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This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.
Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.
Measurements of Cilia Beat Frequency (CBF) and pattern have been established as diagnostic tools for respiratory diseases such as Primary Ciliary Dyskinesia (PCD)1. In Cystic Fibrosis (CF), dysfunction of the CF Transmembrane conductance Regulator (CFTR) chloride channel causes dehydration of the airway surface liquid and impaired mucociliary clearance2. Ciliary function has been investigated in vitro in primary airway cell models as an indicator of CFTR channel activity3. However, considerable patient-to-patient variability exists in CBF in response to CFTR-modulating drugs, even for patients with the same CFTR mutations3. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of CBF.
Nasal epithelial sheets isolated from nasal mucosal brushings are directly used for measurements of ciliary function for PCD diagnosis4. Yet, while there is no control over the size or quality of the nasal epithelial sheets obtained, CBF varies depending on whether it is measured on single cells or cell sheets and on epithelial sheet ciliated edges that are disrupted or undisrupted5. As such, secondary dyskinesias caused by damage to cells during the collection of nasal mucosal brushings may influence CBF. Primary cell culture of nasal epithelial cells and their differentiation at Air-Liquid Interface (ALI) or in three-dimensional basement membrane matrix into ciliated airway epithelial organoids give rise to cilia that are free from secondary dyskinesias4,6,7,8. Airway epithelial cells differentiated at ALI (henceforth termed ALI models) have been deemed an important secondary diagnostic aid that replicate the ciliary beat patterns and frequency of ex vivo nasal mucosal brushings6 and enable analysis of ciliary ultrastructure, beat pattern, and beat frequency while retaining patient-specific defects9. Yet, discrepancies exist in the methodologies used to create these pseudostratified, mucociliary differentiated cell models. Different culture expansion or differentiation protocols could induce distinct epithelial phenotypes (ciliated or secretory)10 and result in significant differences in CBF11. CBF has been quantified in nasal epithelial brushings4,6,12,13,14,15,16, airway epithelial organoids14,17,18 and ALI models3,4,6,13,19,20,21. Yet, amongst these protocols, there are large variabilities, and often many parameters are not controlled for. For example, in some studies, CBF is imaged in situ while the cells of the ALI model remain on the permeable support insert3,19,20,21, yet others scrape the cells from the permeable support insert and image them suspended in media4,6,13.
Furthermore, the wider application of techniques that measure ciliary function is limited by the extreme susceptibility of ciliary function to changes in environmental factors. Environmental factors such as temperature22, humidity23,24, and pH25,26 influence ciliary function and must be regulated to quantify CBF accurately. The various physiological parameters used across different laboratories and how they influence CBF has been reviewed previously27.
Various imaging technologies and approaches to CBF measurements are reported in the literature. For PCD diagnostics, video microscopy is used to measure ciliary function28,29. Recently, a video analysis algorithm based on differential dynamic microscopy was used to quantify both CBF and cilia coordination in airway epithelial cell ALI models3,30. This method enables the characterization of ciliary beating in airway epithelial cells in a fast and fully automated manner, without the need to segment or select regions. Various methods for imaging and quantification of CBF may add to the differences reported in CBF in the literature (Supplementary File 1).
A protocol from culture to quantification to streamline existing methods, standardization of culture conditions, and image acquisition, performed in strict environmentally controlled conditions, would enable consistent, reproducible quantification of CBF within and between individuals.
This protocol provides a complete description of the collection of epithelial cells, expansion and differentiation culture conditions, and quantification of CBF in three different airway epithelial cell model systems of nasal origin: 1) native epithelial sheets, 2) ALI models imaged on permeable support inserts and 3) Extracellular Matrix (ECM)-embedded three-dimensional organoids (Figure 1). Nasal epithelial cells obtained from nasal inferior turbinate brushings are used as representatives of the airway epithelium since they are an effective surrogate for bronchial epithelial cells31 while overcoming the invasive procedure associated with collecting bronchial brushings. The Conditional Reprogramming Cell (CRC) method is used to expand primary airway epithelial cells for the creation of ALI models and three-dimensional organoids. Conditional reprogramming of airway epithelial cells to a stem cell-like state is induced by co-culture with growth-arrested fibroblast feeder cell system and Rho-associated kinase (ROCK) inhibitor32. Importantly, the CRC method increases population doubling in airway epithelial cells while retaining their tissue-specific differentiation potential33,34. In all airway epithelial cell models, the ciliary function is captured in a temperature-controlled chamber using a high-speed video camera with standardized image acquisition settings. Custom-built scripts are employed for the quantification of CBF.
