Pneumacult ali 10 supplement

Bioprinted and manually prepared Transwell inserts on 24-well plates were cultured for 37 d as shown in figure 1(B). The media consisted of PneumaCult-ALI basal medium (Stemcell Technologies) supplemented with PneumaCult-ALI 10× supplement, PneumaCult-ALI Maintenance Supplement (100×), Hydrocortisone stock solution, Heparin solution with the Rock Inhibitor (all from Stemcell Technologies) were mixed and added to both the apical (0.2 ml) and basal (0.65 ml) compartments. The samples were submerged for the first 10 d, which were referred as Day −9 to Day 0 (figure 1(B)). On Day 0, the apical culture medium was removed from the ALI culture and the basal chamber medium was replaced with the same PneumaCult ALI media but lacking the Rock Inhibitor. This medium was replaced every 2 d until Day 28. Excess mucus was removed from the apical surface by washing the cells once with DPBS (1X; Corning) as required but at least once per week to prevent excessive mucus accumulation.

Transwell inserts were coated with dECM solutions using three concentrations and seeded with HBECs at a cell density of 200,000 cells/cm². Once the cells reached ~100% confluency, the apical medium was removed, and the cells were basally fed to establish an air-liquid interface (ALI, this was called day 0 of ALI). After 24 h, the cells were basally fed with 750 µL PneumaCult-ALI Basal medium (StemCell Technologies; catalog number 05001, Vancouver, British Columbia, Canada) with PneumaCult-ALI 10× supplement, PneumaCult-ALI Maintenance 100× Supplement, 1% antibiotic-antimycotic, 0.5% hydrocortisone stock solution, and 0.2% heparin solution (StemCell Technologies; catalog number 7980) to support development and differentiation of a pseudo-stratified epithelial culture (day 1 of ALI). Transwell cell cultures were fed from the basal compartment every day, and TEER was measured after adding 200 µL PBS to the apical compartment [40 (link)]. TEER measurements were conducted every day until a peak was reached, and the measurements started to drop.