Isolation and culture of primary human endometrial epithelial cells and human endometrial epithelial adenocarcinoma Ishikawa cell lines

Human endometrial epithelial cells were isolated using methods previously described with slight modifications (28). Briefly, freshly collected normal endometrial tissues were washed with cold 1× sterile phosphate buffer saline (PBS) and cut into 1–2 mm³ pieces using sterile surgical scissors. The minced tissue was then incubated with 2 mg/mL type II collagenase (0.1%, Sigma-Aldrich) in PBS for 1 hour at 37 °C. After digestion, the suspension was filtered with a 150-µm sterile sieve to remove residual tissue debris and followed by filtering with a 38-µm sterile sieve to separate the human endometrial epithelial cells from the stromal cells. The epithelial cells were collected and resuspended in a 1:1 mixture of Dulbecco’s modified Eagle’s/F12 medium (DMEM/F12; HyClone) supplemented with 20% fetal calf serum (FCS; HyClone, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (HyClone). Cells were plated in 6-well culture plates. Cell immunofluorescence assay was performed to detect the purity of the cells. The purity of the cells was >95%, with no more than 1% stromal cell contamination.

Given that primary human endometrial epithelial cell can only be cultured for 2 passages and cannot be transfected, the human endometrial epithelial adenocarcinoma Ishikawa cell line (Shanghai Fuxiang Biotechnology Co., Ltd., China) was also used in this study. The Ishikawa cells were cultured in RMPI-1640 culture media (HyClone) supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (HyClone) in humidified atmosphere with 5% CO₂ at 37 °C.