Culturing Placental Villous Explants

Placental tissues were obtained from 24 women after elective cesarean section deliveries (36 to 40 weeks of pregnancy). Exclusion criteria included pre-eclampsia, chronic hypertension, infectious disease including toxoplasmosis, chorioamnionitis, chronic renal disease, cardiac disease, connective tissue disease, pre-existing diabetes mellitus and gestational diabetes mellitus. Placental tissues were placed in ice-cold sterile phosphate-buffered saline (PBS), pH 7.2, to remove excess blood then aseptically dissected using a stereomicroscope to remove endometrial tissue and fetal membranes up to 1 h after collection. Floating terminal chorionic villous explants containing five to seven free tips per explant were collected as described previously
[21 (link),22 (link)]. Explants were added to 96-well plates (one per well) and cultured in complete medium containing RPMI 1640 medium (Cultilab, Campinas, SP, Brazil) supplemented with 10% fetal bovine serum (FBS) (Cultilab), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich Co., St. Louis, MO, USA) for 24 h at 37°C and 5% CO₂.