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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 theirΒ 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

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

  1. Media preparation
    1. Bronchial epithelial airway medium (BLEAM) with inhibitors (BLEAM-I): Prepare 500 mL (1 bottle, stored at 4 Β°C) of BLEAM by adding 1.25 mL of HLL supplement (500 Β΅g/mL human serum albumin, 0.6 Β΅M Linoleic acid, 0.6 Β΅g/mL Lecithin), 15 mL of L-Glutamine (6 mM), 2 mL of Extract P (0.4%, bovine pituitary extract), 5 mL of TM1 (1 Β΅M Epinephrine, 5 Β΅g/mL Transferrin, 10 nM Triiodothyronine, 0.1 Β΅g/mL Hydrocortisone, 5 ng/mL epidermal growth factor (EGF), 5 ng/mL insulin), 1 mL of antibiotic solution (Normocin, 0.1 mg/mL). To prevent senescence of the primary cells and improve efficiency of expansion, add the following growth factor inhibitors as previously described39: Y-27632 (Rho-associated protein kinase or ROCK inhibitor) with a final concentration of 5 Β΅M, A83-01 (inhibits Smad signaling) with a final concentration of 1 Β΅M, CHIR 99021 (Glycogen synthase kinase-3 or GSK3 inhibitor) with a final concentration of 0.4 Β΅M and Rapamycin (inhibitor of molecular target of rapamycin or mTOR) with a final concentration of 5 nM. Filter the media through a 0.22 Β΅m pore-size filter. Refer to Table 1 for a list of media components.
    2. Human bronchial/tracheal epithelial cell (HBTEC) ALI medium: Make 500 mL of HBTEC media by thawing the bottle at 37 Β°C and adding 1 mL of antibiotic solution (Normocin, 0.1 mg/mL). Once added, filter the media through a 0.22 Β΅m pore-size filter.
    3. HBTEC media with inhibitors (HBTEC-I): Prepare the HBTEC media as above. Before filtering, add the following inhibitors: Y-27632 with a final concentration of 5 Β΅M and A83-01 with a final concentration of 0.5 Β΅M. Filter the media through a 0.22 Β΅m pore-size filter.
      NOTE: Store BLEAM-I, HBTEC, and HBTEC-I media at 4 Β°C, dated and labeled, in the dark (wrapped in foil), and only warm up aliquots of what is needed (shelf life is 30 days).
    4. HEPES/FBS: Add 500 mL of HEPES-buffered saline and 88 mL of FBS (final concentration is 15%). Once added, filter the solution through a 0.22 Β΅m pore-size filter and store it at 4 Β°C, dated and labeled.
      NOTE: Aliquot and warm BLEAM, HEPES-FBS, and Trypsin-EDTA to 37 Β°C (at least 30 min) prior to use.
  2. Coating culture flasks and ALI cell culture inserts with an 804G-conditioned medium
    1. Prepare 804G cell (rat bladder epithelial cell line) matrix-conditioned media as previously described39.
    2. Coat the cell culture flask and cell culture inserts with 804G-conditioned media for at least 4 h in the 37 Β°C incubator, 5% CO2. Refer to Table 2 for the amounts of media needed to coat different culture flasks and cell culture inserts.

