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Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells
In This Article
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 hyperox....
Protocol
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
- Media preparation
- Bronchial epithelial airway medium (BLEAM) with inhibitors (BLEAM-I): Prepare 500 mL (1 bottle, .......
Representative Results
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.......
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.......
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
| Name | Company | Catalog Number | Comments |
| 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 |
References
- Torres-Cuevas, I., et al. Oxygen and oxidative stress in the perinatal period. Redox Biol. 12, 674-681 (2017).
- Hanidziar, D., Robson, S. C. Hyperoxia and modulation of pulmonary vascular and immune responses in COVID-19.<....
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