This work received funding from the European Union&#8217;s HORIZON 2020 Program for research, technological development, and demonstration under grant agreement no. 642028 H2020-MSCA-ITN-2014, NABBA - Design, and Development of advanced Nanomedicines to overcome Biological Barriers and to treat severe diseases. We thank Dr. Ana Costa and Dr. Jenny Juntke for the great support on the development of the co-culture, Olga Hartwig, for the scientific illustration, Anja Honecker, for ELISA assays, Petra K&#246;nig, Jana Westhues and Dr. Chiara De Rossi for the support on cell culture, analytics, and microscopy. We also thank Chelsea Thorn for proofreading our manuscript.

1. Growth and differentiation of cells in permeable support inserts
<ol>
	<li>Cultivate CFBE41o- in a T75 flask with 13 mL of minimum essential medium (MEM) containing 10% fetal calf serum (FCS), 1% non-essential amino acids and 600 mg/L glucose at 37 &#176;C with 5 % CO2 atmosphere. Add fresh medium to the cells every 2&#8211;3 days.
	<ol>
		<li>Detach the cells after reaching 70% confluence&#160;in the flask with 3 mL of trypsin- Ethylenediaminetetraacetic acid (EDTA) at 37&#176;C for 15&#160;min. Add 7 mL of fresh MEM and centrifuge the cells at 300 x g for 4 min at room temperature (RT). Discard the supernatant and add new 10 mL of MEM while disrupting the clumps by gently pipetting up and down.</li>
		<li>Count the cells with an automated cell counter or hemocytometer chamber. Seed cells with a density of 2 x 105 cells/well in 12-well plates with permeable supports (pore size of 3 &#956;m, see Table of Materials). 
		NOTE: The automated cell counter determines cell number, size distribution, and viability of the cells (see Table of Materials). Permeable supports with a pore size of 0.4 &#956;m could be used here; however, the macrophages, in this condition, should be added directly to the apical side, and their transcellular migration will not be assessed in this case.</li>
		<li>Seed cells at liquid-liquid condition (LLC) by adding 500 &#181;L of the cell suspension on the apical side of the permeable support and 1.5 mL of fresh medium in the basolateral side. Then incubate cells at 37&#176;C under 5% CO2, for 72 h.</li>
		<li>To shift to the air-liquid interface (ALI) culture, on the third day after seeding, remove the medium from the basolateral side first, then from the apical side. To the basolateral side, add 500 &#181;L of fresh MEM and change the medium every second day until cells form a confluent monolayer. 
		NOTE: For the conditions used in this protocol, the CFBE41o- cells usually are confluent after 3-7 days in culture.</li>
		<li>Assess the epithelial barrier properties on day 7 by incubating CFBE41o- cells with 500 &#956;L cell medium in the apical side and 1.5 mL in the basolateral side for 1 h, at 37&#176;C under 5% CO2.</li>
		<li>Measure barrier properties via transepithelial electrical resistance (TEER), with an STX2 chopstick electrode and an epithelial volt-ohmmeter; after 7 days this is higher than 300 &#8486;&#215;cm&#178;. 
		NOTE: Eventually, in some membrane inserts, the cells have low TEER. Therefore permeable inserts with TEER &#60; 300 &#8486;&#215;cm&#178; are not used.</li>
	</ol>
	</li>
	<li>To cultivate THP-1 cells, grow them in a T75 flask using 13 mL of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FCS, and incubate at 37&#176;C under 5% CO2. Split cells every second day by seeding 2 x 106 cells/mL cells in a new T75 flask. 
	NOTE: Non-differentiated THP-1 cells are grown as monocytes in suspension.
	<ol>
		<li>Differentiate the THP-1 cells as follows. Centrifuge contents of a T75 at 300 x g for 4 min. Discard the supernatant, resuspend the pellet in fresh medium and put in a new T75. Add 10 ng/mL Phorbol 12-myristate 13-acetate (PMA) incubatig the cells&#160;in RPMI for 48 h at 37 &#176;C and 5% CO2 atmosphere15. 