2. Isolation and expansion of nTAECs and preparation for subsequent ALI culture

  1. Acquire fresh tracheal aspirate (approximately 1 mL) during routine suction of the endotracheal tube of intubated neonates and collect the aspirate in a mucus trap. If the aspirate volume is insufficient to reach the mucus trap, rinse the suction catheter with 1-3 mL of sterile saline.
  2. Label the sample and store it in a biohazard bag on ice.
    NOTE: The samples can be stored on ice at 4 Β°C for a maximum of 24 h. However, the yield is higher with fresh samples.
  3. Transport the sample from the NICU to the lab.Β Coat a T25 flask with 804G-conditioned media and prepare for inoculation of the sample at the time of transportation of tracheal aspirate to the lab.
  4. Remove the mucus trap from the biohazard bag and clean the outer surfaces thoroughly with 70% ethanol to avoid any contamination. Carefully open the cap on the mucus trap under a sterile cell-culture hood.
  5. Dilute the tracheal aspirate sample with sterile PBS to 5 mL (to dilute mucus) and transfer the whole content into a 50 mL conical tube.
  6. Centrifuge the tube at 250 x g, 20 Β°C-23 Β°C for 5 min, and discard the supernatant (including mucus).
    NOTE: All centrifugations here are done at 250 x g, 20 Β°C-23 Β°C for 5 min unless specified otherwise.
  7. Resuspend the pellet in 5 mL of BLEAM-I.
  8. Remove the T25 flask from the incubator and discard the 804G conditioned media before plating the sample.
  9. Plate the sample in the T25 flask and place in the incubator at 37 Β°C, 5% CO2 (this will be termed Passage 0 or P0).
  10. Change the media with BLEAM-I 24 h after plating to wash out unattached cells and subsequently every 48 h.
    NOTE: After processing the tracheal aspirate sample and incubating it in BLEAM-I media, cuboidal-shaped cells appear 7-10 days post-plating. By around 3 weeks post-plating, the cells are densely packed and require trypsinization for subsequent passaging, expansion, and storage. Early passage (P1 - P2) cells are placed in cryotubes (2.5 x 106 cells per 500 Β΅L of freezing media per cryotube) and stored in liquid nitrogen for long-term storage.
  11. Rapidly thaw frozen cells from liquid nitrogen in a 37 Β°C water bath and transfer the cell suspension to an appropriately sized sterile tube containing BLEAM-I.
  12. Count the cells to determine the total and live cell number utilizing a trypan blue stain and an automated or manual cell counter as described previously48. Dilute the cell suspension with an appropriate volume of BLEAM-I, so the final concentration is 2.1 x 106 cells in 15 mL of cell suspension (for T75 flask).
    NOTE: For a T25 flask, 0.7 x 106 cells in 5 mL of cell suspension are generally used for seeding.
  13. Transfer the 15 mL cell suspension into a T75 flask and incubate overnight at 37 Β°C, 5% CO2.
  14. On the following morning, exchange the media with new BLEAM-I. Then, exchange the media with a new BLEAM-I every 2 days. Once the cells are around 80%-90%, they are ready to be trypsinized for ALI culture.
  15. Apply 5 mL of trypsin-EDTA (T75) to cell monolayer.
    NOTE: Remember to use fresh trypsin. Avoid using trypsin that has been heated multiple times.
  16. Incubate the flask at 37 Β°C for 5 min in the incubator. Then, tap the side of the flask 8x-10x to dislodge the cells. Check under the microscope to confirm that the cells have been dislodged after trypsinization. If >50% of cells remain on the flask, wash with PBS 2x and repeat the trypsinization step with a reduced incubation time of 2-3 min.
  17. Stop the reaction with 15 mL of HEPES-FBS and pipette up and down 4x-5x. Transfer the cells to an appropriate size sterile tube and centrifuge at 250 x g for 5 min to pellet the cells.
  18. After centrifugation, carefully aspirate the supernatant. Dissolve the cell pellet in 1 mL of BLEAM-I by gently pipetting up and down 5x-10x.
  19. Count the cells to determine the total and live cell number. Dilute the cell suspension with an appropriate volume of BLEAM-I, so the final concentration is 1 x 105 live cells per 100 Β΅l of cell suspension.

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 Β°C, 5% CO2 before the next step (see step 1.2).

  1. Remove the 24-well cell culture plate(s) containing the cell culture inserts from the incubator and discard the 804G-conditioned media.
  2. Add 1 mL of BLEAM-I to the basolateral (lower) chamber of each ALI well. Add 100 Β΅L of cell suspension (1 x 105 cells) to the apical chamber of the ALI cell culture inserts and incubate the cells at 37 Β°C, 5% CO2. This stage is defined as ALI day -2.
  3. The following day, change the media in the basolateral (1 mL) and apical (100 Β΅L) chambers with fresh BLEAM-I. When changing the media in the apical chamber, be careful not to disturb the cell monolayer. This stage is defined as ALI day -1.
  4. The following day, remove the media from both the basolateral and apical chambers.
    NOTE: It takes 2 days for the cells on the cell culture membrane to become confluent under submerged conditions. At this stage, ALI can be established.
  5. Add 1 mL of HBTEC-I media to the basolateral chamber but leave the apical chamber media free and exposed to air. This stage is defined as ALI day 0.
  6. Continue to exchange the HBTEC-I media (1 mL) in the basolateral chamber every 48 h from ALI day 0 to 6. Switch to regular HBTEC media (without inhibitors) on ALI day 7 until the desired time point of harvest.
    NOTE: During the first 7 days of ALI culture, media from the basolateral chamber may leak into the apical chamber. This media needs to be aspirated carefully each day to maintain health of the cell-layer and allow them to differentiate.
  7. Harvest cells, cell culture inserts, and basolateral media at different time points for molecular techniques, assays, and analysis.
    1. For this protocol, on ALI days 0, 7, and 28, harvest cells for qPCR gene expression analysis (standard manufacturer protocol) of epithelial differentiation markers. On ALI days 0, 7, 14, and 28, harvest cell culture inserts for testing barrier function (methods described below).
    2. On ALI days 0 and 28, use formalin-fixed cell culture inserts for immunofluorescent staining with epithelial differentiation markers (utilizing previously published protocol)49. On ALI day 14, harvest basolateral media for lactate dehydrogenase (LDH) release (standard manufacturer protocol), cell lysates for qPCR of oxidative stress markers, and immunoblot with caspase-3 and cleaved caspase-3 antibodies (standard manufacturer protocol), and cell culture inserts for oxidative stress assay (method described below).

