In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Abstract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

Introduction

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition with complex characteristics, while patients with COPD gradually tend to be younger¹. Smoking, a primary risk factor for COPD², has a profound impact on airway epithelial cells, which serve as the initial barrier against tobacco smoke. Despite this known association, the detailed mechanisms through which tobacco smoke induces changes in airway epithelial cells remain inadequately explored. A thorough understanding of these molecular alterations is essential for identifying early diagnostic markers and therapeutic targets for COPD.

To address this gap, we developed a novel in vitro model using murine airway epithelial cells. These cells were subjected to long-term stimulation with cigarette smoke extract (CSE), enabling us to monitor dynamic cellular changes and explore the changes in airway epithelial cells under long-term tobacco stimulation and the underlying mechanism. In this model, transwell was used to provide the air-liquid interface of airway epithelial cells, and cigarette smoke extract was stimulated in the early stage of epithelial cell differentiation until the end of differentiation at 28 days. Previous studies investigated only the differentiation and short-term stimulation of airway epithelial cells (cell line dominance). They were limited to a single regulatory pathway^3,4,5,6. However, the protocol presented here uses primary mouse airway epithelial cells and optimizes the cell extraction process for better cell activity than previous airway epithelial cell culture methods. This model focuses on alterations in cellular differentiation alongside comprehensive transcriptomic, proteomic, metabolomic, and epigenomic analyses. Employing immunofluorescence and advanced multi-omics techniques, we aimed to elucidate the cellular responses of airway epithelial cells to chronic tobacco smoke exposure, thereby contributing to a deeper understanding of COPD pathogenesis. This model can be used to explore the changes in airway epithelial cell differentiation pattern and its mechanism caused by long-term stimulation of various pollutants.

Protocol

The overall protocol requires 44 days, including 1 day for preparation of airway epithelial cells isolated from murine tracheas, 15 days for cell proliferation, and 28 days for CSE stimulation at the air-liquid interface. All experimental animals are housed in the SPF Barrier Animal Room of the Animal Experiment Center of Capital Medical University and have been reviewed and approved by the Animal Experiment and Laboratory Animal Ethics Committee of Capital Medical University (AEEI-2020-100) to meet the requirements of ARRIVE guidelines⁷.

1. Isolation of primary murine airway epithelial cells from murine tracheas

1. Preparation
   1. Prepare complete expansion medium by combining 1 mL of 50x supplement and 0.05 mL of hydrocortisone stock with 48.95 mL expansion basal medium. Store at 4 °C and use within 4 weeks.
   2. Dissolve 7.5 mg of proteinase and 5 mg of deoxyribonuclease I in 5 mL of Ham's F-12 to prepare a proteinase solution. Filter it to sterilize and store at 4 °C and use within 4 weeks.
   3. Prepare antibiotic solutions by adding 0.05 mL of 100x penicillin/streptomycin and 0.01 mL of 500x gentamicin/amphotericin to 5 mL of complete expansion medium, PBS, and proteinase solution (100 U/mL Penicillin, 100 U/mL Streptomycin, 10 µg/mL gentamicin and 0.25 µg/mL amphotericin B). Store at 4 °C and use within 4 weeks.
   4. Sterilize surgical instruments and prepare the biological safety cabinet for cell isolation. Simultaneously, ensure that standard cell culture equipment is ready for use.
   5. Prepare rat tail collagen-coated dishes by adding rat tail collagen (100 µg/mL in 0.02 N acetic acid) into 100 mm culture dishes and 24 mm transwells (0.05 mL/cm²). Leave open overnight under UV light for sterilization.
2. Isolation of primary murine airway epithelial cells
   1. Use C57BL/6 mice (6-8 weeks old, male, SPF-raised) for tracheal epithelial cell isolation. Euthanize with 1% pentobarbital sodium solution overdose (about 200 mg/kg). Use five mice for sufficient cell yield.
   2. Sterilize the mouse by immersion in 75% ethanol solution, not immersing the nose and mouth in alcohol to prevent alcohol from flowing into the trachea.
   3. Dissect the mouse.
      1. Use surgical blades and forceps, one set of devices for cutting and opening the epidermis from the lower jaw to the abdominal cavity of the mouse.
      2. Use another set of surgical instruments to tear apart the thyroid glands on both sides and remove the connections between the trachea, the surrounding muscle tissue, and the esophagus.
      3. After that, carefully cut into the chest, use forceps to delve deeper into the chest cavity, take out the entire lung, and find the end of the trachea.
   4. Process the trachea.
      1. Cut out the trachea from the thyroid cartilage to the tracheo-bronchial branch and put it in a pre-cold expansion medium containing four antibiotics.
      2. Shake the tube to rinse as much blood as possible off the surface of the trachea, keep it on ice, and then start the surgery for the next mouse.
   5. Transfer all collected tracheas to pre-cold PBS buffer containing four antibiotics (200 U/mL Penicillin, 200 U/mL Streptomycin, 20 µg/mL gentamicin, and 0.5 µg/mL amphotericin B) for pretreatment before digestion.
   6. Use a set of surgical instruments to remove clots and other tissue from the surfaces of the trachea, and then cut them into 1 cm² size.
   7. Incubate tracheal tissue in proteinase solution at 37 °C for 40 min.
   8. After 40 min, rock the tube several times and use a 40 µm cell strainer to remove the remaining tissues. Rinse the chopped tracheas on a strainer with 5 mL of expansion medium, centrifuge the cell suspension at 400 x g for 5 min at 4 °C, and discard the supernatant.
   9. Resuspend the obtained cells in 8 mL of expansion medium.
   10. Count the viable cells using trypan blue and a hemocytometer.
   11. Plate P0 cell suspension on 100 mm dishes (1 x 10⁴ live cells/cm²) precoated with rat tail collagen, and culture the cells in an incubator at 37 °C, 5% CO₂.

