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

Summary

Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Abstract

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified airway epithelium that mimics the apical side of the lung airway. While the differentiation of primary lung airway cells is expected, the assessment of biophysical properties like ciliary function and membrane impermeability provides a quality assessment of the airway epithelium and ensures the reliability of the experiment. Here, we describe a straightforward protocol for the development of multi-cellular pseudostratified airway epithelium in ALI culture and assess two important biophysical properties: ciliary function and membrane impermeability. To determine ciliary function, we captured the ciliary movement of a 4-week differentiated airway epithelium using a high-speed camera attached to an inverted microscope, followed by quantifying ciliary beat frequency (CBF) using the Simon Amon Video Analysis (SAVA) system. We also measured transepithelial electrical resistance (TEER) using a volt-ohm meter to determine the epithelial barrier integrity of the airway epithelium.

Introduction

Chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis are some of the major health concerns worldwide¹. The prevalence of respiratory diseases caused by viruses such as human influenza A virus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also cause economic and public health burden². Therefore, there is an immense need to develop treatment regimens for respiratory illnesses that lead to irreversible respiratory tissue damage. The respiratory epithelial tissue itself is not only involved in oxygen uptake but also provides a barrier to protect the body from invading pathogens and hazardous chemicals³. The respiratory epithelium has a complex cellular organization composed of three major cell types: ciliated cells, goblet cells, and basal cells. Recently, there has been a report on the presence of novel but rare ionocyte cells in the airway epithelium⁴. The complex tissue functions in a coordinated fashion to provide innate immune responses such as secretion of antimicrobial peptides, cytokines release to activate an adaptive immune response, and mucociliary clearance⁵. The lack of a suitable airway model is one of the obstacles to the study of respiratory infections and the development of treatments.

The air-liquid interface (ALI) model is becoming a key tool for research on respiratory diseases⁶. It is an effective in vitro lung airway model as the primary lung airway cells differentiate into a pseudostratified airway epithelium composed of at least three types of cells that generally reside at the apical side of the airway. First, ciliated cells cover the majority of the apical side of the airway and contribute to mucociliary clearance. Second, goblet cells produce mucus and are the largest cells that co-reside with ciliary cells at the apical side of the airway. Third, basal cells reside at the basal layer of the airway and are the progenitor cells that differentiate into different epithelial cells^5,7. Although ionocytes and tuft cells are rare in the airway epithelium, they may also play a role in ciliary function and membrane permeability⁴. This technique differs from the traditional submerged cell culture, which does not mimic the in vivo lung environment. Since ALI is produced from primary epithelial cells derived from healthy people, patients with asthma, and COPD, it offers a more diverse platform for studying responses to infection and disease pathophysiology.

The health assessment of the ALI-developed cultures is an essential aspect to monitor, ensuring the cultured cells' viability, functionality, and physiological relevance. It enhances the confidence in the integrity, reliability, and reproducibility of ALI cultures. As previously published, our group has been evaluating the ALI culture integrity by assessing two biophysical parameters: ciliary function by quantifying ciliary beat frequency (CBF) and epithelial barrier integrity by determining transepithelial electrical resistance (TEER)^6,7,8. Mucociliary clearance (MCC) is one of the important features of the airway epithelium carried by ciliated cells. Specialized organelles called cilia on the surface of ciliated cells beat in metachronal waves to clear the airways of infections and inhaled particles stuck in the mucous layer of the airway epithelium. Effective MCC is totally dependent upon proper ciliary activity⁹. A good CBF is indicative of a healthy, intact airway epithelium; hence, monitoring of the CBF for ALI cultures provides valuable insight into the epithelium's integrity¹⁰. Although there are multiple ways to quantify CBF, for example, high-speed video microscopy¹¹ and phase-resolved Doppler optical coherence tomography¹², each method needs technical expertise and specialized equipment. In this protocol, we provide an easier and less -technical way of quantifying CBF of the differentiated airway epithelium. We also described a method for quantifying TEER of the same epithelium, which indicates epithelial tissues' integrity and barrier function¹³.

