Cigarette Smoke Exposure on BBB Endothelium

The hCMEC/D3 cells donated by Dr. Couraud (INSERM, Paris) (passage 29–30) were seeded on collagen coated cell culture flasks (2.0 × 10⁴/cm²) and maintained at 37°C with 5% CO₂ in EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), Fibroblast Growth Factor (Sigma Aldrich Inc), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA and HEPES (10 mM). Medium was changed once after 2 days and then every other day until cells formed a continuous confluent monolayer. BBB endothelial monolayers were then chronically exposed (3 times/24 h) to a final 5% CSE concentration [8 (link)] derived from freshly prepared smoke extracts diluted in EBM-2 culture media. Following CSE exposure cells were processed for RNA and protein collection. Please note that a 5% CSE final concentration (using 3R4F cigarettes as reference) yields a nicotine output of ≈100 ng/mL, which is comparable to the steady state blood nicotine concentration measured in an average chronic smoker (≈20 cigarettes/day) [8 (link), 49 (link)–51 (link)]. Experiments related to nicotine exposure as a standalone agent where performed by directly diluting nicotine in the culture media at the desired concentration (100 ng/mL).