Figure 1: Schematic of workflow. Following brushing the participants' nasal inferior turbinate, airway epithelial cells are utilized in one of two ways. Either airway epithelial sheets are isolated, and cilia beat frequency is imaged immediately, or airway epithelial cells are expanded via the conditional reprogramming cell method. CRC-expanded airway epithelial cells are differentiated to establish airway epithelial cells at an air-liquid interface or airway epithelial organoid cultures. Imaging of ciliary beat frequency is acquired using a live-cell imaging microscope with a heating and humidity environmental chamber and a fast frame rate (>100Hz) scientific camera. Data analysis is performed using custom-built scripts. Please click here to view a larger version of this figure.
Study approval was received from the Sydney Children's Hospital Network Ethics Review Board (HREC/16/SCHN/120). Written consent was obtained from all participants (or participants' guardian) prior to the collection of biospecimens.
1. Preparations for establishing airway epithelial cell models
Component | Volume |
DMEM, high glucose | 156.7 mL |
DMEM/F-12, HEPES | 313.3 mL |
Hydrocortisone | 55.6 µL |
Insulin | 1.25 mL |
Cholera toxin | 21 µL |
Adenine | 1.2 mL |
HI-FBS | 25 mL |
Penicillin-Streptomycin | 5 mL |
Human epidermal growth factor | 1 µL/mL |
ROCK inhibitor | 1 µL/mL |
Fungizone | 2 µl/ml |
Tobramycin | 2 µL/mL |
Ceftazidime hydrate | 4 µL/mL |
Gentamicin solution | 1 µL/mL |
Table 1: Components for 500 mL of conditional reprogramming cell media
2. Collection of nasal inferior turbinate brushings
NOTE: This section of the protocol requires a collection tube (50 mL) with nasal cell collection media, cytology brushes, tissues, and appropriate Personal Protective Equipment. Avoid brushing during an upper respiratory tract infection. There is a small risk of bleeding, which is increased if inflammation is present. If the purpose of the brushing is to obtain airway epithelial sheets for ex vivo CBF measurements brushing should occur a minimum of 6 weeks post any upper respiratory infection; ideally, more than 10 weeks post infection35.
Figure 2: Collection of nasal epithelial cells. Illustration of the location of the cytology brush at the mid to posterior part of the inferior turbinate. This position is reached by inserting the brush through the nares, pivoting the brush to a 90° angle to the face and guiding the brush along the nasal passage below the inferior turbinate. Please click here to view a larger version of this figure.
3. Preparation of airway epithelial sheets
NOTE: This section of the protocol requires collection tube (cytology brush(es) + 1 mL of nasal cell collection media) (section 2) and 96-well flat-bottomed plate. If collecting nasal turbinate brushings for the purpose of imaging airway epithelial sheets, only use 1 mL of antibiotic-free nasal cell collection media; otherwise, epithelial sheets will be too dispersed for imaging.
4. Airway epithelial cell expansion and maintenance
5. Seeding and differentiation of airway epithelial cells and maintenance of differentiated ALI models
6. Three-dimensional airway epithelial organoids
Component | Volume |
Advanced DMEM/F-12 | 500 mL |
HEPES | 5 mL |
Alanyl-glutamine | 5 mL |
Penicillin-Streptomycin | 5 mL |
Table 2: Components of airway organoid basal media
Number of wells | Number of cells | Number of domes | Vol of Matrigel ECM | Vol of AOSM |
1 | 10,000 cells | 1 | 45 µL x 1.1 | 5 µL x 1.1 |
2 | 20,000 cells | 2 | 90 µL x 1.1 | 10 µL x 1.1 |
5 | 50,000 cells | 5 | 225 µL x 1.1 | 25 µL x 1.1 |
……… | ………cells | ……… | ………µL x 1.1 | ………µL x 1.1 |
Table 3: Calculations for seeding airway epithelial cells in ECM domes
7. Imaging cilia beat frequency
NOTE: This section of the protocol requires a live-cell imaging microscope with a heating and humidity environmental chamber, a fast frame rate (>100 Hz) scientific camera, a 20x long working distance objective, and imaging software (refer to Table of Materials for recommended equipment used in this protocol).
Figure 3: Stabilisation of ciliary beat frequency in live-cell imaging microscope. Dot plots of mean cilia beat frequency (CBF) in airway epithelial cells at the air-liquid interface (ALI models) following transfer into a live-cell imaging microscope with an environmental chamber. Chamber was equilibrated and maintained at 37 °C, 5% CO2 and relative humidity of 85% for 30 min prior to opening the chamber door and placing the culture plate into the microscope plate insert. Cell models were imaged for 60 min at indicated intervals. ALI models were derived from two participants with CF. Six field of view (FOV) images were acquired per ALI model. Each dot (blue) represents the mean CBF in 12-36 FOV images. Data are represented as mean ± SEM, with mean connected by a dotted line. One-way analysis of variance (ANOVA) was used to determine statistical differences. **** P < 0.0001, ns: no significance. Please click here to view a larger version of this figure.