		NOTE: After the differentiation with PMA, cells do not proliferate anymore and attach to the flask.</li>
		<li>To detach THP-1 macrophage-like cells, wash once with phosphate-buffered saline (PBS) at 37 &#176;C and incubate with 3 mL of cell detachment solution (e.g.&#160;accutase) containing 0.5 mM EDTA for 10 min at room temperature.</li>
		<li>Inspect the cells under an inverted microscope to look for cell detachment. Add 7 mL of fresh medium and centrifuge at 300 x g for 4 min at RT. 
		NOTE: Macrophages can also be detached with trypsin-EDTA, 37 &#176;C for 20 min. However, trypsin is harsher to macrophages than the chosen cell detachment solution (see Table of Materials).</li>
		<li>After removing the supernatant, re-suspend macrophage cells in 3 mL of THP-1 medium into a 15 mL conical tube, count the cells as described in 1.1.2. and incubate for a maximum 1 h at 37 &#176;C under 5% CO2 before setting up the co-culture. 
		NOTE: THP-1 cells in suspension can be stained with viability dyes to image the co-culture further. At this step, use the procedure below (step 1.2.5).</li>
		<li>Stain macrophages with 10 &#181;M of a cell viability dye (based on the conversion of acetate moieties by intracellular esterases, see Table of Materials) in which 3 &#181;L of the cell viability dye is applied to the cell suspension. Incubate cells for 20 min at 37 &#176;C, 5% CO2, then wash 1x with PBS 37&#176;C to remove the dye. 
		NOTE: Centrifuge the cells to remove the dye at 300 x g for 4 min at room temperature (RT).</li>
	</ol>
	</li>
</ol>
2. Establishment of an epithelial-macrophage co-culture on permeable supports
<ol>
	<li>Use CFBE41o- monolayers at ALI with TEER &#8805; 300 &#8486;&#215;cm&#178; (step 1.1.6.). Remove the medium from the lower chamber, carefully invert the support inside a sterile glass Petri dish (50 mm x 200 mm), and remove the cells overgrown through the membrane pores on the bottom side of the membrane using a cell scraper. 
	NOTE: Due to the pore size of 3 &#181;m, epithelial cells tend to grow through the pores toward the basolateral side. Therefore, one needs to remove them before adding the macrophages on this side. CFBE41o- lung epithelial cells can be stained at this step. The procedure in step 1.2.5 can be used; however, instead of a cell suspension, the dye solution in MEM is applied (500 &#181;L apical side only) on the adhered cells on the permeable support.</li>
	<li>Use 2 x 105 cells/well (in 200 &#181;L of RPMI) from the cell suspension of PMA-differentiated THP-1 macrophages and place the cells on the basolateral side of the inverted inserts.</li>
	<li>Close the Petri dishes carefully and incubate for 2 h at 37 &#176;C under 5% CO2.</li>
	<li>Place the inserts back into the 12-well microplates and add 500 &#181;L of MEM medium in the basolateral side of the permeable insert to maintain ALI conditions. The cells are now ready for infection.</li>
</ol>
3. Infection by P. aeruginosa
NOTE: All following steps from here must be done in a biosafety level 2 (BSL2) laboratory.
<ol>
	<li>Inoculate 15 mL of lysogeny broth (LB) supplemented with 300 &#956;g/mL ampicillin in an Erlenmeyer flask (50 mL) with a single colony of P. aeruginosa PAO1-GFP. 
	NOTE: Other strains of P. aeruginosa could also be used here, for instance, PAO1 wild type, PA14, or clinical strains, following their own cultivation protocols.</li>
	<li>Incubate the bacteria for 18 h at 37&#176;C, shaking at 180 rpm.</li>
	<li>Transfer the contents after the 18 h to a 50 mL conical tube and centrifuge at 3850 x g for 5 min. Discard the supernatant and add 10 mL of sterile PBS at 37&#176;C.</li>
	<li>Measure optical density on a spectrophotometer at wavelength 600 nm and adjust the concentration of bacteria using the cell culture medium to a final concentration of 2 x 105 CFU/mL. This corresponds to a multiplicity of infection (MOI) of one bacterium per epithelial cell.</li>
