*4.7. Cell Lines*

Day 16 TE from 3 non-infected pregnancies was minced in DMEM-F-12 (1:1) medium supplemented with 10% bovine serum albumin, 1x penicillin-streptomycin-amphotericin B solution, 10 µg/mL insulin, 0.1 mM non-essential amino acids, 2 mM glutamine, and 1 mM sodium pyruvate. The minced tissue was spun down at 1000 rpm for 5 min and the supernatant was incubated in a 100-mm collagen-treated tissue culture dish at 37 ◦C and 5% CO2. After 24 h, the cells attached to the plate were washed and incubated with fresh complete medium. After 48–72 h, the cells were passaged and later collected at passage number 4–6 at 70–80% confluency to extract mRNA, miRNA, and proteins for further analysis. Western blot analysis for cytokeratin-7 (CK-7) was done to confirm the phenotype of OTR cells. To generate immortalized ovine trophoblast cells (iOTR cells), the OTR cells were infected with pLV-hTERT-IRES-hygro based lentiviral particles resuspended in complete DMEM-F12 (1:1) medium supplemented with 8 µg/mL polybrene transfection reagent. The media with viral particles was replaced with fresh media after 24 h. The cells were selected in complete DMEM-F12 (1:1) medium supplemented with 300–500 µg/mL hygromycin B (Sigma-Aldrich, St. Louis, MO, USA).
