2.3. Co‐culture of endothelial cells and trophoblast cells

Glass bottom (10 mm) confocal culture dishes (Jing'an, China) were coated with 80 μL Matrigel (BD Biosciences) and incubated for 30 minutes to solidify. HUVEC (2 × 10⁴) cells stained with 1 μmol/L CellTracker^™ Green CMFDA Dye (Thermo FisherScientific) were then added into the Matrigel‐coated dishes. After 4 hours, HUVECs formed tubular structures, then the medium was removed and carefully washed with PBS. Thereafter, 2 × 10⁴ treated HTR8/SVneo cells that were stained with 1 μmol/L CellTracker^™ Red CMTPX Dye (Thermo Fisher Scientific) were added to the HUVEC layer. After co‐culture for 4 hours, the medium was removed and fixed with 4% paraformaldehyde for 30 minutes. Images of random regions were taken from each dish using a confocal microscope (Olympus FV1000). Quantification was performed using the Image J software (National Institutes of Health). Images were converted to 8‐bit gray scale with a defined preset threshold. The tubular area was then calculated as the number of pixels. The percentage of HTR8/SVneo in the tube green fluorescence area/red fluorescence area.