The authors would like to thank Lisa Dailey for the helpful inputs. 
This work was supported by grants from the National Institutes of Health (ES013611 and HL095163), the Flight Attendant Medical Research Institute (FAMRI), and the Environmental Protection Agency (CR83346301) and declares no conflict of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Although the research described in this article has been funded in part by the U.S. Environmental Protection Agency through cooperative agreement CR83346301 with the Center for Environmental Medicine, Asthma and Lung Biology at the University of North Carolina-Chapel Hill, it has not been subjected to the agency's required peer and policy review and therefore does not necessarily reflect the views of the agency, and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Loretta M&uuml;ller is supported by the Swiss National Science Foundation with a personal grant.

1. Prepare Plastic Ware: Coat Plate
<ol>
	<li>Add 500 &mu;l PureCol (1:100 diluted with sterile water) to each well of a 12-well plate.</li>
	<li>Incubate at 37 &deg;C in an incubator for 30 min, remove the PureCol and rinse with HBSS right before the cells are added to the plate (see step 3.1 below).</li>
</ol>
2. Obtaining and Pretreating Biopsy of Human NECs
<ol>
	<li>Nasal scrape biopsy
	<ol>
		<li>Obtain superficial ECs lining the nasal turbinates under direct vision through a 9 mm reusable polypropylene nasal speculum (Model 22009) on an operating otoscope with speculum (Model 21700). This device provides optimal visualization of the nasal turbinates and flexibility of motion.</li>
		<li>Perform the biopsies with the subject seated upright in a straight-backed chair with head tilted back slightly or lying in a supine position on an examination table.</li>
		<li>Insert the otoscope speculum into the nostril and the inferior turbinate visualized with illumination.</li>
		<li>Insert a sterile thermoplastic curette through the speculum with the tip extended distally to the back of the turbinate.</li>
		<li>Using gentle pressure on the inferior surface of the turbinate, the curette is drawn across the mucosal surface 5x and retracted. A successful retrieval of mucosal cells held by capillary action will be evident in the cup of the curette.</li>
	</ol>
	</li>
	<li>Place sample in a 15 ml conical tube containing 8 ml of RPMI 1640, transport on ice.</li>
	<li>Use forceps to retrieve the probe, dislodge tissue from rhino probe with a P-1000 pipette, discard probe</li>
	<li>Centrifuge to pellet (4 &deg;C, 400 x g, 5 min), aspirate supernatant (attention: careful not to aspirate the pellet).</li>
	<li>Resuspend the pellet in 1 ml BEGM with 10 &mu;l of 100x DNase 1.</li>
	<li>Incubate at RT for 20 min.</li>
	<li>Centrifuge (4 &deg;C, 400 x g, 5 min), do NOT aspirate the supernatant!</li>
</ol>
3. Seed the Cells on Plastic (Day 0)
<ol>
	<li>Remove the PureCol from the plate and rinse with HBSS (see also step 1.2).</li>
	<li>Add a minimum volume of BEGM++ media (80-100 &mu;l, until a meniscus of media appears in the center of the well) into 1-4 wells (depending on biopsy size) of the coated plate.</li>
	<li>Use a P-1000 pipette to carefully remove the pellet of the biopsy tissue from the tube, without aspirating too much media.</li>
	<li>Transfer the pellet into the middle of the wells, keep the biopsy together as a cluster of cells, and do not break up to make a single cell suspension. A good biopsy yields up to 4 wells that can be seeded. Put the plate into tissue culture incubator (37 &deg;C, 5% CO2, &gt;90% relative humidity).</li>
	<li>Check after two hours to assure that there is enough media (without disturbing the meniscus).</li>
	<li>Day1, 24 hr post-seeding: collect all non-adherent cells of all wells (tilt plate and collect media with cells in a P-1000, collect all wells in a 1.5 ml centrifuge tube).</li>
	<li>Centrifuge (4 &deg;C, 400 x g, 5 min).</li>
	<li>While centrifuging cell suspension: add about 250 &mu;l of BEGM++ and 250 &mu;l of BEGM+ media mixture to the cells seeded on day 0.</li>
	<li>Repeat steps 3.1-3.5 for the non-adherent cells.</li>
	<li>Day 2-5: change media of the cells daily; add 500 &mu;l media to each well, reduce the amount of BEGM++ media stepwise (day 2 after seeding: 125 &mu;l BEGM++ plus 375 &mu;l BEGM+, day3 after seeding and the following days: 73 &mu;l BEGM++ plus 427 &mu;l BEGM+).</li>
</ol>
4. Expanding the Cells in the Flasks
<ol>
	<li>Monitor cells daily; once they have reached 80-90% confluency (usually 5-7 days after the biopsy, if they take longer they won't grow successfully) lift cells as following: carefully aspirate media; add 500 &mu;l of 0.25% trypsin per well, keep them at 37 &deg;C until the cells are detached (it usually takes 2-3 min).</li>
	<li>Prepare the needed volume of Soy Bean Trypsin Inhibitor (SBTI) (750 &mu;l per 1 ml Trypsin) in a 15 ml tube.</li>
	<li>Dislodge cells by repeatedly pipetting up and down the trypsin solution; transfer detached cells in the 15 ml conical tube with SBTI.</li>
