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>

<ol>
	<li>Cutting, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+genetics:+from+molecular+understanding+to+clinical+application.">Cystic fibrosis genetics: from molecular understanding to clinical application.</a> Nature Reviews Genetics. 16 (1), 45-56 (2015).</li><li>Lyczak, J. B., Cannon, C. L., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+Pseudomonas+aeruginosa+infection:+Lessons+from+a+versatile+opportunist.">Establishment of Pseudomonas aeruginosa infection: Lessons from a versatile opportunist.</a> Microbes and Infection. 2 (9), 1051-1060 (2000).</li><li>Wilke, M., Buijs-Offerman, R. M., 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=Mouse+models+of+cystic+fibrosis:+Phenotypic+analysis+and+research+applications.">Mouse models of cystic fibrosis: Phenotypic analysis and research applications.</a> Journal of Cystic Fibrosis. 10, 152-171 (2011).</li><li>Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway.">Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.</a> Pulmonary Pharmacology and Therapeutics. 21 (4), 595-599 (2008).</li><li>Yu, Q., 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=In+vitro+evaluation+of+tobramycin+and+aztreonam+versus+Pseudomonas+aeruginosa+biofilms+on+cystic+fibrosis-derived+human+airway+epithelial+cells.">In vitro evaluation of tobramycin and aztreonam versus Pseudomonas aeruginosa biofilms on cystic fibrosis-derived human airway epithelial cells.</a> Journal of Antimicrobial Chemotherapy. 67 (11), 2673-2681 (2012).</li><li>Moreau-Marquis, S., Redelman, C. V., Stanton, B. a., Anderson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+models+of+Pseudomonas+aeruginosa+biofilms+grown+on+live+human+airway+cells.">Co-culture models of Pseudomonas aeruginosa biofilms grown on live human airway cells.</a> Journal of visualized experiments : JoVE. (44), e2186 (2010).</li><li>Stanton, B. A., Coutermarsh, B., Barnaby, R., Hogan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+reduces+VX-809+stimulated+F508del-CFTR+chloride+secretion+by+airway+epithelial+cells.">Pseudomonas aeruginosa reduces VX-809 stimulated F508del-CFTR chloride secretion by airway epithelial cells.</a> PLoS ONE. 10 (5), 1-13 (2015).</li><li>Lambrecht, B. N., Prins, J., Hoogsteden, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+dendritic+cells+and+host+immunity+to+infection.">Lung dendritic cells and host immunity to infection.</a> European Respiratory Journal. (18), 692-704 (2001).</li><li>Murgia, X., De Souza Carvalho, C., Lehr, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+the+pulmonary+barrier:+New+insights+to+improve+the+efficiency+of+inhaled+therapeutics.">Overcoming the pulmonary barrier: New insights to improve the efficiency of inhaled therapeutics.</a> European Journal of Nanomedicine. 6 (3), 157-169 (2014).</li><li>Ding, P., Wu, H., Fang, L., Wu, M., Liu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmigration+and+phagocytosis+of+macrophages+in+an+airway+infection+model+using+four-dimensional+techniques.">Transmigration and phagocytosis of macrophages in an airway infection model using four-dimensional techniques.</a> American Journal of Respiratory Cell and Molecular Biology. 51 (1), 1-10 (2014).</li><li>Hartl, D., Tirouvanziam, R., 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=Innate+Immunity+of+the+Lung:+From+Basic+Mechanisms+to+Translational+Medicine.">Innate Immunity of the Lung: From Basic Mechanisms to Translational Medicine.</a> Journal of Innate Immunity. 10 (5-6), 487-501 (2018).</li><li>Esposito, S., 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=Manipulating+proteostasis+to+repair+the+F508del-CFTR+defect+in+cystic+fibrosis.">Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.</a> Molecular and cellular pediatrics. 3 (1), 13 (2016).</li><li>Hein, S., Bur, M., Schaefer, U. F., Lehr, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+Pharmaceutical+Aerosol+Deposition+Device+on+Cell+Cultures+(PADDOCC)+to+evaluate+pulmonary+drug+absorption+for+metered+dose+dry+powder+formulations.">A new Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) to evaluate pulmonary drug absorption for metered dose dry powder formulations.</a> European Journal of Pharmaceutics and Biopharmaceutics. 77 (1), 132-138 (2011).