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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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK+inhibitor+improves+survival+of+cryopreserved+serum/feeder-free+single+human+embryonic+stem+cells.">ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells.</a> Human Reproduction. 24 (3), 580-589 (2008).</li><li>Neuberger, T., Burton, B., Clark, H., Van Goor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+primary+cultures+of+human+bronchial+epithelial+cells+isolated+from+cystic+fibrosis+patients+for+the+pre-clinical+testing+of+CFTR+modulators.">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, 39-54 (2011).</li><li>Laselva, O., 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=Emerging+pre-clinical+modulators+developed+for+F508del-CFTR+have+the+potential+to+be+effective+for+ORKAMBI+resistant+processing+mutants.">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 across the epithelium, which was shown to significantly correlate to CFTR in vivo activity assessed by nasal potential difference in patients with various genotypes9. The rescue of CFTR activity in HNE cells upon VX-809 treatment also significantly correlated to the improvement of clinical outcome FEV1 in lumacaftor-ivacaftor treated F508del homozygous patients12.
Importantly, this study shows that freshly harvested HNE cells easily tolerate transport and standard shipping conditions, (i.e., they survive 72 h at room temperature) which allows the testing of patient&#39;s cells originating from various geographical locations. The critical factors for successful culture are mainly the quality of nasal brushing and the number of living cells at sampling. A number of protocols have been published on growing and differentiating HNE cell cultures; some with no conditional reprogramming during the expansion phase13,14,15,16,17,18,19,20, some with conditional reprogramming in feeder9,11,12,21,22,23,24,25,26 or feeder-free5,8,27,28,29 conditions, and several comparing different methods7,8. They all allow obtaining a relatively large number of ALI cultures with correct differentiation and polarization as well as good TEER values. Similar to this study, many used cryopreserved HNE cells frozen at passage 113,28. Interestingly, several authors report that ROCKi can improve survival after cryopreservation when added to the post-thaw culture medium and increase the number of cells that remain attached5,29, suggesting that the amplification medium used in this study is particularly appropriate for cryopreservation.
Here, the study shows that HNE cells can be successfully biobanked, the critical factor again being the quality of the nasal brushing. A good correlation between the outcome of fresh samples and frozen-thawed ones is shown, suggesting that in order to optimize the quality of HNE cells that are going to be biobanked for further studies, a preliminary study could be performed on fresh samples if they fail to achieve correct ALI differentiation and acceptable TEER value; a second nasal brushing would be preferable rather than freezing cells that have a high probability of poor outcome. A second critical factor for cryopreservation is cell number; 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. Freezing at least 3 x 106 cells per cryovial is recommended.
The results in this study suggest that freeze-thawing does not significantly modify HNE cells&#39; electrophysiological properties or response to CFTR modulators. The range of measures in HNE cell cultures from the same F508del-homozygous patient obtained without freezing or after thawing was not different upon VX-809 + VX-770 treatment. Similar results were previously reported for HBE cells where Forskolin-stimulated Isc&#160;were stable after several thaw/culture cycles of non-treated F508del cells30. This provides evidence that HNE cells can be frozen and stored in a biobank and studied later.
An increasing number of studies show that HNE cell cultures can be used to test the efficacy of different therapies in the context of personalized medicine. The analysis showed that dual therapies combining a corrector with a potentiator all have comparable correction efficacy for CFTR-dependent Cl- secretion in F508del-homozygous HNE cells. However, an important interpatient variability was noticed, confirming published results9,12,20,25. By contrast, VX-445 + VX-661 + VX-770 tripled correction of CFTR activity compared to VX-809 + VX-770. A similar level of correction of F508del-CFTR in HNE cells upon triple combination was also observed by Laselva et al.28. As reported by Veit et al.26, correction of Cl- channel function upon VX-445 + VX-661 + VX-770 treatment was even higher, reaching 62% of mean WT-CFTR activity (range of 19-36 &#181;A/cm2 in corrected F508del-HNE and 14-50 &#181;A/cm2 in WT-HNE)26.
