2.2. Placental villous explants
The term placentas (n=7) were collected from normal pregnancies and used immediately after delivery by cesarean section at the University Hospital in Hradec Kralove. The study was approved by the institutional Ethical Committee (No. 201006S15P) and informed consent was provided by all participants involved in the study. Within 1 h after delivery placental villous explants were dissected and cultured with CMRL-1066 medium under conditions of 37 °C, 5% CO2 using a protocol adopted from dr. Sue Greenwood33,34 and optimized for the current study. Briefly, chorionic villi were dissected out from three different places of villous placental tissue and carefully rinsed in sterile Dulbecco’s phosphate-buffered saline with calcium chloride and magnesium chloride (Sigma-Aldrich) to wash from maternal blood residues. The tissue was cut into small pieces weighing ~ 5 mg. Three small pieces were cultured in individual Costar Netwell (Corning, Corning, NY) supports in 1.5 ml of culture medium [CMRL-1066, NaHCO3 (2.2 mg/ml), streptomycin sulphate (100 µg/ml), penicillin (100 IU/ml), insulin (1  µg/ml), retinol acetate (1 µg/ml), L-glutamine (100 µg/ml), hydrocortisone (0.1 µg/ml) and 5 % heat-inactivated fetal bovine serum; Sigma-Aldrich] on 12-well plate. Culture medium was collected and replaced every 24 h during the 8-day period. Explants were treated on the day 5 with pro-inflammatory cytokines/inflammation stimulants (IFN-γ 50 ng/ml; IL-6 50 ng/ml; TNF-α 20 ng/ml; HMGB1 10 ng/ml and LPS 100 ng/ml) for following 24 or 72 h. Supernatants were collected and stored at -80 °C for hCG analysis. Explants viability and reconstituted trophoblast were evaluated based on hCG production by total beta hCG method at the Institute of Clinical Biochemistry and Diagnostics, University Hospital Hradec Kralove (data not shown), following the approach of dr. Sue Greenwood33,34. Only explants showing an increase in the hCG production higher than 3-fold between the days 2 and 5 in culture were used for further experiments.