Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Center for Pregnancy and Newborn Health, University of Oklahoma Health Sciences Center.
Department of Medicine, Section of Pulmonary, Critical Care & Sleep Medicine, University of Oklahoma Health Sciences Center.
J Vis Exp. 2024 Jul 12(209). doi: 10.3791/65913.
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.
早产儿的呼吸道上皮组织经常会暴露于环境压力源中。患有肺部疾病的新生儿所面临的其中一个压力源是，其吸入的氧气（O2）分压高于周围大气 - 即高氧（>21% O2）。高氧对呼吸道的影响取决于多种因素，包括气道的发育阶段、高氧的程度和暴露时间，不同的暴露条件可能导致不同的表型。尽管已经有大量研究关注高氧对新生儿肺泡和气道高反应性的影响，但对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。造成这种情况的一个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细胞的发育和功能。在这里，我们描述了一种从人新生儿气管抽吸物中分离和扩增人新生儿气管气道上皮细胞（nTAEC）的方法，并在气液界面（ALI）培养中培养这些细胞。我们证明 nTAEC 在 ALI 培养中形成成熟的极化细胞单层，并发生黏液纤毛分化。我们还提出了一种在 ALI 培养中使用专用培养箱对细胞单层进行适度高氧暴露的方法。此外，我们描述了一种使用荧光定量法测量 ALI 培养中高氧暴露后细胞氧化应激的测定方法，该方法证实适度高氧暴露会诱导细胞氧化应激，但不会导致显著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）中新生儿气道所遇到的临床相关高氧暴露，并用于研究 O2 对新生儿气道上皮细胞编程的短期和长期影响。使用该模型的研究可以用于探索减轻早期生命氧化损伤对发育中气道的方法，这与前早产儿长期气道疾病的发展有关。
