Isolation of Amniotic Membrane Cells

Fetal membranes were washed with ice-cold phosphate-buffered saline (PBS, Corning, NY, USA) with 1% penicillin–streptomycin solution (10,000 U/mL penicillin, 10,000 U/mL streptomycin, Corning, Steuben County, NY, USA). The amniotic membrane was mechanically peeled off the underlying chorion layer to remove any blood clots. Then the tissues were incubated for 10 min at room temperature with PBS/ethylenediaminetetraacetic acid (EDTA) 0.5 mM. The amniotic membrane was then minced into small pieces (4 cm² approximately) and digested twice for 30 min at 37 °C using trypsin-EDTA 0.25% (Corning, Steuben County, NY, USA) with gentle shaking. For both digestion steps, trypsin was inactivated with fetal bovine serum (FBS, Gibco, Life Technologies, Carlsbad, CA, USA) and the cell suspension was centrifuged for 10 min at 390× g. The cell pellet was resuspended in basal culture medium, Dulbecco’s Modified Eagle’s Medium–high glucose (DMEM H., Corning, Steuben County, NY, USA) containing 10% FBS, 1% penicillin-streptomycin solution, and 10 ng/mL of epithelial growth factor (EGF, Sigma-Aldrich, St. Louis, MO, USA). The single-cell suspension was counted and tested for viability using erythrosin B (Sigma-Aldrich, St. Louis, MO, USA). Only samples with >90% viability were used for further assays.