HNE cell cultures also served to test the efficacy of CFTR modulators for other class II mutations. An important improvement of CFTR activity upon VX-445 + VX-661 + VX-770 was observed in HNE ALI cultures harboring G85E/G85E, V520F/1717-1G&#62;A, Y569/Y569D, M1101K/M1101K, and N1303K/N1303K genotypes26. Similarly, Laselva et al.28 showed a significant correction of CFTR with M1101K and N1303K mutations but no significant correction in G85E HNE cells. Galapagos/AbbVie correctors were evaluated for these lumacaftor-ivacaftor-resistant mutants31. AbbVie corrector AC2-1 and especially bi-corrector AC1+AC2-1 effectively improved Cl- secretion in M1101K and G85E HNE cells, whereas N1303K derived cells were corrected by a combination of AC1+AC2-2.
Measures of CFTR activity in HNE cells are promising pre-clinical biomarkers predictive for the patient&#39;s response to tested treatment. Evidence that freezing-thawing cycles do not alter the correction level provides evidence that these cells can be biobanked and used as a powerful tool in the context of personalized medicine programs. A unique nasal brushing could therefore allow for sufficient material to compare drug efficacies, and patient&#39;s HNE cells could be thawed as new drugs become available with no need to resample the patients; this is particularly interesting in infants.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;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>Mg2+ and Ca2+-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 immunostaining). The overall epithelium presents tight junctions (assessed by ZO-1, zona-occludens-1 staining). Strong apical CFTR staining is visible in wild-type (WT) HNE cells, and decreased CFTR staining both at the apical surface and in the cytoplasm is observed in F508del/F508del HNE.
We considered positive outcomes as samples not only successfully expanding but also successfully differentiating into a pseudo-stratified epithelium after 3 weeks at air-liquid interface and presenting a TEER higher than 200 &#8486;&#183;cm2,&#160;allowing short-circuit current measurements. As only a few dedicated labs perform CFTR-related Cl- secretion assays by short-circuit current measurements, samples often require transport from hospitals to dedicated facilities.
In the 78 fresh HNE cell samples, success rates were compared in nasal brushings seeded immediately and in brushings seeded after 1-7 days of transport in flushing medium at room temperature. The majority of samples (55%) were seeded 1 day after brushing, and 82% of samples were seeded within 48 h. The mean percentage of successful cultures in all 78 samples was 82% and reached 87% in samples seeded between day 0 and day 5 (Table 2).
The success and failure rates for identical samples differentiated in both fresh and freeze-thawed conditions were compared. 83% of samples that differentiated successfully in fresh conditions also were successful in cryopreserved samples. Likewise, in samples that failed to differentiate in fresh conditions, 71% were also unsuccessful in freeze-thaw conditions (Table 2). All these data suggest that the quality of nasal brushing is the key factor to a successful culture.
We also wanted to determine the optimal number of cells per cryovial. Samples frozen at passage 0, from a single 25 cm2 generally only allow to reach 1-1.5 x 106 cells per flask. Samples frozen at passage 1 and amplified to three 75 cm2 flasks generally allowed to reach 10 x 106 cells, thus allowing to freeze several vials containing at least 3 x 106 cells. In the culture conditions described in this protocol, 50% of samples frozen between 2 x 106 and 3 x 106 cells per cryovial failed to grow when thawed, reaching 80% when below 2 x 106 cells per cryovial (Table 2).
CFTR-related Cl- secretion reflects CFTR function and can be measured by short circuit current (Isc) experiments in Ussing chambers. The Isc variation in response to Forskolin (&#916;IscForskolin) and CFTR inhibitor inh172 (&#916;IscCFTRinh172)&#160;are the main endpoints for CFTR corrector efficacy assessment.
Short-circuit-current experiments were performed on either fresh HNE, expanded in amplification medium and seeded onto filters at passage 2 and grown at air-liquid interface for 3 weeks in air-liquid medium, or from HNE that had been previously frozen at passage 1 in enriched freezing medium, thawed, expanded, and seeded on porous filters at passage 3. Cryopreservation times ranged from 3 months to 16 months, with a mean of 9.8 months. In the culture and cryopreservation conditions detailed in this study, no significant changes in growth or morphology were observed between different passages or fresh or frozen-thawed F508del/F508del HNE cultures.
For both mean &#916;IscForskolin (Figure 2A) and mean &#916;IscCFTRinh172 (Figure 2B) in DMSO treated HNE cells, no significant difference was observed between fresh or frozen-thawed HNE. To assess whether functional correction of CFTR was influenced by cryopreservation, mean Isc changes were measured in VX-809 + VX-770 treated cells compared to DMSO treated HNE cells in fresh (HNE cells derived from n =78 patients) or frozen-thawed HNE (HNE cells derived from n = 9 patients) (Figure 2). Both frozen-thawed and fresh cells displayed significant correction upon treatment, with a significant increase in &#916;IscForskolin (p &#60; 0.05) (Figure 2A), and a significant decrease in the &#916;IscCFTRinh172 (p &#60; 0.05) (Figure 2B). The mean fold increase for &#916;IscForskolin (&#916;IscForskolin VX-809 + VX-770/&#916;IscForskolin DMSO) reached 2.9 &#177; 1.6 in frozen-thawed HNE cells and was not significantly different from the fold increase in fresh HNE cells (2.5 &#177; 2.0). Similarly, the fold decrease in &#916;IscCFTRinh172 (&#916;IscCFTRinh172 VX-809 + VX-770/&#916;IscCFTRinh172 DMSO) was not significantly affected by cryopreservation: 1.49 &#177; 0.9 in frozen-thawed HNE compared to 2.5 &#177; 3.7 in fresh HNE.
