Dmem f12 hyclone

Cells were selected solely on the ability to adhere to plastic. Isolated cells attached
to plastic culture dishes more readily (Fig.
1Fig. 1.

Karyotype analysis of AF-MSCs obtained from the ovine fetus. (A) Primary cultured
cells of the sample. (B) Morphology of oAF-MSCs at passage 5. (C) Morphology of
oAF-MSCs after culture in vitro for 20 passages. (D) Karyotype
analysis of passage 20 diploid cells. The normal chromosome complement of 54 pairs
was detected.

). Under anesthesia, amniotic fluid samples were obtained by cesarean section from
pregnant sheep at the full-term stage of gestation. Samples were centrifuged at 230 ×
g for 5 min. Cells were then resuspended at a density of 5 ×
10⁴/ml in MSC-specific medium containing DMEM-F12 (HyClone; Thermo
Scientific, Beijing, China), 10% FBS (Gibco, Carlsbad), 1% GlutaMAX (Gibco), 1
µM dexamethasone (DSMS; Solarbio, Beijing, China), 2 ng/ml fibroblast
growth factor-basic (bFGF; PeproTech Inc., Rocky Hill, NJ, USA), 10 ng/ml epidermal growth
factor (EGF, Sigma, St. Louis, MO, USA), and 1% penicillin-streptomycin and plated in 9 cm
diameter dishes in a humidified atmosphere with 5% CO₂ at 37°C. The culture
medium was replaced every 3 days. Once adherent cells reached 80–90% confluency, they were
harvested using 0.25% trypsin/1 mM EDTA solution (Sigma) and subcultured at a ratio of
1:2. Third-passage cells were frozen for testing.

HESCs were isolated from the mid-secretory phase, and processed according to Sun et al. [20] (link) with minor modifications. First, the endometrial tissues were minced and enzymatically digested with 0.15% (w/v) collagenase I (Worthington Biochemical Corp, Lakewood, NJ, USA) for 30 min at 37 °C. Next, the digested tissues were filtered through a 30 μm-sieve gauze to separate the stromal cells from the glands. The endometrial stromal cells were maintained in DMEM/F12 (HyClone, Thermo Scientific, South Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Thermo Scientific, South Logan, UT, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin, seeded into culture dishes and incubated at 37 °C in 5% CO2. After 48 h, decidualization was induced in HESCs as described by Brosen et al. [21] (link). HESCs were cultured in phenol red-free DMEM/F12 (Gibco BRL/Invitrogen, Carlsbad, CA, USA) containing 2.5% charcoal/dextran-treated FBS (Thermo Scientific, South Logan, UT, USA), 100 IU/ml penicillin, and 100 μg/ml streptomycin. Treatment included 0.5 mM 8-Br-cAMP (B7880, Sigma-Aldrich, St. Louis, Mo, USA), 1 μM methoxyprogesterone acetate (MPA) (M1629, Sigma-Aldrich, St. Louis, Mo, USA) and/or 10 μM Wnt4 agonist (AG-L-67051, sc-222416, Santa Cruz Biotechnology, Dallas, TX, USA) .