Primary trophoblast cells were isolated from healthy term placental villous tissues. Placental tissue was carefully minced, and approximately 30 g was digested three times for 30 min at 37°C in saline Hanks/HEPES solution including DNase I (Sigma‐Aldrich, USA) and trypsin (Thermo Fisher Scientific, USA) as previously described.
41 (link) After digestion and centrifugation at 1000 RCF, the cellular pellets were resuspended in DMEM and separated by centrifugation (1500 RCF, 20°C, 20 min) using a 10%–70% Percoll gradient (Sigma‐Aldrich, USA). Trophoblast cells were obtained from Percoll gradient fractions between 35% and 55%. Cells were plated at a density of 200 000 cells/cm2 in DMEM high‐glucose (4.5 g/L glucose) medium supplemented with 10% fetal bovine serum and 1x antibiotic–antimycotic (Gibco, USA). After isolation, the cells were cultured for 12 h to allow them to attach before being exposed to different oxygen conditions.
The purity of the isolated trophoblast was evaluated by staining with the specific cell markers anti‐cytokeratin 7 (CK7), anti‐vimentin, or anti‐von Willebrand factor (vWF) (Novus Biologicals). Cells were acquired by flow cytometry (BD FACS LSRII; BD Biosciences, USA) as described.
38 (link) The purity of the isolated trophoblasts was 91%–96%.
41 (link) After digestion and centrifugation at 1000 RCF, the cellular pellets were resuspended in DMEM and separated by centrifugation (1500 RCF, 20°C, 20 min) using a 10%–70% Percoll gradient (Sigma‐Aldrich, USA). Trophoblast cells were obtained from Percoll gradient fractions between 35% and 55%. Cells were plated at a density of 200 000 cells/cm2 in DMEM high‐glucose (4.5 g/L glucose) medium supplemented with 10% fetal bovine serum and 1x antibiotic–antimycotic (Gibco, USA). After isolation, the cells were cultured for 12 h to allow them to attach before being exposed to different oxygen conditions.
The purity of the isolated trophoblast was evaluated by staining with the specific cell markers anti‐cytokeratin 7 (CK7), anti‐vimentin, or anti‐von Willebrand factor (vWF) (Novus Biologicals). Cells were acquired by flow cytometry (BD FACS LSRII; BD Biosciences, USA) as described.
38 (link) The purity of the isolated trophoblasts was 91%–96%.
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