Investigating PDGF-D Effects on Brain Cells

Primary human brain vascular pericytes (HBVP) (ScienCell Research Laboratories, CA, USA; 1200) were used to investigate the molecular mechanisms underlying PDGF-D action. Moreover, immortalized human brain endothelial cells (iHBEC) (Cedarlane Laboratories, ON, Canada; CRL-3245) displaying major BBB features [43 (link), 44 (link)] were used to assess pericyte-endothelial cell interaction. HBVP were cultured at 37 °C in 5% CO₂, 95% air in a Dulbecco’s modified Eagle’s medium (DMEM) glucose-normal medium (Multicell; Wisent, QC, Canada) containing 2% fetal bovine serum (FBS), 100 U/mL pericytes growth serum (PGS) and 100 U/mL streptomycin/penicillin. For iHBEC, the surface of flasks/wells was pre-coated with 0.1% gelatin diluted in water (STEMCELL Technologies, BC, Canada) for at least 1 h before iHBEC were seeded. iHBEC were cultured at 37 °C in 5% CO₂, 95% air in endothelial cell medium (ECM; ScienCell) containing 2% FBS, 100 U/mL endothelial cell growth serum (ECGS), and 100 U/mL streptomycin/penicillin. In all experiments, cells were grown to 80% confluence and subjected to a maximum of 7 passages. Cells were treated with saline, 40 or 80 ng/mL of PDGF-D (R&D Systems). At the end of each experiment, cell culture medium was collected, and cells were harvested for protein extraction for further analysis.