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

The scope of our research is to develop and characterize an in vitro model using airway epithelial cells to study the effects of chronic CSE exposure. We aimed to understand the cellular response that occurred in airway epithelial cells and the long-term cigarette smoke exposure, which is relevant to the pathogenic of COPD. The recognition of intricate epithelium immune cell interaction in COPD is considered a pivotal recent development in COPD research.

We have established a novel in vitro model that mimic chronic CSE exposure in airway epithelial cells. Our findings open up new avenue of investigating the long-term effect of chronic cigarette smoke exposure on airway epithelial cells and the role in COPD progression. In the future, our laboratory will focus on exploring specifics pathway that regulate epithelial cells differentiation and the role of every epithelial cells heterogenotic in COPD treatment response.

To begin, arrange freshly prepared expansion medium, filter sterilized protease and antibiotic solutions, and sterilized surgical instruments on the working platform of the biosafety cabinet. Add rat tail collagen into 100 millimeter culture dishes and 24 millimeter transwells. Leave the dishes open overnight under ultraviolet light for sterilization.

Immerse the euthanized mouse in 75%ethanol for sterilization. Do not immerse the nose and mouth. Using surgical scissors and forceps, cut and open the epidermis from the lower jaw to the abdominal cavity of the mouse.

Using another set of surgical scissors and forceps, tear apart the thyroid glands on both sides and remove the connections between the trachea, the surrounding muscle tissue, and the esophagus. Next, carefully cut into the chest and use forceps to delve deeper into the chest cavity. Remove the entire lung and find the end of the trachea.

Cut out the trachea from the thyroid cartilage to the tracheobronchial branch and put it in a pre-cold expansion medium containing four antibiotics. Shake the tube to rinse as much blood as possible off the surface of the trachea before placing it on ice. Now, transfer the trachea to pre-cold PBS containing four antibiotics.

Using surgical scissors and forceps, remove clots and other tissue from the surface of the trachea and cut them into one square centimeter size. Incubate tracheal tissue in proteinase solution at 37 degrees Celsius. After 40 minutes, rock the tube several times and use a 40 micron cell strainer to remove the remaining tissues.

Rinse the chopped trachea on a strainer with five milliliters of expansion medium. Centrifuge the tracheal suspension at 400g for five minutes at four degrees Celsius. And resuspend the pellet in eight milliliters of expansion medium.

Count the viable cells using Trypan Blue and a hemocytometer. Plate P0 cell suspension on a 100 millimeter dish pre-coated with rat tail collagen. Culture the cells at 37 degrees Celsius with 5%carbon dioxide.

Once the murine tracheal epithelial cells form an intact monolayer with a cobblestone appearance, gently rinse the cells with three milliliters of pre-warmed PBS. Add three milliliters of animal component-free enzymatic dissociation solution to the dish and incubate at 37 degrees Celsius for five minutes. Gently pipette to dislodge the cells and transfer the suspension to three milliliters of animal component-free enzyme inhibition solution.

Centrifuge the cell suspension at 400g for five minutes at four degrees Celsius. And resuspend the pellet in one milliliter of expansion medium. Count the viable cells using Trypan Blue and a hemocytometer.

Plate P1 cell suspension on a 100 millimeter dish pre-coated with rat tail collagen. Culture the cells at 37 degrees Celsius with 5%carbon dioxide. Once the primary murine airway epithelial cells reach 80%confluency, seed cells on a glass slide coated with rat tail collagen.

Then add paraformaldehyde to fix the cells for 12 hours. Wash the slide three times with PBS for five minutes each and incubated with 3%bovine serum albumin and 0.1%Triton X-100 in PBS for one hour. Add anti-pancytokeratin antibody at a dilution of one to 500 onto the slide and incubate overnight at four degrees Celsius.

The next day, wash this slide three times with PBS solution for five minutes each before incubating with secondary antibody at a dilution of one to 1, 000 for two hours in the dark. After washing the slide with PBS, add DPI at a dilution of one to 1, 000 and incubate for 15 minutes. Wash the slide once with PBS for five minutes.

Observe the slide under a fluorescence microscope to compare expression patterns to a positive control and a negative control. After detachment and centrifugation, resuspend the P2 phase cells in an appropriate volume of expansion medium. Pipette one milliliter of cell suspension onto the apical chamber of the transwell polycarbonate membrane insert.

Add 1.5 milliliters of proliferation media to the basal compartment of the transwell. Incubate the cells at 37 degrees Celsius until confluence is reached. Prepare 50 milliliters of complete differentiation medium using the following components.

Bubble one cigarette through 12.5 milliliters of differentiation medium. And then filter it through a 0.22 micron pore filter to prepare cigarette smoke extract, or CSE. To ensure standardization between experiments and batches of CSE, measure the absorbance at 320 nanometers on a spectrophotometer.

Next, remove the expansion medium from the apical and basal chamber of transwells. Add a differentiation medium containing an appropriate concentration of CSE to stimulate primary murine airway epithelial cells for 28 days. During incubation, wash the apical chamber twice a week with PBS and replace the medium in the basal chamber with freshly prepared differentiation medium containing CSE Murine airway epithelial cells successfully differentiated at an air-liquid interface in 28 days.

The presence of ciliated and goblet cells was demonstrated by immunofluorescence assay of cilia marker, acetylated alpha-tubulin, and the goblet cell marker, mucin 5AC, respectively. Cell viability at 24 hours with varying CSE concentrations demonstrated that epithelial cells declined when the concentration was higher than 6%Cell activity under long-term stimulation showed no significant cell death with 2%4%and 6%CSE concentrations. But changes in transmembrane resistance and exfoliated cells were noted.

In addition, an overall reduction in differentiated cells and ciliated cells was noted when murine airway epithelial cell cultures were chronically exposed to CSE.