Isolation and Culture of Human Endometrial Stromal Cells (ESCs) and Endometrial Epithelial Organoids (EEOs)

Human endometrial stromal cells (ESCs) and epithelial cells were obtained from tissue biopsies according to previously described methods [80], with slight modifications. Briefly, endometrial biopsies were washed three times with wash buffer (1640 medium with Penicillin-Streptomycin) and then minced into small fragments (approximately 1 mm3) using scissors. The tissue fragments were then digested for 20 minutes in a Collagenase/dispase solution with DNase I. After centrifugation to remove the supernatant, the pellet was rinsed three times. The cell pellet was resuspended in wash buffer and filtered through a 100-micron filter to eliminate tissue debris. The suspension was subsequently filtered through a 40-micron filter. The endometrial epithelial organs on the top of the filter were collected for Endometrial epithelial organoids (EEOs) culture. Meanwhile, the cells in the filtered supernatant were centrifuged and cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS). After 24 hours of culture, the adherent cells were digested with trypsin and reseeded in a new culture dish to obtain ESCs.