Journal of extracellular biology最新文献

As extracellular vesicles (EVs) are increasingly recognized for their superior functions for therapeutics, the need for large-scale EV isolation technology is becoming more critical for clinical and industrial applications. Most existing EV isolation methods are optimized for small-scale laboratory samples, limiting their efficiency and scalability for large-scale production. Here, an electrokinetic-assisted filtration system (ExoFilter), which introduces charge interaction into physical mesh flow filtration, is proposed as a new candidate to address the challenges of scalable EV isolation. The hybrid filtration system demonstrates outstanding high-throughput EV isolation performance (a flux of ∼750 mL/min) using only a coarse physical filter by electrokinetically arresting EVs flowing through the filter lattice. Furthermore, the recovery efficiency of ExoFilter, analysed based on the ELISA results, was found to be approximately 98%, demonstrating the filter's exceptional efficiency in EV isolation. Additionally, ExoFilter enables the rapid isolation of EVs from small samples as little as 200 µL, facilitating quick and easy blood-based EV research. Furthermore, low-molecular-weight albumin from plasma samples was effectively removed. The high-throughput and high-efficiency characteristics of ExoFilter make it well-suited for scalable EV production, offering greater convenience for various clinical applications.

Despite biomolecule delivery is a natural function of extracellular vesicles (EVs), low loading of exogenous macromolecules such as proteins into EVs limits their interest as convincing protein delivery systems for health applications. In this context, lipid-anchorage of exogenous cargo into EV membrane recently emerged as a promising option to enable their vectorisation into cells. Nevertheless, this option was not explored for protein intracellular delivery, and further characterisation of critical parameters governing the association of a lipid-anchored cargo protein to EVs is still needed to confirm the relevance of this anchorage strategy. Therefore, we sought to identify these parameters in a precise and quantitative manner, using bulk and single nanoparticle analysis methods to identify protein loading capacity and subsequent intracellular delivery. We identified incubation temperature, cargo concentration, lipid anchor (LA) structure (lipid nature, linker) and EV origin as critical factors influencing maximal EV loading capacity. Precise control of these parameters enabled to load cargo protein close to EV saturation without hindering cellular delivery. The structural properties of LA influenced not only cargo protein/EV association but also intracellular delivery into different carcinoma cell lines. By thoroughly characterising Lipid-PEG-protein anchorage, this study evidences the interest of this tunable and controllable approach for efficient EV protein delivery.

Nanoflow cytometry (nanoFCM) is an increasingly important analytical procedure in every aspect of extracellular vesicle (EV) research, particularly in the development of EV-based therapeutics. The main objective of this study was to evaluate and optimise the key determinant factors of nanoFCM in the quantification analysis of EVs to ensure its consistency and reliability in the development of EV therapeutic drugs, thereby serving as a potential quality control measure. Our investigation followed the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q14 guideline. We revisited the day-to-day practice of nanoFCM measurement for HEK293 cell-derived and milk-derived EVs (mEVs), focusing on optimising particle quantification and identifying risk factors. Initial evaluation of the procedure revealed a considerable lack of consistency and reliability, which was then subjected to extensive optimisation. The key outcomes of this study include: (1) an optimised analytic procedure incorporating Tween-20, which significantly enhanced the precision and accuracy of the nanoFCM measurement and expanded the reportable range; (2) an analytical target profile (ATP) which provides a preliminary standard for future validation of nanoFCM procedures. Overall, this study serves as a foundation for future efforts towards the standardisation of analytical procedures for EV therapeutics.

Extracellular vesicles (EVs) are small particles released by various cells, including cancer cells. They play a significant role in the development of different cancers, including brain metastasis. These EVs transport biomolecular materials such as RNA, DNA, and proteins from tumour cells to other cells, facilitating the spread of primary tumours to the brain tissue. EVs interact with the endothelial cells of the blood–brain barrier (BBB), compromising its integrity and allowing metastatic cells to pass through easily. Additionally, EVs interact with various cells in the brain's microenvironment, creating a conducive environment for incoming metastatic cells. They also influence the immune system within this premetastatic environment, promoting the growth of metastatic cells. This review paper focuses on the research regarding the role of EVs in the development of brain metastasis, including their impact on disrupting the BBB, preparing the premetastatic environment, and modulating the immune system. Furthermore, the paper discusses the potential of EVs as diagnostic and prognostic biomarkers for brain metastasis.

Human milk serves the sole nutritional role for the developing infant. During lactation, nano-sized extracellular vesicles (EVs) in milk containing a multitude of biologically active components are transferred from mother to offspring. Infant formula (IF) based on cow milk-derived ingredients has been reported to contain reduced levels of EVs as compared to human milk. There is therefore an unmet need to produce large-scale volumes of milk EVs to improve IF composition.

Here, we report a scalable industrial production protocol for a bovine whey-derived ingredient that is highly enriched in EV material using a large-scale sequential ceramic membrane filtration setup. Furthermore, we demonstrate a robust and generally applicable analytical approach to determine the relative contributions of EVs and milk fat globule membrane (MFGM) using molar ratios of the membrane-bound proteins butyrophilin (BTN) and CD9 as surrogate markers for MFGM and EVs, respectively. Taken together, our findings provide a basis for comparing bovine milk-containing foods and aid in developing specialized ingredients that can minimize the compositional difference between infant formula and human milk.

