation agents, and secondly, using both plasma and cell supernatant, four EV-extraction methods including (i) ultracen-trifugation (UC), (ii) size exclusion chromatography (SEC), (iii) centrifugal filtration (LoDF), and (iv) accousto-sorting (AcS). We benchmarked them by characterizing integrity, size-distribution, concentration, purity and the expression profiles for nine proteins of EVs, as well as overall throughput, time-to-result and cost. We found that the difference between EDTA and citrate anticoagulants vary with the extraction method. In our hands, ultracentrifugation produced a high yield of EVs with low contamination; SEC is low-cost, fast, and easy to implement, but the purity of EVs is lower; LoDF and AcS are both compatible with process automation, small volume requirement, and rapid processing times. When using plasma, the LoDF was susceptible to clogging and sample contamination, while the AcS featured high purity but a lower yield of extraction. Analysis of protein profiles suggest that extraction methods extract different sub-population of EVs. Our study highlights the strength and weakness of sample preprocessing methods, and the variability in concentration, purity, and EV expression profiles of the extracted EVs. Pre-analytical parameters such as collection or pre-processing protocols must be considered as part of the entire process in order to address EV diversity and their use as clinically actionable indicators.

Human milk-derived extracellular vesicles (HMEVs) are crucial functional components in breast milk, contributing to infant health and development. Maternal conditions could affect HMEV cargos; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. This study evaluated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. Milk samples (9 prenatal SARS-CoV-2 vs. 9 controls) were retrieved from the IMPRINT birth cohort. After defatting and casein micelle disaggregation, 1 mL milk was subjected to a sequential process of centrifugation, ultrafiltration, and qEV-size exclusion chromatography. Particle and protein characterizations were performed following the MISEV2018 guidelines. EV lysates were analyzed through proteomics and miRNA sequencing, while the intact EVs were biotinylated for surfaceomic analysis. Multi-Omics was employed to predict HMEV functions associated with prenatal SARS-CoV-2 infection. Demographic data between the prenatal SARS-CoV-2 and control groups were similar. The median duration from maternal SARS-CoV-2 test positivity to milk collection was 3 months (range: 1-6 months). Transmission electron microscopy showed the cup-shaped nanoparticles. Nanoparticle tracking analysis demonstrated particle diameters of <200 nm and yields of >1e11 particles from 1 mL milk. Western immunoblots detected ALIX, CD9 and HSP70, supporting the presence of HMEVs in the isolates. Thousands of HMEV cargos and hundreds of surface proteins were identified and compared. Multi-Omics predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming and mucosal tissue development, while mitigating inflammation and lower EV transmigration potential. Our findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era.