We warmly thank all patients and their families for participation in the study. This work was supported by grants from French Association Vaincre la Mucoviscidose; French Association ABCF 2 and Vertex Pharmaceuticals Innovation Awards.

All experiments were performed following the guidelines and regulations described by the Declaration of Helsinki and the Huriet-Serusclat law on human research ethics.
1. Preparation of flasks and different media
<ol>
	<li>Prepare amplification, air-liquid, and freezing medium as described in Table 1.</li>
	<li>Prepare a stock solution of human collagen IV by dissolving 50 mg of collagen in 100 mL of 0.2% glacial acetic acid. Mix on a magnetic stirrer for at least 1 h, and filter sterilize the solution. Store at 4 &#176;C for up to 6 months in a glass bottle, protected from light.</li>
	<li>Prepare a working solution of collagen IV by diluting the stock solution 1:5 in double-distilled water. Store at 4 &#176;C for up to 6 months.</li>
	<li>Coat plastic cell culture flasks or transwell filters with the collagen working solution. Evenly distribute 100 &#181;L for 0.33 cm2 transwell filters, 500 &#181;L for 25 cm2 flasks, and 1 mL for 75 cm2 flasks on the whole growth surface of the flask. 
	NOTE: This can be achieved by gently inclining the flask from side to side. Alternatively, to facilitate coating the flasks, an increased volume can be used, and when the entire surface is evenly covered, collagen solution is aspirated to leave only the indicated volume. The aspirated collagen solution can then be used immediately to coat the next flask (i.e., for three 75 cm2 flasks, distribute 3 mL of collagen solution evenly in the first flask, aspirate 2 mL, which are then used for the next flask, and so on).</li>
	<li>Leave the cell culture flasks in the incubator for a minimum of 2 h or overnight. Aspirate the collagen thoroughly and leave the flasks to dry in the incubator or under the hood for a minimum of 20 min or overnight.</li>
	<li>Wash with 10 mL of Mg2+- and Ca2+-free DPBS, allow to dry in the incubator, and store in aluminum foil to protect from light. Store the flasks for up to 6 months at room temperature (RT).</li>
</ol>
2. Nasal brushing
NOTE: Ensure that nasal brushing is performed while the participant does not have an acute infection. If CF patients present a pulmonary infection, refer to the patient&#39;s antibiogram and add additional antibiotics to the amplification medium. Human nasal epithelial cells were collected by nasal brushing from F508del/F508del patients as previously described9.
<ol>
	<li>Ask the patient to blow their nose, then topically anesthetize the mucosa of both nostrils using cotton meshes soaked in xylocaine 5% with naphazoline solution.</li>
	<li>Gently brush the medial wall and the inferior turbinate of both nostrils using a cytology brush. Soak the brush in a 15 mL tube with 2 mL of commercially available flushing medium; shake gently to detach cells. Repeat brushing in the second nostril.</li>
	<li>Add the following antibiotics to the flushing medium: Penicillin (100 U/mL), Streptomycin (100 &#181;g/mL), Tazocillin (10 &#181;g/mL), Amphotericin B (2.5 &#181;g/mL) and Colimycin (16 &#181;g/mL). 
	&#8203;NOTE: Nasal brushings can be shipped at RT to dedicated labs for expansion and culture and can be seeded up to 72 h after brushing.</li>
</ol>
3. Isolation of HNE
NOTE: HNE were isolated from the cytology brush, washed with Mg2+- and Ca2+-free DPBS, dissociated with 0.25% Trypsin, and seeded onto a 25 cm2 plastic flask as previously described10.
<ol>
	<li>Detach cells from the cytology brush by repeatedly passing the brush through a 1000 &#181;L pipette cone and rinsing with 2 mL of Mg2+- and Ca2+-free DPBS. Centrifuge at 500 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the pellet in 0.25% Trypsin for 8-12 min to dissociate cells. Stop the enzymatic reaction by adding 5 mL of the amplification medium.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C and discard the supernatant. Resuspend the pellet in 8 mL of the amplification medium and seed onto a 25 cm2 collagen-coated flask. Grow at 37 &#176;C and 5% CO2. This corresponds to passage 0.</li>
	<li>The following day, replace the medium with 5 mL of fresh amplification medium and monitor the cells daily for attachment, morphology (cells should have a cohesive cobblestone appearance), and number. 
	&#8203;NOTE: The initial seeding density varies depending on the quality of the nasal brushing and the proportion of epithelial cells. Freshly isolated HNE cells usually reach 80%-90% confluency within 3-10 days. A slower growth rate is suggestive of insufficient epithelial cells in the sample and should be discarded.</li>
</ol>
4. Amplification and passaging of human nasal epithelial cells
<ol>
	<li>Grow human nasal epithelial cells in the amplification medium at 37 &#176;C and 5% CO2 on collagen-coated flasks until 80%-90% confluency, changing medium every 48-72 h. For 25 cm2 flasks, use 5 mL of amplification medium and 10 mL for 75 cm2 flasks.</li>
	<li>When cells reach 80%-90% confluency, wash the cells with 10 mL of Mg2+- and Ca2+-free DPBS, aspirate, and discard.</li>
	<li>Add 1 mL of 0.25% Trypsin for a 25 cm2 flask (or 2 mL of Trypsin for a 75 cm2 flask) and return to the incubator (37 &#176;C, 5% CO2) for 8-12 min.</li>
	<li>Tap the flask firmly, with the palm of the hand, to help the cells detach.</li>
	<li>Add 10 mL of the amplification medium to stop the enzymatic reaction. Vigorously rinse the flask, using the 10 mL pipette to draw up and expel the amplification medium over the flask surface to rinse and detach cells.</li>
	<li>Centrifuge at 500 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the pellet in 5-10 mL of the amplification medium.</li>
	<li>Count the cells using a hemocytometer. 
	&#8203;NOTE: Cells can be frozen at this point (see steps 5.1-5.3). If further amplification is required, re-seed onto collagen-coated flasks (a 25 cm2 flask can be expanded into three 75 cm2 flasks, a greater dilution is not recommended).</li>