Protocol

The protocol should be performed in a sterile condition under a biosafety level 2 laminar flow hood (biosafety cabinet). All the medium and solutions should be thawed at 37 °C prior to use. Centrifugation should be performed at 4 °C. All the materials used in the experiment must be sterile. The primary cells (deidentified donor) were obtained in collaboration with Dr. Kristina Bailey at the University of Nebraska Medical Center (UNMC), Omaha, NE. The primary cells are from deidentified donors, and the cells do not fall under the NIH Human Subject. The cells were obtained under an approved Material Transfer Agreement (MTA) between the University of North Dakota, Grand Forks, ND, and UNMC, Omaha, NE. Here, we used passaged 4 NHBE cells. Although higher passage NHBE cells (up to passage 8) were also shown to differentiate into airway epithelium, we routinely use passage four NHBE cells because there is no obvious difference in whole genome transcription profiles among passages 1 to 4^6,14.

1. Primary cell culture

NOTE: Careful handling of the primary cells is crucial. The whole protocol should avoid harsh treatment and excessive pipetting of the primary cells.

1. Using a sterile serological pipette add 5 mL of collagen to a 100 mm tissue culture (TC) dish to pre-coat the dish. Incubate the TC dish with collagen at room temperature (RT) for 20 min.
2. Using a sterile serological pipette, aspirate the collagen from the TC dish and replace it with 8 mL of pre-thawed complete airway epithelial cell (AEC) medium supplemented with 2% (v/v) penicillin-streptomycin and 1% (v/v) amphotericin B (Table 1).
3. Thaw one vial (containing 5 x 10⁵ cells) of primary normal human bronchial epithelial (NHBE) cells in a water bath at 37 °C.
4. With a 5 mL sterile serological pipette, add 1 mL of pre-thawed complete AEC medium to the cells dropwise to acclimatize the NHBE cells, and collect all the cells in a sterile 15 mL conical tube.
5. Pellet the cells by centrifugation at 212 x g for 5 min at 4 °C. Gently discard the medium from the tube and resuspend the cells in the leftover medium drops in the tube by gentle tapping.
   NOTE: Excessive and harsh tapping should be avoided.
6. Using a 5 mL sterile serological pipette, slowly add 2 mL of complete AEC medium to the resuspended cells and collect all the cells.
7. Add the cells to the plate pre-added with 8 mL of complete AEC medium. Move the TC dish sideways to distribute the cells in the dish uniformly.
8. Incubate the cells at 37 °C in an 85%-95% humid CO₂ cell culture incubator. After 24 h, observe the NHBE cells under the microscope at 10x or 20x for their attachment to the TC dish and growth. If the cells are suspended in the medium, this shows dead cells.
9. After 48 h, aspirate the old medium from the TC dish with a sterile serological pipette.
10. Using a sterile serological pipette, add 10 mL of pre-warmed, complete AEC medium to the cells. Add the media slowly to one corner; avoid direct pouring over the top of the cells.
11. Change the complete AEC media every 48 h until the cells reach 95%-100% confluency.

