Critical Points for Culturing Human Bronchial Epithelial Cells at Air-Liquid Interface

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

Contact STEMCELL Technologies

Our Customer Service, Sales, and Product and Scientific Support departments in North America are available between 6 am and 5 pm Pacific Time (9 am and 8 pm Eastern Time). One of our representatives will be happy to help you by telephone or email. Please complete the form to contact us by email. A representative will get back to you shortly.

StemCell Technologies Inc. and affiliates ("STEMCELL Technologies") does not share your email address with third parties. StemCell Technologies Inc. will use your email address to confirm your identity and send you newsletters, transaction-related emails, promotional and customer service emails in accordance with our privacy policy. You can change your email preferences at any time.