</li><li>Kletting, S., Barthold, S., 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=Co-culture+of+human+alveolar+epithelial+(hAELVi)+and+macrophage+(THP-1)+cell+lines.">Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines.</a> Altex. 35 (2), 211-222 (2018).</li><li>Schwende, H., Fitzke, E., Ambs, P., Dieter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+state+of+differentiation+of+THP-1+cells+induced+by+phorbol+ester+and+1,25-dihydroxyvitamin+D3.">Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3.</a> Journal of leukocyte biology. 59 (4), 555-561 (1996).</li><li>Castoldi, A., Empting, M., 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=Aspherical+and+Spherical+InvA497-Functionalized+Nanocarriers+for+Intracellular+Delivery+of+Anti-Infective+Agents.">Aspherical and Spherical InvA497-Functionalized Nanocarriers for Intracellular Delivery of Anti-Infective Agents.</a> Pharmaceutical Research. 36 (1), 1-13 (2019).</li><li>Ebensen, T., Delandre, S., Prochnow, B., Guzm&#225;n, C. A., Schulze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Combination+Vaccine+Adjuvant+System+Alum/c-di-AMP+Results+in+Quantitative+and+Qualitative+Enhanced+Immune+Responses+Post+Immunization.">The Combination Vaccine Adjuvant System Alum/c-di-AMP Results in Quantitative and Qualitative Enhanced Immune Responses Post Immunization.</a> Frontiers in cellular and infection microbiology. 9, 31 (2019).</li><li>Brockman, S. M., Bodas, M., Silverberg, D., Sharma, A., Vij, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-based+selective+autophagy-induction+rescues+&#948;F508-CFTR+and+inhibits+Pseudomonas+aeruginosa+infection+in+cystic+fibrosis.">Dendrimer-based selective autophagy-induction rescues &#948;F508-CFTR and inhibits Pseudomonas aeruginosa infection in cystic fibrosis.</a> PLoS ONE. 12 (9), 1-17 (2017).</li><li>Anderson, G. G., Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+analysis+of+tobramycin-treated+Pseudomonas+aeruginosa+biofilms+on+cystic+fibrosis-derived+airway+epithelial+cells.">In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells.</a> Infection and Immunity. 76 (4), 1423-1433 (2008).</li><li>Moreau-Marquis, S., Bomberger, J. M., 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=The+DeltaF508-CFTR+mutation+results+in+increased+biofilm+formation+by+Pseudomonas+aeruginosa+by+increasing+iron+availability.">The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.</a> American journal of physiology. Lung cellular and molecular physiology. 295, 25-37 (2008).</li><li>Braakhuis, H. M., Kloet, S. 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=Progress+and+future+of+in+vitro+models+to+study+translocation+of+nanoparticles.">Progress and future of in vitro models to study translocation of nanoparticles.</a> Archives of Toxicology. 89 (9), 1469-1495 (2015).</li><li>Bosshart, H., Heinzelmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THP-1+cells+as+a+model+for+human+monocytes.">THP-1 cells as a model for human monocytes.</a> Annals of Translational Medicine. 4 (21), 4-7 (2016).</li><li>Daigneault, M., Preston, J. a., Marriott, H. M., Whyte, M. K. B., Dockrell, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+of+markers+of+macrophage+differentiation+in+PMA-stimulated+THP-1+cells+and+monocyte-derived+macrophages.">The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.</a> PLoS ONE. 5 (1), (2010).</li><li>Bismarck, P. V., Schneppenheim, R., Schumacher, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+treatment+of+pseudomonas+aeruginosa+respiratory+tract+infection+with+a+sugar+solution+-+A+case+report+on+a+lectin+based+therapeutic+principle.">Successful treatment of pseudomonas aeruginosa respiratory tract infection with a sugar solution - A case report on a lectin based therapeutic principle.</a> Klinische Padiatrie. 213 (5), 285-287 (2001).</li><li>Klinger-Strobel, M., Lautenschl&#228;ger, C., 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=Aspects+of+pulmonary+drug+delivery+strategies+for+infections+in+cystic+fibrosis+-+where+do+we+stand.">Aspects of pulmonary drug delivery strategies for infections in cystic fibrosis - where do we stand.</a> Expert Opinion on Drug Delivery. 5247, 1-24 (2015).</li><li>Ehrhardt, C., Collnot, E. -. M., 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=Towards+an+in+vitro+model+of+cystic+fibrosis+small+airway+epithelium:+characterisation+of+the+human+bronchial+epithelial+cell+line+CFBE41o-.">Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-.