Evaluation of Endothelial Regeneration and Vascular Formation by EO and HEO Complexes in a Mouse Model

To assess the regenerative capacities of EO and HEO complexes on injured endothelium, we established an endometrial injury model in mice and administered intrauterine injections of the complexes. Female SCID mice, aged 6-8 weeks, were utilized for this experiment. Initially, the mice were anesthetized, and a laparotomy was performed to fully expose the left uterus. The uterine horns near the oviductal end and cervical end were ligated. Subsequently, 95% ethanol was injected into the uterine cavity from the oviductal end, followed by a 15-second pause. Then, the uterine cavity was flushed with PBS injected from the oviductal end. EO and HEO complexes, cultured in eCHIP with estradiol for 2 days, were collected. Using a 1 ml syringe, 75 ul of the cell complexes were injected into the damaged uterine cavity from the oviductal end. An equal volume of Matrigel or PBS was injected into other endometrial injury model mice as the Matrigel-treated group and the natural repair group, respectively. Three minutes after injection, the ligatures at both ends were removed, and the uterus was repositioned in the abdomen and sutured. After 3 and 7 days post-surgery, the mice were euthanized, and the uteri were collected for pathological staining and immunohistochemical staining. After 14 days post-surgery, the mice were cohabitated with male mice at reproductive age to observe the fertility of the model mice.

To investigate the vascular formation ability of EO and HEO complexes in mice, we subcutaneously transplanted 30 ul of the cell complexes on the dorsum of female SCID mice. After 14 days of transplantation, fluorescent substances were injected into the tail vein of the mice. After 30 minutes, the transplants were retrieved under a stereomicroscope for confocal microscopy fluorescence imaging.