4. Testing barrier function during ALI differentiation

  1. Measuring Trans-Epithelial Electrical Resistance (TEER)
    NOTE: Measure TEER using Epithelial Volt/Ohm Meter (EVOM) during ALI differentiation to assess cell-layer integrity50.
    1. Before measuring test filter resistance values, use an 804G-conditioned media-coated cell culture (devoid of any cells) to measure background resistance value in Ohms (Ξ©)
    2. Subtract the background resistance value from test filter resistance values and multiply the difference by the cell growth surface area to obtain TEER value for each test filter.
      TEERΒ = (Ξ©T - Ξ©B) X C
      TEER = Trans Epithelial Electrical Resistance (Ξ©.cm2)
      Ξ©T = Test filter resistance value (Ξ©)
      Ξ©B = Background resistance value (Ξ©)
      C = Cell growth surface area (cm2)
      NOTE: Cell culture inserts used for TEER measurements are subsequently fixed with 10% buffered formalin and used immediately for immunofluorescent staining and fluorescent microscopy (method described below) or stored at 4 Β°C for staining later.
  2. Fluoresceine Isethionate Dextran (FITC-dextran) epithelial permeability assay
    NOTE: Epithelial permeability assay was performed utilizing two different molecular weights of FITC-dextran (10 kD and 20 kD) during ALI differentiation.
    1. Prepare FITC-dextran working solution (1 mg/mL): measure 5 mg of FITC and mix in 1 mL of DMSO (5 mg/mL). Resuspend 5 mg/mL stock in HBTEC media warmed to 37 Β°C to a concentration of 1 mg/mL. Protect the solution from light.
    2. Transfer test filters (containing cells) to a new 12-well plate and add 1 mL of HBTEC media in the basolateral compartment.
    3. Aspirate apical compartment media (if any) and replace with 250 Β΅L of FITC-dextran working solution. Incubate at room temperature protected from light for 60 min.
    4. End the FITC-dextran assay by removing the test filters (containing FITC-dextran working solution) from the 12-well plate.
    5. Collect the basolateral media from the 12-well plate in 1.5 mL microcentrifuge tubes and vortex.
    6. Transfer 100 Β΅L aliquots from each tube to a 96-well clear bottom black polystyrene microplate in triplicates. Transfer 100 Β΅L of HBTEC media to additional wells in the 96-well plate as a negative control to measure background fluorescence.
    7. Measure fluorescence intensity using a spectrophotometer using 490 nm excitation and 520 nm emission maxima.
    8. Subtract the average background fluorescence value from the test filter fluorescence values to obtain corrected fluorescence and express the values as a percentage against the corrected fluorescence values on ALI day 0 for FITC 10 kD and 20 kD.

5. Hyperoxia exposure using TriGas incubator

  1. On ALI day 7, determine the number of cell culture inserts to be put in hyperoxia based on experimental and harvest needs.
  2. Set the O2 level in the TriGas incubator to 60% O2. It generally takes ~30-45 min for the O2 level to reach 60%.
  3. Once the O2 level stabilizes at 60%, place the 24-well cell culture plate with the cell culture inserts assigned to the hyperoxia group inside the incubator and close the incubator door.
    NOTE: This step needs to be performed as efficiently as possible to prevent a significant drop in O2 level inside the incubator.
  4. Change the media in the hyperoxia-exposed cell culture inserts following the same schedule as the control (21% O2 exposed) wells. Check the cell culture inserts every day for media leakage and gently aspirate any leakage into the cell culture chamber.
  5. Continue hyperoxia exposure for 7 days (ALI days 7 to 14). Once hyperoxia exposure is completed, harvest cells or perform assays utilizing cell culture inserts on ALI day 14.