</a> Cell and tissue research. 323 (3), 405-415 (2006).</li><li>Anderson, G. G., Kenney, T. F., Macleod, D. L., Henig, N. R., O'Toole, G. 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.&#160;human CF cell line and macrophages) with acute bacterial infection (i.e.&#160;P. aeruginosa). The acute infection in the epithelial cells is demonstrated to be controlled by an antibiotic (i.e.&#160;tobramycin). Besides the use of a human CF cell line, the entire model is set up at ALI conditions, which is considerably closer to the physiological conditions in CF. The use of a CF cell line implements some of the characteristics of the disease in the model. The mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is directly related to the dysregulation of epithelial fluid transport in the lungs. Furthermore, mutations in the CF gene, such as the &#916;F508, result in thick mucus, with inflammation and severe lung damage upon infection with P. aeruginosa4. These pathological manifestations caused by dysfunctional CFTR potentially involve autophagy impairment as an important cellular mechanism associated with the pathogenesis of CF lung disease18. However, the CFBE41o- fail&#160;to secret mucus, which is a limitation of this cell line. If it is intended to study the role of mucus more specifically, the protocol can be adapted by using other bronchial cell lines (e.g., Calu-3).
One critical step to set up this protocol is the combination of epithelial and immune cells and the subsequent infection with P. aeruginosa at ALI. The infection of CFBE41o- by P. aeruginosa in vitro has already been described, mainly using a flow-cell chamber, supplemented with arginine in the culture media, to improve epithelial cell survival and support biofilm formation19. The present protocol aimed for a new model using only human cells, which moreover, could be grown at ALI on permeable well plate inserts for higher sample throughput. The inclusion of THP-1 differentiated macrophages as a human immortalized cell line, instead of being dependent on obtaining reproducible primary cells from donors, is another advantage of our model. By adding these macrophages to the basolateral side of permeable membrane support, it was observed that macrophages protruded and eventually transmigrated to the apical side of the filter-grown epithelial barrier. A variation of this protocol could be the addition of macrophages directly on the apical side on top of the epithelial cells, as described by Kletting et al.14. The co-culture of non-human immune and lung cells has already been described before. Ding et al.10 used mouse Lewis lung carcinoma cells on permeable insert supports in combination with macrophages on the basolateral side and infected with S. aureus, another critical pathogen of chronic infection in CF patients. However, in this study, there was no focus on cystic fibrosis or to use the co-culture as a platform for the evaluation of drug efficacy. Our protocol can be adapted for other bacterial infections, such as Staphylococcus aureus, Mycobacterium abscessus, or Burkholderia cepacia&#8212; important pathogens in CF lung.
Another critical step is the addition of THP-1 macrophages to the cells by flipping the permeable inserts upside-down (section 2). This is crucial to assess macrophage transmigration through the well to the side of infection. The later imaging process from the 3D models with z-stacks, and cross-section view, can be performed to observe the inside of macrophages and detect bacteria uptake (Figure 3). At 1 hpi, bacteria applied on the apical side migrate through the membrane, while macrophage migration and uptake only take place at 3 hpi. Therefore after 1 h of infection, it was appropriate to start the treatment with tobramycin and have the possibility to address both host cell and bacteria survival for a long period (20 h). In the course of the protocol, maintaining sterility is a critical issue due to the multitude of steps that each carry the risk of contamination. Nevertheless, experienced cell-culture personnel will be able to follow this protocol after appropriate preparation and training. Cell medium should be regularly checked for contamination, preferably after all critical steps.
