Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

We describe the collection of airway stem cells from nasal mucosa. These cells are expanded and differentiated into a pseudostratified epithelium in the lab. This is in vitro expansion and differentiation, which is similar to our airways in vivo, which means that there are specialized cells, such as ciliated cells, present that can be used for quantifying cilia beat frequency.

To enable consistent and reproducible quantification of cilia beat frequency between different individuals and in response to different drugs, we standardize culture conditions and image acquisition. Cilia beat frequency measurements are used as clinical tools in disease such as primary ciliary dyskinesia. We hope to establish this as a clinical marker for cystic fibrosis.

Demonstrating the procedure is Katelin Allan and Laura Fawcett, PhD students from my lab, and Dr.Sharon Wong, a postdoc from my laboratory. Begin by asking the participant to breathe through their mouth. Then taking a cytology brush in the dominant hand and resting the fifth digit on the participant's chin to anchor the hand, insert the cytology brush into the participant's nasal passage at a 45-degree angle to the participant's face, and pass it through the nasal meatus.

After pivoting the brush upright, so that it is perpendicular to the participant's face, advance the brush gently, but firmly, against the lateral wall of the nose beneath the inferior turbinate until it is at the mid to posterior part of the inferior turbinate. Rotate the brush 360 degrees up to three times. Then remove it gently, reversing the insertion maneuver, so that the cells are not dislodged from the brush.

Place the brush into the prepared collection tube with nasal cell collection media, and place the collection tube on ice. After gentle vortexing to dislodge cells from the brush, transfer the collection tube on ice back to the biosafety cabinet. Using a serological pipette, transfer the media from the collection tube to a new tube, leaving behind the cytology brushes.

Then centrifuge the sample. After centrifugation, resuspend the cell pellet in one milliliter of conditional reprogramming cell media. Then using a five-milliliter serological pipette, pass the cells through a cell sieve placed on top of a 50-milliliter tube in a circular motion.

To obtain a single cell suspension, collect the suspension from the bottom of the tube, and pass it through the sieve multiple times. Then discard the cell sieve. To seed the airway epithelial cells onto the prepared permeable support inserts, mix the cells well without creating bubbles, ensuring that they are homogenous and in suspension.

Then add 150 microliters of the cell suspension to the apical side of the prepared permeable support inserts. To differentiate the airway epithelial cells at the air-liquid interface, culture them in differentiation, or ALI media for two days. Then aspirate the media, and add 750 microliters of ALI media only to the basal compartment to create an air-liquid interface.

To seed the airway epithelial cells in ECM domes, resuspend dissociated airway epithelial cells with the appropriate volume of 90%ECM. Then holding the pipette vertically at a 90-degree angle as close to the bottom of the well as possible, dispense 50 microliters of the ECM cell suspension to the center of the well. Incubate the plate at 37 degrees Celsius for 20 minutes until the ECM solidifies.

While the ECM is solidifying, warm the Airway Organoid Seeding Media, or AOSM, to room temperature to prevent it from causing re-liquification, and disintegration of the ECM dome upon addition. After the incubation, add 500 microliters of warmed AOSM to each well by dispensing down the wall of the well. Do not pipette media directly onto the ECM dome.

Change the media every two days for four to seven days. To aspirate the media, tilt the plate at a 45-degree angle and aspirate from the bottom edge of the well away from the ECM dome. After four to seven days, initiate organoid differentiation by adding 500 microliters of warmed Airway Organoid Differentiation Media, or AODM, to each well, and change media every two days for seven days.

Place the culture plate containing the airway epithelial cell models into the microscope plate insert, and close the microscope environmental chamber. Allow the sample to equilibrate in the pre-warmed, 37 degrees Celsius, 5%carbon dioxide-filled microscope chamber for 30 minutes. At the microscope eyepiece, focus on the cell model.

Then using the acquisition software, click on L100 to switch the light path to the port where the camera is mounted. Click the green Play button to visualize the microscope field of view via the software. Check that the cilia are in focus, and adjust if required.

To acquire time-lapse images from the menu, click on Acquire, and then on Fast TimeLapse. In the pop-up window, select a save location, and file name, acquire 1, 000 frames. To preview the cilia in the microscope field of view, click on Apply, followed by the green Play button.

Adjust the Z focus if required. Then click Run now to capture the Fast TimeLapse. After installing the necessary software and toolboxes, Copy the appropriate custom analysis scripts and the support scripts folder to the local drive of the computer.

Next, upon the computing software, click on the Home tab, followed by Set Path. In the pop-up window, click on Add with Subfolders. Under the MATLAB search path, select the folder shown.

Then click on Save and Close. Confirm that the analysis scripts are linked to the computing software by checking that they appear in the left-hand panel. Next, transfer the example raw image files acquired to the computer's local drive.

Then click on the BeatingCiliaBatchOMEfiles_jove. m analysis script file in the computing software. To run the script, click on the Editor tab, followed by the green Play button.

In the prompt window that appears, select the raw image files to be analyzed. Enter the exposure time for acquisition time per frame. Then click on OK.Run the GetFirstAmplitude.

m script on the folder that contains the AveSpectrum files. Wait for the script to output the FirstAmplitudeStacked. xlsx file, which contains the highest amplitude frequency, and is within the physiological range of airway epithelial cilia beating.

Copy the frequency values from the FirstAmplitudeStacked. xlsx file, and plot them using scientific analysis software. The results of cilia beat frequency measured in airway epithelial cell ALI models derived from three participants with cystic fibrosis and three healthy control participants are presented.

On day 14 of culture differentiation, beating cilia were present. From day 14, day 21 of culture differentiation, a statistically significant increase in cilia beat frequency was not observed between cohorts. On day 21 of culture differentiation, the mean cilia beat frequency for healthy control participants was significantly higher than that of participants with cystic fibrosis.

Further, when cilia beat frequency was imaged in the same cell models following the removal of mucus there was a statistically significant increase in cilia beat frequency when mucus was removed in ALI models of both healthy individuals and those with cystic fibrosis. The imaging environment must be strictly regulated, since cilia function is extremely susceptible to changes in environmental factors. This platform has the potential to be established for patient-specific drug screening to be able to identify drugs that modulate CFTR function.