Pro ox 110 oxygen controlling regulator

The human JEG–3 choriocarcinoma cell line was a kind gift from Professor Gregory Rice (University of Queensland, Brisbane, Australia). This cell line is derived from metastatic lesions of choriocarcinoma, and has an extravillous trophoblast phenotype [30 (link)]. JEG–3 cells were cultured in phenol red-free RPMI 1640 medium (Life Technologies, USA), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Scientific, Logan, USA), 1% (v/v) non-essential amino acids (Biological Industries, Israel) and 1mmol/L sodium pyruvate (Life Technologies, USA). The cell line was maintained at 37°C in a humidified incubator with 5% CO₂. For hypoxia experiments, JEG–3 cells were seeded in 10 cm² dishes and maintained in a humidified incubator (21% O₂, 5% CO₂, 37°C) for 16h prior to treatment. Cells were then incubated for different times (4, 8, 16 and 24h) either at 8% O₂ or at 1% O₂ in hypoxic C–474 chambers equipped with Pro-Ox 110 oxygen controlling regulator (Biospherix, Lacona, USA). The 0h time-point corresponded to cells left at 21% O₂ for 16h before hypoxia exposure.

The human HTR8/Svneo first trimester trophoblast cell line was purchased from the American Type Culture Collection (CRL-3271; Lot #70016636, ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium (GE Healthcare, Piscataway, NJ, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mmol/L sodium pyruvate, and 1% penicillin/streptomycin (P/S) (all Gibco, ThermoFisher, Waltham, MA, USA). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. For hypoxia experiments, cells were seeded in the appropriate cell culture plastic wells and maintained at 21%, 8%, 3%, or 1% O₂ for 24 h to 72 h prior to experiments. Hypoxic chamber C-474 equipped with Pro-Ox 110 oxygen controlling regulator (BioSpherix, Parish, NY, USA) was used.

To obtain a cell culture enriched in CSCs, we cultured ovarian cancer cells in spheres conditions under hypoxia [66 (link),67 (link),68 (link)]. Briefly, single HeyA8 cells were cultured in ultra-low attachment Petri dishes (Corning Incorporated, Corning, NY, USA) at a density of 10⁵ cells/mL in the stem cell medium (SCM): DMEM/F12 (1:1), 20 ng/mL epidermal growth factor (EGF; Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF; Sigma-Aldrich, San Luis, MO, USA), 5 ug/mL insulin (Sigma-Aldrich, San Luis, MO, USA), 1% antibiotic-antimycotic solution, and under hypoxic conditions (1% O₂) for 7 days in the hypoxic chambers C474 equipped with Pro-Ox 110 oxygen controlling regulator (BioSpherix, Parish, NY, USA).