</ol>
5. Cryopreservation of primary nasal epithelial cells
NOTE: Grow HNE cells until passage 1 before biobanking to obtain enough cells to facilitate cell growth when thawed. Biobanking is, however, possible at initial passage 0 or passage 2. The following cryopreservation steps are adapted to all passages.
<ol>
	<li>After cell counting, centrifuge at 500 x g for 5 min at 4 &#176;C, and discard the supernatant</li>
	<li>Resuspend the pellet in the enriched freezing medium (Table 1) to obtain 3 x 106-5 x 106 cells per mL and per cryovial.</li>
	<li>Slowly freeze the cells by reducing the temperature at ~1 &#176;C per min in an appropriate cryo-freezing container at -80 &#176;C. The next day, move the preserved sample to a nitrogen storage container for long-term storage (the present study is limited to 17 months storage).</li>
</ol>
6. Thawing frozen amplified HNE cells
<ol>
	<li>Warm up the water bath to 37 &#176;C. Prepare the amplification medium as described in Table 1 and warm the medium to 37 &#176;C.</li>
	<li>Remove cryovials from the nitrogen storage tank and rapidly place them in the water bath, taking care not to submerge the whole vial in the water. Remove the cryovials from the water bath when only a small frozen droplet remains. Wipe the vial with 70% alcohol, wipe dry, and place under the hood.</li>
	<li>Using a 1 mL pipette, transfer the thawed cells to an empty 15 mL tube.</li>
	<li>In a drop-wise manner, add 1 mL of warm amplification medium. After 1 min, add another 1 mL of amplification medium and wait an additional minute.</li>
	<li>Add 10 mL of the amplification medium and centrifuge for 2 min at 500 x g.</li>
	<li>Aspirate and discard the supernatant. Resuspend the cell pellet in a volume of amplification medium required to achieve a cell density of at least 1 x 106 cells/mL.</li>
	<li>Seed the cells onto a collagen-coated flask containing the amplification medium.
	<ol>
		<li>If the cryovial contains more than 4 x 106 cells, seed onto a 75 cm2 flask, in 10 mL of the amplification medium</li>
		<li>If cryovial contains less than 4 x 106 cells, seed cells onto a 25 cm2 flask, in 5 mL of the amplification medium</li>
	</ol>
	</li>
	<li>Incubate at 37 &#176;C, 5% CO2, and visually observe cell expansion over the next 2-3 days.</li>
	<li>Then, perform cell amplification as detailed in section 4. 
	&#8203;NOTE: If few cells were harvested in step 4.7. (i.e., if freezing at passage 0 before expansion), steps 6.5. and 6.6. can be omitted, and cells can be seeded directly onto a 25 cm2 collagen-coated flask without centrifugation. The medium must then be changed the following day to remove dimethyl sulfoxide (DMSO).</li>
</ol>
7. Differentiation of human nasal epithelial cells at the air-liquid interface
NOTE: HNE were differentiated at the air-liquid interface as previously described10.
<ol>
	<li>Seed the cells at a density of 330,000 cells/filter on 0.33 cm2 collagen-coated porous filters and supplement with 300 &#181;L of the amplification medium at the apical side and 900 &#181;L of the air-liquid medium at the basolateral side.</li>
	<li>After 3 days, aspirate the apical medium and culture the cells at the air-liquid interface for 3-4 weeks in the air-liquid medium to establish a differentiated epithelium. Change the basal medium every 48-72 h.</li>
</ol>
8. CFTR modulators
<ol>
	<li>Prepare air-liquid medium containing CFTR modulators. Use VX-445, VX-661, and VX-809 at a final concentration of 3 &#181;M and VX-770 at 100 nM. Use corrector ABBV-2222 at 1 &#181;M final concentration and potentiator ABBV-974 at 10 &#181;M. Use 100% DMSO to dissolve all CFTR modulators.</li>
	<li>Prepare air-liquid medium containing the same amount of 100% DMSO as in corrector medium.</li>
	<li>Add the medium containing drugs or DMSO to the basolateral side of polarized HNE cells grown at an air-liquid interface and incubate for 24 h in 5% CO2 at 37 &#176;C.</li>
	<li>After 24 h, aspirate and discard the medium and replace with fresh medium prepared as in steps 8.1-8.2., and further incubate for a 24 h period in 5% CO2 at 37 &#176;C.</li>
</ol>
9. Ussing chamber studies
<ol>
	<li>Measure short-circuit current measurements (Isc) under voltage-clamp conditions as previously described9. 
	NOTE: Transepithelial electrical resistance (TEER) of cultures was measured with a chopstick voltmeter, and only cultures reaching at least 200 &#8486;&#183;cm2 were considered for the following experiments. Filters of polarized HNE cells were mounted in Ussing chambers, and short-circuit current measurements (Isc) were measured under voltage-clamp conditions.</li>
</ol>
10. Immunocytochemistry
<ol>
	<li>Perform Immuno-detection as previously described9. 
	&#8203;NOTE: CFTR immuno-detection was performed using the anti-CFTR C-terminal (24-1) monoclonal antibody overnight at a 1/100 dilution. Zona-occludens-1 (ZO-1) (1/500 dilution), alpha-tubulin (1/300 dilution), Muc 5AC (1/250 dilution) and cytokeratin 8 (1/250 dilution) staining were done on additional filters. After washing with PBS-Triton-X100 0.1%, goat secondary antibodies conjugated to Alexa 488 or 594 were added for 30 min in 10% goat serum (1/1000 dilution). After a final wash, filters were cut from support and mounted with Vectashield mounting medium containing DAPI. A confocal microscope (63x/1.4 oil differential interference contrast &#955; blue PL APO objective) was used to capture images, which were analyzed with the ImageJ software.</li>
</ol>
11. Statistical analysis
<ol>
	<li>Perform statistical analysis using appropriate software. 
	NOTE: Statistical analysis was performed using S.A.S software. As several HNE filters were obtained per patient and per condition, quantitative parameters were expressed as median values (&#177; SEM) per patient. Comparisons (mean &#177; SD) were carried out using Wilcoxon matched pairs signed rank test or unpaired t-test.</li>
</ol>