	<li>Rinse all wells with in total 1 ml HBSS, add HBSS to the tube with the cells.</li>
	<li>Centrifuge to pellet (4 &deg;C, 400 x g, 5 min), aspirate the media, resuspend in 1 ml BEGM+ media and vortex the tube.</li>
	<li>Expand cells in a T25 or T75 flask depending on pellet size (this is p1; approximately two confluent wells go into one T75 flask, one confluent well is enough for one T25, usually 2 confluent wells yield one T75).
	<ol>
		<li>Add BEGM+ to the flasks (5 ml each for a T75, 3 ml each for a T25).</li>
		<li>Add the cell suspension to the flasks. Transfer the flasks into a tissue culture incubator.</li>
	</ol>
	</li>
	<li>24 hr post-flask-seeding: add additional BEGM+ media to the flask (3 ml to a T25, 10 ml to a T75).</li>
	<li>maintain the cells in the flasks: Remove media (aspirate from the corner) and add BEGM+ media (5 ml/T25, 20 ml/T75). The media needs to be changed every 2-3 days. Maintain the cells in the flask until they are about 80% confluent (confluency may not be even throughout the entire flask; normally this takes between 7-10 days; if they are not confluent after 10 days they won't grow successfully).</li>
</ol>
5. Seed the Cells on Tissue Culture Inserts
<ol>
	<li>Remove the media from the flask and add Trypsin (3 ml for T25, 4 ml for T75 flask).</li>
	<li>Incubate at 37 &deg;C until the cells are detached (about 2-3 min).</li>
	<li>Prepare SBTI (750 &mu;l per 1 ml Trypsin &gt; 2.25 ml for T25, 3 ml for T75) in a 15 ml tube.</li>
	<li>Dislodge cells by taping the flask and pipetting up and down and transfer to conical tube with the SBTI.</li>
	<li>Rinse the flask with HBSS (1 ml for T25, 2 ml for T75), add the HBSS to the 15 ml tube.</li>
	<li>Centrifuge to pellet (4 &deg;C, 400 x g, 5 min), remove supernatant carefully.</li>
	<li>Prepare the plates: decide how many plates are going to be seeded, label the plates with sample code and number, passage number (i.e. p2) and date. Add 700 &mu;l BEGM+ media to the basal chamber.</li>
	<li>Resuspend the cell pellet in 1 ml BEGM ALI media by vortexing the tube.</li>
	<li>Count the cells with a hemocytometer (a T25 usually produces about 4 million cells; a T75 can have up to 20 million cells depending on the islet; use 1-2 million of cells for one 12-well plate) and dilute the cell suspension with BEGM ALI media to the total volume needed for the amount of plates that should be seeded (1.2 x 106 cells in 2.5 ml media per plate).(Note: If part of the cells want to be frozen, label the tube and remove cells here: we usually freeze 2 x 106 cells in 2 ml of freezing media)</li>
	<li>Add 200 &mu;l cell suspension to the apical side of each uncoated Corning 12well cell insert (&gt;this will be about 80,000-150,000 cells/insert; 12 mm transwells with 0.4 &mu;m pore polyester (Polyethylene Terephthalate (PET)) membrane insert); transfer into tissue culture incubator.</li>
	<li>After 24 hr, change the apical media (200 &mu;l expansion medium).</li>
	<li>Maintaining the cell cultures: Change the media (BEGM ALI media; 700 &mu;l basolateral and 200 &mu;l apical) every other day. Inserts have to be maintained submerged until cells are totally confluent (no holes in monolayer are visible). Depending on cell number this usually takes between 2-6 days, if it takes longer the cells will most likely not be viable.</li>
</ol>
6. Establish Air Liquid Interface Once the Cells are Completely Confluent (When the Cells on the Transwells are Completely Confluent)
<ol>
	<li>Once the cells are completely confluent replace both apical and basolateral media with BEGM ALI media supplemented with 500nM retinoic acid for 48 hr.</li>
	<li>48 hr after adding the retinoic acid supplemented BEGM ALI media: Prepare PneumaCult-ALI Maintenance Medium (following manufacturer's instructions): Calculate the volume of medium needed (for one plate usually 8.5 ml for basal compartments); add per 1 ml PneumaCult Complete Base Medium:
	<ul>
		<li>10 &mu;l PneumaCult 100x Maintenance Supplement</li>
		<li>2 &mu;l Heparin (of a 2 mg/ml stock solution)</li>
		<li>5 &mu;l Hydrocortisone (of a 96 &mu;l/ml stock solution).</li>
	</ul>
	</li>
	<li>Remove all media and add 700 &mu;l PneumaCult-ALI maintenance media to the basolateral side only (no medium on the apical side).</li>
	<li>Maintain the cell cultures for 4 weeks at the ALI:
	<ol>
		<li>Change the media every other day (Monday, Wednesday and Friday schedule works well for us).</li>
		<li>After one week at ALI, once a week (Wednesdays) the apical surface is rinsed: add 200 &mu;l HBSS to the apical side, keep them for 10 min in the incubator, carefully aspirate the HBSS (use a 9 in glass pipette attached to the vacuum trap in the Biological safety cabinet, use 200 &mu;l tips on the tip of the pipette to suction off the HBSS).</li>
	</ol>
	</li>
</ol>
Note: Change tips between plates, as well as changing the tips when going from apical surface versus basolateral. After about 2 weeks at the ALI, cells start to differentiate and cilia appear. Cell cultures reach full differentiation after about 4 weeks at the ALI.