As with any model, the infected co-culture also has some limitations; for instance, the integration of the macrophage-like cells. Here, it was important to have macrophages on the basolateral side; however, the manipulation of the insert with a previously grown epithelial layer may have provided early damage and disturbances to the co-culture. Although, the permeable support model provided high-throughput characteristics, which has not been observed in previous co-cultures of the CF infected lung20,21. With that, further experiments need to assess the limitations of using THP-1 as macrophage substitute. While this cell line is widely used, it is less responsive to LPS22 and it lacks full activation and the entire population is not differentiated from monocytes to macrophage-like cells23. Another limitation is the lack of other key components in CF infection and drug delivery. The CFBE41o- cell line does not possess cilia nor does it produce mucus, which usually happens 20-30 days of cell culture at ALI. As this was not the case for CFBE41o- cell line, we used the cells after seven days when a tight epithelial barrier was formed. Mucociliary clearance alters the residence conditions for either microbes24 or drug particles9,25 and in vitro models assessing lung deposition should take this into account. Differently from what is observed by other cells, the tissue culture inserts coating with an extracellular matrix material (like fibronectin or collagen I) do not show a significant difference for CFBE41o-, for instance in TEER26. Therefore, the permeable filters were not coated with an extracellular matrix material in this protocol.
With the protocol described here, mono and co-cultures after 6 h infection provide sufficient cytokine release to be used as a measurement in future drug testing. The co-culture brings an advantage of cell cooperation in modeling immune response. The inefficacy of tobramycin in reducing inflammation was expected since not all bacteria were eliminated during the treatment (Figure 4E, F). Nevertheless, modeling the response to tobramycin in a CF model is crucial, as tobramycin (in higher concentrations) can be effective in P. aeruginosa inhibition, even on biofilm19,27. One possibility for further use of this protocol is to integrate anti-inflammatory drugs in the treatment. The overall recommendation regarding inflammatory responses would be to use the short duration treatment (6 h), which still has the host cell and bacteria present. After this time point, the host cells are destroyed in untreated samples. Both ELISA and&#160;FACS could be used to measure the release of cytokines. Finally, if the samples are stored longer than 15 days at -80 &#176;C, it is recommended to check the reliability of the cytokines by using, for instance, positive control of fresh samples (e.g.&#160;cells stimulated with LPS).
Some modifications of the protocol are possible. For example, the current protocol can be expanded to the application of nebulized drugs (step 3.6). This is necessary to model pulmonary drug delivery via oral inhalation. Nebulization of water-soluble drugs, like tobramycin, or nano-carriers thereof, such as liposomal colistin, is relatively straight forward by commercially available devices routinely used in the clinic. Also, there are several commercially available devices to deposit aerosols onto cell culture inserts. In addition, as the model described here is based on permeable membrane supports, it could also be adopted to some contemporary microfluidic (e.g.&#160;&#8220;lung on a chip&#8221;) devices, for example, to study the influence of breathing and the related mechanical stretching and changes in the airflow. Moreover, this protocol could be modified by the addition of mucus or replacement by primary cells depending on the scientific question to be addressed. Another interesting next step would be the testing of nanomedicines, especially as nanotechnology is making progress in the development of novel anti-infectives28, CF correctors29 and co-delivery of antibiotics and pathoblockers30. Overall, the current protocol may be perceived as useful in assessing bacterial survival upon antibiotic treatment in a complex system, together with some host-related readouts: cell cytotoxicity, epithelial barrier integrity, macrophage transmigration&#160;and inflammatory response. These are essential parameters for drug development.

