Name: Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency (Video) | JoVE Journal
Uploaded: 2021-11-10T13:00:00.000+00:00
Duration: 11 min 13 s
Description: Learn to collect, expand, and differentiate primary human nasal epithelial cells into functional airway models for quantifying cilia beat frequency. This standardized protocol enables reproducible measurement of ciliary function for respiratory disease research.

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5 minutes

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.

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<li>Brewington, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brushed+nasal+epithelial+cells+are+a+surrogate+for+bronchial+epithelial+CFTR+studies%5BTitle%5D)+AND+%22JCI+Insight%22%5BJournal%5D)">Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies.</a> JCI Insight. 3 (13), (2018).</li>
<li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ROCK+inhibitor+and+feeder+cells+induce+the+conditional+reprogramming+of+epithelial+cells%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.</a> The American Journal of Pathology. 180 (2), 599-607 (2012).</li>
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<li>Delmotte, P., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ciliary+beat+frequency+is+maintained+at+a+maximal+rate+in+the+small+airways+of+mouse+lung+slices%5BTitle%5D)+AND+%22American+Journal+of+Respiratory+Cell+and+Molecular+Biology%22%5BJournal%5D)">Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices.</a> American Journal of Respiratory Cell and Molecular Biology. 35 (1), 110-117 (2006).</li>
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</ol>

The authors declare that they have nothing to disclose.

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 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="Airway epithelial reprogramming process via nasal brushing; diagram includes cilia imaging setup, analysis." class="xfigimg" src="/files/ftp_upload/63090/63090fig01.jpg" data-altupdatedat="1788282455000" data-alt="Figure 1"> 
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 (&gt;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><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 (&gt;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 &amp;micro;g/mL</td></tr><tr><td>Corning Gel Strainer 40 UM</td><td>Sigma-Aldrich</td><td>CLS431750</td><td>Pore size 40 &amp;mu;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 &amp;mu;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&amp;prime;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 &amp;micro;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&amp;nbsp;mg streptomycin/mL</td></tr><tr><td>Dulbecco&amp;prime;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 &amp;micro;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

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

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 ...

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

Research

JoVE Journal

Medicine

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described.  This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

Using Primary Neurosphere Cultures to Study Primary Cilia

Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Culture and Imaging of Human Nasal Epithelial Organoids

Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine

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.

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

Learn to collect, expand, and differentiate primary human nasal epithelial cells into functional airway models for quantifying cilia beat frequency. This standardized protocol enables reproducible measurement of ciliary function for respiratory disease research.

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency (Video) | JoVE Journal

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

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