Sterile flask

The normal immortalized human endometrial stromal cell line (HESC) was purchased from Cell Bank of Chinese Academy of Science (Shanghai, China) and authenticated by Genetic Testing Biotechnology Corporation (Suzhou, China) with STR profiling method. HESC and primary endometrial stromal cells (ESC) isolated from fresh specimens were all cultured by DME/F12 medium (Hyclone, USA), supplemented with 10% fetal bovine serum (FBS) (Sciencell, USA) and 1% antibiotics (NCE, China). The culture medium was exchanged every other day and cells were digested by 0.25% EDTA trypsin (NCE, China) when they grew up to 80%~90% confluence, then the digested cells were resuspended, planted in two sterile flask (Corning, USA) and cultured in incubator of 37℃, 5% CO₂ atmosphere.

A total of 1‐2 mL bone marrow samples were taken with a heparinized syringe from the lateral tibial tubercle of six newborn male SD rats (2‐weeks‐old). Low‐glucose DMEM (low‐DMEM) containing 10% foetal bovine serum (FBS) (Gibco) was used to wash the samples of bone marrow, we then centrifuged the samples at 200 g for 5 minutes, and the supernatants was discarded. Complete medium (Low‐DMEM mixture 10% FBS and 1% penicillin‐streptomycin) was also used to resuspend the cell pellets. BMSCs were isolated using density gradient centrifugation. Then we incubated the cells in a sterile flask (5 × 5 cm; Corning) at a cell culture chamber (37°C, 5% CO₂). After 3 days of culture, the medium was replaced with new culture medium and the unattached cells were discarded. After the cell's reached satisfactory growth, then cells were washed with PBS, the cells were digested using trypsinase. Three lineage differentiations method was used to identify the BMSCs. Detailed on the specific method are in our previous study.²⁵