Smooth muscle cell medium

Pulmonary arterial smooth muscle cells were isolated as described previously (Shimoda et al., 1998 ). Lungs were placed in cold HBSS and intrapulmonary arteries (PAs; 200–600 μm outer diameter) were isolated under a dissecting microscope and cleaned of adventitia and endothelium. The arteries were allowed to recover for at least 30 min on ice before being transferred to reduced-Ca²⁺ (20 μM CaCl₂) HBSS at room temperature for at least 20 min. The tissue was digested for 20–25 min at 37°C in reduced-Ca²⁺ HBSS containing collagenase (type I; 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml) and dithiothreitol (1 mM). PASMCs were obtained by gentle trituration in Ca²⁺-free HBSS using a modified P1000 pipette tip and cultured in Smooth Muscle Cell Medium supplemented with SmGM™-2 bullet kit (Lonza). PASMC phenotype and purity were validated by assessing the presence of smooth muscle-specific actin (SMA) using immunofluorescence in cells incubated with anti-SMA (1:1,000, Sigma) primary antibody and counterstained with DAPI. PASMCs were used at passages 0–3.

Intracranial aneurysm tissues were obtained from the brain tissues of 3 IA patients during microsurgery. The modified isolation protocol was based on a previously described study (Bygglin et al., 2011 (link); Huang et al., 2017 (link)). After clipping, the samples were immediately collected into DMEM supplemented with 5% penicillin/streptomycin. The tissue segments were washed three times with phosphate-buffered saline (PBS) supplemented with 1% penicillin/streptomycin. The surrounding connective tissue was separated, and the endothelial cell layer was scratched gently. Then, the tissues were dissected into 1 mm × 1 mm fragments that were evenly arranged on a dish and incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C in a medium comprising DMEM supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin. After 1–2 weeks, the cells grown from the IA explants reached semiconfluence and were subcultured in smooth muscle cell medium (Lonza, Basel, Switzerland) after trypsinization. Control human brain vascular SMCs were obtained from ScienCell Research Laboratories (Carlsbad, CA, United States) and cultured in smooth muscle cell medium. We used the markers anti-myosin-11 and SMA to identify the SMCs.