We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.
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.
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 development2,3,4,5,6. 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 infants6,7,8,9,10,11. 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 birthday12,13. 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 disease12,14,15,16,17,18,19. 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 models20,21,22,23,24. However, most of these models focused on the role of mesenchymal tissue, alveolar epithelium, and airway smooth muscle25,26,27,28.
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 lines33,34. 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 infants14,36,37. 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 development39,40,41,42. 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 previously39,42, 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.
Neonatal tracheal aspirate samples were collected only after informed consent from parents, and the protocol used for collection, transport, and storage has been approved by the Institutional Review Board (IRB) of the University of Oklahoma Health Sciences Center (IRB 14377).
1. Preparation for isolation, passaging, and ALI culture of nTAEC
2. Isolation and expansion of nTAECs and preparation for subsequent ALI culture
3. ALI culture of nTAECs
NOTE: Cell culture inserts should be coated with 804G-conditioned media and incubated for at least 4 h at 37 °C, 5% CO2 before the next step (see step 1.2).
4. Testing barrier function during ALI differentiation
5. Hyperoxia exposure using TriGas incubator
6. Oxidative stress assay to assess the effects of hyperoxia
NOTE: We used the CM-H2DCFDA assay kit and measured fluorescence intensity on ALI day 14 as a marker of oxidative stress in cell culture inserts following O2 exposure. H2DCFDA is a chemically reduced and acetylated form of 2′,7′-dichlorofluorescein (DCF) which is a live cell-permeant indicator of reactive oxygen species (ROS). CM-H2DCFDA is the thiol-reactive chloromethyl derivative of H2DCFDA, which promotes further covalent binding to intracellular components, allowing longer retention of the dye within the cell. These molecules are nonfluorescent until the acetate groups are removed by the action of intracellular esterases, and oxidation occurs in the cell51. Following intracellular oxidation, the resultant increase in fluorescence can be measured with a fluorescent microscope as a surrogate measure of cellular oxidative stress52. The reagent is light and air-sensitive and thus needs to be protected from light and kept airtight as far as possible.
To isolate nTAECs, we collected tracheal aspirates from intubated neonates in the NICU and transported the aspirates on ice to the lab for further processing (Figure 1A). After seeding the tracheal aspirate samples in airway epithelial growth medium (BLEAM-I containing Rho/Smad, GSK3, and mTOR inhibitors), cuboidal cells appeared within 7-10 days. By 14 days, the cells were 50%-60% confluent, and around 21 days post-plating, the cells were densely packed and required trypsinization for subsequent passaging and expansion. Serial passages of these cells (up to P3) were cryopreserved in liquid nitrogen for long-term storage. The cells (≤ passage 3) were then thawed out for ALI culture.
nTAECs were cultured in ALI once they reached around 80%-90% confluency in a T75 plate in submerged conditions. We performed immunofluorescence staining of the nTAEC monolayer on ALI day 0 with the basal cell-specific markers – cytokeratin-5 (KRT5)53 and tumor protein p63 (TP63)54 (Figure 1B). On ALI day 0, nearly all the cells are KRT5 and TP63 positive, suggesting airway basal cell phenotype with minimal differentiation. After ALI was established, TEER was measured using EVOM (Figure 1C) and rapidly increased in the early phase of ALI culture (ALI day 0 to 7) as nTAECs started forming a mature polarized cell monolayer, with subsequent plateauing of TEER between ALI day 7 and day 28 (Figure 1D). Further, FITC-dextran of two molecular weights (10 kD and 20 kD) were used to assess paracellular permeability during ALI differentiation and showed an overall gradual decrease of fluorescence over time (Figure 1E). This correlates with the formation of a mature cell-monolayer during ALI differentiation with decreasing permeability to solutes.
