Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

Therapeutic oxygen (O₂) is one of the most used therapies in the neonatal intensive care unit (NICU)¹. Consequently, hyperoxia exposure (>21% O₂) is a common atmospheric stressor encountered by neonates with and without significant lung disease. Lung responses to hyperoxia can vary depending on the intensity and/or duration of exposure and the anatomical location, cell type, and stage of lung development^2,3,4,5,6. The bulk of the research in neonatal hyperoxia lung injury has been focused on the effect of hyperoxia exposure in the context of postnatal alveolarization to model bronchopulmonary dysplasia (BPD) – the most common chronic lung disease affecting preterm infants^{6,7,8,9,10,11}. BPD severity is classified by the amount of respiratory support, and O₂ needed at 36 weeks post-menstrual age⁹. Most babies with BPD improve clinically over time as the lungs continue to grow, with the majority weaning off respiratory support before their first birthday^12,13. Regardless of the severity of BPD after birth, significant morbidities that affect former preterm babies include a 5-fold increased risk of preschool wheezing, asthma, recurrent respiratory infections throughout childhood, and early onset chronic obstructive pulmonary disease^{12,14,15,16,17,18,19}. The effect of hyperoxia on long-term airway disease and pulmonary infections in preterm infants has been investigated using in vitro and in vivo animal models^{20,21,22,23,24}. However, most of these models focused on the role of mesenchymal tissue, alveolar epithelium, and airway smooth muscle^25,26,27,28.

The airway surface epithelium lines the entire path of the respiratory system, extending from the trachea down to the terminal bronchiole, ending just before the level of the alveoli²⁹. Airway basal cells are the primary stem cells in the airway epithelium, with the capacity to differentiate into the entire repertoire of mature airway epithelial lineages, which include ciliated and secretory cells (club cells: non-mucus producing, and goblet cells: mucus-producing)^29,30,31,32. Cell culture studies in the context of neonatal hyperoxic lung injury have mostly used adult human or mouse cancer cell lines^33,34. Additionally, most in vitro experiments have used submerged culture systems, which do not permit differentiation of the cells into a mucociliary airway epithelium resembling the in vivo airway epithelium in humans³⁵. Consequently, there is a gap in knowledge regarding the effects of hyperoxia-induced lung injury in developing airway epithelial cells of human neonates. One reason is the scarcity of translational models to study the effects of atmospheric exposure on human neonatal airway epithelium. Hyperoxic lung injury early in life can lead to long-term airway disease and increased risk of infection, resulting in life-altering consequences in former preterm infants^14,36,37. In non-surviving infants with severe BPD, airway surface epithelium has distinct abnormalities, including goblet cell hyperplasia and disordered ciliary development, denoting abnormal mucociliary clearance and increased epithelial height compromising airway caliber³⁸. In the last decade, there has been increased interest in culturing primary airway epithelial cells at the air-liquid interface (ALI) to study postnatal airway epithelial development^39,40,41,42. However, ALI models of neonatal airway epithelial cells have not been used in the context of atmospheric redox perturbation models such as hyperoxia exposure.

Using a previously published method³⁹, we have utilized neonatal tracheal aspirate samples obtained from intubated neonates in the NICU and successfully isolated and expanded primary neonatal tracheal airway epithelial cells (nTAECs). We have utilized inhibitors of Rho, Smad, Glycogen synthase kinase (GSK3), and mammalian target of rapamycin (mTOR) signaling to increase the expansion capacity and delay senescence in these cells, as described previously^39,42, which allows for efficient and later passaging of nTAECs. The protocol describes methods for establishing 3D ALI cultures using nTAECs and performing hyperoxia exposure on the nTAEC monolayers. Rho and Smad inhibition is used for the first 7 days of ALI culture (ALI days 0 to 7), after which these inhibitors are removed from the differentiation media for the rest of the ALI culture duration. The apical surface of the ALI-cultured airway epithelial cell monolayer stays exposed to the environment⁴³, which enables atmospheric perturbation studies and closely resembles the pathobiology of a developing neonatal airway exposed to hyperoxia in vivo. The concentration of O₂ used in previous cell culture studies (regardless of immortalized or primary cells) of neonatal hyperoxic lung injury varies significantly (ranging from 40% to 95%), as does the duration of exposure (ranging from 15 min to 10 days)^{36,44,45,46,47}. For this study, the ALI cell monolayer was exposed to 60% O₂ for 7 days from ALI day 7 to 14 (after removal of Rho/Smad inhibitors from the differentiation media). Hyperoxia exposure was performed during the early-mid phase of mucociliary differentiation (ALI day 7 to 14) as opposed to the fully differentiated mature epithelium and thus simulates the in vivo developing airway epithelium in preterm infants. This exposure strategy minimizes the risk of acute O₂ toxicity (which is expected with higher concentrations of O₂) while still exerting oxidative stress within a physiologically relevant range and resembles the critical window of transition from the relatively hypoxic intrauterine environment to a hyperoxic external environment in preterm human neonates.