Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells
Summary
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.
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.
Introduction
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.
Protocol
Representative Results
Discussion
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, immunofluore…
Declarações
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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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