	<li>Add 100 &#956;L of bacterial suspension to the apical side of the permeable support (step 2.4.) and incubate at 37 &#176;C under 5% CO2 for 1 h, to allow bacteria attachment to the cells. Then, remove apical liquid carefully with a pipette to restore ALI conditions. Keep some samples uninfected as a control. 
	NOTE: At this stage, the bacteria attached should be plated in LB agar (see steps 5.4/5.5) to determine the initial bacteria inoculum.</li>
	<li>Incubate the drug of interest&#160;after bacterial adhesion in the cells. For treatment experiments, add 500 &#956;L of a drug solution diluted in cell medium (in this protocol tobramycin 6 &#956;g/mL was used) to the apical side. Add 1,500 &#181;L of cell medium without drug on the basolateral side. 
	NOTE: Instead of instilling the drugs as a solution, the model can also be adapted to aerosol deposition. For such purposes, the cells at ALI are fed from the basolateral side with 500 &#181;L of cell medium. The drug is then first nebulized and allowed to deposit in the apical compartment by an appropriate device (not described here). The infected and treated sample can be checked for the endpoints outlined in the sections 4&#8211;7. From this step on, permeable supports can be used to create either images (section 4) or to get results of bacteria growth and mammalian cell viability, amongst others (sections 5&#8211;7).</li>
</ol>
4. Sample preparation for confocal laser-scanning microscopy (CLSM)
<ol>
	<li>After the establishment of the co-culture, infection and drug treatment, remove all medium from the apical and basolateral side. Wash 1x with PBS at 37&#176;C&#160;and then fix the cells with 3% paraformaldehyde (PFA) for 1 h at RT (300 &#181;L on apical/600 &#181;L on basolateral). Cell nuclei are stained with 5 &#181;g/mL of DAPI-PBS for 30 min at room temperature. 
	CAUTION: PFA is hazardous.</li>
	<li>Cut the membranes using a scalpel and place them between two 12 mm microscopy cover slides using a mounting medium (see Table of Materials). Let it dry inside the flow bench for 30 min before storage at 4 &#176;C. Visualize by confocal scanning microscopy. 
	NOTE: After the co-culture and before the mounting, tight junctions immunostaining can be performed. For that, cells are fixed with paraformaldehyde 3% for 30 min, washed again with PBS, and permeabilized with saponin 0.05%/BSA 1% in PBS. In this protocol, the zonula occludens protein (ZO-1) was detected via mouse anti-human ZO-1 antibody (1:400, incubation at 4 &#176;C overnight). The samples were then incubated for 2 h at RT with goat anti-mouse IgG antibody Alexa Fluor 633 (1:2000 in red). Nuclei were stained with DAPI (1 &#181;g/mL) and mounted with mounting medium on coverslips.</li>
	<li>Use a confocal microscope for imaging the stored membranes. Choose 25x or 63x water-immersion objectives and lasers at 405, 488, 505 or 633 nm for detection. Images should have a 1024&#8201;x&#8201;1024 pixel resolution. 
	NOTE: The lasers are chosen according to the stain used.</li>
	<li>Acquire apical and cross-section views, and use zeta-stack mode (10&#8211;15 stacks) for the construction of a three-dimensional model using imaging software.</li>
</ol>
5. Measurement of bacterial proliferation via colony-forming units (CFU)
<ol>
	<li>Collect the apical and basolateral medium (containing bacteria) to assess CFU of non-attached bacteria. Collect&#160;500 &#181;L from the apical and basolateral sides and pool them. 
	NOTE: Use this suspension directly to count bacteria (step 5.4) or centrifuge at 21,250 x g for 10 min to evaluate lactate dehydrogenase (LDH) from the supernatant (section 6) and/or re-suspended&#160;in PBS to count extracellular bacteria (step 5.4).</li>