2. Expansion culture and passage of primary murine airway epithelial cells

1. Replace the medium for the P0 cell culture every 3 days until cells reach 70%-80% confluency, usually within 6 days. At this point, murine tracheal epithelial cells can form an intact monolayer with a cobblestone appearance (Figure 1A-D).
2. Pre-warm sufficient volumes of PBS, complete expansion medium, and animal component-free (ACF) cell dissociation kit to 37 °C.
3. Gently rinse the cells with 3 mL of PBS.
4. Perform enzymatic dissociation.
   1. Add 3 mL of ACF enzymatic dissociation solution to the dish and incubate at 37 °C for 5 min. After pipetting, dislodge the cells gently and transfer the cell suspension to 3 mL of ACF enzyme inhibition solution.
   2. Repeat the addition of 3 mL of ACF enzymatic dissociation solution and incubate again for 5 min to ensure maximum cell detachment.
5. Spin the cell suspension at 400 x g for 5 min at 4 °C. Discard the supernatant.
6. Resuspend the cells in 1 mL of expansion medium.
7. Count the viable cells using trypan blue and a hemocytometer.
8. Plate P1 cell suspension on several 100 mm dishes (5 x 10⁴ live cells/cm²) pre-coated with rat tail collagen, and culture the cells in an incubator at 37 °C, 5% CO₂.

