Isolation of exosomes
The human umbilical cord was taken from a full-term infant, cut into
small pieces, and cultured. Cells were split when the cells reached
80–90% confluence. In the following procedures, only P5 generation
cells were used. After the cells grew to 70% confluence, the culture
medium was discarded, the cells were washed thoroughly with PBS, and the
medium was replaced with serum-free medium for 48 h. Next, the
conditioned medium was collected, centrifuged at 1000 ×g for 20
min to remove cell debris, and centrifuged at 2000 ×g for 20 min,
and the supernatant was filtered using a 220-nm filter. Next, the
filtered supernatant was centrifuged at 10,000 ×g for 60 min at
4°C, and the supernatant was filtered using a 220-nm filter again. The
filtered supernatant was centrifuged at 100,000 ×g for 3 h at
4°C, the supernatant was discarded, the pelleted exosomes were diluted
in 1 ml of pulse medium, and the effective diameter of exosomes was
assessed by dynamic light scattering (DLS, Brookhaven Instruments).