We thank the study participants and their families for their contributions. We appreciate the assistance from Sydney Children&#39;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&#39;s Hospital Foundation. The authors would like to acknowledge Luminesce Alliance - Innovation for Children&#39;s Health for its contribution and support. Luminesce Alliance - Innovation for Children&#39;s Health is a not-for-profit cooperative joint venture between the Sydney Children&#39;s Hospitals Network, the Children&#39;s Medical Research Institute, and the Children&#39;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&#39;s Hospital Foundation and UNSW University postgraduate award scholarships.

Study approval was received from the Sydney Children&#39;s Hospital Network Ethics Review Board (HREC/16/SCHN/120). Written consent was obtained from all participants (or participants&#39; guardian) prior to the collection of biospecimens.
1. Preparations for establishing airway epithelial cell models
<ol>
	<li>Prepare nasal cell collection media by combining 80% Dulbecco&#39;s Modified Eagle Medium and 20% Fetal Bovine Serum. Supplement with 1 &#181;L/mL of Penicillin/Strep...

<ol>
	<li>Barbato, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ciliary+dyskinesia:+a+consensus+statement+on+diagnostic+and+treatment+approaches+in+children.">Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children.</a> European Respiratory Journal. 34 (6), 1264-1276 (2009).</li><li>Cutting, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+genetics:+from+molecular+understanding+to+clinical+application.">Cystic fibrosis genetics: from molecular understanding to clinical application.</a> Nature Reviews Genetics. 16 (1), 45-56 (2015).</li><li>Chioccioli, M., Feriani, L., Kotar, J., Bratcher, P. 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A., Rutman, A., Williams, G., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliated+air-liquid+cultures+as+an+aid+to+diagnostic+testing+of+primary+ciliary+dyskinesia.">Ciliated air-liquid cultures as an aid to diagnostic testing of primary ciliary dyskinesia.</a> Chest. 138 (6), 1441-1447 (2010).</li><li>Thomas, B., Rutman, A., O'Callaghan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupted+ciliated+epithelium+shows+slower+ciliary+beat+frequency+and+increased+dyskinesia.">Disrupted ciliated epithelium shows slower ciliary beat frequency and increased dyskinesia.</a> European Respiratory Journal. 34 (2), 401-404 (2009).</li><li>Coles, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+protocol+for+culture+of+airway+epithelial+cells+as+a+diagnostic+tool+for+primary+ciliary+dyskinesia.">A revised protocol for culture of airway epithelial cells as a diagnostic tool for primary ciliary dyskinesia.</a> Journal of Clinical Medicine. 9 (11), (2020).</li><li>Pifferi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+cell+culture+method+for+the+diagnosis+of+atypical+primary+ciliary+dyskinesia.">Simplified cell culture method for the diagnosis of atypical primary ciliary dyskinesia.</a> Thorax. 64 (12), 1077-1081 (2009).</li><li>Pifferi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+diagnosis+of+primary+ciliary+dyskinesia:+cell+culture+and+soft+computing+analysis.">Rapid diagnosis of primary ciliary dyskinesia: cell culture and soft computing analysis.</a> European Respiratory Journal. 41 (4), 960-965 (2013).</li><li>Lee, D. 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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...

