Transwell insert with a 0.4 μm pore size membrane

Human endometrial mesenchymal stem cells (hEnSCs) were isolated from the menstrual blood of a 40-year-old Chinese woman according to a protocol previously described [23 (link)]. Briefly, menstrual blood was collected, and hEnSCs were acquired by density-gradient centrifugation. The procedure was approved by the Institutional Ethics Committee of the International Peace Maternity and Child Health Hospital (Shanghai, China). hEnSCs were cultured in Chang Medium (S-Evans Biosciences, Hangzhou, China) with 5% CO₂ at 37 °C. For western blot assay and morphological assessment, 2 × 10⁵ hEnSCs were seeded in the lower compartment of a 6-well plate. H₂O₂ was added to a terminal concentration of 1 μM to simulate IUA characteristics in vitro according to previous reports [24 (link)]. After 2.5 h, the former medium was discarded and replaced with fresh medium. For the hAEC-treated group, 10⁵ hAECs were seeded onto a Transwell insert with a 0.4-μm pore size membrane (Corning) and placed above the lower compartment for coculture. After 24 h, the hEnSCs were collected for protein extraction.

To imitate indirect cell-cell communication between EnSCs and EOC cells, transwell system or EnSCs conditioned medium (EnSC-CM) were used in ex vivo experiments. 1 × 10⁵ EnSCs were seeded on the transwell insert with a 0.4 μm pore size membrane (Corning) and 2 × 10⁵ EOC cells were seeded in a 6-well culture plate at the lower compartment. In direct co-culture system, 2 × 10⁵ EOC cells were co-seeded with 1 × 10⁵ EnSCs in a 6-well culture plate. All groups were cultured with Chang Medium and incubated with 5% CO₂ at 37 °C. After 48 hours, cancer cells were collected for subsequent experiments.