Extracellular vesicles (EVs) are cell-produced, membrane-surrounded vesicles that harbour the biological features of donor cells. In the current study, we are the first to isolate and characterize EVs isolated from human precision-cut liver slices (PCLS), obtained from both healthy and metabolic dysfunction-associated steatohepatitis (MASH) cirrhotic livers. PCLS derived from patients can faithfully represent disease conditions in humans. EVs were isolated from human PCLS after incubating in normal medium or modified medium that mimics the pathophysiological environment of metabolic dysfunction associated liver disease (MASLD). MASH PCLS produced higher amounts of EVs compared to healthy PCLS (p < 0.001). Mass spectrometry revealed that around 300 proteins were significantly different in EVs derived from MASH PCLS versus healthy PCLS (FDR < 0.05), irrespective of the type of medium. Significantly changed EV proteins were largely involved in signalling receptor binding function and showed potential in promoting fibrosis. In the liver, these ligand-associated receptors are highly expressed in hepatic stellate cells, and the MASH EVs functionally promoted the activation of hepatic stellate cells. Furthermore, the amounts of EpCAM and ITGA3 in EVs were positively associated with the progression of MASLD, which suggests the use of liver-derived EVs as potential biomarkers for MASLD. Characterization of EVs derived from human PCLS may assist future studies in investigating the pathogenesis and identifying liver-specific EVs as biomarkers of MASLD.

Natural killer (NK) cells are exploited in cellular therapies for cancer. While NK cell therapies are efficient against haematological cancers, it has been difficult to target solid tumours due to low tumour infiltration and a hostile tumour microenvironment. NK-cell derived extracellular vesicles (NK-EVs) target and kill cancer cells in vitro and represent an alternative treatment strategy for solid tumours. To exploit their potential, it is necessary to standardize NK-EV production protocols. Here, we have performed a comparative analysis of EVs from the human NK-92 cell line cultured in five serum-free commercial media optimized for growth of human NK cells and one serum-free medium for growth of lymphocytes. The effect of growing the NK-92 cells in static cell cultures versus shaking flasks was compared. EVs were purified via ultracentrifugation followed by size-exclusion chromatography. We found that there were no significant differences in EV yield from NK-92 cells grown under static or dynamic conditions. However, we found clear differences between the different culture media in terms of EV purity as assessed by the enrichment of the CD63 and CD81 markers in the isolates that translated into their capacity to induce apoptosis of the colon cancer cell line HCT 116. These findings will be instructive for the design of future production protocols for therapeutic NK-cell derived EVs.

Extracellular vesicles (EVs) are crucial for intercellular communication and are found in various biological fluids. The identification and immunophenotyping of such small particles continue to pose significant challenges. Here, we have developed a workflow for the optimisation of a next-generation panel for in-depth immunophenotyping of circulating plasma EVs using spectral flow cytometry. Our data collection followed a multistep optimisation phase for both instrument setup and 21-colour panel design, thus maximising fluorescent signal recovery. This spectral approach enabled the identification of novel EV subpopulations. Indeed, besides common EVs released by erythrocytes, platelets, leukocytes and endothelial cells, we observed rare and poorly known EV subsets carrying antigens related to cell activation or exhaustion. Notably, the unsupervised data analysis of major EV subsets revealed subpopulations expressing up to five surface antigens simultaneously. However, the majority of EVs expressed only a single surface antigen, suggesting they may not fully represent the phenotype of their parent cells. This is likely due to the small surface area or the biogenesis of EVs rather than antibody steric hindrance. Finally, we tested our workflow by analysing the plasma EV landscape in a cohort of systemic lupus erythematosus (SLE) patients. Interestingly, we observed a significant increase in CD54⁺ EVs, supporting the notion of elevated circulating ICAM under SLE conditions. To our knowledge, these are the first data highlighting the importance of a spectral flow cytometry approach in deciphering the heterogeneity of plasma EVs paving the way for the routine use of a high-dimensional immunophenotyping in EV research.

The ‛stickiness’ of extracellular vesicles (EVs) can pose challenges for EV processing and storage, but adhesive properties may also be exploited to immobilise EVs directly on surfaces for various measurement techniques, including super-resolution microscopy (SRM). Direct adhesion to surfaces may allow the examination of broader populations of EVs than molecular affinity approaches, which can also involve specialised, expensive affinity reagents. Here, we report on the interaction of EVs with borosilicate glass and quartz coverslips and on the effects of pre-coating coverslips with poly-L-lysine (PLL), a reagent commonly used to facilitate interactions between negatively charged surfaces of cells and amorphous surfaces. Additionally, we compared two mounting media conditions for SRM imaging and used immobilised EVs for a B-cell interaction test. Our findings suggest that borosilicate glass coverslips immobilise EVs better than quartz glass coverslips. We also found that PLL is not strictly required for EV retention but contributes to the uniform distribution of EVs on borosilicate glass coverslips. Overall, these findings suggest that standard lab materials like borosilicate glass coverslips, with or without PLL, can be effectively used for the immobilisation of EVs in specific imaging techniques.