3. Differentiation and CSE stimulation of primary murine airway epithelial cells at air-liquid interface

1. Confirm the cell type.
   1. Verify the epithelial origin of isolated cells using an immunofluorescence assay (IFA) with antibodies against cytokeratins. Seed cells on glass slides coated by rat tail collagen and use paraformaldehyde to fix them for at least 12 h after cells reach 80% confluency.
   2. Wash slides 3 times with PBS solution for 5 min and incubate them with 3% BSA and 0.1% triton X-100 in PBS solution at room temperature (RT) for 1 h.
   3. Incubate slides overnight at 4 °C with commercial anti-pan cytokeratin antibody at a dilution of 1:500.
   4. The next day, wash slides 3 times with PBS solution for 5 min and incubate them with secondary antibody buffer at a dilution of 1:1000 at RT for 2 h in the dark.
   5. After incubation, wash slides 3 times with PBS (5 min per wash), and add DAPI at a dilution of 1:1000 to them. After incubation at RT for 15 min, wash slides once with PBS for 5 min.
   6. Observe slides under a fluorescence microscope and compare expression patterns to a positive control (A549 cell line) and a negative control (Raw264.7 cell line) (Figure 2).
2. Seed the cells for differentiation.
   1. Detach and centrifuge P2 cells as previously described, and resuspend the cell pellet in an appropriate volume of expansion medium to facilitate plating of 1 x 10⁴ live cells/cm².
   2. Pipette 1 mL of cell suspension onto the apical chamber of the transwell polycarbonate membrane insert. To the basal compartment of the transwell, add 1.5 mL of proliferation media.
3. Incubate the cells at 37 °C and fully change all medium in the basal and apical chambers every 3 days using expansion medium until confluence is reached. This typically takes 3-6 days.
4. Prepare 50 mL of complete differentiation medium by adding 5 mL of ALI 10x Supplement, 500 µL of ALI Maintenance Supplement, 100 µL of heparin solution, and 250 µL of hydrocortisone stock solution to 44.15 mL of ALI Basal Medium.
5. Prepare CSE.
   1. Bubble one cigarette through 12.5 mL of differentiation medium and then filter it through a 0.22 µm pore filter to prepare CSE. To ensure standardization between experiments and batches of CSE, measure the absorbance at 320 nm on a spectrophotometer and define optical density (OD) of 1 as 100%⁸.
6. Use the differentiation medium containing an appropriate concentration of CSE to stimulate primary murine airway epithelial cells to detect effects on the differentiation of the cells.
7. Allow cells to differentiate for 28 days, during which the basal chamber media is changed, and the apical side is washed twice a week by removing the apical chamber media and replacing the basal chamber media with the differentiation medium containing the appropriate concentration of CSE.
8. Analyze the cells post-exposure.
   1. After exposure to differentiation medium containing CSE, harvest the cells and the culture supernatants at any time point (i.e., 0-28 days) for detecting morphological changes (i.e., immunofluorescence assay (IFA) or hematoxylin-eosin staining (HE) or for bioinformatics analytical procedures (i.e., transcriptomics, proteomics, metabolomics, etc.).

Representative Results

Differentiation
Murine airway epithelial cells successfully differentiated after culturing at an air-liquid interface with a differentiation medium for 28 days. The presence of ciliated and goblet cells was demonstrated by immunofluorescence assay of cilia marker acetylated α-Tubulin (green; Figure 3A) and the goblet cell marker Mucin5AC, respectively6 (red; Figure 3B).

Determination of CSE concentration for cell differentiation
Stimulate the isolated epithelial cells with different concentrations of CSE for 24 h. The results showed that the epithelial cells declined in cell viability when the concentration of CSE was higher than 6% (p <0.05, Figure 4A). In order to maintain cell activity under long-term stimulation, culture mediums containing 2%, 4%, and 6% CSE were used to stimulate differentiation of epithelial cells for 28 days. Furthermore, the results showed that with the increase in CSE concentration, there was no significant cell death in the epithelial cells (Figure 4B), but there was a gradual change in the number of intercellular transmembrane resistance (TEER) (Figure 4C) and exfoliated cells (Figure 4D). In addition, an overall reduction in differentiated cells and ciliated cells was noted when murine airway epithelial cell cultures were chronically exposed to CSE (Figure 4E-H). Therefore, the differentiation medium containing 6% CSE was chosen to dysregulate epithelial barrier function and reduce ciliated cell numbers without a decrease in cell viability while differentiating epithelial cells for 28 days.

Multi-omics analysis
Two groups of the cells stimulated with differential medium containing 0% or 6% CSE for 28 days were harvested. Total mRNA was extracted for transcriptomics analysis. After library preparation, sequencing, quality control, reads mapping to the reference genome, and quantification of gene expression, the differential expression of genes between the two groups was analyzed. The clusterProfiler R package was utilized to conduct an enrichment analysis of the Gene Ontology (GO) for genes that exhibited differential expression, with adjustments made to account for gene length discrepancies (Figure 5A). The Kyoto Encyclopedia of Genes and Genomes (KEGG) serves as a comprehensive database that facilitates the comprehension of complex biological system functionalities and roles at various levels, including cellular, organismal, and ecological, by integrating molecular-level data derived from extensive datasets such as those produced by genomic sequencing and high-throughput experimental methodologies (Figure 5B). For proteomics, total proteins were extracted from two groups of cells at 28 days after cultures with differential mediums containing 0% or 6% CSE. After protein quality test, salt removal, protein enzymatic hydrolysis, Tandem Mass Tags (TMT) labeling of peptides, separation of fractions, LC-MS analysis, protein identification, and protein quantitation, differentially expressed proteins between two groups can be defined. GO analysis was conducted using the interproscan program against the non-redundant protein database (Figure 6A), and KEGG was used to analyze the pathway (Figure 6B). For metabolomics, the culture supernatants were collected from two groups of airway epithelial cells at 28 days after cultures with differentiation medium containing 0% or 6% CSE. After HPLC-MS/MS analysis, identification, and quantification of metabolites, the differential metabolites between the two groups can be defined. The KEGG Database (Figure 7A), Human Metabolome Database (Figure 7B), and Lipidmaps Database (Figure 7C) were used to annotate all metabolites. The KEGG database was used to analyze the functions of these differential metabolites and metabolic pathways (Figure 7D). For epigenomics, ATAC-seq was performed as previously reported⁹. Nuclei were extracted from two groups of cells at 28 days after culture with differential medium containing 0% or 6% CSE, and the nuclei pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was incubated at 37 °C for 30 min. After adding an adapter, library preparation, quality assessment, clustering, and sequencing, differential epigenomics between two groups can be defined. The GO enrichment analysis of differentially peak-related genes directly reflects the number distribution of differentially peak-related genes in GO items enriched in biological processes, cellular components, and molecular function (Figure 8A). The KEGG database was used to analyze the functions of these differential genes and pathways (Figure 8B).

