Abstract
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.
Highlights
Maintenance of normal phenotypic characterization and function of airway epithelial cells when passaging primary cells is essential
In an attempt to understand how the culture conditions affect cell growth, epithelial phenotype and function, we investigated the effect of expansion media and passage numbers on primary normal human bronchial epithelial cells (NHBE) cells cultured at air-liquid interface (ALI)
NHBE cells grown in M2 media, or variations thereof, were only grown until passage four (P4) since it was already clear that M1 + Rho-associated kinase (ROCK) and M3 were more proficient for cell growth and expansion
Summary
Maintenance of normal phenotypic characterization and function of airway epithelial cells when passaging primary cells is essential. Due to the key function of the ion channel CFTR in airway fluid homeostasis and mucociliary clearance, assessment of its function will further enhance our knowledge of the primary 3D airway epithelium in vitro model[19,20,21]. To date, it is unknown whether passaged primary 3D airway epithelial cultures retain functioning CFTR channels. In an attempt to understand how the culture conditions affect cell growth, epithelial phenotype and function, we investigated the effect of expansion media and passage numbers on primary NHBE cells cultured at air-liquid interface (ALI). Several epithelial features were assessed, including bronchial epithelial phenotype, culture characteristics, cell integrity, differentiation, and function of ion channels
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