	<li>Assess survival of bacteria attached and/or internalized in the cells by adding 500 &#181;L of sterile deionized cold water in each compartment of the permeable support. Incubate cells for 30 min at room temperature. 
	NOTE: The samples can either be plated on LB agar (see step 5.4) or frozen (as whole insert plate) at -20 &#176;C for plating later on.</li>
	<li>For assessing CFU of adherent/internalized bacteria, thaw samples at 37&#176;C for 10 min (if frozen). Using pipette tips for each well, scrape the membrane surface and pipette up and down to remove all adhered content. 
	NOTE: At this step, all the epithelial cells are lysed and adherent/internalized bacteria are available as a suspension to be plated.</li>
	<li>With the bacterial suspension from both fractions, perform a 1/10 serial dilution using Tween-80 0.05% in PBS and plate the bacteria on LB agar plates. 
	NOTE: Dilutions in between 1 to 10 are recommended. The bacteria should be counted in the highest dilution, where single colonies are first identified.</li>
	<li>Incubate agar plates at 30&#176;C for 16&#8211;72 h to count colonies, and calculate CFU accordingly. 
	NOTE: A temperature of 30&#176;C at the time of plate incubation is essential for treated-samples and to observe delayed-growth of colonies.</li>
</ol>
6. Evaluation of cell cytotoxicity via lactate dehydrogenase assay
<ol>
	<li>Use the supernatant of infected cells containing bacteria (from step 5.1) for cell viability assessment for LDH assay16. Centrifuge the supernatant at 21,250 x g for 10 min to pellet the bacteria and eventually rest of the cells. Use the bacteria-free supernatant to measure LDH release. 
	NOTE: The supernatant should not be frozen before measuring LDH by this assay.</li>
	<li>Transfer 100 &#181;L of the supernatant to a 96-well plate, and add 100 &#181;L of the LDH assay solution (see the Table of Materials). Incubate at room temperature for 5 min in the dark, then read absorbance at 492 nm.</li>
</ol>
7. Assessing the release of human cytokines
<ol>
	<li>For cytokine quantification, use either ELISA or cytometric bead array immunoassay17(see the Table of Materials).&#160;For this, centrifuge supernatant from step 5.1 at 21,250 x g for 10 min and measure either immediately or store -80 &#176;C for up to 15 days until analysis.</li>
	<li>Evaluate supernatants with a commercially available ELISA kit. 
	NOTE: The procedure follows the manufacture instructions, which include the coating of plates with the capture antibody, addition of the samples (100 &#181;L) and cytokine standards, incubation, washing, and addition of detection antibody to provide a colorimetric measurement of cytokine presence.&#160;Alternatively, flow cytometry can be used to measure cytokines via commercially available kits (see Table of Materials).</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+Pseudomonas+aeruginosa+biofilms+on+cultured+airway+cells+by+a+fosfomycin/tobramycin+antibiotic+combination.">Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination.</a> Pathogens and Disease. 67 (1), 39-45 (2013).</li><li>Cavalieri, F., Tortora, M., Stringaro, A., Colone, M., Baldassarri, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomedicines+for+antimicrobial+interventions.">Nanomedicines for antimicrobial interventions.</a> Journal of Hospital Infection. 88 (4), 183-190 (2014).</li><li>Savla, R., Minko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+approaches+for+inhalation+treatment+of+fibrosis.">Nanotechnology approaches for inhalation treatment of fibrosis.</a> Journal of Drug Targeting. 21 (10), 914-925 (2013).</li><li>Ho, D. -. K., 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=Challenges+and+strategies+in+drug+delivery+systems+for+treatment+of+pulmonary+infections.">Challenges and strategies in drug delivery systems for treatment of pulmonary infections.</a> European Journal of Pharmaceutics and Biopharmaceutics. 144, 110-124 (2019).</li></ol>