During ALI culture, basal cells differentiate into mature epithelial lineages, which include ciliated cells, non-mucus-secreting club cells, and mucus-secreting goblet cells39. Gene expression analysis of mature epithelial cell-specific markers on ALI day 0 showed minimal to no expression of forkhead box protein J155 (FOXJ1, ciliated cell marker), uteroglobin56 (SCGB1A1, club cell marker) and mucin-5AC57 (MUC5AC, goblet cell marker; Figure 2A), with subsequent increase (ALI day 7 and 28) suggesting differentiation into these mature airway epithelial subtypes during ALI culture. Additionally, we performed immunofluorescent staining on ALI day 28 utilizing mature epithelial cell-specific markers and quantified epithelial differentiation consisting of acetylated-tubulin58 (TUBA4A) positive ciliated cells (~22%), SCGB1A1 positive club cells (~13%) and MUC5AC positive goblet cells (~3%) with the rest being KRT5 positive basal cells (~51%) and other rare cell types (Figure 2B).
Rho and Smad inhibitors were used during the first 7 days of ALI culture (ALI days 0 to 7) and then removed from the differentiation media for the rest of the ALI culture duration (Figure 3A). The apical surface of the cell monolayer during ALI culture is exposed to air, enabling atmospheric perturbation studies. Hyperoxia exposure of the cell monolayer was performed with 60% O2 continuously for 7 days from ALI day 7 to 14 utilizing the TriGas Incubator. The control group was maintained in a separate incubator with room air (21% O2) exposure. Given the future intent to study long-lasting consequences (1-2 weeks following completion of O2 exposure) of O2 exposure on airway epithelial development and regeneration, it was essential that the O2 exposure did not acutely result in severe cytotoxicity or membrane damage to the extent that it significantly disrupts the ALI cell monolayer. To assess cell membrane damage, we measured LDH release in the basolateral media on ALI day 14, which showed no significant difference between hyperoxia and room air-treated cultures (Figure 3B). To further assess if cytotoxic effects from O2 were significant enough to induce apoptosis in nTAECs, we performed immunoblot for total caspase-3 and cleaved caspase-359 on cell lysates harvested from hyperoxia and room air exposed groups on ALI day 14 (Figure 3C). Total caspase-3 expression was comparable between the two groups, and cleaved caspase-3 expression was not detected, suggesting that 7 days of 60% O2 exposure did not induce apoptosis in nTAECs on ALI day 14. Additionally, a positive control was designed with overnight incubation of nTAECs under submerged conditions with increasing concentrations of staurosporine (0.5 µM, 1 µM, and 2 µM), a cytotoxic agent, and an apoptosis inducer. Immunoblotting for total caspase-3 and cleaved caspase-3 expression confirmed successful induction of apoptosis. To measure oxidative stress induced by 60% O2 exposure, a cellular oxidative stress assay with fluorescent intensity measurement was performed on the nTAEC cell monolayers on ALI 14 (Figure 3D). Hyperoxia exposure caused a significant increase in fluorescent intensity (calculated by corrected total cell fluorescence or CTCF) compared to room air exposed cells, suggesting increased cellular oxidative stress. Gene expression analysis of antioxidant genes showed that 60% O2 caused upregulation of these antioxidant genes on ALI day 14, with catalase, SOD1, SOD2, and GPX3 being significantly upregulated (Figure 3E).
Figure 1: Neonatal tracheal airway epithelial cells (nTAECs) are successfully isolated from neonatal tracheal aspirates and cultured in air-liquid interface (ALI). (A) nTAECs isolated utilizing tracheal aspiration from intubated neonates were passaged in submerged condition with subsequent ALI culture. (B) Cells were harvested on ALI day 0 for immunofluorescent staining with airway basal cell markers – KRT5 (red) and TP63 (red). Nuclei are stained blue with DAPI. Scale bar = 50 µm. (C) Measurement of trans-epithelial electric resistance (TEER) in cell culture inserts using an Epithelial Volt/Ohm Meter (EVOM). (D) TEER was measured during ALI differentiation. Data presented as mean (n=2 donors, 5-10 wells per donor per timepoint). Error bars indicate SEM. (E) Fluorescein isothiocyanate-dextran (FITC-dextran) trans-epithelial permeability assay was performed during ALI differentiation utilizing two different molecular weights of FITC-dextran (10 kD and 20 kD). FITC-dextran solution (10 kD or 20 kD) was added to the apical chamber, and the cell culture inserts were incubated at room temperature for 60 min. Fluorescence in the basolateral chamber media was measured spectrophotometrically in 96-well plates. Background fluorescence (HBTEC media) was subtracted from the test measurements to obtain a corrected fluorescence value (expressed as a percentage compared to day 0 values). Data presented as mean (n=2 donors, three wells per donor per timepoint for each molecular weight of FITC-dextran). Error bars indicate SEM. Please click here to view a larger version of this figure.
