Isolation and culture of human endometrial stromal and glandular cells

Endometrial tissues were separated and conserved in a monolayer culture, as described previously⁽³⁴⁾. The isolated endometrial cells were separated by filtration through a wire sieve (73 mm diameter pore, Sigma). The endometrial glands (largely undispersed) were retained by the sieve, whereas the dispersed stromal cells passed through the sieve into the filtrate. The stromal cells were plated in plastic flasks (75 cm², Falcon, Franklin Lakes, NJ), maintained at 37 °C in a humidified atmosphere (5% CO₂ in air), and allowed to replicate to confluence. Thereafter, the stromal cells were passed by standard methods of trypsinization, plated in culture dishes (100 mm diameter), and allowed to replicate to confluence. ESCs after the first passage were characterized as described previously⁽³⁴⁾ and were found to contain 0-7% epithelial cells, no detectable endothelial cells, and 0.2% macrophages. Experiments were commenced 1-3 days after the cells reached confluence. The confluent cells were treated with serum-free, phenol red-free media for 24 h before treatment with test agents. Stromal cells reached confluence in 7-10 days.

Experiments with glandular cells were performed using a well-differentiated endometrial adenocarcinomacell line (Ishikawa cell) provided to us by Dr. R. Hochberg (Departmentof Obstetrics and Gynecology, Yale University, New Haven, CT)from a frozen stock. Thawed cells were maintained in T75 flasks(BD Biosciences, Franklin Lakes, NJ) until passage.The cells were treated with serum-free phenol red-free media for 24 h before treatment with test agents. Cells were treated with E₂ (Sigma) for 3-90 min and immunocytochemistry and Western blot analysis were performed as described.