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Extracellular vesicles (EVs) are highly heterogeneous, and different EV subpopulations from various origins mediate different biological effects. The separation of different subpopulations of EVs from mixtures is critical but challenging. Epididymosomes are secreted by the epididymal epithelium and play a key role in sperm maturation and function. However, limited access to human epididymal tissue and epididymal fluid has hampered further study of epididymosomes and their potential clinical applications. Here, we established a novel strategy based on flow cytometry sorting to isolate human CD63‐positive epididymosomes from ejaculate. We identified CD52, a membrane‐located protein expressed exclusively in the epididymis, as the sorting marker for human epididymosomes. Then, CD63‐positive epididymosomes were isolated from human semen using a flow cytometry sorting instrument and concentrated. Additionally, we observed that isolated CD63‐positive epididymosomes improved sperm function more than other CD63‐positive seminal EV subpopulations did, demonstrating the successful isolation of a subpopulation of epididymosomes from human semen and their potential application in the clinic.

Circulating cell-free nucleic acids (NAs), in particular plasma-derived cell-free DNA, have evolved into promising clinical analytes for prenatal diagnostics, cancer analysis, and cancer surveillance and therapy monitoring. Nevertheless, salivary extracellular and extracellular vesicle (EV)-derived DNA and microRNA have recently gained attention as potential non-invasive biomarkers for a variety of diseases, including cancer, cardiovascular, autoimmune, and infectious diseases. Our goal in this study was therefore to evaluate and optimize commercially available approaches for cell-free nucleic acid isolation, focusing specifically on DNA and miRNA present in cell-free saliva or saliva-derived EVs. Along these lines, we investigated various commercially available kits, which enable parallel isolation of cell-free DNA and RNA in separate fractions from cell-free saliva and salivary EVs, respectively, and compared them to single analyte extraction kits. The efficiency of all tested nucleic acid extraction methods was determined by comparing DNA and RNA fluorescence spectroscopy measurements and quantitative PCR values obtained from a selection of different DNA- and microRNA targets. We found the Norgen Plasma/Serum RNA/DNA Purification Mini kit in combination with the miRCURY exosome isolation kit to work best in our hands and to provide the highest yields of EV-derived nucleic acids. Having tested and identified effective protocols for isolating salivary extracellular nucleic acids, we present with this comparison study, among others, a sound basis for future circulating small nucleic acid and epigenetic biomarker research aiming for early disease diagnosis, prognosis, and prediction from cell-free saliva, representing an easy-to-collect and readily available diagnostic fluid.

The large-scale automated manufacture of extracellular vesicles (EVs) is crucial for meeting the growing demand for EV-based therapies, enabling the standardized, efficient, and scalable production of EVs for clinical translation.We developed a fully automated collection technology and optimized machinery (FACTORY), achieving large-scale automated manufacture of EVs.The EVs manufactured via FACTORY maintain normal biological activity while meeting stringent quality standards: sterility, mycoplasma-free status, low endotoxin levels, and high consistency.The FACTORY platform allows researchers across disciplines to obtain EVs through a one-click operation, ensuring batch-to-batch consistency in isolation technology and comparability of research outcomes, thereby accelerating the clinical translation of EV-based applications. Extracellular vesicles (EVs) hold significant potential as therapeutic agents and drug carriers. However, current isolation techniques severely limit their clinical application, due to heavy reliance on manual operation, making large-scale isolation of EVs impractical and failing to meet the requirements for clinical translation. Here, we set up the fully automated collection technology and optimum machinery (FACTORY) platform, allowing the efficient collection of high-quality EVs. The platform integrates continuous flow centrifugation and tangential flow filtration (TFF) technologies, achieving a seamless process for the removal of impurities and collection of EVs, thereby ensuring that large scale-manufactured EVs are sterile, mycoplasma free, and low in endotoxins, and exhibit good consistency. We successfully obtained a substantial quantity of EVs utilizing FACTORY, and systematically characterized their EV-specific markers, biological functions, and therapeutic effects. Results indicated that FACTORY significantly promotes the clinical translation of EVs, thereby laying a solid foundation for their application in drug delivery and beyond. [Display omitted] The FACTORY platform addresses the urgent need for scalable, automated, and efficient methods for collecting EVs, which hold great promise in clinical therapeutics. Currently, FACTORY is assessed at Technology Readiness Level (TRL) 6, indicating its readiness for real-world implementation. By integrating continuous flow centrifugation and tangential flow filtration (TFF), FACTORY enables large-scale EV isolation with high yield, sterility, and batch-to-batch consistency. Experimental results show that FACTORY produces a higher yield of EVs compared with traditional ultracentrifugation, with comparable purity and morphology. In addition, FACTORY-isolated EVs meet critical safety criteria, including sterility, mycoplasma-free status, and minimal endotoxin levels. The FACTORY platform holds significant promise for advancing EV-based therapeutics, with potential applications in regenerative medicine, drug delivery, and immunomodulation. Its scalability, automation, and ability to produce high-quality EVs position it as a transformative tool for clinical and industrial use, paving the way for broader adoption in emerging medical fields, including medical aesthetics and targeted therapy development. This study developed the fully automated collection technology and optimum machinery (FACTORY) platform for extracellular vesicles (EVs), integrating continuous flow centrifugation and tangential flow filtration. The platform allows one-click, large-scale isolation of high-quality EVs with exceptional consistency, accelerating the clinical translation of EV-based applications.

