Optimized Isolation and Culture of Human Endometrial Stromal Cells and Epithelial Organoids from Biopsies

Human endometrial stromal cells (ESCs) and epithelial cells were isolated from tissue biopsies utilizing a previously established method [80] with minor modifications. Initially, endometrial biopsies underwent three washes with wash buffer, comprising 1640 medium supplemented with Penicillin-Streptomycin. Subsequently, the biopsies were mechanically dissected into small fragments measuring approximately 1 mm3 using sterile scissors. These tissue fragments were then subjected to enzymatic digestion in a solution containing Collagenase/dispase and DNase I for a duration of 20 minutes. Following centrifugation to separate the supernatant, the resulting cell pellet was washed three times with wash buffer. The cell pellet was resuspended in wash buffer and passed through a 100-micron filter to eliminate tissue debris. The resulting cell suspension was further filtered through a 40-micron filter. Endometrial epithelial cells retained on the 40-micron filter were collected for the establishment of Endometrial epithelial organoids (EEOs) in culture. Concurrently, the cells present in the filtered supernatant were collected by centrifugation and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS). After a 24-hour incubation period, the adherent stromal cells were detached using trypsin and subsequently reseeded in a new culture dish to obtain a purified population of ESCs.