Genes encoding tight junction proteins
Transcriptomic data showed that after long-term stimulation of CSE, there was decreased expression of numerous genes encoding tight junction proteins, including Cgn, Cldn2, Cldn3, Cldn8, Cldn10, Cldn20, Cldn23, Jam2 and Ocln (Figure 9A), in which reduced expression of Cldn3 and Ocln were further confirmed by Western-Blot and immunohistochemistry (IHC) (Figures 9B-E). These data suggest that exposure to CSE markedly reduces the expression of tight junction proteins of airway epithelial cells.

Cytokine and chemokine expression
In addition to the breakdown of tight connections, transcriptomics also showed that after long-term stimulation of CSE, the expression of some cytokines and chemokines, including Cxcl5, Csf3, Il1a, Il34, Ccl20, and Il33 increased, but expression of Ccl5 decreased (Figure 10A). Luminex test further confirmed that there were increased concentrations of CXCL-5, CSF-3, and IL-1α (Figure 10B-D) but decreased CCL-5 (Figure 10E) in the supernatant of murine airway epithelial cells. These results suggest that exposure to CSE selectively acts on the expression of cytokines and chemokines.

Figure 1: Growth of primary murine airway epithelial cells. (A) Newly isolated cells appear round and transparent. (B) By day 2 post-isolation, expanded cells formed into small cell islands. (C) By day 4, the formation of larger cell islands. (D) By day 6, over 90% fusion with cobblestone morphology. (4x magnification; scale bars: 250 µm). Please click here to view a larger version of this figure.

Figure 2: Immunofluorescence assay of epithelial cell markers. Cytokeratin expression in primary murine airway epithelial cells, A549 (positive control), and Raw264.7 (negative control). Nuclei stained with DAPI (63x magnification; scale bars: 40 µm). Please click here to view a larger version of this figure.

Figure 3: Immunofluorescence assay for markers of ciliated and goblet cells. The markers used were acetylated (A) α-Tubulin and (B) Mucin 5AC for confirming cilia cells and goblet cells, respectively (100 magnification; scale bars: 10 µm in panel A and 50 µm in panel B). Please click here to view a larger version of this figure.

Figure 4: Impact of CSE concentrations on epithelial cells. (A) Cell viability at 24 h at varying CSE concentrations. (B) Cell viability at 28 days. (C) TEER measurement. (D) Count of exfoliated cells. (E-H) Immunofluorescence for ciliated (green) and goblet cells (purple) at 0%, 2%, 4%, and 6% CSE concentrations (20x magnification; scale bars: 50 µm). Please click here to view a larger version of this figure.

Figure 5: GO and KEGG enrichment analyses for gene expression. (A) GO term significance in enriched biological process, cellular component, and molecular function. (B) Proportion of differentially expressed genes in KEGG pathways. Please click here to view a larger version of this figure.

Figure 6: Protein expression analysis. (A) GO enrichment bar chart showing differential protein expression. (B) KEGG pathway analysis based on differentially expressed proteins. Please click here to view a larger version of this figure.