Studying specific subpopulations of cancer-derived extracellular vesicles (EVs) could help reveal their role in cancer progression. In cancer, an increase in reactive oxygen species (ROS) happens which results in lipid peroxidation with a major product of 4-hydroxynonenal (HNE). Adduction by HNE causes alteration to the structure of proteins, leading to loss of function. Blebbing of EVs carrying these HNE-adducted proteins as a cargo or carrying HNE-adducted on EV membrane are methods for clearing these molecules by the cells. We have referred to these EVs as Redox EVs. Here, we utilize a surface tension-mediated extraction process, termed exclusion-based sample preparation (ESP), for the rapid and efficient isolation of intact Redox EVs, from a mixed population of EVs derived from human glioblastoma cell line LN18. After optimizing different parameters, two populations of EVs were analyzed, those isolated from the sample (Redox EVs) and those remaining in the original sample (Remaining EVs). Electron microscopic imaging was used to confirm the presence of HNE adducts on the outer leaflet of Redox EVs. Moreover, the population of HNE-adducted Redox EVs shows significantly different characteristics to those of Remaining EVs including smaller size EVs and a more negative zeta potential EVs. We further treated glioblastoma cells (LN18), radiation-resistant glioblastoma cells (RR-LN18), and normal human astrocytes (NHA) with both Remaining and Redox EV populations. Our results indicate that Redox EVs promote the growth of glioblastoma cells, likely through the production of H2O2, and cause injury to normal astrocytes. In contrast, Remaining EVs have minimal impact on the viability of both glioblastoma cells and NHA cells. Thus, isolating a subpopulation of EVs employing ESP-based immunoaffinity could pave the way for a deeper mechanistic understanding of how subtypes of EVs, such as those containing HNE-adducted proteins, induce biological changes in the cells that take up these EVs.

Acute gastroenteritis (AGE) remains a significant global health concern, with noroviruses among the most prevalent viral pathogens. However, other enteric viruses also contribute substantially to the public health burden. This study provides the first molecular characterization of a co-infection involving a rarely reported enterovirus A76 (EV-A76) and a norovirus GI.6[P11] in a patient from Thailand. Metagenomic sequencing successfully identified complete viral genomes, revealing unique genetic variations. Phylogenetic analysis demonstrated that the EV-A76 strain shares high nucleotide similarity with a recently reported strain from Nepal, distinguishing it from previously identified recombinant strains. The amino acid sequence alignment of the complete EV-A76 genome revealed several distinctive amino acid substitutions compared to the most closely related strains. Notably, variations in the VP1 C-terminus and VP2 EF loop, known for high variability, were observed. These regions, crucial for epitope formation, are particularly susceptible to high-frequency mutations. This study reports the first documented co-infection of EV-A76 and norovirus GI.6[P11] in a single sample, identified through metagenomic sequencing in an AGE case in Thailand in 2023. The observed genetic variations highlight the necessity for ongoing monitoring of viral diversity to strengthen genomic surveillance and inform prevention strategies, especially for emerging pathogens with significant public health implications.

Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.

