In Vitro Analysis of Trophoblast and Arterial Endothelial Cell Responses to TCDD

Blastocyst-derived rat trophoblast stem (TS) cells previously generated in our laboratory (Asanoma et al. 2011) were cultured in rat TS cell medium [RPMI 1640, 20% (vol/vol) fetal bovine serum (FBS; ThermoFisher), 100μm 2-mercaptoethanol (M7522; Sigma-Aldrich), 1 mM sodium pyruvate (11360-070; ThermoFisher), 50μM penicillin (15140122; ThermoFisher), and 50U/mL streptomycin (15140122; ThermoFisher)] supplemented with 70% rat embryonic fibroblast conditioned medium prepared as previously described (Asanoma et al. 2011), fibroblast growth factor 4 (37.5 ng/mL; 100-31; Peprotech), and heparin (1.5μg/mL; H3149; Sigma-Aldrich). Rat arterial endothelial cells were purchased from VEC Technologies, Inc. and maintained in MCDB-131 complete culture medium. Cells were plated in 962-mm2 wells at ∼50–60% confluence and treated 12 h after plating. Cells were exposed to vehicle control (i.e., DMSO) or TCDD at 10 or 100μM, concentrations known to induce CYP1A1 in vitro (Knutson and Poland 1980). The DMSO concentration in the cell cultures was 0.05%. After 24 h, cells were harvested, medium removed, and total RNA isolated.