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.