fig2

Figure 2. Schematic overview of the upstream workflow for EV production, isolation, purification, and characterization, divided into four key stages: (A) EV source: Involves controlled selection of EV sources, with critical considerations encompassing phenotype, biological variation, and contamination risks; (B) Expansion: Centers on cell expansion strategies, prioritizing the optimization of cultivation modes and scalable production conditions; (C) Isolation and purification: Utilizes complementary techniques for EV isolation and purification, including UC, TFF, AF4, MC, UF, DGC, SEC/AEC, PP, and AP; (D) Characterization: Integrated characterization using (i) bulk analytical methods, such as WB, ELISA, and DLS, together with (ii) single-particle techniques, including NTA, TEM, SRFM, and nFCM, to evaluate EV size, morphology, molecular composition, purity, and heterogeneity. Figure was created with https://BioRender.com/nm03i7i. AF4: Asymmetrical flow field-flow fractionation; AP: affinity-based precipitation; DGC: density gradient centrifugation; DLS: dynamic light scattering; ELISA: enzyme-linked immunosorbent assay; EV: extracellular vesicle; EVs: extracellular vesicles; MC: membrane chromatography; nFCM: nano-flow cytometry; NTA: nanoparticle tracking analysis; PP: polymer-based precipitation; SEC/AEC: size exclusion chromatography/anion exchange chromatography; SRFM: super-resolution fluorescence microscopy; TEM: transmission electron microscopy; TFF: tangential flow filtration; UC: ultracentrifugation; UF: ultrafiltration; WB: Western blotting.