8. Data analysis and quantification of CBF
Figure 4: Setting up computing software for data analysis. (A) Open the Home tab. (B) Select Set Path. (C) Select Add with Subfolders. (D) Select folders containing the analysis scripts. (E) Select Save. (F) Select Close. (G) The analysis scripts will appear in the left-hand panel. Please click here to view a larger version of this figure.
Figure 5: Running analysis scripts using computing software. (A) Open the script for analysis of CBF ('BeatingCiliaBatchOMEfiles_JOVE.m') or creation of cilia beating movie ('LoadRawDataExportFilteredMovies_JOVE.m'). (B) Open the Editor tab. (C) Select the green play (Run) button to run the analysis script. (D) A prompt window will require the selection of files for analysis or movie creation. (E) While running the 'BeatingCiliaBatchOMEfiles_JOVE.m' script, a prompt will appear to manually input the acquisition time per frame (s) in case the file-reading script does not read the metadata properly. (F) Progress bar indicating cilia beat frequency being computed. (G) While running the 'LoadRawDataExportFilteredMovies_JOVE.m' script, a prompt will appear to manually input the type of movie to be outputted (mp4 or avi), the movie frame rate (fps), whether the immobile component is removed from the movie data ('y' or 'n'), the frame time (s), and the pixel size (microns) of the data exported into the movie. It is recommended to use 'y' for immobile filtering as it will remove mucus or any other obstructing immobile layers in the data. (H) Progress bar for indicating movie being exported. Please click here to view a larger version of this figure.
Movie inputs | Description |
File type | Input the file type you would like to export (mp4 or avi). |
Frame rate | Input the frame rate at which the movie should be exported. If you have ~1000 frames per time series acquired, it is recommended to set frame rate ~30 fps. |
Immobile filtering | Options are ‘y’ or ‘n’. Default is ‘y’, and the time filtering script removes, using Fourier space, any immobile components from movie data. Typically, any layers of cells under cilia or immobile mucus will contribute a zero-frequency offset component or time invariant component in the signal that can be filtered out. |
Acquisition time per frame | The acquisition time per frame of acquired data. It is used to display a time stamp in the movie in seconds. |
Pixel size | The pixel size in micrometres is used to display a scale bar in the movie in micrometres. |
Table 4: Input settings for movie creation
To demonstrate the efficiency of this protocol in quantifying CBF, the results of CBF measured in airway epithelial cell ALI models derived from three participants with CF and three healthy control participants are presented. On Day 14 of culture differentiation, beating cilia were present (Figure 6). From Day 14 to 21 of culture differentiation, a statistically significant (P < 0.0345) increase in CBF was observed within both cohorts. On Day 21 of culture differentiation, the mean CBF f...
There are multiple factors that could obscure the quantification of CBF in nasal epithelial sheets. Epithelial sheets should be imaged within 3-9 hrs of sample collection since the ciliary function is most stable during this time37. Less red blood cells and debris are most optimal for imaging since these interfere with data acquisition. When selecting an ROI for imaging, it is important to select an epithelial sheet that edge has not been damaged or disrupted during the collection of the sample, a...
The authors declare that they have nothing to disclose.
We thank the study participants and their families for their contributions. We appreciate the assistance from Sydney Children's Hospitals (SCH) Randwick respiratory department in the organization and collection of patient biospecimens - special thanks to Dr. John Widger, Dr. Yvonne Belessis, Leanne Plush, Amanda Thompson, and Rhonda Bell. We acknowledge the assistance of Iveta Slapetova and Renee Whan from the Katharina Gaus Light Microscopy Facility within the Mark Wainwright Analytical Centre at UNSW Sydney. This work is supported by National Health and Medical Research Council (NHMRC) Australia (GNT1188987), CF Foundation Australia, and Sydney Children's Hospital Foundation. The authors would like to acknowledge Luminesce Alliance - Innovation for Children's Health for its contribution and support. Luminesce Alliance - Innovation for Children's Health is a not-for-profit cooperative joint venture between the Sydney Children's Hospitals Network, the Children's Medical Research Institute, and the Children's Cancer Institute. It has been established with the support of the NSW Government to coordinate and integrate pediatric research. Luminesce Alliance is also affiliated with the University of Sydney and the University of New South Wales Sydney. KMA is supported by an Australian Government Research Training Program Scholarship. LKF is supported by the Rotary Club of Sydney Cove/Sydney Children's Hospital Foundation and UNSW University postgraduate award scholarships.
