Abstract：
The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (>21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.
摘要：
早产新生儿气道上皮不断暴露于环境应激源。患有肺病的新生儿中的这些应激源之一包括高于周围大气的氧气(O2)张力-称为高氧(>21%O2)。高氧对气道的影响取决于各种因素，包括气道的发育阶段，高氧的程度,以及暴露的持续时间，可变的暴露可能导致独特的表型。虽然对高氧对新生儿肺泡化和气道高反应性的影响进行了广泛的研究，关于高氧对人新生儿气道上皮细胞的短期和长期潜在影响知之甚少。其主要原因是缺乏有效的体外模型来研究人新生儿气道上皮发育和功能。这里,我们描述了一种利用人新生儿气管抽吸物分离和扩增人新生儿气管气道上皮细胞（nTAEC）并在气液界面（ALI）培养物中培养这些细胞的方法。我们证明nTAEC在ALI培养物中形成成熟的极化细胞单层，并经历粘膜纤毛分化。我们还提出了一种使用专门的培养箱在ALI培养物中适度高氧暴露细胞单层的方法。此外,我们描述了一种使用荧光定量测量ALI培养物中高氧暴露后细胞氧化应激的测定法，这证实了中度高氧暴露会诱导细胞氧化应激，但不会引起明显的细胞膜损伤或细胞凋亡。该模型可用于模拟新生儿重症监护病房（NICU）中新生儿气道遇到的临床相关高氧暴露，并用于研究O2对新生儿气道上皮编程的短期和长期影响。使用该模型的研究可用于探索减轻早期气道氧化损伤的方法。这与前早产儿长期气道疾病的发展有关。