The authors have nothing to disclose.

This paper describes a protocol for a 3D co-culture of the infected airways, constituted by the human cystic fibrosis bronchial epithelial cell line CFBE41o- and the human monocyte-derived macrophage cell line THP-1. The protocol allows the assessment of epithelial barrier integrity, macrophage transmigration, bacteria survival, and inflammation, which are important parameters when testing drug efficacy and host-responses simultaneously. The novelty in the model lies within the incorporation of epithelial cells (i.e.&#16...

Pseudomonas aeruginosa is a relevant pathogen in cystic fibrosis (CF) that contributes to lung tissue impairment1. The production of polysaccharides, such as alginate and other mucoid exopolysaccharides, coordinates the progress of the disease, which leads to tenacious bacterial adherence, limits the delivery of antibiotics to bacteria and protects the bacteria against the host immune system2. The transition of P. aeruginosa from the planktonic stage to biofilm formation is a critical issue in this context, also facilitating the occurrence of antibiotic tolerance.
In the context of CF, the mouse has primarily been used as a model. Mice, however, do not spontaneously develop this disease with the introduction of CF mutations3. Most of the bacterial biofilm development and drug susceptibility studies have been performed on abiotic surfaces, such as Petri dishes. However, this approach does not represent the in vivo complexity. For instance, important biological barriers are absent, including immune cells as well as the mucosal epithelium. Though P. aeruginosa is quite toxic to epithelial cells, some groups&#160;have managed to co-cultivate an earlier P. aeruginosa biofilm with human bronchial cells. These cells originated from cystic fibrosis patients with CFTR mutation (CFBE41o- cells)4 and allowed to assess antibiotic efficacy5 or assess the correction of the CFTR protein during infection6. Such a model was shown to improve the predictability of drug efficacy, in addition to enabling characterization of issues with drugs that failed in later phases of drug development7.
However, in the lung, the mucosal epithelium is exposed to air. Moreover, immune cells present in the airways, like tissue macrophages, play an essential role against inhaled pathogens or particles8. Macrophages migrate through the different cell layers to reach the bronchial lumen and fight the infection. Furthermore, inhaled drugs also have to cope with the presence of mucus as an additional non-cellular element of the pulmonary air-blood barrier9. Indeed, several complex three-dimensional (3D) in vitro models have been developed, aiming to increase the in vivo relevance. Co-culture systems not only increase the complexity of in vitro systems for drug discovery but also enable to study cell-cell interactions. Such complexity has been addressed in studies about macrophage migration10, the release of antimicrobial peptides by neutrophils11, the role of mucus in infection9, and the epithelial cell reaction to excessive damage12. However, a reliable CF-infected in vitro model that features the genetic mutation in CF, that is exposed to the air (increased&#160;physiological condition), and integrates immune cells is still lacking.
To bridge this gap, we describe a protocol for stable human 3D co-culture of the infected airways. The model is constituted of human CF bronchial epithelial cells and macrophages, infected with P. aeruginosa and capable of representing both a diffusional and immunological barrier. With the goal of testing anti-infectives at reasonably high throughput, this co-culture was established on the permeable filter membrane of well plate inserts, using two human cell lines: CFBE41o- and THP-1 monocyte-derived macrophages. Moreover, to eventually study the deposition of aerosolized anti-infectives13, the model was established at the air-liquid interface (ALI) rather than liquid covered conditions (LCC).
As we report here, this model allows assessing not only bacterial survival upon an antibiotic treatment but also cell cytotoxicity, epithelial barrier integrity, macrophage transmigration, and inflammatory responses, which are essential parameters for drug development.
This protocol combines two relevant cell types for inhalation therapy of the pulmonary airways: macrophages and CF bronchial epithelium. These cells are seeded on opposite sides of permeable support inserts, allowing cell exposure to air (called the air-liquid interface (ALI) conditions). This co-culture of host cells is subsequently infected with P. aeruginosa. Both host cell lines are of human origin: the epithelial cells represent the cystic fibrosis bronchial epithelium, with a mutation on the CF channel (CFBE41o-), and the THP-114 cells are a well-characterized macrophage-like cell line. A confluent epithelial layer is first allowed to form on the upper side of well plate inserts before the macrophage-like cells are added to the opposite compartment. Once the co-culture is established at ALI, the system is inoculated with P. aeruginosa at the apical side. This infected co-culture system is then used to assess the efficacy of an antibiotic, e.g.&#160;tobramycin. The following end-points are analyzed: epithelial barrier integrity in terms of transepithelial electrical resistance (TEER), visualization of cell-cell and cell-bacteria interactions by confocal laser scanning microscopy (CLSM), bacterial survival by counting of colony-forming units (CFU), host cell survival (cytotoxicity) and cytokine release.

