The apical compartment was washed with phosphate buffered saline to remove residual growth factors of the media. On day three after the transfer to transwell inserts, when the cells were fully confluent, the apical and basolateral medium was removed and 1.5 ml Pneumacult-ALI-S Medium (Stemcell Technologies, Vancouver, Canada) was added basolaterally. On the following day, apical and basolateral media was exchanged using PneumaCult™-Ex Plus Medium. Subsequently, 100.000 cells per insert were seeded in 300 µl on the transwell membrane. Dissociation of SAEC was performed using the Animal Component-Free Cell Dissociation Kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer’s instructions.

The basolateral compartments of the transwells were filled with 1.5 ml PneumaCult™-Ex Plus Medium. Collagen solution was aspirated and transwell inserts were washed with phosphate buffered saline. The Transwells (#3460, Corning Life Sciences B.V., Amsterdam, the Netherlands) were coated with 300 µl rat tail collagen type 1 solution (Corning Life Sciences B.V., Amsterdam, the Netherlands, 30µg/ml in phosphate buffered saline) for 45 minutes at 37 ☌. 80 %) to be harvested for seeding onto transwell inserts. On the fourth day after seeding, the cells were sufficiently confluent (max. On the following day, the medium was exchanged, to remove residuals from the cryoprotectant cocktail. Human Small Airway Epithelial Cells (SAEC, CC-2547, Lonza, Basel, Switzerland) were thawed and approximately one million cells were seeded into a T175 cell culture flask with 50 ml pre-warmed PneumaCult™-Ex Plus Medium (Stemcell Technologies, Vancouver, Canada). Readouts were performed 24 hours post last exposure. The cells were exposed to CS three times a week during the differentiation phase (28 days) beginning on day 0 (= day of air-lift).

The smoke was diluted with ambient air (dilution rate: 0.5 l/min). Four cigarettes were smoked in parallel in compliance to ISO 3308, drawing every 15 seconds a puff from the sequentially smoked cigarettes (9 puffs per cigarette). (Professional In-Vitro Technologies) ExpoCube in the experimental setup developed by Fraunhofer Institute for Toxicology and Experimental Medicine. SAECs were exposed to whole cigarette smoke of K3R4F reference cigarettes (University of Kentucky, Lexington, KY, USA) using an automated cigarette smoking machine (In-Expose smoking robot, Scireq Montreal, QC, Canada) and the P.R.I.T. GEO help: Mouse over screen elements for information.Ĭell type: Small Airway Epithelial Cells (SAECs)

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