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 2β€²,7β€²-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.

  1. Preparation of CM-H2DCFDA assay solution:Β Each vial contains 50 Β΅g of reagent dye in powdered form. To make a 1 mM stock solution, add 80 Β΅L of sterile DMSO to the vial, mix well with a pipette, and vortex gently. For 1 Β΅M final concentration of the dye in cell culture inserts, add 0.1 Β΅L of the 1 mM stock per 100 Β΅L PBS (e.g., add 0.6 Β΅L stock solution to 600 Β΅L PBS for 6 cell culture inserts).
    NOTE: The final concentration of the dye can vary between 1-5 Β΅M. The dose needs to be optimized for each culture condition.
  2. Add 100 Β΅L of the 1 Β΅M dye solution in each cell culture for both the hyperoxia-exposed or normoxia-exposed group. Use at least 1 cell culture as a negative control from each treatment group (100 Β΅L of PBS without dye). Incubate at 37 Β°C for 30 min, protected from light. Wash 1x with PBS.
  3. Slide preparation: Drain any excess PBS. Carefully hold the cell culture inserts and use a blade to slowly cut out the cell culture membrane. Pour 1-2 drops of mountant liquid on the slide. With flat-tipped forceps, carefully hold the corner of the membrane, place it cell-side down on the mountant, and cover it with a cover slip. Remove excess mountant liquid and leave to air dry for 15 min at room temperature protected from light. The slides are now ready to be imaged.
    NOTE: Preparation of the slide and fluorescent microscopy should be performed as fast as possible as the fluorescent intensity fades significantly over 2-3 h.
  4. Fluorescent microscopy: Capture images using a fluorescent microscope utilizing the Cy2 (cyanine-2) channel. Capture images from three separate, non-overlapping areas per cell culture.
  5. Fluorescent detection of oxidative stress
    NOTE: Use the images from fluorescent microscopy to measure corrected total cell fluorescence (CTCF) utilizing the ImageJ software (https://imagej.nih.gov/ij/) and the workflow described below. CTCF serves as a marker for oxidative stress in the cell culture inserts following hyperoxia exposure and is measured by eliminating the background fluorescence with the help of ImageJ software.
    1. Open the microscopy image in ImageJ and select the area of interest using the rectangle tool. Save the selection area (Ξ±) by right clicking and select Add to ROI (region of interest) manager. Use this selection area across images.
    2. Select the parameters for measurement under Analyze > Set Measurements and select Area, Integrated Density and Mean Gray Value within the settings tab.
    3. For each image, use the same selection area from the ROI manager and select Analyze > Measure. The integrated density value represents the total fluorescence (Ft) of the selected area. Note down the measurement values from the pop-up window.
    4. To correct the background fluorescence (Fb) in each image, select a small area of non-fluorescent region with the rectangle tool. Select Analyze > Measure for this region. The mean intensity value represents Fb. Note down the measurement values from the pop-up window.
    5. Calculate CTCF by multiplying the mean fluorescence intensity of background readings with the total area of the ROI and subtracting this number from the integrated density value of the ROI. Repeat this workflow for all images for both treatment groups (Control and Hyperoxia).
      CTCF = FtΒ - (Fb x Ξ±)
      CTCF = Corrected total cell fluorescence (A.U.)
      Ft = Total fluorescence
      Fb = Background fluorescence
      Ξ±Β = Selection area

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...

Disclosures

The authors have nothing to disclose and no conflicts of interest to report.