Background The isolation and proteomics characterization of extracellular vesicles (EVs) from body fluids is challenging due to their vast heterogeneity. We have recently demonstrated that Fluorescence-activated Cell Sorting (FACS) efficiently isolates the whole EV circulating compartment directly from untouched body fluids enabling a comprehensive EV proteomics analysis. Results Here, we characterized, for the first time, a single-phenotype EV subset by sorting leukocyte-derived EVs (Leuko EVs) from peripheral blood and tears of healthy volunteers. Using an optimized and patented staining protocol of the whole EV compartment we identified and excluded non-EV particles, debris and damaged EVs. We further isolated, using an anti-CD45 antibody, Leuko EVs (CD45+ EVs), reaching a high level of purity (> 90%). Purified Leuko EVs were characterized using atomic force microscopy, nanoparticle tracking, and shotgun proteomics analysis revealing a similar coded protein cargo in both biological fluids. Subsequently, the same workflow was applied to tears from Relapsing–Remitting Multiple Sclerosis (RRMS) patients, revealing a Leuko EVs protein cargo enrichment that reflects the neuroinflammatory condition characteristics of RRMS. This enrichment was evidenced by the activation of upstream regulators TGFB1 and NFE2L2 , which are associated with inflammatory responses. Additionally, the analysis identified markers indicative of endothelial cell proliferation and the development of enhanced vascular networks, with AGNPT2 and VEGF emerging as activated upstream regulators. These findings indicate the complex interplay between inflammation and angiogenesis in RRMS. Conclusions In conclusion, our combined FACS-Proteomics strategy offers a promising approach for biomarker discovery, analysing cell-specific EV phenotypes directly from untouched body fluids, advancing the clinical value of tears EVs and improving the understanding of EV-mediated processes in vivo. Data are available via ProteomeXchange with the identifier PXD049036 and in EV-TRACK knowledgebase with ID: EV240150.

Extracellular vesicles (EVs) can be isolated and purified from cell cultures and biofluids using different methodologies. Here, we explored a novel EV isolation approach by combining superabsorbent polymers (SAP) in a dialysis membrane with size exclusion chromatography (SEC) to achieve high concentration and purity of EVs without the use of ultracentrifugation (UC). Suspension HEK293 cells transfected with CD63 coupled with Thermo Luciferase were used to quantify the EV yield and purity. The 500 mL conditioned medium volume was initially reduced by pressure ultrafiltration, followed by UC, SAP or a centrifugal filter unit (CFU). Using either of these methods, the EVs were concentrated to a final volume of approximately 1 mL, with retained functionality. The yield, quantified by luciferase activity, was highest with UC (70%–80%), followed by SAP (60%–70%) and CFU (50%–60%). Further purification of the EVs was performed by iodixanol density cushion (IDC) or SEC (Sepharose CL‐2B or 6B, in either 10 or 20 mL columns). Although the IDC and Sepharose CL‐2B (10 mL) achieved the highest yields, the purity was slightly higher (30%) with IDC. In conclusion, combining SAP concentration with CL‐2B SEC is an alternative and efficient way to isolate EVs without using UC.

The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study to isolate and characterize EVs from human conjunctival epithelial (IM-HConEpiC) and human conjunctival mesenchymal stromal cell (Conj-MSCs) secretomes using different isolation methods: differential ultracentrifugation (UC), and a combination of ultrafiltration (UF) with precipitation or size exclusion chromatography (SEC). EVs were characterized by total protein content, size, morphology, and expression of protein markers. EV functional effect was tested in an in vitro oxidative stress model. We successfully recovered EVs with the three methods, although significantly higher yields were obtained with UF-precipitation. Dynamic light scattering analysis confirmed the presence of nano-sized particles, being UC-isolated EVs larger than those isolated by UF-precipitation and UF-SEC. Atomic Force Microscopy showed EVs with a slightly ellipsoidal morphology. EVs enriched with UF-precipitation method were further analyzed, confirming the expression of Alix, CD63, TSG101, and Syntenin-1 by Western blotting and showing that Conj-MSC-derived EVs significantly reduced oxidative stress on IM-HConEpiC. Therefore, we conclude that UF-precipitation is the most efficient method for conjunctival EV enrichment.

The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next‐generation aqueous two‐phase system (next‐gen ATPS) for the isolation of EVs regardless of scale and volume that is superior to conventional methods such as ultracentrifugation (UC) and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG) and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX‐rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX‐rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry (nFC) when using various pre‐conjugated antibodies specific for CD9, CD63 and CD81. TRIzol RNA isolation is further enabled by the addition of dextranase, a critical component of this next‐gen ATPS method. RNA yield of next‐gen ATPS‐isolated EVs is superior to UC and other commercial kits. This negates the use of specialized EV RNA extraction kits. The use of dextranase also enables more accurate immunoreactivity of pre‐conjugated antibodies for the detection of EVs by nFC. Transcriptomic analysis of EVs isolated using the next‐gen ATPS revealed a strong overlap in microRNA (miRNA), circular RNA (circRNA) and small nucleolar RNA (snoRNA) profiles with EV donor cells, as well as EVs isolated by UC and the exoRNeasy kit, while detecting a superior number of circRNAs compared to the kit in human samples. Overall, this next‐gen ATPS method stands out as a rapid and highly effective approach to isolate high‐quality EVs in high yield, ensuring optimal extraction and analysis of EV‐encapsulated nucleic acids.