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The authors have nothing to disclose.

Respiratory ECs provide not only a physical barrier to protect the human body from exogenous stimuli20, but also act as a switchboard to initiate and regulate respiratory immune defense responses1-3 via secretion of soluble mediators or direct receptor-ligand cell-cell interactions. In order to further study the role of ECs in the immune response, to examine potential differences among ECs from diseased populations, or to study the effects of exogenous stressors on ECs, it is important to recapitula...

Goal of the technique
Epithelial cells (ECs) in the airways are among the first sites to be directly exposed to inhaled environmental stressors, such as pathogens, allergens or air pollutants. ECs are more than a structural barrier: They act as a switchboard to initiate and regulate the respiratory immune response1-3 via the release of soluble mediators4-6 and the expression of ligands/receptors on their surfac 7-9. While immortalized cell lines can be used as models to study respiratory ECs in vitro, they fail to differentiate into heterogeneous cell populations composed of polarized ciliated and mucus producing cells, similar to what is observed in vivo. To recapitulate important characteristics of the respiratory epithelium observed in vivo, primary airway ECs can be used. Therefore, our goal was to optimize our method utilizing nasal ECs (NECs) obtained from human volunteers. The NECs are expanded and cultured in vitro, yielding a cell culture model of differentiated NECs which mimics many of the features of the nasal epithelium seen in vivo.
Rationale
The use of in vitro models with human primary ECs mimicking in vivo situation - as described in this study - gives unique possibilities to study disease specific differences of ECs, physiological parameters associated with the airway epithelium, or the interactions between ECs and other cell types, such as dendritic cell 10, natural killer cells11 or macrophages. Several existing methods utilize bronchial ECs, which can be obtained invasively via brush biopsies during bronchoscopies, or from otherwise discarded lung tissue12-17. In addition, commercial sources to obtain fully differentiated human airway epithelial cell cultures exist (EpiAirway model from MatTek, Ashland, MA , Clonetics from Lonza, Basel, Switzerland, MucilAir from Epithelix Sars, Geneva Switzerland), where investigators can choose from different donors. However, because of the limited pool of commercially available epithelial cells, limited or prescribed set of parameters, the cost associated with commercially obtaining primary human airway ECs, and the limited access to discarded lung tissue or freshly obtained human bronchial biopsy tissue, prohibit many investigators from conducting studies in fully differentiated human airway ECs. Therefore, we set out to develop a technique that will 1) utilize non-invasively obtained human NECs, 2) provide a protocol yielding cultures of differentiated human airway epithelium, and 3) be reproducible in most lab settings with an existing tissue culture infrastructure.
Advantages over Existing Techniques/Models
