α modified minimum essential medium α mem

Human chorion (n=3) was obtained from placentas from term third-trimester pregnancies following delivery at Seoul St. Mary's Hospital. The chorionic tissue was washed in Dulbecco's phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA) and cut into small pieces (2ⅹ2 cm). The tissue was then incubated with 0.3% collagenase type I (Gibco) at 37 ℃ for 15-30 min. The digested tissue was subsequently passed through a 100 µm cell strainer (BD Falcon, Bedford, MA, USA) and the filtered cells were collected by centrifugation at 2500 rpm for 5 min. The cells were resuspended in α-modified minimum essential medium (α-MEM; Gibco), supplemented with 16.5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA), and cultured in T25 flasks (Nunc, Roskilde, Denmark) at 37 ℃ in 5% CO₂. This medium was changed twice weekly. When the primary cells (passage 0; P0) reached 70% confluency, they were trypsinized with 0.5% trypsin-EDTA (Gibco) and resuspended in T75 flasks (SPL, Pocheon, Kyounggi, Korea). The cells were subcultured repeatedly and were deemed ready for experimental use after passage 3.