Dmem hg

Villous trophoblast cells were isolated from term placental tissue by enzymatic digestion and Percoll gradient separation, as previously described [13 (link)] with minor modifications. In brief, approximately 50 g of villous tissue was washed in 0.9% NaCl (Sigma, Saint Louis, MO, USA) four times for 5 min. Thereafter the tissue was minced and digested four times with 0.25% trypsin (Sigma, USA) and 300 IU/mL deoxyribonuclease I (Sigma, Saint Louis, MO, USA) at 37 °C (20 min each). The cell suspension was filtered and overlayed on fetal bovine serum (FBS, Seraglob, Schaffhausen, Switzerland). After centrifugation at 1000× g for 15 min at 10 °C, the cell pellet was collected in Dulbecco’s modified Eagle’s medium containing 4.5 g/L glucose (DMEM-HG, Gibco, Paisley, UK) basic medium (without FBS) and filtered through 100 µm strainer (BD Biosciences, Durham, NC, USA). Next, cells were overlayed on a discontinuous Percoll^® (Sigma, Saint Louis, MO, USA) density gradient. After centrifugation, CTBs were located at the layer corresponding to 1.046–1.065 g/mL (35% to 50%) density [26 (link)]. The isolated cells were cultured at a density of 0.2 × 10⁶ cells/cm² and 0.5 × 10⁶ cells/cm² in 6-well or 24-well CellBIND plates (Costar, Kennebunk, ME, USA), respectively, using complete DMEM-HG medium (including 10% FBS and 1× antibiotic-antimitotic (Gibco, Grand Island, NY, USA).

Primary sensory neurons were cultured in vitro according to a protocol described previously.¹⁶ Briefly, the Wistar rats were anaesthetized with chloral hydrate (10%, 3.3 mL/1 kg) and sterilized with 75% alcohol. Then, the DRG tissues were extracted and cut into pieces in cold DMEM/HG (Gibco, Waltham, MA, USA). The DRG tissues were incubated with 0.125% trypsin (Gibco, Waltham, MA, USA) in DMEM/HG for 30 minutes. After centrifugation (5 minutes, 1000 rpm/min), the cells were suspended in Neurobasal Medium (Invitrogen, Carlsbad, USA). The culture medium was supplemented with NGF (50 ng/mL, Sigma‐Aldrich, St. Louis, USA), B‐27 (20 μL/mL, Invitrogen, Carlsbad, USA) and L‐glutamine (1%, 0.2 mol/L, Gibco, USA). The cells were seeded into poly‐L‐lysine coated plates. The next day, NogoA‐Fc (4 mg/mL, R&D Systems, Minneapolis, USA) was used to mimic the inhibitory environment in vitro. Then, the miR‐30b agomir/antagomir or negative control and sema3A siRNA were added for transfection. The sema3A (100 ng/mL, R&D Systems, Minneapolis, USA)²⁴ and Y‐27632 (10 μmol/L, Selleckchem, Munich, Germany) were added into the culture medium immediately after transfection.

Primary cultures of cerebral neurons were obtained as described previously [32 (link)]. Briefly, mouse foetuses (embryonic day [E] 17) were removed from the uterus, and the individual foetuses were freed from the embryonic sac. Brain and cortical tissues were dissected and placed in high-glucose Dulbecco’s modified Eagle’s medium without phenol red (DMEM-HG; Gibco, Grand Island, NY, USA; Cat. No. 31053028). Papain solution (10 U/mL; Sigma-Aldrich, St. Louis, MO, USA; Cat. No. LS003126) was added to these cerebral tissues, which were then incubated for 15 min at 37°C in a 5% CO₂ incubator. Dissociated cortical cells were plated on poly-L-lysine (Sigma-Aldrich; Cat. No. P4832)-coated cell culture dishes and cultured in DMEM-HG (Gibco, Grand Island, NY, USA; Cat. No. 31053028) containing 10% foetal bovine serum (Gibco, Australia; Cat. No. 10099141) and 1% penicillin/streptomycin (P/S; Invitrogen, Carlsbad, CA, USA; Cat. No. 15140148) at a density of 1.0 × 10⁶ cells/mL. After 4 h of seeding, the medium was changed to neurobasal medium (NM; Gibco, Carlsbad; Cat. No. 21103049) supplemented with B-27 (Gibco, Grand Island, NY; USA Cat. No. 17504044). The cells were cultured in a humidified incubator at 37°C with 5% CO₂. The medium was changed every 3 days. Cultures were used for in vitro experiments after 7 days.