Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Our research aims to measure two critical biophysical properties of pseudostratified airway epithelium, which can be obtained by differentiating normal human bronchial epithelial cells in an air liquid interface. We identified different features of different respiratory viruses by using pseudostratified airway epithelium. For example, respiratory syncytial virus-driven cytoskeletal inflammation, which is a non-canonical mechanism of bronchiolitis.

We also identified goblet cell hyperplasia increases SARS-CoV-2 replication in chronic obstructive pulmonary disease airway epithelium. We focused on elucidating the mechanism of respiratory syncytial virus-driven modulation of cytoskeletal signaling to identify novel therapeutic targets to combat RSV infection and virus-induced bronchiolitis. To begin, take normal human bronchial epithelial, or NHBE, cell suspension.

With a P200 pipette, add 50, 000 resuspended cells in 200 microliters of complete airway epithelial cell, or AEC, medium to the apical side of the insert. Incubate the cells at 37 degrees Celsius in an 85%to 95%humidified carbon dioxide cell culture incubator. Once the cells are confluent, transfer the inserts to an empty well using sterile forceps.

Using a P1000 pipette, aspirate all the basal AEC medium. Then, add 500 microliters of fresh complete ALI differentiation medium supplemented with 2%penicillin-streptomycin and 1%amphotericin B to the basal well. Using sterile forceps, transfer the inserts to the basal well with the added complete ALI medium.

With a P200 pipette, gently aspirate the apical AEC medium from the well and incubate it. After 48 hours, transfer the inserts to an empty well with sterile forceps. Using a P1000 pipette, aspirate the old medium and add 500 microliters of fresh complete ALI medium.

Place the inserts back in the well with fresh medium using sterile forceps. On day 26, using a P200 pipette, add 100 microliters of DPBS to the apical side of the inserts. Incubate at 37 degrees Celsius in a humidified cell culture incubator for 30 minutes.

Then, aspirate the DPBS using a P200 pipette. To begin, turn on the microscope, the attached computer, and the environment chamber. Open the chamber door and place the plate containing the inserts with NBHE cells on the microscope stage.

Once the plate is on the stage, close the chamber. Then, use the microscope stage operating knob to move the plate and bring the inserts into view. On the computer screen, click on the SAVA system icon to open the SAVA program.

On the software interface, click on Configure Experiment. In the next tab, click on Create Experiment. Then, in the new tab, enter the directory where the data will be stored in the Enter Experiment Directory Name field and provide details in the Enter Experiment Description field, then click OK.Now, click on the experiment that was created and is shown in the directory under the Experiments section.

Afterward, click on Modify and provide sample details in the Sample Description field. Click Add, then click Exit to leave the tab. On the main software interface, select the created experiment from the drop-down menu in the Experiment slot.

Then, click on Record Video to monitor the ciliary beat frequency on the computer screen. The screen will show focused ciliary movement. For each insert, record six different random fields for 2.1 seconds at 120 frames per second.

Click on Save Video to monitor the ciliary beat frequency data. To analyze data, click on Analyze Video on the software interface. And in the next tab, click OK.Then go to the following tab and click on Analyze All.

Download the spreadsheet generated for each experiment in the computer's SAVA Data Acquisition folder. Take the Gaussian mean frequency for data presentation. The ciliary beat frequency reached at least three hertz for both the airway epithelium of healthy adults and those with COPD, showing comparable ciliary function.

To begin, turn on the EVOM2 volt ohmmeter using the power switch. Connect the test resistor to the EVOM2 at the input slot. Monitor the reading on the EVOM2 screen.

If the reading is above or below 1, 000, turn the ADJ switch on the EVOM2 using forceps until it shows a uniform 1, 000 reading to calibrate the device. Now, take an empty insert for the experimental control reading. Using a P200 pipette, add 100 microliters of DPBS to the apical side of the empty insert.

Then, add 500 microliters of DPBS to the basal side with a P1000 pipette. Afterward, disconnect the test resistor and connect the STX2 electrode to the input slot on the EVOM2. Then, gently clean the STX2 electrode with 70%ethanol.

Insert the STX2 electrode into the empty insert with DPBS, keeping the shorter electrode leg on the apical part and the longer electrode leg on the basal part. Now, record the stable reading on the EVOM2 screen. Next, using a P200 pipette, add 100 microliters of DPBS to the apical side of the insert.

Insert the STX2 electrode into the insert with the 26-day differentiated airway epithelium, as shown earlier. Then, record the stable reading on the EVOM2 screen. Transepithelial electrical resistance values increased in adults with COPD, indicating enhanced membrane impermeability in COPD.