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.

中文翻译：

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正常人支气管上皮细胞（NHBE）细胞3D培养物的优化，用于体外肺模型研究。

需要强大的体外肺部模型来进行风险评估，以测量导致呼吸系统疾病的关键事件。原代正常人支气管上皮细胞（NHBE）代表良好的肺部模型，但获得分化良好的3D培养物可能具有挑战性。在这里，我们评估了在不同培养条件下扩展原代NHBE细胞同时保持其纤毛和杯状细胞以及离子通道功能等3D培养特征的能力。使用PneumaCult-Ex Plus（扩展培养基）/ PneumaCult-ALI（分化培养基）可以最佳地获得分化的培养物。原代细胞传代达四次，维持气道上皮的特征，纤毛的伪分层柱状上皮具有杯状细胞，跨上皮电阻（TEER）（> 400 Ohms.cm2），和囊性纤维化跨膜电导调节剂介导的短路电流（> 3 µA / cm2）。直到第6代，均未观察到纤毛搏动频率（CBF）或气道表面液（ASL）弯月面长度的变化。这项研究首次证明在传代的NHBE原代细胞中，CFTR离子通道功能和正常的上皮表型特征得以维持。此外，这项研究强调了在开发体外肺模型时，评估扩增和分化条件以实现最佳表型和功能终点（CBF，ASL，离子通道功能，分化细胞的存在，TEER）的重要性。直到第6代，均未观察到纤毛搏动频率（CBF）或气道表面液（ASL）弯月面长度的变化。这项研究首次证明在传代的NHBE原代细胞中，CFTR离子通道功能和正常的上皮表型特征得以维持。此外，这项研究强调了在开发体外肺模型时，评估扩增和分化条件以实现最佳表型和功能终点（CBF，ASL，离子通道功能，分化细胞的存在，TEER）的重要性。直到第6代，均未观察到纤毛搏动频率（CBF）或气道表面液（ASL）弯月面长度的变化。这项研究首次证明在传代的NHBE原代细胞中，CFTR离子通道功能和正常的上皮表型特征得以维持。此外，这项研究强调了在开发体外肺部模型时评估扩增和分化条件以实现最佳表型和功能终点（CBF，ASL，离子通道功能，分化细胞的存在，TEER）的重要性。