Figure 7: Metabolite KEGG pathway analysis. (A) Annotating all metabolites using the KEGG Database. (B) Annotating all metabolites using the Human Metabolome Database. (C) Annotating all metabolites using the Lipidmaps Database. (D) The distribution of differentially expressed metabolites in various KEGG pathways. Please click here to view a larger version of this figure.

Figure 8: Differentially Peak-related gene expression analysis. (A) GO enrichment bar chart showing differential protein expression. (B) KEGG pathway analysis based on differentially expressed proteins. Please click here to view a larger version of this figure.

Figure 9: Expression of tight junction protein in murine airway epithelial cells with or without CSE stimulation. (A) Heat map of tight junction protein expression in the transcriptome. (B-E) The expression levels of (B,C) Cldn3 and (D,E) Ocln in murine airway epithelial cells were detected by Western Blot and immunohistochemistry for Cldn3 and Ocln (80 magnification; scale bars: 50 µm). Please click here to view a larger version of this figure.

Figure 10: Cytokine expression analysis. (A) Transcriptome heatmap of cytokine expression. (B-E) Luminex assay for CXCL-5, CSF-3, IL-1α, and CCL-5 levels in cells with/without CSE stimulation. Please click here to view a larger version of this figure.

Discussion

COPD is a common chronic airway inflammatory disease. Exposure to tobacco smoke leads to chronic airway inflammation, airway remodeling, and lung structural destruction, which is the result of the interaction of various structural cells and immune cells¹⁰. As the front line of the innate immune system in the lung, airway epithelial cells play a very important role during the development of the disease¹¹. In this point of view, clarifying how epithelial cells change and regulate the downstream cells under the stimulation of harmful substances is a scientific problem that needs to be solved urgently.

Recently, several animal models of COPD that have overcome some limitations have been reported¹². Moderate levels of neutrophilic airway inflammation, a Th1/Th17 inflammatory signature, marked mucus hyperproduction, increased airway remodeling, declined lung function, and enlarged airspaces can be observed in these models. Although these models require only 1 month to establish COPD-like changes, what happened in the mice exposed to cigarette smoke within 1 month is still unclear. Using long-term CSE stimulation has the advantage of being more easily calibrated, which means airway epithelial cells can be exposed to as severe cigarette stimulus as possible without death, just like airway epithelial cells in smokers. In this case, monitoring dynamic pathological changes leading to airway inflammation and the formation of emphysema is critical for further understanding the pathogenesis of COPD relevant to smoking. Therefore, we established an in vitro model to mimic smoke status and tried to monitor dynamic changes during the exposure of CSE to airway epithelial cells. Furthermore, relatively purified airway epithelial cells make the multi-omics study easier than that of whole tissues, which can help us to find the molecular and cellular responses in airway epithelial cells exposure to CSE and to identify the key pathways and therapeutic targets of COPD in high throughput.

Here, the protocol describing the isolation of murine tracheal epithelial cells is adapted from the protocols of Hilaire et al.¹³, and You et al.¹⁴ with some modifications. In the protocol used in this study, murine airway epithelial cells were propagated and differentiated into a pseudostratified structure containing ciliated and goblet cells using an air-liquid interface and medium containing CSE, thus establishing a chronic CSE acclimation model. The following events are the key points of the present model.

Considering that cervical dislocation could mechanically strain the neck and physically damage the epithelial cells, and chloral hydrate anesthesia could also impact the airways, pentobarbital sodium was used for euthanasia¹⁵. During surgical procedures on mice, immersing only the neck and lower areas to prevent alcohol from entering the nasal could increase the viability of airway epithelial cells¹⁶. Preliminary data from the present study demonstrated that airway epithelial cells subjected to tracheal intubation or alcohol immersion had low viability and failed to expand. In addition, to get airway epithelial cells without erythrocytes, one must be careful in surgical procedures to avoid blood contamination when isolating the main airways.

Even in specific-pathogen-free mice, the airways colonized significant microbes. The addition of appropriate antibiotics in the expansion medium of P0 primary murine airway epithelial cells is essential for avoiding contamination from bacterial or fungal pathogens. A cocktail solution of penicillin, streptomycin, amphotericin B, and gentamicin¹⁷ effectively ensures the expansion of P0 murine airway epithelial cells in a sterile in vitro environment. Preliminary experiments demonstrated that if there is no effective inhibition of bacterial contamination, P0 murine airway epithelial cells will die before the first passage.

