Rat Trophoblast Stem Cell Culture and Exposure Assay

Blastocyst-derived rat trophoblast stem (TS) cells previously generated in our laboratory (Asanoma et al. 2011 (link)) were cultured in rat TS cell medium [RPMI 1640, 20% (vol/vol) fetal bovine serum (FBS; ThermoFisher), $100\mathrm{\mu }\mathrm{m}$ 2-mercaptoethanol (M7522; Sigma-Aldrich), $1\text{\hspace{0.17em}mM}$ sodium pyruvate (11360-070; ThermoFisher), $50\mathrm{\mu }\mathrm{M}$ penicillin (15140122; ThermoFisher), and $50\mathrm{U}/\mathrm{mL}$ streptomycin (15140122; ThermoFisher)] supplemented with 70% rat embryonic fibroblast conditioned medium prepared as previously described (Asanoma et al. 2011 (link)), fibroblast growth factor 4 ( $37.5\text{\hspace{0.17em}ng}/\mathrm{mL}$ ; 100-31; Peprotech), and heparin ( $1.5\mathrm{\mu }\mathrm{g}/\mathrm{mL}$ ; 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 $962{\text{-mm}}^2$ wells at $\sim 50–60\%$ confluence and treated 12 h after plating. Cells were exposed to vehicle control (i.e., DMSO) or TCDD at 10 or $100\mathrm{\mu }\mathrm{M}$ , concentrations known to induce CYP1A1 in vitro (Knutson and Poland 1980 (link)). The DMSO concentration in the cell cultures was 0.05%. After 24 h, cells were harvested, medium removed, and total RNA isolated.