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><tbody><tr><td>Accutase</td><td>Accutase</td><td>AT104</td><td></td></tr><tr><td>Ampicillin</td><td>Carl Roth, Germany</td><td>HP62.1</td><td></td></tr><tr><td>CASY TT Cell Counter and Analyzer</td><td>OLS Omni Life Sciences</td><td>-</td><td></td></tr><tr><td>CellTrace Far Red</td><td>Thermo Fischer</td><td>C34564</td><td></td></tr><tr><td>Centrifuge Universal 320R</td><td>Hettich, Germany</td><td>1406</td><td></td></tr><tr><td>CFBE41o&lt;sup&gt;-&lt;/sup&gt; cells</td><td>1. Gruenert Cell Line Distribution Program&lt;br/&gt; 2. Sigma-Aldrich</td><td>1. gift from Dr. Dieter C. Gruenert&lt;br/&gt; 2. SCC151</td><td></td></tr><tr><td>Chopstick Electrode Set for EVOM2, 4mm</td><td>World Precision Instruments, Sarasota, USA</td><td>STX2</td><td></td></tr><tr><td>Confocal Laser-Scanning Microscope CLSM</td><td>Leica, Mannheim, Germany</td><td>TCS SP 8</td><td></td></tr><tr><td>Cytokines ELISA Ready-SET-Go kits</td><td>Affymetrix eBioscience, USA</td><td>15541037</td><td></td></tr><tr><td>Cytokines Panel I and II</td><td>LEGENDplex Immunoassay (Biolegend, USA).</td><td>740102</td><td></td></tr><tr><td>Cytotoxicity Detection Kit (LDH)</td><td>Roche</td><td>11644793001</td><td></td></tr><tr><td>D-(+) Glucose</td><td>Merck</td><td>47829</td><td></td></tr><tr><td>Dako Fluorescence Mounting Medium</td><td>DAKO</td><td>S3023</td><td></td></tr><tr><td>DAPI (4&amp;prime;,6-diamidino-2-phenylindole)</td><td>Thermo Fischer</td><td>D1306</td><td></td></tr><tr><td>Epithelial voltohmmeter</td><td>World Precision Instruments, Sarasota, USA</td><td>EVOM2</td><td></td></tr><tr><td>Falcon Permeable Support for 12 Well Plate with 3.0&amp;mu;m Transparent PET Membrane, Sterile</td><td>Corning, Amsterdam, Netherlands</td><td>353181</td><td></td></tr><tr><td>Fetal calf serum</td><td>Lonza, Basel, Switzerland</td><td>DE14-801F</td><td></td></tr><tr><td>Goat anti-mouse (H+L) Cross-adsorbed secondary Antibody, Alexa Fluor 633</td><td>Invitrogen</td><td>A-21050</td><td></td></tr><tr><td>L-Lactate Dehydrogenase (LDH), rabbit muscle</td><td>Roche, Mannheim, Germany</td><td>10127230001</td><td></td></tr><tr><td>LB broth</td><td>Sigma-Aldrich, Germany</td><td>L2897-1KG</td><td></td></tr><tr><td>MEM (Minimum Essential Medium)</td><td>Gibco Thermo Fisher Scientific Inc.</td><td>11095072</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution (100X)</td><td>Gibco Thermo Fisher Scientific Inc.</td><td>11140050</td><td></td></tr><tr><td>P. aeruginosa strain PAO1</td><td>American Type Culture Collection</td><td>47085</td><td></td></tr><tr><td>P. aeruginosa strain PAO1-GFP</td><td>American Type Culture Collection</td><td>10145GFP</td><td></td></tr><tr><td>Paraformaldehyde Aqueous Solution -16%</td><td>EMS DIASUM</td><td>15710-S</td><td></td></tr><tr><td>Phosphate buffer solution buffer</td><td>Thermo Fischer</td><td>10010023</td><td></td></tr><tr><td>Petri dishes</td><td>Greiner</td><td>664102</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma, Germany</td><td>P8139-1MG</td><td></td></tr><tr><td>Precision Cover Glasses</td><td>ThorLabs</td><td>CG15KH</td><td></td></tr><tr><td>Purified Mouse anti-human ZO-1 IgG antibody</td><td>BD Transduction Laboratories</td><td>610966</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 medium</td><td>Gibco by Lifetechnologies, Paisley, UK</td><td>11875093</td><td></td></tr><tr><td>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><td>Saponin</td><td>Sigma-Aldrich, Germany</td><td>S4521</td><td></td></tr><tr><td>T75 culture flasks</td><td>Thermo Fischer</td><td>156499</td><td></td></tr><tr><td>THP-1 cells</td><td>Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany)</td><td>No. ACC-16</td><td></td></tr><tr><td>Tobramycin sulfate salt</td><td>Sigma</td><td>T1783-500MG</td><td></td></tr><tr><td>Trypsin-EDTA 0.05%</td><td>Thermo Fischer</td><td>25300054</td><td></td></tr><tr><td>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- monoculture could be seen in the uninfected epithelial-macrophage co-cultures.
To model a bacterial infection, P. aeruginosa was inoculated at a multiplicity of infection (MOI) of 1:1 on CFBE41o- cells. Six hours after infection (Figure 2A), macrophages were observed on the apical side of the co-culture. After the infection, the TEER dropped from 834 to 250 &#8486;&#215;cm2, indicating a compromised epithelial barrier, as also visualized by ZO-1 staining (Figure 2B).
