Critical Points for Culturing Human Bronchial Epithelial Cells at Air-Liquid Interface
Primary human bronchial epithelial cells (HBECs) can be differentiated to a physiologically relevant mucociliated phenotype using PneumaCult™-ALI
Submerged Cell Culture in PneumaCult™-Ex in Flasks Prior to Plating in Inserts:
- Start with viable, low passage HBEC cells. Cells that have been passaged for up to three passages are suitable for culture at the air-liquid interface (ALI culture), but please note that differentiation potential to ciliated and mucus-producing cells may decline as early as passage 4.
- When the submerged cultures reach approximately 80% confluence, the cells are ready to be passaged.
- Start with viable, low-mid passage HBEC cells. Cells that have been cultured for two additional passages, compared to other commercially available expansion media, are suitable for culture at the air-liquid interface (ALI culture). HBECs cultured in PneumaCult™-Ex Plus experience at least two more population doublings compared to those cultured in either PneumaCult™-Ex or BEGM™.
- When the submerged cultures reach approximately 50-70% confluence, the cells are ready to be passaged.
- When passaging cells into the Transwell® inserts, avoid the use of serum. We recommend using warm Trypsin-EDTA (Catalog #07910) to harvest the cells, and blocking the Trypsin using warm Soybean Trypsin Inhibitor (such as Sigma Catalog #T6522). Or for PneumaCult™-Ex Plus, use the Animal Component-Free Cell Dissociation Kit (Cat #05426).
- Once plated on the Transwell® inserts, culture HBECs in PneumaCult™-Ex or PneumaCult™-Ex Plus until confluence is reached. Typically, cultures take 2-4 days, but some donors may require extended time in the Expansion phase to reach confluence. Transitioning cultures that are < 80% confluent to air-lift is not recommended.
- Replace the PneumaCult™-Ex or PneumaCult™-Ex Plus medium in both the basal and apical chambers of the wells every 2 days.
- PneumaCult™-ALI medium is only present in the basal chamber. The medium should be replaced every 2 days.
- To avoid weekend feedings, medium can be replenished on Friday afternoons and then again on Monday morning.
- After 2 weeks at ALI, mucus may start to form. Excess mucus can be removed by gently washing the cells with 500uL warm Ca2+/Mg2+-free PBS. If needed, repeat weekly.
- For cultures with thicker mucus, add 500 µL of warm Ca2+/Mg2+-free PBS and incubate at 37°C for 30 minutes before pipetting or aspirating this solution off. A second wash may be required if mucus has been allowed to accumulate.
For further assistance and information, please contact techsupport@stemcell.com.
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