Isolation and Culture of Cytotrophoblasts

Isolation of cytotrophoblasts (CTBs) from term placental specimens obtained from cesarean delivery without labor was performed as described (Tang et al., 2011 (link); Guzeloglu-Kayisli et al., 2020 (link)). Briefly, after removal of decidual tissues, fragments of chorionic villi were subjected to enzymatic digestion in a Hanks’ Balanced Salt Solution (Invitrogen, Carlsbad, CA) solution containing 0.25% trypsin (Invitrogen), 0.2% DNase I (Roche, Indianapolis, IN), 25 mM HEPES, 2 mM CaCl₂ and 0.8 mM MgSO₄ in at 37°C for 90 min. Cell suspension were then separated using a discontinuous (30%, 35%, 45%, 50%) Percoll gradient (GE Healthcare, Piscataway, NJ). Cells in 35%–45% Percoll interface were collected and immune-purified using mouse anti-human CD9 (R&D Inc., Minneapolis, MN) and mouse anti-CD45 (R&D) antibodies for negative selection by removing fibroblasts and immune cells. Then, the unbound cells were collected, washed, and cultured in 6-well plates in DMEM/F12 (Gibco, Life Technologies Corporation, Grand Island, NY) containing 10% FBS (Gemini Bio Products, West Sacramento, CA). Following 24 h incubation in serum-free media, cells were treated with vehicle or recombinant human IL-11 (10 ng/ml, R&D) or IL-6 (10 ng/ml, R&D) or IL-11 + IL-6 for 6 h for microarray analysis (Monzon-Bordonaba et al., 2002 (link); Monhasery et al., 2016 (link); Strickland et al., 2017 (link))