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

Summary

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

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.).

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 concentr...

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 regu...

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