The procedures for in vitro culture and differentiation of primary porcine FTECs differentiation were described previously [20 (link)]. Briefly, porcine FT tissues were purchased from the Yamanashi Meat Logistics Center. FTECs were obtained by digesting and scraping the inside of opened porcine FTs using collagenase type IV (CLS4, Worthington, NJ, USA) and DNase I (9003-98-9, Sigma-Aldrich, St. Louis, MO, USA). To induce differentiation, cells were seeded onto a collagen type I coated 0.4-µm pore transwell (#3470, Corning, NY, USA) and basal media that included agonists or phytoestrogens were applied to the basal side in ALI culture. Reagents were as follows: β-estradiol (#E4389, Sigma-Aldrich); diarylpropionitrile (DPN) (#1428-67-7, Sigma-Aldrich); and PHTPP (#805239-56-9. Sigma-Aldrich). Phytoestrogens including genistein (#446-72-0, Wako, Osaka, Japan), daidzein (#486-66-8, Wako), glycitin (#40246-10-4, Wako), and coumestrol (#479-13-0, Cayman, Ann Arbor, MI, USA) were dissolved in dimethylformamide.
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