2. Air-Liquid interface culture

1. Using a P200 pipette, add 100 µL of collagen to a pre-coated 6.5 mm transwell insert with 0.4 μm-pore polyester membrane (from here on referred as inserts) and incubate at RT for 20 min.
2. With a sterile serological pipette, aspirate the medium from the confluent NHBE cells.
3. Add 5 mL of 1x Dulbecco's Phosphate Buffer Saline (DPBS) with a sterile serological pipette to one corner of the TC dish.
4. Gently swirl the plate to wash the cells and aspirate the 1x DPBS with a sterile serological pipette. Ensure cells do not dry out at any step during the protocol.
5. Using a serological pipette, add 5 mL of TrypsinLE to the cells and incubate at 37 °C in a humid CO₂ cell culture incubator until all the cells are dissociated from the bottom surface of the plate.
6. Using a sterile serological pipette, collect all the cells in a 15 mL conical tube. With a sterile serological pipette, add 3 mL of trypsin to the TC dish and collect any residual cells.
7. Pellet the cells by centrifugation at 212 x g for 5 min at 4 °C. Discard the medium and gently tap the tube to resuspend the cells in the leftover trypsin drops in the tube.
8. Using a serological pipette add 2 mL of complete AEC medium to the resuspended cells.
9. Count the cells using a hemacytometer or automated cell counter.
10. Dilute the cells with complete AEC media, with each insert receiving 5 x 10⁴ cells in a volume of 200 µL.
11. With sterile forceps, transfer the inserts to an empty well in a 24-well culture dish.
12. Using a P1000 pipette, add 500 µL of complete AEC media to the basal well of the plate.
13. Using sterile forceps, transfer the inserts back to the basal well with added complete AEC media.
14. With a P200 pipette, add 5 x 10⁴ resuspended NHBE cells in 200 µL of the complete AEC medium to the apical side of the insert. Incubate the cells at 37 °C in an 85% - 95% humid CO₂ cell culture incubator.
15. After 24 h, observe the cells under the microscope to see if they have formed a confluent layer in the well.
    NOTE: If the cells do not form a confluent layer, change the basal AEC medium every 48 h with fresh, complete AEC medium till the cells form a confluent monolayer. While changing basal media, transfer the inserts to the empty well with sterile forceps. Aspirate the old medium with a P1000 pipette, add fresh medium, and place the inserts back in fresh, complete AEC medium.
16. Once the cells are confluent, transfer the inserts to an empty well with sterile forceps. Using a P1000 pipette, aspirate all the basal AEC medium.
17. With a P1000 pipette, add 500 µL of fresh complete air-liquid interface differentiation (ALI) medium supplemented with 2% (v/v) penicillin-streptomycin and 1% (v/v) amphotericin B to the basal well. 
18. Transfer the inserts to the basal well with added complete ALI medium using sterile forceps. Using a P200 pipette, gently aspirate the apical AEC medium from the well.
    NOTE: The apical side should not be added with the complete ALI medium. Aspirate the AEC medium carefully from the apical side to avoid any cell detachment.
19. Incubate the cells at 37 °C in a humid CO₂ cell culture incubator. After 48 h, transfer the inserts to an empty well with sterile forceps.
20. Using a P1000 pipette, aspirate the old medium and add fresh 500 µL of complete ALI medium.
21. With sterile forceps, place the inserts back in the well with fresh medium. Keep changing the complete ALI medium every 48 h till day 28 (four weeks).
22. On day 28, using a P200 pipette, add 100 µL of 1x DPBS to the apical side of the inserts and incubate at 37 °C in a humid CO₂ cell culture incubator for 30 min. Aspirate the 1x DPBS with a P200 pipette.
    NOTE: DPBS should be added by pouring on the wall instead of directly to the cells. While aspirating DPBS, avoid touching the epithelium and vigorous pipetting because any damage to the epithelium can compromise the epithelium (tissue-like) integrity.
23. From the 2^nd week of differentiation, evaluate mucus production by observing a possible darker layer due to mucus on the epithelium under the microscope. The excessive mucus can be removed by DPBS washing. Briefly, add 200 µL of 1x DPBS on the apical side of the inserts on a 24-well plate and incubate the plate at 37 °C in a humid CO₂ cell culture incubator for 30 min. Remove DPBS and keep the plate at 37 °C in a humid CO₂ cell culture incubator.

3. Determination of ciliary beat frequency

NOTE: For determination of CBF at 37 °C (if necessary), the microscope needs to have an environment chamber incubator providing 5% CO₂ at 37 °C and humidity.

1. Turn on the microscope with its attached computer and the environment chamber. Open the door of the chamber to place the plate containing the inserts on the microscope stage.
2. Once the plate is placed on the stage, close the chamber. Using the microscope stage operating knob, move the plate to bring the inserts into view.
3. Select the 20x magnification lens in the phase-contrast mode. Observe the plate through the eyepiece of the microscope and focus the cell using the tuning knob.
4. On the computer screen, click on SAVA system icon. SAVA program should be installed on the computer connected to the microscope.
5. On the software interface, click on Configure Experiment. On the next tab, click on Create Experiment.
6. A new tab will appear, give details of the directory where the data will be stored in the Enter Experiment Directory Name and experiment details in Enter Experiment Description, then click OK.
7. Click The Experiment that was created and is shown in the Directory under the Experiments.
8. Click on Modify and provide sample details in the Sample Description. Click on Add then on Exit to exit from this tab.
9. On the main software interface on the experiment slot, select the experiment created from the dropdown menu on the main interface.
10. Click on Record Video and monitor the ciliary beat frequency (CBF) on the computer screen. The screen will show focused ciliary movement.
11. For each insert, record 6 different random fields for 2.1 s at 120 frames per second. Click on Save Video to monitor the CBF data.
12. To analyze data, click on Analyse Video on the software interface. On the next tab, click on OK.
13. On the following tab, click on Analyse All. Download the spreadsheet generated for every experiment in the computer SAVA data acquisition folder in the computer.
14. Take Gaussian mean frequency for data presentation.

