Endothelial tube formation assay in normoxic or hypoxic HUVECs. The formation of tubelike structures was observed under dark field using a cell-permeable dye, Calcein AM (green). Endothelial tube formation of HUVECs cultured under normoxic conditions (20% O₂) with (A) RPMI8226^20%O2-exosomes, (B) RPMI8226^1%O2-exosomes, (C) RPMI8226-HR^1%O2-exosomes, (D) half the amount of RPMI8226-HR^1%O2-exosomes, or (E) without exosomes (control; HUVECs^20%O2). (F) Quantitative data for the tubelike structures determined by pixel density. RPMI8226^1%O2-exosomes and RPMI8226-HR^1%O2-exosomes significantly enhanced tube formation of HUVECs^20%O2 compared with control (HUVECs^20%O2) (*P < .01, **P < .001). Because nanoparticle tracking analysis indicated that the HR cells secreted double the amount of exosomes compared with parental cells (Figure 2B), half the amount of RPMI8226-HR^1%O2-exosomes was also assessed (RPMI8226-HR^1%O2-exosomes vs RPMI8226-HR^1%O2-exosome (1/2): ^#P < .01, Student t test). Endothelial tube formation of HUVECs cultured under hypoxic conditions (1% O₂) with (G) RPMI8226^20%O2-exosomes, (H) RPMI8226^1%O2-exosomes, (I) RPMI8226-HR^1%O2-exosomes, (J) half the amount of RPMI8226-HR^1%O2-exosomes, or (K) without exosomes (control; HUVECs^1%O2). Because HUVECs were exposed to hypoxic conditions, the induction of tube formation was enhanced in all treatments. (L) Quantitative data for the tubelike structures determined by pixel density. RPMI8226^1%O2-exosomes and RPMI8226HR^1%O2-exosomes enhanced tube formation of HUVECs^1%O2 (*P < .01, **P < .001). Even when RPMI8226-HR^1%O2-exosomes were reduced to half the dose, induction of tube formation did not decrease (RPMI8226-HR^1%O2-exosomes vs RPMI8226-HR^1%O2-exosome [1/2]; P = .38, Student t test). Values are mean ± SD. The scale bar indicates 500 µm. exo, exosome.