Obtaining fresh NECs, as compared to bronchial or small airway ECs, is much less invasive, individuals can be biopsied multiple times with no significant side effects, and superficial nasal scrape biopsies can be conducted in diseased populations which would otherwise not qualify for research-related bronchoscopies. Noninvasive superficial scrape biopsies to obtain NECs allow the investigator to set donor specification a priori, including age, gender, disease status, etc. in accordance with the research objective to be accomplished. Thus, when designing research studies investigators are not limited by clinically available tissues/epithelial specimen, purchase expensive differentiated cell culture models from commercial vendors, or design invasive bronchoscopy studies. In addition, the ease to obtain NECs increases the ability to conduct experiments in cells from different donors, which enhances statistical validation of observed findings. Lastly, the nose is one of the first sites to be exposed to any kind of air-borne stressors. Thus, generating organotypic in vitro culture systems mimicking the nasal epithelium are useful for studying inhalation of viral particles, pollutants, allergen, or gases, which can be easily achieved by using this cell culture system.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Nasal speculum</td>
 <td>Welch Allyn</td>
 <td>Model 22009</td>
 <td>9 mm, reusable polyproylene</td>
 </tr>
 <tr>
 <td>Operating otoscope</td>
 <td>Welch Allyn</td>
 <td>Model 21700</td>
 <td></td>
 </tr>
 <tr>
 <td>Rhino probe cell cuvette</td>
 <td>Arlington Scientific</td>
 <td>SY-96-092</td>
 <td>bend the head of the cuvette a little more</td>
 </tr>
 <tr>
 <td>12-well culture plates</td>
 <td>Corning</td>
 <td>3512</td>
 <td></td>
 </tr>
 <tr>
 <td>Transwell membrane cell culture inserts</td>
 <td>Corning</td>
 <td>3460</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue culture flask </td>
 <td>Corning</td>
 <td>430639 and 430641</td>
 <td>T25 and T75 vented flask</td>
 </tr>
 <tr>
 <td>RPMI 1640 media (with L-Glutamine)</td>
 <td>Gibco</td>
 <td>11875</td>
 <td></td>
 </tr>
 <tr>
 <td>Bronchial/Tracheal Epithelial Cell Growth Medium</td>
 <td>Cell applications</td>
 <td>511-500</td>
 <td></td>
 </tr>
 <tr>
 <td>BEGM Bronchial Epithelial Cell Growth Medium</td>
 <td>Lonza</td>
 <td>CC-3170</td>
 <td>This comes as a kit of one bottle of medium and vials with supplements.</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate 7.5% solution</td>
 <td>Gibco</td>
 <td>25080</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>NuSerum</td>
 <td>BD Biosciences</td>
 <td>355104</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>BAMBANKER freezing media</td>
 <td>BAMBANKER</td>
 <td>302-14681</td>
 <td>Use as it is.</td>
 </tr>
 <tr>
 <td>CoolCell </td>
 <td>Biocision</td>
 <td>HTBCS-136</td>
 <td>(CryoPrep alcohol-free cell freezing container)</td>
 </tr>
 <tr>
 <td>PureCol</td>
 <td>AdvancedBioMatrix</td>
 <td>5005-B</td>
 <td>dilute 1:100 with sterile water (should be roughly 0.03 mg/ml)use 500 &mu;l/well of a 12-well plate</td>
 </tr>
 <tr>
 <td>HBSS with Ca2 and Mg2+</td>
 <td>Gibco</td>
 <td>14025</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAse 1</td>
 <td>Sigma</td>
 <td>DN25</td>
 <td>Dissolve at 1.5 mg/ml, which is a 100x solution, add 10 &mu;l to each ml of media</td>
 </tr>
 <tr>
 <td>Trypsin, 0.25%, 1x, with phenol red</td>
 <td>Gibco</td>
 <td>25200-056</td>
 <td>Use at it is</td>
 </tr>
 <tr>
 <td>Soy Bean Trypsin Inhibitor (SBTI)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td>Use dissolved in HBSS at a concentration of 1 mg/ml</td>
 </tr>
 <tr>
 <td>Retinoic acid</td>
 <td>Sigma</td>
 <td>R7632</td>
 <td>Make it up 100 &mu;M stock solution (see below), dilute to 10 &mu;M with absolute alcohol, take 5 &mu;l of this and add it to 1 ml BEGM media (our working solution). 
 
