Gibco DMEM/F-12, HEPES

Protocol tips

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	B-27 Supplement (Gibco, 17504-044, 50×), N-2 Supplement (Gibco, 17502-048, 100×) and Glutamax (Gibco, 35050-061, 100×), with mixed feeders

Protocol tips
B-27 Supplement (Gibco, 17504-044, 50×), N-2 Supplement (Gibco, 17502-048, 100×) and Glutamax (Gibco, 35050-061, 100×), with mixed feeders

Publication protocol

The bovine trophoblast like stem cells (bTS) were established using the dual inhibitor system, consisting of DMEM/Ham’s F-12 and Neurobasal™ Medium (Gibco, 21103-049), with 0.5 μM PD0325901 (STEMCELL, 72182), 3 μM CHIR99021 (STEMCELL, 72052), B-27 Supplement (Gibco, 17504-044, 50×), N-2 Supplement (Gibco, 17502-048, 100×) and Glutamax (Gibco, 35050-061, 100×), with mixed feeders .L Wnt-3A/MEF cells (L Wnt-3A cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China, and MEF cells were isolated and stored by the lab) were mixed at a ratio of 1:1 and were treated with mitomycin C (17 mg/mL; Haizheng, Tai Zhou, Zhe Jiang, China) for 2.5–3 h at 37 °C (Huang et al. 2014; Li et al. 2008) were used in coculture with bovine EECs. The culture medium of this system was named 2i culture medium.

EECs(Endometrial Epithelial Cells) were cultured in 4-well plates for 36 h, and then the culture medium (DMEM/Ham’s F-12; 15 % FBS; 0.1 % P.S.) was changed to 2i culture medium. Then, bTS cells (bTS cells need mechanical passaging, the cell colonies were cut into small pieces by the edge of injector needle per well, 0.5 × 0.5 mm2) were transferred into the wells containing the EECs. The 2i cell culture medium was changed 48 h later, after the cells were initially seeded into the plates, and then medium was changed every 24 h.


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