Figure 2: Neonatal tracheal airway epithelial cells (nTAECs) undergo mucociliary differentiation during air-liquid interface (ALI) culture. (A) Cells were harvested on ALI days 0, 7, and 28 for qPCR analysis of epithelial cell-specific markers: cytokeratine-5 (KRT5) for basal, forkhead box protein J1 (FOXJ1) for ciliated, uteroglobin (SCGB1A1) for club and mucin-5AC (MUC5AC) for goblet cells. Relative expression of each gene was determined using the δCt method with GAPDH as the endogenous control. Data presented as mean (from n = 3 donors, three wells per donor per timepoint). Error bars indicate SEM. Statistical analysis was performed utilizing two-way ANOVA with Tukey post-hoc analysis, *p=0.0114, ***p=0.0005, ***p<0.0001. (B) Immunofluorescent staining of basal (KRT5, red), ciliated (acetylated-tubulin or TUBA4A, green), club (SCGB1A1, red), and goblet (MUC5AC, green) cells were performed on ALI day 28 to quantify epithelial differentiation. Nuclei are stained blue with DAPI. Data for percentage (%) positive cells presented as mean (n=2 donors, two cell culture inserts per donor, 6-8 non-overlapping images per slide, minimum of 3000 cells counted per slide). Error bars indicate SEM. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Figure 3: Hyperoxia induces cellular oxidative stress in neonatal tracheal airway epithelial cells (nTAECs) but does not cause significant cell membrane damage or apoptosis. (A) Experimental timeline for hyperoxia exposure of nTAECs using the Trigas incubator. Oxygen exposure was started after inhibitors were removed from the differentiation media on ALI day 7 and continued for 7 days (ALI day 7 to 14). (B) Basolateral media was harvested for LDH release assay to assess cell membrane damage between room air (control) and 60% O2 (hyperoxia) exposed group on ALI day 14. Data presented as individual data points (corrected absorbance) with lines denoting the grand mean for each group, n=3 donors denoted by black circles, squares, and triangles with four wells per donor, respectively. Statistical analysis was performed utilizing an unpaired t-test, p=0.6184. (C) Cells were harvested on ALI day 14, and immunoblotting for markers of apoptosis (caspase-3 and cleaved caspase-3) was performed to compare responses between room air and O2-exposed nTAECs. Positive control for the assay was designed by incubating nTAECs overnight with staurosporine (Stau 0.5 µM, 1 µM, and 2 µM) or vehicle control (Veh Ctrl) in a submerged culture condition. Cells were harvested for immunoblot with total caspase-3 and cleaved caspase-3 antibody. Densitometry was performed with α-tubulin as a housekeeping gene. Data presented as mean (from n=2-3 donors, one to two wells per donor). Error bars indicate SEM. An unpaired t-test was used to compare total caspase-3 expression between room air and the 60% O2 group, p=0.4. One-way ANOVA was used to compare total caspase-3 and cleaved caspase-3 expression between Veh Ctrl and Stau exposed groups, *p<0.05, ***p<0.0005. (D) CM-H2DCFDA assay for cellular oxidative stress was performed on ALI day 14 with cell culture inserts harvested from room air and O2-exposed nTAECs. Representative fluorescent microscopy images of room air (control) and 60% O2 (hyperoxia) exposed cell culture inserts are shown. Corrected total cell fluorescence (CTCF) was calculated to compare cellular oxidative stress between room air and 60% O2-exposed nTAECs. CTCF data presented as individual data points with line denoting the grand mean for each group, 6-7 images/group from 2 individual donors (black circles and squares) with two wells per donor. Statistical analysis was performed utilizing an unpaired t-test, *p<0.05. Scale bar = 50 µm. (E) Cells were harvested on ALI day 14 from room air, and hyperoxia-exposed wells for qPCR analysis of oxidative stress genes, including catalase, superoxide dismutase 1 & 2 (SOD1 & SOD2), glutathione peroxidase 1, 2, and 3 (GPX1, GPX2, and GPX3) was performed. Fold change data of each gene was determined using the δCt method with 18s rRNA as endogenous control. Data presented as mean (from n=3 donors, two wells per donor). Error bars indicate SEM. Statistical analysis was performed utilizing an unpaired t-test, *p<0.05, **p=0.006. Please click here to view a larger version of this figure.
