Extracellular vesicles (EVs) are secreted nano-sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal-reflection microscopy and analyzes their co-localization to investigate EV subpopulations. The single-vesicle imaging and colocalization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.

We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, having beneficial effects for various types of regenerative processes. Although EVs have shown promising effects as therapeutic agents, these effects are difficult to research due to the limitations of EV production. In this study, an EV production method based on a flat-plate bioreactor is introduced. The bioreactor produces approximately seven times more mesenchymal stem cell-derived EVs than static culture conditions. The mechanism underlying the increased production of EVs in a flat-plate bioreactor and its application to acute kidney injury is investigated. This study describes the mechanism of EV production by demonstrating the link between EV biogenesis and increased calcium ion concentration under flow conditions. EVs secreted by cells cultured in the bioreactor have therapeutic efficacy in terms of improving kidney damage, resulting in tissue regeneration in a cisplatin-induced acute kidney injury model. This method will help overcome the limitations of EV production, and the analysis of the application of EVs will increase their reliability as well as the understanding of the use of bioreactor-derived EVs as therapeutic agents.

Extracellular vesicles (EVs) are nano-sized vesicles derived from cells that transport biomaterials between cells through biofluids. Due to their biological role and components, they are considered as potential drug carriers and for diagnostic applications. Today's advanced nanotechnology enables single-particle-level analysis that was difficult in the past due to its small size below the diffraction limit. Single EV analysis reveals the heterogeneity of EVs, which could not be discovered by various ensemble analysis methods. Understanding the characteristics of single EVs enables more advanced pathological and biological researches. This review focuses on the advanced techniques employed for EV analysis at the single particle level and describes the principles of each technique.

Cell-engineered nanovesicles (CNVs) are considered as an alternative to exosomes, because they can be produced efficiently on a large scale and have been successfully reported in several applied research studies. However, CNVs may originate from various organelles, i.e., some of them may cause adverse effects on recipient cells, and their origin has not yet been identified. In this study, we air-sprayed human embryonic kidney 293 (HEK293) cells into lipid-bilayer CNVs. To identify the subcellular origin of the CNVs, we prepared nine different HEK293 cell lines by transfection with organelle-specific fluorescent protein plasmids that target the plasma membrane, peroxisome, lysosome, early endosome, late endosome, nucleus, mitochondrion, Golgi apparatus, and endoplasmic reticulum. The origin of CNVs were identified by measuring fluorescence expressions for organelle-specific markers using fluorescence nanoparticle tracking analysis (NTA). In the results, we found that CNVs derived from the plasma membrane constituted the largest portion, but CNVs derived from the other organelles comprised a non-negligible portion as well. This information will be useful to guide advanced research on outer membrane vesicles and exosome-mimetic nanovesicles engineered from cells.