2.4. Isolation and culture of primary human endometrial stromal cells

We purified primary human endometrial stromal cells as described previously with slight modification.18 Briefly, washed endometrium tissues were minced into 1‐ to 2‐mm pieces with a sterile surgical scissors and digested in PBS containing 2 mg/mL of type II collagenase (0.1%, Sigma‐Aldrich, USA) for 45‐60 minutes at 37°C with constant agitation. The resulting suspension was filtrated through sterile 150 and 37.4 μm sieves in turn to remove undigested epithelial cells and debris. The filtrate was then centrifuged at 1000 g for 5 minutes and then further cultured in Red Blood Cell Lysis Buffer for 10 minutes to remove erythrocytes. After being centrifuged at 1000 g for another 5 minutes, the human endometrial stromal cells were plated in T25 flasks. The stromal cells were subsequently cultured in Dulbecco's modified Eagle's/F12 medium (DMEM/F12; HyClone) and supplemented with 20% fetal bovine serum (FBS; HyClone), 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone) in humidified atmosphere with 5% CO₂ at 37°C. The purity of isolated stromal cells was >95%, and stromal cells were contaminated by less than 1% of epithelial cells, as determined by diffuse and strong cytoplasmic immunostaining for Vimentin (diluted 1:50; Abcam, Cambridge, UK) and negative cellular staining for E‐cadherin (diluted 1:50; Abcam, Cambridge, UK) in immunocytochemistry.