![]() However, resulting cells can have disrupted differentiation or lack crucial biomarkers typical of an in vivo airway epithelium, for example BEAS-2B cells may not express MUC5AC, the goblet cell secreted protein that plays a crucial role in mucociliary clearance 17. It is possible to immortalize primary human adult cells, such as with exogenous human telomerase reverse transcriptase (hTERT), viral oncoproteins (HPV-16 E6 and E7, or SV40 T-antigen), defective SV40 virus genome and additional cellular genes such as cdk4 8, 9, 10, 11, 12, 13, 14, 15, 16. Hence, most of the published studies use cells passage one or two even though some commercial primary cells are guaranteed to reach 10–15 population doublings (about 4 passages) before expansion rates dramatically decline, and differentiation potentially fails. In addition, obtaining well-differentiated cultures can be challenging. However, the use of primary cells for culturing 3D epithelial layers also presents its own limitations including their availability and price, rate of cellular expansion, and the number of time cells can divide. A recent review by Fang and Eglen 7 clearly outlines the limitations of 2D cellular cultures, and highlights the current 3D models. They are also often cancer-derived such as A549 and Calu-3 cells. Airway-derived cell-lines form monolayers (such as 16HBE and Calu-3) but do not consist of multiple differentiated cells, including ciliated and goblet cells, and may be more sensitive to treatment/disruptions. Therefore, development and validation of in vitro models to assess biological responses against disruption of the first line of defense in upper airways is crucial.ģD primary human airway epithelial cultures are increasingly used as an in vitro model for biological assessments to investigate function and mechanisms as well as to minimize the use of in vivo animal models. According to the World Health Organization (WHO), approximately 3 million deaths were due to COPD in 2015 (5% of all deaths globally in that year), and most of these deaths (90%) occur in low or middle-income countries. Disruption to this first line of defense can be caused by air pollution, genetic mutations, cigarette smoking, and pathogenic infections associated with diseases such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) 3, 4, 5, 6. A crucial balance of salt and water must be secreted into the airway surface liquid (ASL) to maintain mucus clearance. Healthy airways consist of a pseudostratified epithelium containing four primary cell types: basal stem cells which anchor the basement membrane, ciliated cells (50–70%) which use motile force to remove trapped particles and pathogens in the mucus, mucus (MUC5AC and MUC5B) producing goblet cells (~25%), and Club cells (~11%) expressing club cell-specific 10 kDa (CC10) secretory protein with anti-inflammatory and immune-modulating properties 1, 2. Mucociliary clearance in the human lungs is the first line of defense to continuously remove harmful aerosols, pathogens and toxins. 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. For the first time, this study demonstrates that CFTR ion channel function and normal epithelial phenotypic characteristics are maintained in passaged primary NHBE cells. No change in ciliary beat frequency (CBF) or airway surface liquid (ASL) meniscus length was observed up to passage six. 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.cm 2), and cystic fibrosis transmembrane conductance regulator-mediated short-circuit currents (>3 ♚/cm 2). Differentiated cultures were optimally obtained with PneumaCult-Ex Plus (expansion medium)/PneumaCult-ALI (differentiation medium). 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. Primary normal human bronchial epithelial cells (NHBE) represent a good lung model but obtaining well-differentiated 3D cultures can be challenging. Robust in vitro lung models are required for risk assessment to measure key events leading to respiratory diseases.
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