Media component | Amount | Final concentration | ||
BLEAM-I | ||||
BLEAM media | 500 mL | |||
HLL supplement | 1.25 mL | 500 µg/mL human serum albumin 0.6 µM Linoleic acid; 0.6 µg/mL Lecithin |
||
L-Glutamine | 15 mL | 6mM | ||
Extract P | 2 mL | 0.4% bovine pituitary extract | ||
TM1 | 5 mL | 1 µM Epinephrine; 5 µg/mL Transferrin 10 nM Triiodothyronine; 0.1 µg/mL Hydrocortisone 5ng/mL Epidermal growth factor (EGF) 5 ng/mL Insulin |
||
Normocin | 1 mL | 0.1 mg/mL | ||
Y-27632 | May vary based on lot number | 5 µM | ||
A83-01 | May vary based on lot number | 1 µM | ||
CHIR 99021 | May vary based on lot number | 0.4 µM | ||
Rapamycin | May vary based on lot number | 5 nM | ||
HBTEC | ||||
HBTEC media | 500 mL | |||
Normocin | 1 mL | 0.1 mg/mL | ||
HBTEC-I | ||||
HBTEC media | 500 mL | |||
Normocin | 1 mL | 0.1 mg/mL | ||
Y-27632 | May vary based on lot number | 5 µM | ||
A83-01 | May vary based on lot number | 0.5 µM |
Table 1: Media components for Bronchial epithelial airway medium with inhibitors (BLEAM-I), Human bronchial/tracheal epithelial cell ALI media with inhibitors (HBTEC-I), and HBTEC.
Cell culture flask/Cell culture inserts | 804G cell-conditioned media (mL) |
T25 flask | 3 mL |
T75 flask | 5 mL |
24-well cell culture insert (0.4 µm pore-size, 0.33 cm2 surface area) | 400 µL |
Table 2: Volumes of 804G cell-derived matrix-conditioned media required for culture flasks and cell culture inserts.
The protocol described here details a method for the collection and processing of neonatal tracheal aspirate samples from intubated neonates in the NICU with subsequent isolation and expansion of live nTAECs from these samples using previously established methods39. Furthermore, we have described a method for culturing nTAECs on ALI and characterizing their differentiation into a polarized mucociliary airway epithelium as a function of time via measurement of TEER, FITC-dextran assay, immunofluorescent staining, and qPCR analysis of cell type (i.e., basal, ciliated, club and goblet) specific markers. The novel aspect of this protocol is the atmospheric redox perturbation performed through moderate hyperoxia (60% O2) exposure on developing nTAEC monolayers. We have shown that 60% O2 exposure for 7 days induces cellular oxidative stress in nTAECs. We further show that the O2 exposure strategy does not acutely induce significant cellular apoptosis or cell membrane damage, which allows maintenance of these nTAECs in ALI culture beyond the time of completion of O2 exposure. Thus, the model can potentially be used to study both short- and long-lasting effects of O2 exposure on neonatal airway epithelial programming.
