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Overall figure description: Create a multi-panel schematic scientific illustration showing the major methods used for isolation and purification of fungal (mushroom-derived) extracellular vesicles (EVs) from culture filtrate, mycelium, or fruiting bodies. Panel A: Source and sample preparation Show: Mushroom fruiting body Fungal mycelium Culture filtrate Indicate: “Fungal growth system” “Cell-free supernatant after filtration” Panel B: Differential ultracentrifugation (DUC) Show stepwise centrifugation workflow: Low-speed spin → removal of cells and debris Medium-speed spin → removal of large vesicles High-speed ultracentrifugation (40,000 × g and 100,000 × g) Label pellets: “40,000 × g EV fraction (higher purity, intact vesicles)” “100,000 × g EV fraction” Add notes: “Most commonly used method” “Limitations: long processing time, protein contamination, vesicle damage” Panel C: Size-exclusion chromatography (SEC) Show: Chromatography column packed with Sepharose CL-2B / CL-4B beads Indicate separation: Large EVs elute first Small proteins elute later Label: “Separation based on hydrodynamic size” “Preserves vesicle integrity” Annotation: “Combination of DUC + SEC reduces protein contamination (~98%)” Panel D: Density gradient centrifugation Show: Centrifuge tube with sucrose or iodixanol gradient Indicate EV band at: “Buoyant density 1.10–1.19 g/mL” Label: “High-purity EV isolation” “Suitable for metabolomics and lipidomics” Panel E: Polymer-based precipitation Show: Addition of polymer reagent Vesicle precipitation Label: “Rapid isolation” “Low equipment requirement” “Co-precipitation of contaminants (limitation)” Panel F: Microfluidic and advanced filtration technologies Show: Microfluidic chip Dual-cyclic tangential flow filtration (dcTFF) device Indicate: “Selective isolation of EVs (30–200 nm)” “High-throughput and automated”