BEBMTM Bronchial Epithelial Cell Growth Basal Medium

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	incubated at 37°C, 5% CO2.

Protocol tips
incubated at 37°C, 5% CO2.

Publication protocol

Cell expansion
Three different initial proliferation culture media were used to revive NHBE (LONZA) cells from cryopreservation, seeded as passage one (P1) into T25 cell culture tissue flasks: BEGM growth media (LONZA, Walkersville MD), PneumaCult-Ex (PnC-Ex) media (StemCell, Tukwila WA) or PneumaCult-Ex Plus (PnC-Ex-PLUS) media (StemCell, Tukwila WA). Cells were seeded at ~100,000 cells/T25 flask, and incubated at 37°C, 5% CO2. Once cells reached 70–80% confluency, they were dissociated using TryplE Express dissociation media (ThermoFisher, MA USA). From passage two (P2), different growth conditions were tested, including the use of a Rho kinase inhibitor (Y-27632; R&D Systems, Minneapolis MN) and/or feeder cells (mouse 3T3 fibroblast cells treated with mitomycyin C)

Cells cultured at Air-Liquid Interface (ALI)
Once cells had reached 70–80% confluency, they were dissociated and seeded on 6.5 mm Transwells (Corning; Fisher Scientific) coated with 0.3 mg/mL Collagen type IV from human placenta (Sigma-Aldrich, St. Louis MO). NHBE cells grown in BEGM + ROCK, PnC-Ex or PnC-Ex-PLUS media were seeded at 50,000 cells/well. Respective growth media was used to feed cells until 4–5 days or 100% confluency was reached. Upon reaching confluency, the apical media was removed and the basal media replaced with PneumaCult-ALI medium for all cell conditions. Medium was changed every second day and apical surfaces washed with HBSS (Ca2+, Mg2+) twice per week. All cells were grown until 4 weeks airlifted (37°C, 5% CO2) before various physiological and characterization tests were performed. Epithelial layers were grown from passaged cells until they failed to expand any further.





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Manufacturer protocol

Download the product protocol from Lonza for BEBMTM Bronchial Epithelial Cell Growth Basal Medium below.

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