Name | Company | Catalog Number | Comments |
Adenine | Sigma-Aldrich | A2786 | 10 mg/mL |
Advanced DMEM/F-12 | Thermo Fisher Scientific | 12634-010 | |
Alanyl-glutamine | Sigma-Aldrich | G8541 | 200 mM |
Andor Zyla 4.2 sCMOS | Oxford Instruments | Fast frame rate (>100 Hz) scientific camera | |
Bottle-top vacuum filter system | Sigma-Aldrich | CLS431098 | |
Ceftazidime hydrate | Sigma-Aldrich | A6987 | 50 mg/mL |
Cell Culture Microscope | Olympus | CKX53 | |
CFI S Plan Fluor ELWD 20XC | Nikon Instruments Inc. | MRH08230 | Long working distance objective lens. NA0.45 WD 8.2-6.9 |
Cholera toxin | Sigma-Aldrich | C8052-1MG | 200 µg/mL |
Corning Gel Strainer 40 UM | Sigma-Aldrich | CLS431750 | Pore size 40 μm |
Corning Matrigel Matrix (Phenol red-free) | Corning | 356231 | Extracellular matrix (ECM) |
Corning bottle-top vacuum filter system | Sigma-Aldrich | CLS431098 | |
Corning CoolCell LX Cell Freezing Container | Sigma-Aldrich | CLS432002 | |
Corning Transwell polyester membrane cell culture inserts | Sigma-Aldrich | CLS3470 | Permeable support inserts. 6.5 mm Transwell with 0.4 μm pore polyester membrane insert. |
Countess Cell Counting Chamber Slides | Thermo Fisher Scientific | C10228 | |
Countess II Automated Cell Counter | ThermoFisher Scientific | AMQAX1000 | Automated cell counter |
Cytology brushes | McFarlane Medical | 33009 | |
DMEM/F12-Ham | Thermo Fisher Scientific | 11330032 | |
DMEM/F12-Ham | Thermo Fisher Scientific | 11330032 | |
DMEM-High Glucose | Thermo Fisher Scientific | 11965-092 | |
Dulbecco′s Phosphate Buffered Saline (PBS) | Sigma-Aldrich | D8537 | |
Eclipse Ti2-E | Nikon | Live-cell imaging microscope. | |
Fetal Bovine Serum, certified, heat inactivated, United States | Thermo Fisher Scientific | 10082147 | |
Fungizone (Amphotericin B) | Thermo Fisher Scientific | 15290018 | 250 µg/mL |
Gentamicin solution | Sigma-Aldrich | G1397 | 50 mg/mL |
Graphpad Prism | Graphpad | Scientific analysis software | |
Greiner Cryo.s vials | Sigma-Aldrich | V3135 | Cryogenic vials |
HEPES solution | Sigma-Aldrich | H0887 | 1 M |
HI-FBS | Thermo Fisher Scientific | 10082-147 | |
Hydrocortisone | Sigma-Aldrich | H0888 | 3.6 mg/mL |
Incubator NL Ti2 BLACK 2000 | PeCon | Microscope environmental chamber. Allows warm air incubation and local CO2 and O2 gassing | |
Insulin | Sigma-Aldrich | I2643 | 2 mg/mL |
Lab Armor 74220 706 Waterless Bead Bath 6L | John Morris Group | 74220 706 | Bead bath |
Lab Armor Beads | Thermo Fisher Scientific | A1254302 | Thermal beads |
MATLAB | MathWorks | Computing software | |
Microsoft Excel | Microscoft | Spreadsheet software | |
NIH/3T3 | American Type Culture Collection | CRL-1658 | Irradiated NIH-3T3 mouse embryonic feeder cells |
NIS-Elements AR | Nikon Instruments Inc. | Image acquisition software | |
Penicillin-Streptomycin | Sigma-Aldrich | P4333 | 10,000 units penicillin and 10 mg streptomycin/mL |
Dulbecco′s Phosphate Buffered Saline (PBS) | Sigma-Aldrich | D8537 | |
PneumaCult Airway Organoid Kit | StemCell Technologies | 5060 | Airway Organoid Kit |
PneumaCult-ALI Medium | StemCell Technologies | 5001 | |
PneumaCult-Ex Plus Medium | StemCell Technologies | 5040 | |
PureCol-S | Advanced BioMatrix | 5015 | Type I Collagen solution |
ReagentPack Subculture Reagents | Lonza | CC-5034 | |
rhEGF (Epidermal Growth Factor, human) | Sigma-Aldrich | E9644 | 25 µg/mL |
Y-27632 2HCl (ROCK inhibitor) | Selleckchem | S1049 | 10 mM |
Tobramycin | Sigma-Aldrich | T4014 | 100 mg/mL |
Trypan blue solution | Sigma-Aldrich | T8154 | 0.4% |
UNO Stage Top Incubator | Okolab | Microscope incubator. Allows temperature, humidity and CO2 conditioning |
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