Fig. 1

Characterization of culture-activated primary rat HSCs and sEVs isolated from the culture media. (A) Schematic representation of the process for rat primary hepatic stellate cell isolation and culture. (B) Morphology of primary hepatic stellate cells of rats at Day 0, Day 3, Day 7 and Day 14, bright field, scale bar = 200 μm. (C) BODIPY 493/503 staining of cytoplasmic lipid droplets and immunofluorescence staining of Desmin and α-smooth muscle actin (α-SMA) of HSCs at the indicated time points. BODIPY 493/503 (green), Desmin and α-SMA (Cy3, red), nuclei were counterstained with DAPI (blue), scale bar = 50 μm. (D) Western blotting analyses of the HSC activation markers, α-SMA and collagen type I (ColI). Cells were collected at the indicated time points. Coomassie blue staining was used as a loading control. (E) Transmission electron microscopy image of the particles isolated from the primary HSC culture medium. Left, sEVs enriched by the ExoQuick Kit; right, sEVs enriched by ultracentrifugation (UC), scale bar = 200 nm. (F) Representative size distribution of isolated particles and their concentrations determined by nanoparticle tracking analyses. (G) The expression of CD63, CD81 and CD9 in the isolated particles detected by western blotting. Coomassie blue staining was used as a loading control. sEV, small extracellular vesicle; 3dHSCs, primary hepatic stellate cells cultured on Day 3; and 14dHSCs, primary hepatic stellate cells cultured on Day 14