 All-trans Retinol has a coefficient of extinction of 52,480 in ethanol at 325 nm. Dissolve 25 mg in 50 ml absolute ethanol. Serial dilutions need to be made, dilute 50 ml of stock in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, take 50 ml of this, dilute in 450 ml absolute ethanol, and again take 50 ml of this, dilute in 450 ml absolute Ethanol, take 100 ml of this and dilute in 900 ml absolute ethanol and read in the spectrophotometer at 325 nm, this number is then multiplied by its' dilution factor, then divided by the extinction coefficient of 52,480, this will give a number in mM. Make a 100 mM stock (store at -20 &deg;C) and from this stock a 10 mM aliquot is made, the daily retinoic acid needed would be 5 ml of this 10 mM aliquot in 995 ml ALI media.
 Example: 325nm reading is 1.15. 1.15 x 10,000 (dilution factor) / 52,480 = 0.219 = 219 mM, make a 100 mM stock from this, in absolute ethanol.</td>
 </tr>
 <tr>
 <td>DMEM high glucose (1x), liquid, with L-glutamine and sodium pyruvate</td>
 <td>Gibco</td>
 <td>11995</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Pituitary Extract</td>
 <td>Gibco</td>
 <td>13028-014</td>
 <td>Used for BEGM ALI media.</td>
 </tr>
 <tr>
 <td>Nystatin</td>
 <td>Sigma</td>
 <td>N4014-50 mg</td>
 <td>Make up a solution of 3.3 mg/ml.</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Fisher Scientific</td>
 <td>BP1600-100</td>
 <td>Make up a solution of 10 mg/ml solution in milliQ water.</td>
 </tr>
 <tr>
 <td>PneumaCult-ALI media</td>
 <td>StemCell</td>
 <td>5001</td>
 <td>This comes as a kit of one bottle of medium and vials with supplements. For details see below.</td>
 </tr>
 <tr>
 <td>Hydrocortisone </td>
 <td>StemCell</td>
 <td>7904</td>
 <td>Dissolve 2.4 mg hydrocortisone in 1 ml ddH2O (used to make the 200x Hydrocortisone stock solution)</td>
 </tr>
 <tr>
 <td>Heparin Sodium Salt in PBS (2 mg/ml)</td>
 <td>StemCell</td>
 <td>7980</td>
 <td>Use at it is. </td>
 </tr>
 <tr>
 <td>Filter flasks (500 ml)</td>
 <td>Corning</td>
 <td>431097</td>
 <td>0.22 &mu;m pore size</td>
 </tr>
 <tr>
 <td>Filter tubes (50 ml)</td>
 <td>Millipore</td>
 <td>SCGP00525</td>
 <td>Steriflip, 0.22 &mu;m pore size</td>
 </tr>
 <tr>
 <td>Pipette tips - ART Barrier Tips</td>
 <td>Molecular Bioproducts</td>
 <td>2139RI, 2069RI, 2179RI</td>
 <td></td>
 </tr>
 <tr>
 <td>Conical tubes, sterile, 15 ml and 50 ml</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>ddH2O, absolute ethanol, ice</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CO2 incubator</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>refrigerated centrifuge</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>biological safety cabinet</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>pipettes (p2, p200, p1000), 9 in glass pipettes</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>gloves</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>lab coat with tight cuffs</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>
 <table border="1">
 <tbody>
 <tr>
 <td>Media and solutions used:</td>
 <td>Recipe</td>
 <td>Used for</td>
 </tr>
 <tr>
 <td colspan="3">General remark: always warm the media to room temperature</td>
 </tr>
 <tr>
 <td rowspan="2">BEGM media</td>
 <td><ul><li> 500 ml BEGM Cell Application media with one aliquot of retinoic acid</li></ul> </td>
 <td>(mix them half-half and filter )</td>
 </tr>
 <tr>