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

NameCompanyCatalog NumberComments
10% Buffered FormalinFisher Scientific23-426796
1X PBS (Phosphate Buffered Saline) Solution, pH 7.4Gibco10010049
A 83-01Tocris29-391-0
ALI Transwell Inserts, 6.5mmCorning3470
Anti-Acetylated Tubulin antibody, Mouse monoclonalSigmaT7451
Anti-alpha Tubulin antibodyAbcamab7291
Anti-Cytokeratin 5 antibodyAbcamab53121
BronchiaLife Epithelial Airway Medium (BLEAM)LifeLine Cell TechnologyLL-0023
CHIR 99021Tocris44-231-0
Cleaved caspase-3 antibodyCell signaling9664T
SCGB1A1 or Club Cell Protein (CC16) Human, Rabbit Polyclonal AntibodyBioVendor R&DRD181022220-01
CM-H2DCFDA (General Oxidative Stress Indicator)Thermo ScientificC6827
Corning Cell Culture Treated T25 FlasksCorning430639
Corning U-Shaped Cell Culture T75 FlasksCorning430641U
CyQUANT LDH Cytotoxicity AssayThermo ScientificC20300
DAPI Solution (1 mg/mL)Fisher ScientificEN62248
Dimethyl sulfoxide [DMSO] Hybri-MaxSigmaD2650
Distilled waterGibco15230162
EVOM Manual for TEER MeasurementWorld Precision InstrumentEVM-MT-03-01
FBS (Fetal Bovine Serum)Gibco10082147
Fluorescein Isothiocyanate Dextran (average mol wt 10,000)Fisher ScientificF0918100MG
Fluorescein isothiocyanate–dextran (average mol wt 20,00)SigmaFD20-100MG
Goat Anti-Mouse IgG(H+L), Human ads-HRPSouthern Biotech1031-05
Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 488InvitrogenA-21141
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546InvitrogenA-11035
Goat Anti-Rabbit IgG(H+L), Mouse/Human ads-HRPSouthern Biotech4050-05
HBTEC Air-Liquid Interface (ALI) Differentiation MediumLifeLine Cell TechnologyLM-0050
HEPESLonzaCC-5024
Heracell VIOS 160i Tri-Gas CO2 Incubator, 165 LThermo Scientific51030411
High-Capacity cDNA Reverse Transcription KitThermo Scientific4368814
HLL supplementLifeLine Cell TechnologyLS-1001
ImageJNIHN/Aimagej.nih.gov/ij/
Invivogen Normocin - Antimicrobial ReagentFisher ScientificNC9273499
L-GlutamineLifeLine Cell TechnologyLS-1013
Normal Goat SerumGibcoPCN5000
NormocinInvivogenant-nr-05
p63 antibodySanta Cruz Biotechnologysc-25268
ProLong Gold Antifade MountantInvitrogenP36930
PureLink RNA Mini KitThermo Scientific12183025
RAPAMYCINThermo ScientificAAJ62473MC
TaqMan Fast Advanced Master MixThermo Scientific4444964
Taqman Gene Exression Assays: 18S rRNAThermo ScientificHs99999901_s1
Taqman Gene Exression Assays: CATThermo ScientificHs00156308_m1
Taqman Gene Exression Assays: FOXJ1Thermo ScientificHs00230964_m1
Taqman Gene Exression Assays: GAPDHThermo ScientificHs02786624_g1
Taqman Gene Exression Assays: GPX1Thermo ScientificHs00829989_gH
Taqman Gene Exression Assays: GPX2Thermo ScientificHs01591589_m1
Taqman Gene Exression Assays: GPX3Thermo ScientificHs01078668_m1
Taqman Gene Exression Assays: KRT5Thermo ScientificHs00361185_m1
Taqman Gene Exression Assays: MUC5ACThermo ScientificHs01365616_m1
Taqman Gene Exression Assays: SCGB1A1Thermo ScientificHs00171092_m1
Taqman Gene Exression Assays: SOD1Thermo ScientificHs00533490_m1
Taqman Gene Exression Assays: SOD2Thermo ScientificHs00167309_m1
Thermo Scientific Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane (0.22 ΞΌm pores, 500 ml)Thermo Scientific5660020
TM-1 Combined SupplementLifeLine Cell TechnologyLS-1055
Total caspase-3 antibodyCell signaling14220S
Triton X-100Sigma9036-19-5
Trypsin-EDTA (0.05%), Phenol redGibco25300062
Y-27632 2 HClTocris12-541-0

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Translational 3D cell CultureHyperoxiaHuman Neonatal Airway Epithelial CellsPreterm Neonatal AirwayOxygen TensionLung DiseasePhenotypesNeonatal AlveolarizationAirway HyperreactivityIn Vitro ModelNTAECsAir liquid Interface CultureMucociliary DifferentiationOxidative StressNICUEarly life Oxidative Injury

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