2.6.3 Airway chip cell loading. 100 µl HMVEC-L cells (Lonza, CC-2527) were loaded into the vascular compartment at a concentration of 500 000 cells per ml EBM-2 media via
syringe assembly. The airway chips were inverted within a T-150 resealable flask housing and incubated overnight at 37 °C to allow attachment of the HMVEC cells to the basal side of the membrane. They were then returned to an upright position and 5 ml EBM-2 media was added to the vascular inlet reservoir to initiate vascular flow. 100 µl of normal human bronchial epithelial cells (NHBE) (Lonza, #CC-25410S) at a concentration of 1 × 106 cells per ml B-ALI basal media (Lonza, 00193516) were subsequently loaded via syringe assembly into the airway compartment and incubated overnight at 37 °C to facilitate attachment. 5 ml B-ALI basal media was then added to the airway chamber inlet reservoir to initiate flow. If the NHBE monolayer failed to achieve 95% confluency within 48 hours of initial seeding, the NHBE loading step was repeated.
2.6.4 Airway chip maintenance. When the NHBE monolayer reached 95% confluency within the airway
compartment, the airway chips were transitioned to airlift culture conditions. All media was removed from the inlet and exit reservoirs, as well as the airway compartment, via gentle suction with a 1000 µl pipet tip. 5 ml of B-ALI differentiation media (Lonza, 00193517) supplemented with 2 µl ml-
inducer was added to the vascular compartment inlet reservoir only. For the first week, differentiation media was
exchanged daily by removing effluent from the vascular exit reservoir and adding fresh media to the vascular inlet
reservoir. Thereafter, media was exchanged every 48 hours for the duration of the experiment. Every 3–4 days, 1× PBS was added to the airway compartment inlet to rinse the NHBE cells of mucous and cellular debris