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<li>Neuberger, T., Burton, B., Clark, H., Van Goor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+primary+cultures+of+human+bronchial+epithelial+cells+isolated+from+cystic+fibrosis+patients+for+the+pre-clinical+testing+of+CFTR+modulators%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Use of primary cultures of human bronchial epithelial cells isolated from cystic fibrosis patients for the pre-clinical testing of CFTR modulators.</a> Methods in Molecular Biology. 741, Clifton, N.J. 39-54 (2011).</li>
<li>Laselva, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+pre-clinical+modulators+developed+for+F508del-CFTR+have+the+potential+to+be+effective+for+ORKAMBI+resistant+processing+mutants%5BTitle%5D)+AND+%22Journal+of+Cystic+Fibrosis%22%5BJournal%5D)">Emerging pre-clinical modulators developed for F508del-CFTR have the potential to be effective for ORKAMBI resistant processing mutants.</a> Journal of Cystic Fibrosis. 20 (1), 106-119 (2021).</li>
</ol>

The authors report no competing financial interests related to this publication or scientific video production.

The use of patient-derived nasal epithelial cells as surrogates for human bronchial epithelial (HBE) cells to measure CFTR activity in the context of personalized medicine has been proposed as HNE reproduce cells&#39; properties in culture9,11. The strong advantage of HNE over HBE cell cultures is that they are easily and non-invasively sampled. Short-circuit current measurements in HNE cell cultures enable assessment of CFTR-dependent Cl- transport ac...

