Enrichment and Proteomic Profiling of Stromal-Associated Extracellular Vesicle Subpopulations From Arthritic Synovial Fluid by Size-Exclusion Chromatography Coupled to CD90-Directed Immunocapture.

Extracellular vesicles (EVs) are emerging as key mediators of disease-associated intercellular communication and as promising biomarker sources across many disease conditions, including joint disorders such as rheumatoid arthritis (RA) and osteoarthritis (OA). Molecular profiling of synovial tissue has uncovered disease-driving cellular states, but tissue biopsies are invasive and not routinely available. EVs in synovial fluid may offer a minimally invasive and complementary window into joint pathobiology. However, the molecular complexity of synovial fluid and the heterogeneity of EV populations have hindered the analysis of defined disease-relevant EV subsets. To address this challenge, we developed a refined size-exclusion chromatography-ultrafiltration (SEC-UF) workflow coupled to magnetic bead-based immunocapture for targeted enrichment of cell type-associated EV subpopulations from arthritic synovial fluid. As proof-of-concept, we targeted a stromal-associated EV population using CD90/THY1, a surface marker expressed by discrete synovial fibroblasts subsets implicated in arthritis pathobiology. In vitro validation using synovial fibroblast-derived EVs confirmed surface-accessible CD90 and demonstrated selective immunocapture of defined EV subpopulations. Application to patient-derived synovial fluid showed that SEC-UF pre-enrichment improves the robustness of CD90