Culturing nTAECs in ALI confers certain advantages over conventional submerged culture as it promotes the formation of a polarized airway epithelial monolayer with tight junction formation and mucociliary differentiation60,61. Previous studies with nTAECs have shown that in the early phase of ALI culture (ALI day 0 to 7), the cells undergo pseudo-stratification (appearing as multi-layered as opposed to single cell layer) and finally form a mature pseudostratified epithelium by the end of second week42,62. Tight junction formation follows a similar trajectory in the early phase of ALI culture accompanied by a rapid increase in TEER with later plateauing63, demonstrated in the model with nTAECs. Further, airway epithelial cells cultured in ALI enhance expression and apico-lateral localization of claudin-1 (a component of tight junction complex), which augments the sealing properties of tight junctions during ALI culture, promoting the formation of a mature cell monolayer63. This correlates with decreased paracellular permeability, which was tested in the model with FITC-dextran assay and demonstrated a generally decreasing trend in monolayer permeability. We have delineated the mucociliary differentiation pattern in nTAECs with immunostaining for ciliated (TUBA4A), club (SCGB1A1), and goblet (MUC5AC) cell-specific markers on ALI day 28. Additionally, we performed gene expression of ciliated (FOXJ1), club (SCGB1A1), and goblet (MUC5AC) cell-specific markers during the ALI time course. Other groups have shown that the transcription factor FOXJ1 is essential for motile ciliogenesis, while TUBA4A is present in both primary and motile cilia64,65. In this model, the gene expression pattern for FOXJ1 during ALI differentiation of nTAECs roughly correlates with the time of appearance of motile cilia in airway epithelial culture from previous studies64,66.
A recent study by Teape et al. assessed proteomic and metabolomic effects of hyperoxia exposure on ALI culture67. However, cells were derived from adult donors. Previous studies on respiratory syncytial virus (RSV)62,68,69,70 and the recent COVID-19 pandemic71 have highlighted the importance of age-related differences in airway epithelial response and function. Zhao et al. utilized similar workflows to isolate neonatal and adult airway epithelial cells and showed that after 21 days of ALI differentiation, there were no significant differences in the cellular composition between neonatal and adult airway epithelial cells62. However, there were distinct differences in their response to RSV infection, with neonatal cells showing more RSV-infected cells and epithelial damage compared to adults. In general, the culture conditions utilized by Zhao et al. produced a more robust differentiation in nTAECs compared to ours, with a higher number of ciliated (41% vs 22%) and goblet (10% vs 3%) cells. These differences could be attributed to the use of different growth media. The PneumaCult-ALI medium used by their group (as opposed to the HBTEC-ALI medium) has been shown to induce more robust differentiation with higher numbers of ciliated and goblet cells72. In this model, gene expression of cell-type specific markers for ciliated, club, and goblet cells showed an initial rapid increase (ALI day 0 to 7) with later decrement around ALI day 28. A possible reason could be the plateauing of differentiation after ALI day 14, which has been demonstrated in previous studies with nTAECs42.
We have described the use of moderate hyperoxia (60% O2) exposure for 7 days, which we think more closely simulates the real-life experiences of preterm neonates in the NICU compared to the use of very high O2 levels (> 80% O2)67 where the risk of acute O2 toxicity is increased1,5. Indeed, 95% O2 exposure of newborn lung tissue was shown to induce cellular apoptosis through caspase activation73. However, this exposure strategy is relevant in only a minor percentage of babies in the NICU who, as a consequence of the O2-induced acute lung injury, develop severe BPD with chronic bronchial pathologies (acute lung injury model)37. In the modern era of judicious use of O2 therapy, most premature babies do not get exposed to such high O2; however, they are still at risk of developing future airway disease (developmental perturbation model). In this latter group, the injury from early-life redox perturbation may be silent initially but manifest later during toddler age or beyond in the form of chronic airway disease37. Our data utilizing cellular oxidative stress assay showed that moderate hyperoxia exposure (60% O2) significantly increased oxidative stress within the nTAEC cell monolayer. Analysis of LDH release and cellular caspase-3 activation suggested that our hyperoxia exposure strategy did not cause significant cell death. The methods and the model system we describe can potentially be utilized to uncover O2-sensitive molecular mechanisms early in life as a link to the later development of pulmonary disease in former preterm infants.