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&#160;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63090/63090fig01.jpg" /> 
Figure 1: Schematic of workflow. Following brushing the participants&#39; 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 (&#62;100Hz) scientific camera. Data analysis is performed using custom-built scripts. <a href="https://www.jove.com/files/ftp_upload/63090/63090fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenine</td>
			<td>Sigma-Aldrich</td>
			<td>A2786</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alanyl-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G8541</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Andor Zyla 4.2 sCMOS</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Fast frame rate (&#62;100 Hz) scientific camera</td>
		</tr>
		<tr>
			<td>Bottle-top vacuum filter system</td>
			<td>Sigma-Aldrich</td>
			<td>CLS431098</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceftazidime hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A6987</td>
			<td>50 mg/mL</td>
		</tr>
		<tr>
			<td>Cell Culture Microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>CFI S Plan Fluor ELWD 20XC</td>
			<td>Nikon Instruments Inc.</td>
			<td>MRH08230</td>
			<td>Long working distance objective lens. NA0.45 WD 8.2-6.9</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052-1MG</td>
			<td>200 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Corning Gel Strainer 40 UM</td>
			<td>Sigma-Aldrich</td>
			<td>CLS431750</td>
			<td>Pore size 40 &#956;m</td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix (Phenol red-free)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Extracellular matrix (ECM)</td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum filter system</td>
			<td>Sigma-Aldrich</td>
			<td>CLS431098</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning CoolCell LX Cell Freezing Container</td>
			<td>Sigma-Aldrich</td>
			<td>CLS432002</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polyester membrane cell culture inserts</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3470</td>
			<td>Permeable support inserts. 6.5 mm Transwell with 0.4 &#956;m pore polyester membrane insert.</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>ThermoFisher Scientific</td>
			<td>AMQAX1000</td>
			<td>Automated cell counter</td>
		</tr>
		<tr>
			<td>Cytology brushes</td>
			<td>McFarlane Medical</td>
			<td>33009</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12-Ham</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12-Ham</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-High Glucose</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td>Live-cell imaging microscope.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, United States</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone (Amphotericin B)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290018</td>
			<td>250 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Gentamicin solution</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>50 mg/mL</td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Scientific analysis software</td>
		</tr>
		<tr>
			<td>Greiner Cryo.s vials</td>
			<td>Sigma-Aldrich</td>
			<td>V3135</td>
			<td>Cryogenic vials</td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>HI-FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>3.6 mg/mL</td>
		</tr>
		<tr>
			<td>Incubator NL Ti2 BLACK 2000</td>
			<td>PeCon</td>
			<td></td>
			<td>Microscope environmental chamber. Allows warm air incubation and local CO2 and O2 gassing</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I2643</td>
			<td>2 mg/mL</td>
		</tr>
		<tr>
			<td>Lab Armor 74220 706 Waterless Bead Bath 6L</td>
			<td>John Morris Group</td>
			<td>74220 706</td>
			<td>Bead bath</td>
		</tr>
		<tr>
			<td>Lab Armor Beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1254302</td>
			<td>Thermal beads</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>Computing software</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microscoft</td>
			<td></td>
			<td>Spreadsheet software</td>
		</tr>
		<tr>
			<td>NIH/3T3</td>
			<td>American Type Culture Collection</td>
			<td>CRL-1658</td>
			<td>Irradiated NIH-3T3 mouse embryonic feeder cells</td>
		</tr>
		<tr>
			<td>NIS-Elements AR</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>Image acquisition software</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>10,000 units penicillin and 10&#160;mg streptomycin/mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult Airway Organoid Kit</td>
			<td>StemCell Technologies</td>
			<td>5060</td>
			<td>Airway Organoid Kit</td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Medium</td>
			<td>StemCell Technologies</td>
			<td>5001</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus Medium</td>
			<td>StemCell Technologies</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr>
			<td>PureCol-S</td>
			<td>Advanced BioMatrix</td>
			<td>5015</td>
			<td>Type I Collagen solution</td>
		</tr>
		<tr>
			<td>ReagentPack Subculture Reagents</td>
			<td>Lonza</td>
			<td>CC-5034</td>
			<td></td>
		</tr>
		<tr>
			<td>rhEGF (Epidermal Growth Factor, human)</td>
			<td>Sigma-Aldrich</td>
			<td>E9644</td>
			<td>25 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Y-27632 2HCl (ROCK inhibitor)</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Tobramycin</td>
			<td>Sigma-Aldrich</td>
			<td>T4014</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>Trypan blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>0.4%</td>
		</tr>
		<tr>
			<td>UNO Stage Top Incubator</td>
			<td>Okolab</td>
			<td></td>
			<td>Microscope incubator. Allows temperature, humidity and CO2 conditioning</td>
		</tr>
	</tbody>
</table>

collection, expansion, and differentiation of primary human nasal epithelial cell models for quantification of cilia beat frequency

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.

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 &#60; 0.0345) increase in CBF was observed within both cohorts. On Day 21 of culture differentiation, the mean CBF f...

Watch this Scientific Journal Video about Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency at JoVE.com

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

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 ...

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.

collection-expansion-differentiation-primary-human-nasal-epithelial

Research

JoVE Journal

Medicine

3.6K vistas.  University of New South Wales. Describimos la colección de células madre de las vías respiratorias de la mucosa nasal. Estas células se expanden y diferencian en un epitelio pseudoestratificado en el laboratorio. Esto es expansión y diferenciación in vitro, que es similar a nuestras vías respiratorias in vivo, lo que significa que hay células especializadas, como las células ciliadas, presentes que se pueden utilizar para cuantificar la frecuencia de latido de los cilios.Para permitir una cuantificación co...