4. Quantification of transepithelial electrical resistance

NOTE: TEER quantification needs liquid at the apical side of the airway epithelium. To establish this protocol, add 100 µL of 1x DPBS at the apical side of the airway epithelium in the inserts that already contain the basal medium (experimental) or 500 µL of 1x DPBS.

1. Turn on the EVOM2 volt ohmmeter using the power switch. Connect the Test Resistor to the EVOM2 at the INPUT slot.
2. Monitor the reading on the EVOM2 screen. If it is above or below 1000, then it needs calibration. Using forceps, turn the ADJ switch at the EVOM2 volt ohmmeter till it shows a uniform 1000 reading.
3. Take an empty insert for experimental control reading. Using a P200 pipette add 100 µL of 1x DPBS at the apical side of the empty insert, while 500 µL of 1x DPBS is added at the basal side with a P1000 pipette.
4. Disconnect the Test Resistor and connect the STX2 electrode at the INPUT slot on EVOM2. Gently clean the STX2 electrode with 70% ethanol.
5. Insert the STX2 electrode in the emptyinsert with 1x DPBS, keeping the shorter electrode leg on the apical part of each insert and the longer electrode leg on the basal part.
6. Record the stable reading on the EVOM2 screen, as the reading can fluctuate due to movement or other undetermined reasons.
7. Insert the STX2 electrode in the insert with 28-day differentiated epithelium, keeping the shorter electrode leg on the apical part of each insert and the longer electrode leg on the basal part in the medium.
8. Record the stable reading on the EVOM2 screen. Take several independent readings to calculate a reliable average. Subtract the background (empty insert reading) from the experimental insert reading for calculation.

Results

We evaluated both ciliary function and membrane impermeability of the airway epithelium obtained from differentiating primary NHBE cells from two independent deidentified donors (two nonsmoking, healthy adults or adults with COPD, above 50-year-old females). For determining ciliary function, we determined the CBF of the 28-day differentiated airway epithelium. We observed CBF reached at least 3 Hz for both airway epithelium, which was comparable to the previously published results (Figure 1)...

Discussion

Human primary airway epithelium (ALI culture) is increasingly used as an in vitro lung model for biological assessments to investigate function and mechanisms and to reduce animal use¹⁴. ALI has several advantages over any monolayer submerged culture. For example, it provides a tissue-like pseudostratified airway epithelium that mimics the apical side of the lung airway^6,7,14. However, the quality and int...

Materials

Name	Company	Catalog Number	Comments
1x DPBS	Thermo Fisher Scientific, USA	14190-144
Airway Epithelial Cell Growth Medium	PromoCell, Germany	C-21060
Amphotericin B	Thermo Fisher Scientific, USA	15290-026
EVOM2 volt Ohmmeter	World Precision Instruments, USA	Not applicable
Heparin Solution	STEMCELL technologies, USA	07980
Hydrocortisone stock	STEMCELL technologies, USA	07926
Microscope: Leica DMI8 (Leica Microscope) with 120f/sec high-speed camera (Basler) associated with the microscope. Microsope also fixed with stage chamber incubator i8.	Leica, USA	Not applicable
Penicillin-Streptomycin	Corning, USA	30-002-CI
PneumaCult-ALI Basal Medium	STEMCELL technologies, USA	05002
PureCol	Advanced BioMatrix, USA	5005
Sisson-Ammons video analysis (SAVA) system. Software V.2.1.15	Amson Engineering, USA	Not applicable