Isolation of First Trimester Cytotrophoblasts

Isolation of villous cytotrophoblasts from first trimester placenta was performed according to Haider et al. [29 (link)] and Vondra et al. [30 (link)] using three consecutive digestion steps followed by Percoll density gradient centrifugation (5–70%; Sigma-Aldrich, Taufkirchen, Germany). Placental tissues were washed with PBS and placental villi were minced. The digestion steps were performed in 50 mL tubes at 37 °C in a shaker with 200 rpm for 8 min, 15 min, and 15 min. The digestion buffer (10× HBSS (Hank’s Balanced Salt) [31 (link)], 7.5% NaHCO₃, 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) was supplemented with 0.25% trypsin (Gibco, Life Technologies, Carlsbad, CA, USA) and 1.25 mg/mL DNase I (Sigma-Aldrich, Taufkirchen, Germany). The second and third digestion steps were pooled and purified with a Percoll gradient as detailed for term placental cytotrophoblasts isolation (see below).