Primary human bronchial epithelial cells (HBECs) can be expanded in submerged culture using PneumaCult™-Ex (Cat# 05008) or PneumaCult™-Ex Plus" (Cat# 05040), and differentiated to a physiologically relevant mucociliated phenotype at air liquid interface using PneumaCult™-ALI (Catalog #05001). Below we provide critical steps for expansion and differentiation of HBECs in our PneumaCult™, a defined, serum- and BPE-free cell culture media system.

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.

Submerged Cell Culture in PneumaCult™-Ex Plus in Flasks Prior to Plating in Inserts:

• 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.

Initiating submerged culture in well plates with Transwell® inserts:

• 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).

PneumaCult™-ALI Expansion Phase, submerged culture in Transwell® inserts:

• 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 Maintenance Phase, maintenance of ALI culture in Transwell® inserts:

• 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 more information about culturing HBECs in PneumaCult™-Ex, PneumaCult™-Ex Plus and PneumaCult™-ALI, please refer to the respective Product Information Sheets found on the product page under the Technical Resources tab.

For further assistance and information, please contact techsupport@stemcell.com.