 <td><ul><li> 500 ml BEGM Lonza media with one aliquot of single quot supplements </li></ul></td>
 <td><ul><li> digestion of the biopsy</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="3">BEGM+ media</td>
 <td><ul><li> 50 ml BEGM media </li></ul></td>
 <td><ul><li> maintaining cells on plastic in the plates (passage0) </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l retinoic acid (working solution)</li></ul> </td>
 <td><ul><li> seeding cells in the flask (passage1; partially)</li></ul> </td>
 </tr>
 <tr>
 <td></td>
 <td><ul><li> maintain the cells in the flask </li></ul></td>
 </tr>
 <tr>
 <td rowspan="3">BEGM++ media</td>
 <td><ul><li> 50 ml Lonza's BEGM media with supplements</li></ul> </td>
 <td><ul><li> seeding fresh cells in 12-well plate on plastic</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 1 ml Sodium BiCarb</li></ul> </td>
 <td><ul><li> maintaining cells in the plates on plastic (passage0; only partially)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 2.5 ml NuSerum</li></ul> </td>
 <td><ul><li> seeding cells in the flask (passage1; partially)</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="5">BEGM ALI media</td>
 <td><ul><li> 250 ml DMEM-H </li></ul></td>
 <td rowspan="5"><ul><li> Maintaining cells on the transwell before they go ALI</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 250 ml BEGM including supplements</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 2 ml Bovine Pituitary Extract (12.5 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 1 ml Nystatin (3.3 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 75 &mu;l BSA (10 mg/ml solution)</li></ul> </td>
 </tr>
 <tr>
 <td rowspan="5">200X Hydrocortisone Stock Solution (96 &mu;l/ml solution)</td>
 <td><ul><li> 200 &mu;l dissolved hydrocortisone</li></ul> </td>
 <td rowspan="5"><ul><li> As a supplement to the PneumaCult-ALI Complete Base Medium for the last day in the flask and when cells are on inserts</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 4250 &mu;l dissolved sodium chloride </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 540 &mu;l ddH2O</li></ul> </td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l absolute ethanol</li></ul> </td>
 </tr>
 <tr>
 <td>Filter solution through 0.22 &mu;m filter, aliquot solution and store at -20 &deg;C, avoid additional freeze/thaw cycles</td>
 </tr>
 <tr>
 <td rowspan="3">PneumaCult-ALI Complete Base Media</td>
 <td><ul><li> 450 ml PneumaCult-ALI Basal Medium</li></ul> </td>
 <td><ul><li> Base medium for all three PneumaCult-ALI media </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 50 ml PneumaCult 10x Supplement </li></ul></td>
 <td><ul><li> The PneumaCult media is light sensitive, thus always keep it in the dark</li></ul> </td>
 </tr>
 <tr>
 <td>(this is stable for 2 weeks at 2-8 &deg;C or for up to 6 months at -20 &deg;C)</td>
 <td></td>
 </tr>
 <tr>
 <td rowspan="4">PneumaCult-ALI Maintenance Medium</td>
 <td>Per 1 ml of PneumaCult-ALI Complete Base Medium add:</td>
 <td rowspan="4">ALI culture on inserts</td>
 </tr>
 <tr>
 <td><ul><li> 10 &mu;l PneumaCult 100x Maintenance Supplement </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 2 &mu;l Heparin (of a 2 mg/ml stock solution) </li></ul></td>
 </tr>
 <tr>
 <td><ul><li> 5 &mu;l Hydrocortisone (of a 96 &mu;l/ml stock solution) </li></ul></td>
 </tr>
 </tbody>
</table>
 </td>
 </tr>
 </tbody>
</table>