<table border="0" cellpadding="0" cellspacing="0" style="width:1308px;" width="1306">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">Accutase</td>
			<td style="width:525px;">Accutase</td>
			<td style="width:205px;">AT104</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Carl Roth, Germany</td>
			<td>HP62.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CASY TT Cell Counter and Analyzer</td>
			<td>OLS Omni Life Sciences</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Far Red</td>
			<td>Thermo Fischer</td>
			<td>C34564</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Universal 320R</td>
			<td>Hettich, Germany</td>
			<td>1406</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">CFBE41o- cells</td>
			<td style="width:525px;">1. Gruenert Cell Line Distribution Program 
			2. Sigma-Aldrich</td>
			<td style="width:205px;">1. gift from Dr. Dieter C. Gruenert 
			2. SCC151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chopstick Electrode Set for EVOM2, 4mm</td>
			<td>World Precision Instruments, Sarasota, USA</td>
			<td>STX2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Laser-Scanning Microscope CLSM</td>
			<td>Leica, Mannheim, Germany</td>
			<td>TCS SP 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokines ELISA Ready-SET-Go kits</td>
			<td>Affymetrix eBioscience, USA</td>
			<td>15541037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytokines Panel I and II</td>
			<td>LEGENDplex Immunoassay (Biolegend, USA).</td>
			<td>740102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytotoxicity Detection Kit (LDH)</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+) Glucose</td>
			<td>Merck</td>
			<td>47829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dako Fluorescence Mounting Medium</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI (4&#8242;,6-diamidino-2-phenylindole)</td>
			<td>Thermo Fischer</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial voltohmmeter</td>
			<td>World Precision Instruments, Sarasota, USA</td>
			<td>EVOM2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Permeable Support for 12 Well Plate with 3.0&#956;m Transparent PET Membrane, Sterile</td>
			<td>Corning, Amsterdam, Netherlands</td>
			<td>353181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal calf serum</td>
			<td>Lonza, Basel, Switzerland</td>
			<td>DE14-801F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse (H+L) Cross-adsorbed secondary Antibody, Alexa Fluor 633</td>
			<td>Invitrogen</td>
			<td>A-21050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lactate Dehydrogenase (LDH), rabbit muscle</td>
			<td>Roche, Mannheim, Germany</td>
			<td>10127230001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB broth</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>L2897-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM (Minimum Essential Medium)</td>
			<td>Gibco Thermo Fisher Scientific Inc.</td>
			<td>11095072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco Thermo Fisher Scientific Inc.</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P. aeruginosa strain PAO1</td>
			<td>American Type Culture Collection</td>
			<td>47085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P. aeruginosa strain PAO1-GFP</td>
			<td>American Type Culture Collection</td>
			<td>10145GFP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde Aqueous Solution -16%</td>
			<td>EMS DIASUM</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffer solution buffer</td>
			<td>Thermo Fischer</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Greiner</td>
			<td>664102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma, Germany</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precision Cover Glasses</td>
			<td>ThorLabs</td>
			<td>CG15KH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified Mouse anti-human ZO-1 IgG antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td>Gibco by Lifetechnologies, Paisley, UK</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soda-lime glass Petri dish, 50 x 200 mm (height x outside diameter)</td>
			<td>Normax, Portugal</td>
			<td>5058561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saponin</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S4521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T75 culture flasks</td>
			<td>Thermo Fischer</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">THP-1 cells</td>
			<td>Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany)</td>
			<td>No. ACC-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tobramycin sulfate salt</td>
			<td>Sigma</td>
			<td>T1783-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA 0.05%</td>
			<td>Thermo Fischer</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween80</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P1754</td>
			<td></td>
		</tr>
	</tbody>
</table>

p. aeruginosa infected 3d co-culture of bronchial epithelial cells and macrophages at air-liquid interface for preclinical evaluation of anti-infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

Figure 1A shows the morphology of the resulting co-culture of human bronchial epithelial cells and macrophages after growing both for 24 h on the apical and basolateral side of permeable supports, respectively. The epithelial barrier integrity is shown by higher TEER (834 &#8486;&#215;cm2) and CLSM by immunostaining for the tight junction protein ZO-1 (Figure 1B). The same results observed in terms of barrier integrity of uninfected CFBE41o-</sup...

Watch this Scientific Journal Video about P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives at Jo

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

p-aeruginosa-infected-3d-co-culture-bronchial-epithelial-cells

Research

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Immunology and Infection

10.6K Views.  Helmholtz Institute for Pharmaceutical Research Saarland (HIPS). We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

Video: P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...

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

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

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

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