M11 25

4 h following the thawing, the zona pellucida was removed by briefly treating the embryos with acidic Tyrode’s solution (T1788, Sigma). Embryos were cultured in pre-equilibrated in vitro culture 1 (IVC1) culture medium (M11-25, Cell Guidance Systems). We introduced a modification to our original IVC1 recipe^{6 (link)} consisting of the addition of 50 ng/mL of Insulin Growth Factor 1 (IGF-1) (78078, STEMCELL Technologies), as this improved the preservation of epiblast and hypoblast cells in culture. Embryos were cultured in 8 well μ-slides tissue culture wells (80826, Ibidi) in approximately 300 μL volume per embryo per well. Half-media changes with IVC1 supplemented with IGF-1 were done every 24 h.

To isolate ICMs, we subjected late blastocysts to immunosurgery (Solter and Knowles, 1975 (link)). In brief, in vitro cultured single and double embryos were incubated with anti-mouse whole serum (M5774-2ML, Sigma-Aldrich) and sera complement guinea pig (S1639-5ML, Sigma-Aldrich) diluted to 20% in M2 medium for 45 min each. After elimination of trophectoderm cells by pipetting with a narrow glass capillary in M2 medium, ICMs were seeded in individual hanging drops of pre-warmed IVC-1 medium (Bedzhov et al., 2014 (link)) (M11-25, Cell Guidance Systems) and cultured for 24, 48, and 72 h at 37°C in 5% CO₂ (Figure 2A).