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.

NECs cultured following the here described protocol re-differentiate into a heterogeneous layer of ECs composed of ciliated and non-ciliated cells, representing the in vivo situation (Figure 1). Some of the differentiated NECs are mucus producing (cells staining in blue using AB/PAS, Figure 1B). Even though the here presented protocol is optimized, differentiation can vary with regards to the distribution of the overall cell populations (i.e. different ratios of ciliate...

Watch this Scientific Journal Video about Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface at JoVE.com

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

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 ...

culturing-of-human-nasal-epithelial-cells-at-the-air-liquid-interface

Research

JoVE Journal

Biology

36.7K Views.  The University of North Carolina at Chapel Hill. 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.

Video: Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

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 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid acquisition require assessing mouthpart structure and function relationships; however, fluid uptake mechanisms are historically inferred from observations of structural architecture, sometimes unaccompanied with experimental evidence. Here, we report a novel method for assessing fluid-uptake abilities with butterflies (Lepidoptera) and flies (Diptera) using small amounts of liquids. Insects are fed with a 20% sucrose solution mixed with fluorescent, magnetic nanoparticles from filter papers of specific pore sizes. The crop (internal structure used for storing fluids) is removed from the insect and placed on a confocal microscope. A magnet is waved by the crop to determine the presence of nanoparticles, which indicate if the insects are able to ingest fluids. This methodology is used to reveal a widespread feeding mechanism (capillary action and liquid bridge formation) that is potentially shared among Lepidoptera and Diptera when feeding from porous surfaces. In addition, this method can be used for studies of feeding mechanisms among a variety of fluid-feeding insects, including those important in disease transmission and biomimetics, and potentially other studies that involve nano- or micro-sized conduits where liquid transport requires verification.

Fluid-feeding insects have the ability to acquire minute quantities of liquids from porous surfaces. This protocol describes a method to directly determine the ability for insects to ingest liquids from porous surfaces using feeding solutions with fluorescent, magnetic nanoparticles.

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

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 ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...