Optimization of Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Studies.

Rayner RE, Makena P, Prasad GL, Cormet-Boyaka E.
Source: Scientific Reports
Publication Date: (2019)
Issue: 9(1): 500
Research Area:
Dermatology/Tissue Engineering
Respiratory Research
Cells used in publication:
Epithelial, bronchial (NHBE), human
Species: human
Tissue Origin: lung

Expansion of primary NHBE cells in three diferent expansion media prior to ALI. NHBE cells were cultured in three diferent expansion media from passage one (P1), BEGM medium (M1), PneumaCult-Ex medium (M2) or PneumaCult-Ex Plus medium (M3). Rho kinase inhibitor (ROCK) and/or 3T3 Swiss albino feeder cells (3T3) were added to growth media conditions from passage two (P2) in only the BEGM (M1) or PnC-Ex media (M2).*Cells were not passaged further in these conditions if they took longer than 7 days to reach ~75% confuency


Robust in vitro lung models are required for risk assessment to measure key events leading to respiratory diseases. Primary normal human bronchial epithelial cells (NHBE) represent a good lung model but obtaining well-differentiated 3D cultures can be challenging. Here, we evaluated the ability to expand primary NHBE cells in different culture conditions while maintaining their 3D culture characteristics such as ciliated and goblet cells, and ion channel function. Differentiated cultures were optimally obtained with PneumaCult-Ex Plus (expansion medium)/PneumaCult-ALI (differentiation medium). Primary cells passaged up to four times maintained airway epithelial characteristics as evidenced by ciliated pseudostratified columnar epithelium with goblet cells, trans-epithelial electrical resistance (TEER) (>400 Ohms.cm2), and cystic fibrosis transmembrane conductance regulator-mediated short-circuit currents (>3 µA/cm2). No change in ciliary beat frequency (CBF) or airway surface liquid (ASL) meniscus length was observed up to passage six. For the first time, this study demonstrates that CFTR ion channel function and normal epithelial phenotypic characteristics are maintained in passaged primary NHBE cells. Furthermore, this study highlights the criticality of evaluating expansion and differentiation conditions for achieving optimal phenotypic and functional endpoints (CBF, ASL, ion channel function, presence of differentiated cells, TEER) when developing in vitro lung models.