Cystic Fibrosis (CF) is an autosomal recessive disorder resulting from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene leading to the absence or dysfunction of the CFTR protein, an anion channel located at the apical surface of epithelia1,2. Recent advances in CFTR therapy have improved the prognosis of the disease, and the last approved drugs combining CFTR correctors and CFTR potentiators led to major improvements in lung function and quality of life for CF patients carrying the most frequent mutation p.Phe508del mutation (F508del)3,4. Despite this promising therapeutic progress, around 10% of CF patients are ineligible as they carry mutations that are unrescuable by these CFTR modulators. For these patients, there is a need to test other drugs or drug combinations to find the most efficient combination for specific mutations, highlighting the importance of personalized therapies.
Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing and allow quantification of cyclic AMP-mediated Chloride (Cl&#8722;) transport as an index of CFTR function. HNE cells yield an accurate model of human airway, but their lifespan is limited in culture. Thanks to the optimization of culture techniques, patient-derived primary HNE cells can be conditionally reprogrammed with Rho-associated kinase inhibitor (ROCKi), amplified, and differentiated into a pseudo-stratified epithelium in air-liquid interface (ALI) conditions on microporous filters5,6. Numerous culture protocols for HNE culture exist (commercially available, serum-free, &#34;homemade&#34;, co-culture with feeder-cells, etc.), and choice of media and culture conditions have been described to impact growth, cell population differentiation and epithelial function7,8. The protocol here presents a simplified, feeder-free, ROCKi amplification method that allows to successfully obtain a large number of HNE cells that are then differentiated at ALI for CFTR function assays.
We have demonstrated that, in differentiated HNE cells, a 48 h treatment with CFTR modulators is sufficient to induce electrophysiological correction of CFTR dependent Cl- current and that the correction observed in vitro may be correlated with the patient&#39;s clinical improvement9. HNE cells, therefore, represent an appropriate model not only for fundamental CF research but for pre-clinical studies with patient-specific CFTR modulator testing. In this context of personalized therapy, the goal of the protocol was to validate that cryopreserved HNE cells from CF patients, grown in our conditions, were an appropriate model for CFTR correction studies, and similar results could be expected when comparing CFTR dependent Cl- transport from fresh and frozen-thawed cells. The study also assessed different CFTR modulators&#39; efficacy when using dual and triple therapies.

<table><tbody><tr><td>ABBV-2222</td><td>Selleckchem</td><td>S8535</td><td></td></tr><tr><td>ABBV-974</td><td>Selleckchem</td><td>S8698</td><td></td></tr><tr><td>Advanced DMEM/F-12</td><td>Life Technologies</td><td>12634010</td><td></td></tr><tr><td>Alexa 488 goat secondary antibody</td><td>Invitrogen</td><td>A11001</td><td></td></tr><tr><td>Alexa 594 goat secondary antibody</td><td>Invitrogen</td><td>A11012</td><td></td></tr><tr><td>Amphotericin B</td><td>Life Technologies</td><td>15290026</td><td></td></tr><tr><td>Anti-alpha-tubulin antibody</td><td>Abcam</td><td>ab80779</td><td></td></tr><tr><td>Anti-CFTR monoclonal antibody (24-1)</td><td>R&amp;amp;D Systems</td><td>MAB25031</td><td></td></tr><tr><td>Anti-cytokeratin 8 antibody</td><td>Progen</td><td>61038</td><td></td></tr><tr><td>Anti-Muc5AC antibody</td><td>Santa Cruz Biotech</td><td>sc-20118</td><td></td></tr><tr><td>Anti-ZO-1 antibody</td><td>Santa Cruz Biotech</td><td>sc-10804</td><td></td></tr><tr><td>Ciprofloxacin</td><td></td><td>provided by Necker Hospital Pharmacy</td><td></td></tr><tr><td>Colimycin</td><td>Sanofi</td><td>provided by Necker Hospital Pharmacy</td><td></td></tr><tr><td>Collagen type IV</td><td>Sigma-Aldrich Merck</td><td>C-7521</td><td></td></tr><tr><td>cytology brush</td><td>Laboratory GYNEAS</td><td>02.104</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich Merck</td><td>D2650</td><td></td></tr><tr><td>EGF</td><td>Life Technologies</td><td>PHG0311</td><td></td></tr><tr><td>Epinephrin</td><td>Sigma-Aldrich Merck</td><td>E4375</td><td></td></tr><tr><td>F12-Nutrient Mixture</td><td>Life Technologies</td><td>11765054</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>10270106</td><td></td></tr><tr><td>Ferticult</td><td>Fertipro NV</td><td>FLUSH020</td><td></td></tr><tr><td>Flasks 25</td><td>Thermo Scientific</td><td>156.367</td><td></td></tr><tr><td>Flasks 75</td><td>Thermo Scientific</td><td>156.499</td><td></td></tr><tr><td>Glacial acetic acid</td><td>VWR</td><td>20104.298</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich Merck</td><td>H3375</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich Merck</td><td>SLCJ0893</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich Merck</td><td>I0516</td><td></td></tr><tr><td>Mg&lt;sup&gt;2+ &lt;/sup&gt;and Ca&lt;sup&gt;2+&lt;/sup&gt;-free DPBS</td><td>Life Technologies</td><td>14190094</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15140130</td><td></td></tr><tr><td>Tazocillin</td><td>Mylan</td><td>provided by Necker Hospital Pharmacy</td><td></td></tr><tr><td>Transwell Filters</td><td>Sigma-Aldrich Merck</td><td>CLS3470-48EA</td><td></td></tr><tr><td>Triton-X100</td><td>Sigma-Aldrich Merck</td><td>T8787</td><td></td></tr><tr><td>Trypsin 0,25%</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>VX-445</td><td>Selleckchem</td><td>S8851</td><td></td></tr><tr><td>VX-661</td><td>Selleckchem</td><td>S7059</td><td></td></tr><tr><td>VX-770</td><td>Selleckchem</td><td>S1144</td><td></td></tr><tr><td>VX-809</td><td>Selleckchem</td><td>S1565</td><td></td></tr><tr><td>Xylocaine naphazoline 5%</td><td>Aspen France</td><td>provided by Necker Hospital Pharmacy</td><td></td></tr><tr><td>Y-27632</td><td>Selleckchem</td><td>S1049</td><td></td></tr></tbody></table>