In digestion methods, both 4 °C and 37 °C were tested. Results from preliminary experiments indicated that digest in 37 °C outperformed 4 °C in terms of time efficiency, cell viability, and cell yield.

Before the seeding of epithelial cells, rat tail collagen was dissolved in acetic acid and used for culture dish coating to improve epithelial cell adhesion¹⁸. However, the density of the collagen coating should not be too high or too low; an insufficient density fails to provide adequate adhesion conditions, while an excessive density results in crystalline residue on the culture surface.

A critical step is to keep more epithelial cells alive and avoid fibroblast contamination. To achieve these, we recommend that 1) using the animal component-free cell dissociation kit can gently isolate epithelial cells and reduce unnecessary cellular damage and 2) usage of differential medium containing hydrocortisone can inhibit the growth of fibroblasts and promote the proliferation of epithelial cells.

Traditional submerged monolayer cultures of airway epithelial cells have been a staple in respiratory research and toxicological studies. However, these methods fall short of accurately replicating the differentiated characteristics of airway epithelial tissues as found in vivo. To address this limitation, we have opted for an alternative airway epithelial model. This model is predicated on the cultivation of primary airway cells on microporous membrane scaffolds, such as those provided by transwell systems, at an air-liquid interface¹⁹. The exposure of the apical surface to the ambient air in this ALI culture setup is instrumental in fostering cellular differentiation, thereby more closely mimicking the in vivo conditions²⁰.

If differentiation is not effective, it is crucial to evaluate potential reasons. This includes the possibility that the airway epithelial cells may have undergone excessive passaging, which can compromise their differentiation capacity. Additionally, it is imperative to ensure that mucus and fluid accumulation within the apical chamber are completely eliminated during the bi-weekly media exchange process, as residual content may impede the differentiation process.

While this model presents certain limitations, it remains a valuable tool for research. One limitation is that the use of a CSE-containing medium to stimulate airway epithelial cells may not fully replicate the in vivo conditions experienced by the airway epithelial cells in smokers. Additionally, the viability of differentiated ciliated epithelial cells in vitro is typically limited to a month. Despite these constraints, the model effectively integrates several key aspects: it facilitates the study of airway epithelial cell differentiation at the air-liquid interface, long-term acclimation to CSE, and the application of multi-omics analysis over a certain time frame. This comprehensive approach offers a sophisticated and holistic framework for future investigations into the physiological and pathological mechanisms of airway epithelial cells. Furthermore, it aids in the identification of potential biomarkers and therapeutic targets for airway diseases. We hope that our experience will provide useful information for those who would like to use this model for relevant studies.

Materials

Name	Company	Catalog Number	Comments
100x Penicillin/Streptomycin solution	Gibco	15140122
24 mm Transwell with 0.4 µm Pore Polyester Membrane Insert, Sterile	BIOFIL	TCS016012
40 µm Cell Strainer	Falcon	352340
500x Gentamicin/Amphotericin Solution	Gibco	R01510
acetylated α-Tubulin	CST	#5335
Acetyl-α-Tubulin (Lys40) (D20G3)XP Rabbit mAb	cellsignal	#5335
Animal Component Free Cell Dissociation Kit	Stemcell	05426
Anti-pan Cytokeratin antibody	abcam	ab7753
Cigarette	Marlboro
Claudin3	immunoway	YT0949
Deoxyribonuclase I from bovine pancreas	Sigma-Aldrich	DN25
Deoxyribonuclase I from bovine pancreas	Sigma	DN25
Ham’s F-12	Sigma-Aldrich	N6658
Heparin Solution	Stemcell	07980
Hydrocortisone Stock Solution	Stemcell	07925
Mucin 5AC	abcam	ab212636
Occludin	proteintech	27260-1-AP
PBS	Cytosci	CBS004S-BR500
Penicillin-Streptomycin Solution	Gibco	15140122
PneumaCult-ALI Basal Medium	Stemcell	05002
PneumaCult-ALI 10x Supplement	Stemcell	05003
PneumaCult-ALI Maintenance Supplement	Stemcell	05006
PneumaCult-Ex Plus 50x Supplement	Stemcell	05042
PneumaCult-Ex Plus Basal Medium	Stemcell	05041
Pronase E	Sigma-Aldrich	P5147
Rat tail collagen	Corning	354236
Trypan Blue	Stemcell	07050