The functional correction activity of several modulator therapies was further investigated to assess whether this HNE cell model could discriminate between different treatments. The results here combine HNE cells from both fresh and frozen-thawed conditions as the response to treatment was not significantly altered by cryopreservation (Figure 2).
We first assessed the corrector response of two approved CFTR modulator therapies on Cl- transport after a 48 h treatment with either VX-809 (3 &#181;M) + VX-770 (100 nM) or VX-661 (3 &#181;M) + VX-770 (100 nM) in HNE cells from 10 F508del/F508del patients (Figure 3). As compared to DMSO treated HNE cells, &#916;IscForskolin&#160;(Figure 3A) and &#916;IscCFTRinh172&#160;(Figure 3B) were significantly improved by both VX-809 + VX-770 and VX-661 + VX-770. Both treatments functionally corrected CFTR-related Cl- secretion to a similar level, with a significant mean fold increase in &#916;IscForskolin&#160;of 1.75 &#177; 1.39 for VX-809 + VX-770 (p &#60; 0.001) and 0.97 &#177; 0.93 for VX-661 + VX-770 (p &#60; 0.01). Mean &#916;IscCFTRinh172 fold decrease was 1.79 &#177; 2.04 for VX-809 + VX-770 (p &#60; 0.001) and was not significantly different from the mean &#916;IscCFTRinh172 fold decrease of 1.16 &#177; 1.44 for VX-661 + VX-770 (p &#60; 0.001 vs. DMSO).
Three different CFTR modulator therapies in HNE from six F508del/F508del patients (Figure 4) were further compared. The results here combine HNE cells from both fresh and frozen-thawed conditions. Both Vertex and Abbvie CFTR modulators, when used as a combination of a CFTR corrector and a CFTR potentiator, corrected &#916;IscForskolin&#160;(Figure 4A) and &#916;IscCFTRinh172&#160;(Figure 4B) to a similar extent. The partial correction observed in Figure 3 by VX-809 + VX-770 was confirmed in these HNE cells with a mean &#916;IscForskolin fold increase of 2.87 &#177; 1.67 (p &#60; 0.05). ABBV-2222 (1 &#181;M) + ABBV-974 (10 &#181;M) treatment induced a similar correction representing 91% of VX-809 + VX-770 correction for &#916;IscForskolin&#160;(mean &#916;IscForskolin fold increase of 2.6 &#177; 1.4, p &#60; 0.05 vs. DMSO) and 85% of VX-809 + VX-770 for &#916;IscCFTRinh172&#160;(mean &#916;IscCFTRinh172 fold decrease of 4.2 &#177; 5.7, p &#60; 0.05 vs. DMSO).
Interestingly, when HNE were treated with the triple combination VX-445 (3 &#181;M) + VX-661 (3 &#181;M) + VX-770 (100 nM), correction efficacy significantly improved, reaching 312% of VX-809 + VX-770 &#916;IscForskolin correction (mean &#916;IscForskolin fold increase of 9.0 &#177; 4.1, p &#60; 0.05 vs. VX-809 + VX-770) and 372% of &#916;IscCFTRinh172 (mean &#916;IscCFTRinh172 fold decrease of 15.5 &#177; 10.5, p &#60; 0.05 vs. VX-809 + VX-770).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63409/63409fig01.jpg" /> 
Figure 1: Differentiation markers in HNE cultures. Representative confocal microscopy images of immune-fluorescent staining of CFTR (green), Zona-Occludens-1 (ZO-1, red), alpha-tubulin (green), Muc5AC (red) and cytokeratin 8 (K8, green) in wild-type (WT) (first panel) and F508del (panels 2 -4) HNE cells grown at air-liquid interface for 3 weeks. Nuclei were stained blue with DAPI.&#160;Projections of confocal microscopy images, top view (upper panels) and side view (lower panels) of three-dimensional (3D) Z stacks reconstitutions. Scale bar (20 &#181;m) applies to both upper and lower panels. <a href="https://www.jove.com/files/ftp_upload/63409/63409fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63409/63409fig02.jpg" /> 
Figure 2: Effect of freezing-thawing on CFTR-related Cl- secretion in HNE cells. Summary of short-circuit current (Isc) recordings (mean &#177; SD) in HNE cultures derived from F508del/F508del patients and grown at air-liquid interface for 3 weeks. Cells were treated for 48 h with vehicle alone (DMSO) or VX-809 (3 &#181;M) + VX-770 (100 nM) in fresh (light grey, patient-derived HNE cells from 78 patients) or frozen-thawed (dark grey, patient-derived HNE cells from 9 patients). Data represent the mean (&#177; SD) response to acutely added (A) Forskolin (10 &#181;M)/3-isobutyl-1-methylxanthine (IBMX, 100 &#181;M), &#916;IscForskolin or (B) CFTR inhibitor inh172 (5 &#181;M) &#916;IscCFTRinh172. A minimum of two filters were analyzed per patient and per condition. Asterisks indicate the statistical difference between correctors and control (DMSO-treated) * p &#60; 0.05 (unpaired t-test). <a href="https://www.jove.com/files/ftp_upload/63409/63409fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63409/63409fig03.jpg" /> 
