Wound healing assay and Transwell migration assay

For wound healing assays, HUVECs in logarithmic growth phase were seeded to form confluent monolayers in six-well plates. A straight line was scraped with a pipette tip to form a gap across the cell monolayer. Cell debris left near the gaps was removed by washing with PBS three times. hAMSC-CM or control medium was then added with 2% FBS. Microscopic images were obtained at 0 and 16 h. For each image, the distance of gaps was measured using ImageJ software. For each group, five random locations of each gap were measured.

The migration of HUVECs grown in logarithmic phase in vitro was measured using 24-well Transwell inserts with a 6.5-mm diameter and 8- nm pore (Corning, NY, USA). A total of 1.5 × 10⁴ cells/well were suspended in 200 μL of control medium without FBS, and loaded into the upper chambers. Next, 600 μL of hAMSC-CM or control medium (both containing 10% FBS) was added into the lower chambers. After 12-h incubation, migrated cells that had attached to the lower surface of the filter were fixed with 4% paraformaldehyde and then stained with crystal violet. Cells were counted and photographed in five random fields under a digital microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Wound healing and Transwell migration assays were replicated three times independently.