Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform

Abstract

Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches.

Extracellular Vesicles: Techniques and Biomedical Applications Related to Single Vesicle Analysis

“…Breitwieser et al built an SP-IRIS-based ExoView R100 platform to create a SEV phenotype library with a multidimensional data set (size, quantity, tetraspanin composition). This platform enabled a deeper understanding of the complexity and heterogeneity of SEVs . In order to distinguish different EV subgroups more efficiently, a multiplex-bead-based platform was introduced.…”

“…This platform enabled a deeper understanding of the complexity and heterogeneity of SEVs. 155 In order to distinguish different EV subgroups more efficiently, a multiplex-bead-based platform was introduced. The specific conjugation of fluorescently labeled detection antibodies to distinct EV subpopulations allowed visualization of SEVs via STED microscopy, with up to 39 different surface proteins detected in one sample.…”

“…Techniques for SEV analysis can be broadly divided into two categories: label-free methods and label-based methods. Label-free approaches, such as cryo-electron microscopy (cryo-EM), atomic force microscopy (AFM), Raman spectroscopy, and single-particle interferometric reflectance imaging sensor (SP-IRIS), perform noninvasive SEV detection based on the physical characteristics of EVs. − In label-based SEV analysis, fluorescently labeled antibodies or aptamers are widely applied due to their high specificity, particularly when combined with flow cytometry (FCM), fluorescence imaging techniques, digital methods, etc. − …”

Next‐generation liquid biopsy instruments: Challenges and opportunities

“…Conventional tissue biopsy, as the key of the diagnostic pathology, can make a definite histopathological diagnosis over the vast majority of the cancer cases [26]. However, ExoView Exosomes Specific capture based on immunostaining Fluorescence Non-small cell lung and cervical cancers [31] Abbreviations: CTC, circulating tumor cell; cfDNA, cell-free DNA.…”

“…To achieve rapid and real-time cancer diagnosis, LB instruments have been developed to reduce biochemical operations, lower the economic burden for training hospital personnel, including pathologists, nurses, and physicians, and simplify the interpretation of the test results. For example, the CEA device (developed by Massachusetts General Hospital, USA) and the ExoView (NanoView Technology, USA) instrument were developed to realize the isolation and detection of target biomarkers at the same time [30,31]. Nevertheless, there is a lot of labor-intensive biochemical operations during the isolation or detection processes, such as specific capture for biomarkers based on immunostaining.…”