Figure 3: Correction of CFTR function by dual therapies. Summary of short-circuit current (Isc) recordings (mean &#177; SD) in HNE cultures derived from 10 F508del/F508del patients and grown at air-liquid interface for 3 weeks (combined results from both fresh and frozen-thawed cultures). Cells were treated for 48 h with vehicle alone (DMSO); VX-809 (3 &#181;M) + VX-770 (100 nM) or VX-661 (3&#181;M) + VX-770 (100 nM). Data represent the mean (&#177; SD) response to acutely added (A) Forskolin (10 &#181;M)/IBMX (100 &#181;M), &#916;IscForskolin or (B) CFTR inhibitor inh172 (5 &#181;M), &#916;IscCFTRinh172. A minimum of two filters were analyzed per patient and per condition. Asterisks indicate a statistical difference between correctors and control (DMSO-treated) ** p &#60; 0.01, *** p &#60; 0.001, **** p &#60; 0.0001 (Wilcoxon matched pairs signed rank test). <a href="https://www.jove.com/files/ftp_upload/63409/63409fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63409/63409fig04.jpg" /> 
Figure 4: Efficiency of triple-therapy vs. dual therapies on CFTR correction. Summary of short-circuit current (Isc) recordings (mean &#177; SD) in HNE cultures derived from 6 F508del/F508del patients and grown at air-liquid interface for 3 weeks (combined results from both fresh and frozen-thawed cells). Cells were treated for 48 h with vehicle alone (DMSO); VX-809 (3 &#181;M) + VX-770 (100 nM); ABBV-2222 (1 &#181;M) + ABBV-974 (10 &#181;M); or VX-661 (3 &#181;M) + VX-445 (3 &#181;M) + VX-770 (100 nM). Data represent the mean (&#177; SD) response to acutely added (A) Forskolin (10 &#181;M)/IBMX (100 &#181;M), &#916;IscForskolin or (B) CFTR inhibitor inh172 (5 &#181;M), &#916;IscCFTRinh172. A minimum of two filters were analyzed per patient and per condition. Asterisks indicate a statistical difference between treatments. * p &#60; 0.05 (Wilcoxon matched pairs signed rank test). <a href="https://www.jove.com/files/ftp_upload/63409/63409fig04large.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>Media Component</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td colspan="2">AMPLIFICATION MEDIUM</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12 Nutrient mixture</td>
			<td>86%</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>100 U/mL</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Tazocillin</td>
			<td>10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>2.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Colimycin</td>
			<td>16 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>3 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>0.2 &#181;M</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Epinephrine</td>
			<td>0.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Y-27632 (Rho-associated kinase inhibitor)</td>
			<td>10 &#181;M</td>
		</tr>
		<tr>
			<td colspan="2">AIR-LIQUID MEDIUM</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12 Nutrient mixture</td>
			<td>94%</td>
		</tr>
		<tr>
			<td>UltroserG</td>
			<td>2%</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>100 U/mL</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Tazocillin</td>
			<td>10 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>2.5 &#181;g/mL</td>
		</tr>
		<tr>
			<td>Colimycin</td>
			<td>16 &#181;g/mL</td>
		</tr>
		<tr>
			<td colspan="2">ENRICHED FREEZING MEDIUM</td>
		</tr>
		<tr>
			<td>F12 Nutrient Mixture</td>
			<td>77%</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>10%</td>
		</tr>
	</tbody>
</table>
Table 1: Amplification, air-liquid, and enriched freezing medium composition. List of reagents and corresponding final concentrations.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Length of conservation of nasal brushing before seeding (n = 78)</td>
		</tr>
		<tr>
			<td>Days before seeding</td>
			<td>Success rates (%)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>83</td>
		</tr>
		<tr>
			<td>1</td>
			<td>84</td>
		</tr>
		<tr>
			<td>2</td>
			<td>67</td>
		</tr>
		<tr>
			<td>3</td>
			<td>100</td>
		</tr>
		<tr>
			<td>4</td>
			<td>89</td>
		</tr>
		<tr>
			<td>5</td>
			<td>100</td>
		</tr>
		<tr>
			<td>7</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="2">Outcomes from the same initial nasal brushing grown in both fresh and frozen-thawed conditions (n = 13)</td>
		</tr>
		<tr>
			<td>Outcome in fresh conditions</td>
			<td>Success rates upon freeze-thawing (%)</td>
		</tr>
		<tr>
			<td>&#160;success</td>
			<td>83</td>
		</tr>
		<tr>
			<td>failure</td>
			<td>29</td>
		</tr>
		<tr>
			<td colspan="2">Impact of cell number on success rates (n = 36)</td>
		</tr>
		<tr>
			<td>Number of cells per cryovial</td>
			<td>Success rates upon freeze-thawing (%)</td>
		</tr>
		<tr>
			<td>&#60;2 x 106</td>
			<td>20</td>
		</tr>
		<tr>
			<td>2&#8211;3 x 106</td>
			<td>50</td>
		</tr>
		<tr>
			<td>3&#8211;4 x 106</td>
			<td>67</td>
		</tr>
		<tr>
			<td>&#62;4 x 106</td>
			<td>79</td>
		</tr>
	</tbody>
</table>
Table 2: Parameters influencing success rates in fresh and frozen-thawed HNE cell cultures. Success rates were defined as nasal brushings that successfully expanded and differentiated into a pseudo-stratified epithelium after 3 weeks at the air-liquid interface and presented a TEER higher than 200 &#8486;&#183;cm2. Before seeding, samples were shipped at room temperature in flushing medium with antibiotics. Results represent data from 78 fresh samples and 36 frozen-thawed samples. 13 nasal brushings were analyzed in both fresh and frozen-thawed conditions.