Figure 3 shows macrophage transmigration through the permeable membrane&#160;pores and bacteria uptake by THP-1 cells on the apical side. The samples were fixed at 1, 3, and 6 h post-incubation from independent experiments. In the THP-1 monocultures (Figure 3A&#8211;C), macrophages migration was observed as early as 1 h, while in the co-culture (Figure 3D&#8211;F), this was seen after 3 h infection. Bacteria uptake in THP-1 was observed after 3 h of infection, in both monoculture and co-culture. No bacterial uptake by CFBE41o- could be seen. Cross-sectional views were placed such that the permeable membrane support was in the middle as a separation of the apical and basolateral compartment.
Figure 4 shows confocal scanning laser microscopy pictures of infected co-cultures (CFBE41o- + THP-1) treated with or without tobramycin for 6 h (Figure 4A, B) or 20 h (Figure 4C,D). Without treatment, both the epithelial cells and the macrophages died after 20 h of infection (Figure 4C). However, upon tobramycin treatment, the host cells are preserved after 20 h; still, some bacteria can be observed in the culture. Despite being seen after 6 h of treatment in the microscopy pictures (Figure 4B), the bacteria did not proliferate as observed in CFU assays in Figure 4E. Nevertheless, after 20 h treatment, the bacteria recovered the proliferation capability, as seen by the colonies in the CFU assay (Figure 4F). The cell lysis protocol with cold water and scraping can release bacteria attached and possibly internalized in cells. At the same time, the cells are destroyed (Supplementary Figure S1A, B). The centrifugation steps used in this paper for epithelial cells (300 x g) or bacteria (21,250 x g) did not hamper the viability of both (Supplementary Figures S1C, D). All CFU assays were performed by freezing the samples at -20&#176;C, followed by thawing and plating. This procedure reduced the number of bacteria by 2-logs, compared to fresh samples (Supplementary Figure S1E). As this procedure is done simultaneously for all experimental groups (treated and untreated) at different time points, this reduction will be incorporated in the final results (Supplementary Figure S1E). Moreover, the concentration of tobramycin used here showed no toxicity for the uninfected cells (Supplementary Figure S2A) and also no further inflammatory response (Supplementary Figure S2B). However, it was within the range of the minimum inhibitory concentration to kill P. aeruginosa.
Figure 5 shows the transepithelial electrical resistance (TEER) and cell viability. Figure 5A&#8211;B illustrates the TEER of monocultures and co-cultures. The co-culture of CFBE41o- cells with THP-1 did not induce any change in the epithelial barrier integrity compared to the monoculture (red bars). Upon the infection, the TEER value dropped (green bar). After 1 h of infection, some samples were treated with the antibiotic tobramycin (blue bar), for 6 or 20 h. The treatment preserved the epithelial barrier integrity, as observed by the higher TEER. Figure 5C shows the percentage of LDH release as an indication of cell toxicity upon infection and tobramycin treatment after 6 h. The co-culture itself induced a release of LDH, which was the same for the infected cells (around 20%). After 20 h of infection, no signal of LDH could be detected. To prove LDH reliability for long-term infection, PAO1-GFP was incubated in medium with and without LDH 1 U/mL compared to respective uninfected controls (Supplementary Figure S2C). The LDH signal was lost after prolonged incubation (20 h) with P. aeruginosa, indicating that LDH is only stable in shorter incubation times in infected cultures.
Figure 6 shows the kinetics of pro-inflammatory cytokines detected via ELISA. The advantage of an infected co-culture of CFBE41o- and THP-1 cells was observed with the higher secretions of pro-inflammatory cytokines. The secretion of some pro-inflammatory cytokines was either similar (IL-6) or higher (IL-8, TNF-&#945;, IL-1&#946;) in the infected co-culture (Figure 6C) than in the corresponding monocultures (Figures 6A-B). Unexpectedly, some cytokines in THP-1 monocultures (Figure 6B) are downregulated in infected samples (Il-8, TNF&#945;, IL-1&#946;).
