Enhanced visualization of articular cartilage chondroprogenitor-derived extracellular vesicles using cytospin centrifugation for confocal imaging

Abstract

Characterizing Extracellular Vesicles (EVs) poses a challenge due to their complex composition and small size. Efforts to visualize fluorescently labeled EVs post-isolation with confocal microscopy encounter a significant obstacle in congregating the isolates on slides. While cytospin is a well-established method for cells, its application with EVs remains unexplored. This study aimed to isolate EVs and evaluate the efficiency of cytospin preparation to enhance their visualization using confocal microscopy. Following informed consent, cartilage shavings from a non-diseased human tibiofemoral joint were digested to obtain cartilage-resident chondroprogenitors through a migratory-assay (MCP). The MCPs were characterized for MSC markers (positive: CD105, CD73, CD90; negative: HLA-DR, CD34, CD45) using FACS. The conditioned medium was utilized to isolate EVs using the cutoff filter and precipitation technique, and their confirmation for transmembrane markers CD63 and CD81 was performed via FACS. Subsequently, the isolated EVs were subjected to confocal microscopy with and without (control) cytospin preparation. The MCPs exhibited high expression of positive and low expression of negative MSC markers. The study effectively employed cytospin centrifugation and confocal imaging to robustly identify EVs isolated from MCPs. Fluorescently labeled with CD63 and CD81 markers, the EVs isolated using cytospin centrifugation were concentrated on glass slides, and compared to control slides; the experimental setup facilitated a 100 times concentration of EVs, thereby enabling easy identification. Confocal analysis confirmed positive expressions of CD63 and CD81, aligning with FACS results. This study introduces a novel approach that enhances the visualization of MCP-derived EVs using cytospin centrifugation.