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 ...

primary-human-nasal-epithelial-cells-biobanking-context-precision

Nanyang Technological University

Research

JoVE Journal

Biology

2.7K Views.  Institut Necker Enfants Malades. 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. 

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

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 ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Nasal Potential Difference to Quantify Trans-epithelial Ion Transport in Mice

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful in cases of attenuated, oligo- or mono-symptomatic forms of CF usually diagnosed later in life, and of CF-related disorders such as congenital bilateral absence of vas deferens, idiopathic chronic pancreatitis, allergic bronchopulmonary aspergillosis, and bronchiectasis. In both clinical and preclinical settings, the test has been used as a biomarker to quantify responses to targeted therapeutic strategies for CF. Adapting the test to a mouse is challenging and can entail an associated mortality. This paper describes the adequate depth of anesthesia required to maintain a nasal catheter in situ for continuous perfusion. It lists measures to avoid broncho-aspiration of solutions perfused in the nose. It also describes the animal care at the end of the test, including administration of a combination of antidotes of the anesthetic drugs, leading to rapidly reversing the anesthesia with full recovery of the animals. Representative data obtained from a CF and a wild-type mouse show that the test discriminates between CF and non-CF. Altogether, the protocol described here allows reliable measurements of the functional status of trans-epithelial chloride and sodium transporters in spontaneously breathing mice, as well as multiple tests in the same animal while reducing test-related mortality.

Here, we present a protocol to measure nasal potential difference in mice. The test quantifies the function of transmembrane ion transporters such as the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel. It is valuable to evaluate the efficacy of novel therapies for cystic fibrosis.

The nasal potential difference test has been used for almost three decades to assist in the diagnosis of cystic fibrosis (CF). It has proven to be helpful ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...