During fetal life, airway epithelial cells are submerged in lung fluid and exist in the relatively hypoxic intrauterine environment26. Neonatal airway epithelial cells undergo rapid turnover and differentiation and form a critical barrier between the lung and the outside world. As newborns transition to postnatal life, airway epithelial cells are exposed to a relatively hyperoxic postnatal environment. This transition is rendered more abrupt in preterm and clinically ill newborns as they also routinely require therapeutic O2 as part of their care40,74, placing them at high risk of oxidative injury after birth1. While the effects of hyperoxia on postnatal lung alveolarization have been well characterized using animal models75, the effects of hyperoxia exposure on developing airway epithelial cells are relatively understudied38. The use of human neonatal-derived airway epithelial cells with physiologically relevant hyperoxia exposure during ALI adds translational relevance to our model. Additionally, the use of hyperoxia exposure during the early-mid phase of active differentiation and cell monolayer formation rather than fully differentiated mature airway epithelium67 potentially simulates the clinical scenario in extremely preterm newborns (< 28 weeks of gestation) who are born during the earlier stages (canalicular to saccular) of lung development rather than the alveolar stage7.
Our model has limitations that must be considered when applying it to human development and disease. We have used conditional reprogramming of primary neonatal airway epithelial cells previously published in an ALI model of nTAECs39. The use of growth factor inhibitors (Rho/Smad/GSK3/mTOR) enables faster and later passaging of these cells. We have attempted to culture nTAECs in both submerged and ALI conditions without inhibitors in the growth medium; however, expansion and passaging of these cells were significantly slower, and the cell monolayer in ALI became leaky by the end of the second week (data not shown). Another group utilizing the same inhibitors for culturing primary neonatal airway epithelial cells42 performed single-cell RNA-sequencing to show that the presence of these inhibitors did not cause significant differential expression of genes involved in oxidative stress, epithelial-mesenchymal transition, or cellular senescence. Nonetheless, we considered this limitation in our model and decided to perform hyperoxia exposure after Rho and Smad inhibitors were removed from the differentiation media. For this study, we have only tested moderate hyperoxia (60% O2) effects on nTAECs. However, future studies intend to utilize graded hyperoxia exposure (40%, 60%, and 95% O2) and compare the responses in nTAECs. While the model described here has translational appeal given the use of cells from human neonates admitted in the NICU, this is an in vitro cell culture model, and thus, significant findings utilizing this model would warrant confirmation with a complementary in vivo animal model.
In conclusion, we have described the step-by-step method for establishing an in vitro 3D ALI culture model utilizing neonatal airway epithelial cells isolated from tracheal aspirates of intubated neonates in the NICU. We have used this model to examine the effects of moderate hyperoxia exposure, a common environmental insult in premature neonates in the NICU. Hyperoxia exposure induced cellular oxidative stress in the cell monolayer without causing significant cell membrane damage or cell death, allowing the continuation of ALI culture to study the long-lasting effects of O2 exposure on neonatal airway epithelial development and function. Future applications utilizing these methods could potentially uncover early disease mechanisms involved in hyperoxia-induced long-term development of pulmonary diseases frequently seen in former preterm infants.
The authors have nothing to disclose.
This work is supported by funding from Presbyterian Health Foundation (PHF) and Oklahoma Shared Clinical and Translational Resources (U54GM104938 with an Institutional Development Award (IDeA) from NIGMS) to AG. We would like to thank Dr. Paul LeRou and Dr. Xingbin Ai at Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, for providing neonatal donor cells used in some of the experiments. Figures were created with Biorender. Statistical analysis was performed with GraphPad Prism.