Figure 7 demonstrates the release of cytokines in mono- and co-cultures upon infection and treatment with tobramycin measured via fluorescence activated cell sorting (FACS). The secretion of the pro-inflammatory cytokine IL-8 (Figure 7A) and the anti-inflammatory cytokine IL-10 (Figure 7F) was higher in the co-cultures of epithelial cells and macrophages, compared to the monocultures. However, for all other cytokines (IL-1 &#945;, IL-12p40, IL-23 and GM-CSF) (Figures 7B&#8211;E), the levels of cytokine secretion were not higher in the co-culture that in the respective monocultures.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61069/61069fig1.jpg" /> 
Figure 1: Cross-sections and apical views of the uninfected epithelial-macrophage co-culture. (A) Cross views of uninfected 24 h epithelial-macrophage co-culture. CFBE41o- stained red, THP-1 macrophages in yellow&#160;and nuclei in blue (DAPI). (B) Apical views of the uninfected CFBE41o- monolayer immuno-stained for ZO-1 (red). DAPI: nuclei. Scale bars: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61069/61069fig2.jpg" /> 
Figure 2: Cross-sections and apical views of the infected epithelial-macrophage co-culture. (A) Cross views and (B) apical view of epithelial-macrophage co-culture at 6 h post-infection (hpi) with P. aeruginosa PAO1-GFP. CFBE41o- stained in red, THP-1 macrophages in yellow, nuclei in blue (DAPI) and P. aeruginosa PAO1-GFP in green. (B) Apical views of the 6 h infected CFBE41o- monolayer. Scale bars: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61069/61069fig3.jpg" /> 
Figure 3: Kinetics of macrophage transmigration and bacteria uptake visualized by cross-section of the 3D model. PAO1-GFP infection kinetics in monocultures of&#160;THP-1 macrophages (A&#8211;C)&#160;or co-culture (D&#8211;F). THP-1 macrophages are stained in red, nuclei of epithelial cells in blue&#160;(DAPI), and P. aeruginosa&#160;in green&#160;(GFP). Each figure is divided into apical and basolateral sides, the space in between is considered to be the membrane, which is empty or occupied by the CFBE41o- confluent layer (D&#8211;F). Inserts in the figures show bacteria uptake by macrophages at different times (A&#8211;F). Scale bars: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61069/61069fig4.jpg" /> 
Figure 4: Characterization of PAO1-GFP survival in tobramycin-treated co-culture. (A&#8211;D) Confocal micrographs co-cultures with and without treatment. (A) Untreated co-culture after 6 h of infection. (B) Infected co-culture treated with tobramycin 6 &#181;g/mL (Tob) for 6 h. (C) Untreated co-culture 20 h post-infection, (D) infected co-culture treated with tobramycin 6 &#181;g/mL for 20 h. Nuclei stained with DAPI (blue), macrophages&#160;(red) and P. aeruginosa GFP (green). (E) Colony-forming units (CFU) of adherent/internalized bacteria after 6 and (F) 20 h with tobramycin 6 &#181;g/mL treatment. Empty membrane insert was used as an abiotic substrate to grow PAO1-GFP. Two-way ANOVA with Tukey&#8217;s multiple comparisons test (# no colonies) was used, *p &#60; 0.05; ***p &#60; 0.001; ****p &#60; 0.0001; ns: not significant. Error bars indicate standard deviation, n = 9&#8211;27 replicates of 3&#8211;9 independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61069/61069fig5.jpg" /> 
Figure 5: Barrier integrity and evaluation of the viability of mono- and co-culture. The following co-culture conditions were assessed: uninfected (gray bars), infected (green bars), or infected and treated with tobramycin (blue bars). (A) Transepithelial electrical resistance after 6 h and (B) 20 h of infection in mono-cultures (CFBE41o- and THP-1) and co-culture. (C) Cytotoxicity of mono- and co-culture measured via LDH release 6 h post-infection. Two-way ANOVA with Tukey&#8217;s multiple comparisons test was used; *p &#60; 0.05; ****p &#60; 0.0001; ns: not significant. Error bars indicate standard deviation; n = 9 replicates of three independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61069/61069fig6.jpg" /> 
Figure 6: Kinetics of cytokine release of uninfected and infected mono- and co-culture supernatants assessed via ELISA. ELISA was done according to the kit manufacturer&#39;s protocol. (A) CFBE41o-, (B) THP-1, and (C) co-culture releasing IL-8, TNF-&#945;, IL-1&#946;, and IL-6. Error bars indicate standard deviation. n = 6 replicates of 2 independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61069/61069fig7.jpg" /> 
