Gelatin coated culture flasks

Human brain pericytes (ScienCell Research Laboratories, Carlsbad, CA, USA) were plated and expanded in Stemline medium (Sigma-Aldrich) supplemented with 2% foetal bovine serum (Invitrogen), 1% penicillin/streptomycin (Gibco), 20 ng/ml bFGF (Invitrogen) on gelatin-coated culture flasks (Nunc), and incubated at 37 °C in 5% CO₂ conditions (Heraeus HERAcell 150 CO₂ incubator, Thermo Scientific). Cells grew exponentially with a doubling time of approximately 48 h, reaching ca. 85% confluence after 48–72 h. The cells were seeded in six-well culture plates at 100,000 cells/well for the following experiments. The cells were expanded and used between passages 2–5. As previously described, human brain pericyte–conditioned medium was collected from non-treated pericyte-conditioned medium (CM) and pericytes treated with PDGF-BB (20 ng/ml, RD systems) (CM^PDGFBB) for 72 h (Gaceb et al. 2018 (link)). Briefly, cell medium was centrifuged at 1500g for 5 min, and the supernatant free from cell debris was collected and used for the following experiments. At 72 h, human brain pericyte–conditioned medium shows only a very low contamination with the added PDGF-BB (Gaceb et al. 2018 (link)).

We previously established and characterized an adult human brain pericytes line, obtained from brain tissue harvested from individuals undergoing ventriculostomy or surgery for intractable temporal lobe epilepsy as described [23 (link)]. All procedures were performed with informed written consent by the patient for the donation of brain tissue and approved by the ethical committee of the Scania University Hospital, Lund, Sweden. Using flow cytometry, our previous studies show that human brain pericytes express the key pericyte markers including PDGFRb (CD140b), CD146, CD105, and CD13[24 ] [25 ]and are negative for monocyte/macrophage markers CD14, the microglial marker CD11b and the endothelial marker CD31[10 (link),24 ,25 ]. The human brain pericytes were expanded in Stemline medium (Sigma-Aldrich) supplemented with 2% fetal bovine serum (FBS, Invitrogen), 1% Penicillin/Streptomycin (P/S, Gibco), 20 ng/ml basic fibroblast growth factor (bFGF, Invitrogen) on gelatin coated culture flasks (Nunc) and incubated at 37°C in 5% CO₂ conditions (Heraeus HERAcell 150 CO₂ incubator, Thermo Scientific). The cells grew exponentially with a doubling time of approximately 48 hs, reaching approximately 85% confluence after 48-72hs. Commercially available pericytes (ScienCell) were expanded in pericyte medium (ScienCell) according to the manufacturers instructions.

CI-muMECs (murine microvascular endothelial cells; #INS-CI-1004, InSCREENeX) were cultured in gelatin-coated culture flasks (Thermofisher Scientific) in high-glucose DMEM containing 20% FBS, 0.5% endothelial cell growth supplement from bovine neural tissue, 1% sodium pyruvate, 1% non-essential amino acids and antibiotics (all Sigma-Aldrich). For experiments, cells were plated in collagen type I BioFlex plates (Flexcell^® International) and transfected with control or zyxin siRNA (Qiagen) using MATra siRNA reagent (IBA) according to the manufacturer’s instructions. Following 72 h of culture, transfected cells were either exposed to cyclic stretch (15% elongation, 0.5 Hz) using a Flexcell^® FX-5000™ Tension System or incubated under static conditions for 6 h. Cells were then harvested and processed for protein analysis or RNA isolation.