10% Buffered Formalin | Fisher Scientific | 23-426796 | |
1X PBS (Phosphate Buffered Saline) Solution, pH 7.4 | Gibco | 10010049 | |
A 83-01 | Tocris | 29-391-0 | |
ALI Transwell Inserts, 6.5mm | Corning | 3470 | |
Anti-Acetylated Tubulin antibody, Mouse monoclonal | Sigma | T7451 | |
Anti-alpha Tubulin antibody | Abcam | ab7291 | |
Anti-Cytokeratin 5 antibody | Abcam | ab53121 | |
BronchiaLife Epithelial Airway Medium (BLEAM) | LifeLine Cell Technology | LL-0023 | |
CHIR 99021 | Tocris | 44-231-0 | |
Cleaved caspase-3 antibody | Cell signaling | 9664T | |
SCGB1A1 or Club Cell Protein (CC16) Human, Rabbit Polyclonal Antibody | BioVendor R&D | RD181022220-01 | |
CM-H2DCFDA (General Oxidative Stress Indicator) | Thermo Scientific | C6827 | |
Corning Cell Culture Treated T25 Flasks | Corning | 430639 | |
Corning U-Shaped Cell Culture T75 Flasks | Corning | 430641U | |
CyQUANT LDH Cytotoxicity Assay | Thermo Scientific | C20300 | |
DAPI Solution (1 mg/mL) | Fisher Scientific | EN62248 | |
Dimethyl sulfoxide [DMSO] Hybri-Max | Sigma | D2650 | |
Distilled water | Gibco | 15230162 | |
EVOM Manual for TEER Measurement | World Precision Instrument | EVM-MT-03-01 | |
FBS (Fetal Bovine Serum) | Gibco | 10082147 | |
Fluorescein Isothiocyanate Dextran (average mol wt 10,000) | Fisher Scientific | F0918100MG | |
Fluorescein isothiocyanate–dextran (average mol wt 20,00) | Sigma | FD20-100MG | |
Goat Anti-Mouse IgG(H+L), Human ads-HRP | Southern Biotech | 1031-05 | |
Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | A-21141 | |
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 | Invitrogen | A-11035 | |
Goat Anti-Rabbit IgG(H+L), Mouse/Human ads-HRP | Southern Biotech | 4050-05 | |
HBTEC Air-Liquid Interface (ALI) Differentiation Medium | LifeLine Cell Technology | LM-0050 | |
HEPES | Lonza | CC-5024 | |
Heracell VIOS 160i Tri-Gas CO2 Incubator, 165 L | Thermo Scientific | 51030411 | |
High-Capacity cDNA Reverse Transcription Kit | Thermo Scientific | 4368814 | |
HLL supplement | LifeLine Cell Technology | LS-1001 | |
ImageJ | NIH | N/A | imagej.nih.gov/ij/ |
Invivogen Normocin – Antimicrobial Reagent | Fisher Scientific | NC9273499 | |
L-Glutamine | LifeLine Cell Technology | LS-1013 | |
Normal Goat Serum | Gibco | PCN5000 | |
Normocin | Invivogen | ant-nr-05 | |
p63 antibody | Santa Cruz Biotechnology | sc-25268 | |
ProLong Gold Antifade Mountant | Invitrogen | P36930 | |
PureLink RNA Mini Kit | Thermo Scientific | 12183025 | |
RAPAMYCIN | Thermo Scientific | AAJ62473MC | |
TaqMan Fast Advanced Master Mix | Thermo Scientific | 4444964 | |
Taqman Gene Exression Assays: 18S rRNA | Thermo Scientific | Hs99999901_s1 | |
Taqman Gene Exression Assays: CAT | Thermo Scientific | Hs00156308_m1 | |
Taqman Gene Exression Assays: FOXJ1 | Thermo Scientific | Hs00230964_m1 | |
Taqman Gene Exression Assays: GAPDH | Thermo Scientific | Hs02786624_g1 | |
Taqman Gene Exression Assays: GPX1 | Thermo Scientific | Hs00829989_gH | |
Taqman Gene Exression Assays: GPX2 | Thermo Scientific | Hs01591589_m1 | |
Taqman Gene Exression Assays: GPX3 | Thermo Scientific | Hs01078668_m1 | |
Taqman Gene Exression Assays: KRT5 | Thermo Scientific | Hs00361185_m1 | |
Taqman Gene Exression Assays: MUC5AC | Thermo Scientific | Hs01365616_m1 | |
Taqman Gene Exression Assays: SCGB1A1 | Thermo Scientific | Hs00171092_m1 | |
Taqman Gene Exression Assays: SOD1 | Thermo Scientific | Hs00533490_m1 | |
Taqman Gene Exression Assays: SOD2 | Thermo Scientific | Hs00167309_m1 | |
Thermo Scientific Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane (0.22 μm pores, 500 ml) | Thermo Scientific | 5660020 | |
TM-1 Combined Supplement | LifeLine Cell Technology | LS-1055 | |
Total caspase-3 antibody | Cell signaling | 14220S | |
Triton X-100 | Sigma | 9036-19-5 | |
Trypsin-EDTA (0.05%), Phenol red | Gibco | 25300062 | |
Y-27632 2 HCl | Tocris | 12-541-0 |
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