Figure 7: Supernatant results of cytokine panel measured via FACS with and without tobramycin 6 &#181;g/mL for 6 h post-infection. Supernatants of mono- and co-culture after 6 h post-infection used to analyze the respective cytokines IL-8 (A), IL-1&#945; (B), IL12p40 (C), IL-23 (D), GM-CSF (E) and IL-10 (F). Error bar indicates standard deviation, n = 9 replicates of 3 independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1: Control experiments for critical steps of the protocol. (A, B) Micrographs of CFBE41o- cells in 24-well plates grown for 2 days at density of 2 x 105 cells/well. (A) CFBE41o- cells in PBS for 30 min, and (B) water-treated cells after 30 min after scraping with a pipette. (C) Viability of mammalian cells after centrifugation. CFBE41o- cells were removed from T75 cell culture flask as described in step 1.1.1 and 1.1.2. 100 &#181;L of resulting cell suspension was analyzed in 10 mL isotonic solution. An automated cell counter was used to assess the viability of single cells. Then, respective cell suspensions were centrifuged at 300 x g for 4 min, re-suspended and counted again. Error bars indicate standard deviation, n = 6 different flasks of 2 individual experiments. (D) Viability of PAO1-GFP after centrifugation. PAO1-GFP bacteria were diluted to OD = 0.01 in cell medium. CFU was assessed via a 10-fold dilution row and LB plates incubated overnight at 30 &#176;C. Respective plastic tubes were centrifuged at 21,250 x g for 10 min and re-suspended in medium. CFU was assessed accordingly again. Two-tailed student&#39;s t-test, * p &#60; 0.033. Error bar indicates standard deviation, n = 6 of 2 experiments. (E) Viability of bacteria after freezing. PAO1-GFP bacteria were prepared as in (D) and CFU was analyzed, then plastic tubes were frozen for one day at -20 &#176;C and thawed to assess CFU again. Two-tailed student&#180;s t-test, *** p &#60; 0.001. Error bars indicate standard deviation, n = 6 of two experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069supfig1large.jpg" target="_blank">Please click here to download this file.</a>
Supplementary Figure S2: Control experiments to assess LDH behavior and influence of tobramycin. (A) Control experiment to assess cytotoxicity after 20 h of incubation with 6 &#181;g/mL tobramycin. Mono- and co-culture was done as described in the protocol, but cells were grown for 2 days on 24-well plates and THP-1 cells were seeded apically. Cells with 6 &#181;g/mL tobramycin or controls were incubated for 20 h. One-Way ANOVA, Tukey&#39;s multiple comparisons test, *** p &#60; 0.001. Error bar indicates standard deviation, n = 6 of 2 experiments (CFBE41o-), n = 3 of one experiment (THP-1 and co-culture). (B) Control-ELISA of supernatants of mono- and co-culture with/without tobramycin. Cell culture was done according to (A) to show no cytokine release compared to controls for all conditions. ELISA was done in step 7.1 and 7.2, 10 &#181;g/mL (LPS) was added as control, IL-8 release for LPS-treated controls containing THP-1 was higher than detectable. Two-Way ANOVA, Tukey&#39;s multiple comparisons test, ns p &#62; 0.12; * p &#60; 0.033; *** p &#60; 0.001. Error bars indicate standard deviation, n = 6 of two experiments, n = 3 of one experiment (LPS control). (C) LDH degradation due to excessive PAO1-GFP proliferation. LDH was added at concentration of 1 U/mL to the MEM Medium. Either LDH medium or&#160;control medium was&#160;used to dilute cells to OD = 0.01 (corresponds to 1 x 108 CFU/mL) and then incubated for 20 h. LDH assay was done as described in section 6. One-Way ANOVA, Tukey&#180;s multiple comparisons test, ns p &#62; 0.12; ***p &#60; 0.001. Error bar indicates standard deviation, n = 8&#8211;9 of three individual experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61069/61069supfig2large.jpg" target="_blank">Please click here to download this file.</a>

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K Views.  Helmholtz Institute for Pharmaceutical Research Saarland (HIPS). Dus als we nieuwe anti-infecties evalueren, moeten we twee dingen aanpakken. Een kant is de bacterie. Als het planktonische of geïnformeerde biofilms zijn, is de andere kant de aanwezigheid van gastheercellen, vooral epitheliale gastheercellen, die strakke biologische barrières vormen.Als deze barrières worden uitgevoerd, hoe de monologische respons zou kunnen zijn, en met dit nieuwe model, kunnen we eigenlijk beide parameters te controleren op hetzelfde moment. Bij de behandeling v...

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 ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

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 ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video