primary human nasal epithelial cells: biobanking in the context of precision medicine

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells&#39; electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Fresh HNE cells cultured at the air-liquid interface display typical features of the polarized and differentiated respiratory epithelium as assessed by immunostaining (Figure 1). HNE cells re-differentiate into a heterogeneous layer of epithelial cells (positive keratin 8 immunostaining) that mimic the in vivo situation of a pseudo-stratified respiratory epithelium composed of ciliated (positive alpha-tubulin staining) and non-ciliated mucus-producing goblet cells (positive Muc5Ac i...

Watch this Scientific Journal Video about Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine at JoVE.com

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

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.

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and ...

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Research

JoVE Journal

Biology

2.8K Views.  Institut Necker Enfants Malades. Dit protocol belicht de belangrijkste stappen voor het versterken, differentiëren en cryopreserveren van primaire neusepitheelcellen. In onze kweekomstandigheden bootsen primaire neusepitheelcellen het longepitheel na. Dit maakt patiënt afgeleide culturen en gepersonaliseerde geneeskundestudies van verse of biobankcellen mogelijk.De patiënt-afgeleide nasale epitheliale culturen kunnen worden gebruikt om CFTR-activiteit te evalueren en te helpen bij cystische fibrosetherapie door de ...

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

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

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

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).

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Developmental Biology

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Neuroscience

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

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

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3',5'-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.

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.

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

Medicine

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

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

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.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

Immunology and Infection

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

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.

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public health crises in the past 20 years. Four additional HCoVs cause a significant portion of common cold cases each year (HCoV-NL63, -229E, -OC43, and -HKU1), highlighting the importance of studying these viruses in physiologically relevant systems. HCoVs enter the respiratory tract and establish infection in the nasal epithelium, the primary site encountered by all respiratory pathogens. We use a primary nasal epithelial culture system in which patient-derived nasal samples are grown at an air-liquid interface (ALI) to study host-pathogen interactions at this important sentinel site. These cultures recapitulate many features of the in vivo airway, including&#160;the cell types present, ciliary function, and mucus production. We describe methods to characterize viral replication, host cell tropism, virus-induced cytotoxicity, and innate immune induction in nasal ALI cultures following HCoV infection, using recent work comparing lethal and seasonal HCoVs as an example1. An increased understanding of host-pathogen interactions in the nose has the potential to provide novel targets for antiviral therapeutics against HCoVs and other respiratory viruses that will likely emerge in the future.

The nasal epithelium is the primary barrier site encountered by all respiratory pathogens. Here, we outline methods to use primary nasal epithelial cells grown as air-liquid interface (ALI) cultures to characterize human coronavirus-host interactions in a physiologically relevant system.

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public ...

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

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.

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

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