Placental explants and cell culture
Placental explants culture was performed as described previously(Hu et al., 2015). Briefly, cotyledons from the central part of the placenta were removed under sterile conditions. Explants (approximately 30-50mg) were dissected and cultured in 24-well plates containing DMEM/F12 (Gibco, Thermo Fisher Scientific, Rockford, IL) with 10% FCS at 37℃ in 5% CO2-95% air. Culture medium was replaced every 24h. On day 4, explants were treated with increasing concentration of cortisol (10-8-10-6M) in absence and presence of CBX (10-6-10-5) in FCS-free DMEM/F12 for 24h. Culture media and tissues were collected and stored at -80ºC.
Primary placental cell cultures were performed by a modified Kliman’s method (He et al., 2014). Briefly, cotyledons were removed from the maternal side and dispersed with trypsin (Sigma-Aldrich) and deoxyribonuclease I (Sigma-Aldrich). A purified fraction of cytotrophoblasts was obtained following Percoll (GE healthcare, Uppsala, Sweden) gradient centrifugation. The cells were then plated into 12-well plates (Corning-Costar Inc., Corning, NY) at a density of 1.2×106/well and grown in phenol red-free DMEM (Gibco) with 10% FCS at 37℃ in 5% CO2-95% air. After 48 h of plating, cells were treated with cortisol (Sigma-Aldrich) in absence and presence of CBX at the indicated concentrations for 24h. Control cultures were maintained without additives. Each treatment was performed in triplicate for each preparation of cells. Culture media and cells were collected and stored at -80ºC.
HTR8, an extravillous cytotrophoblasts (EVT) cell line, was a gift from Prof. Charles H. Graham (Queen’s University, Canada). HIPEC65, a cell line established from a primary culture of transformed by T-SV40, was kindly provided by Dr. Thierry Fournier (INSERM, France). The above cells were grown in DMEM/F12 supplemented with 10% FCS, 100 UI/ml penicillin and 100 mg/ml streptomycin at 37ºC in 5% CO2-95% air.