Rat Trophoblast Stem Cells under Hyperglycemia

Blastocyst-derived rat TS cells15 (link) were cultured in basal culture medium (RPMI 1640 (Cellgro), 20% fetal bovine serum (Sigma-Aldrich), 100 µM 2-mercaptoethanol (M7522; Sigma-Aldrich), 1 mM sodium pyruvate (ThermoFisher, 11 360–070), 50 µM penicillin (15140122; ThermoFisher), and 50 U/mL streptomycin (15140122; ThermoFisher)) supplemented with 70% rat embryonic fibroblast-conditioned medium, fibroblast growth factor 4 (25 ng/mL; Sigma-Aldrich), and heparin (1 µg/mL; Sigma-Aldrich). To model hyperglycemia, 25 mM glucose (Sigma-Aldrich) was added to the culture medium. D-mannitol (Sigma-Aldrich) was added to the culture medium to control for osmolality. Rat TS cells were exposed to ambient or low oxygen (0.5% or 1.5% O₂) tensions and 5% CO₂ at 37°C in an NAPCO 8000 incubator (ThermoFisher) for 24 hours and then harvested for immunocytochemical or biochemical analyses.