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;mi...

<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. 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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. 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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 ...

So when we are evaluating novel anti-infectives, we have to address two things. One side is the bacteria. If they are planktonic or informed biofilms, the other side is the presence of host cells, especially epithelial host cells, which form tight biological barriers.If these barriers are effected, how the monological response might be, and with this new model, we can actually monitor both parameters at the same time. When treating bacterial infections, we have to realize what organ is affected. For instance, if it is the lung, we can take advantage, and topically administer the drug as an aerosol.However, if we want to have a model for studying this, we need a model that also allows the position of aerosols which is the case in our model. The other advantage is that it's based on human cells, so that we can avoid problems due to differences to animals and species differences. This system could give you more insight to the field of infection research by shedding light on molecular and cellular events during the interaction of host cells and bacteria.When attempting this protocol for the first time, remember to check the cell medium for sterility and cell morphology and to be careful when handling the permeable supports. Cultivate CFBE41 O minus cells in a T 75 flask with minimum essential medium containing 10%FCS, 1%non-essential amino acids, and 600 milligrams per liter glucose at 37 degrees Celsius with 5%carbon dioxide. Add fresh medium to the cells every two to three days.After the cells reached 70%confluency, wash them with 10 milliliters of PBS and detach with three milliliters of trypsin EDTA at 37 degrees Celsius for 15 minutes. Then, add seven milliliters of fresh MEM and centrifuge the cells at 300 times G for four minutes. After discarding the supernatant, at 10 milliliters of MEM, while disrupting the clumps with gentle pipetting.Count the cells with an automated cell counter or Hemocytometer chamber, then dilute them to a concentration of 200, 000 cells per 500 microliters for seeding in 12 well plates with permeable supports. Add 500 microliters of the cell suspension per well to the apical side of the permeable support. Then add 1.5 milliliters of fresh medium to the basal lateral side.Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 72 hours. On the third day after seeding, create an air liquid interface by removing the medium from the basal lateral side first then from the apical side. Add 500 microliters of fresh MEM to the basal lateral side and change the medium every second day until cells form a confluent monolayer.To assess the epithelial barrier properties of the cells, add 500 microliters of cell medium to the apical side, and 1.5 milliliters to the basal lateral side, then returned the plate to the incubator for an hour. Measure the transit material electrical resistance or TEER with ann STX2 chopstick electrode and an epithelial volt ohm meter. To cultivate THP1 cells, grow them in the T 75 flask using RPMI 1640 medium, supplemented with 10%FCS at 37 degrees Celsius and 5%carbon dioxide.Split the cells every second day by seeding 2 million cells per milliliter in a new T75 flask. To differentiate the THP1 cells, centrifuge the contents of the flask at 300 times G for four minutes, discard the supernatant. Resuspend the pellet in fresh medium and put in a new T75 flask.Add 10 nanograms per milliliter PMA to the cells and return them to the incubator. To detach the macrophage like cells, wash them once with PBS at 37 degrees Celsius, and incubate them with three milliliters of cell detachment solution containing 0.5 millimolar EDTA for 10 minutes at room temperature. Check for cell detachment under an inverted microscope.When the cells have detached, add seven milliliters of fresh medium and centrifuge them at 300 times G for four minutes. After removing the supernatant, resuspend the macrophage cells in three milliliters of THP1 medium in a 15 milliliter conical tube, count the cells, and incubate them for a maximum of one hour before setting up the co-culture. To establish an epithelial macrophage co-culture, remove the medium from the lower chamber of the CFBE41 O minus monolayers and carefully invert the support inside a sterile glass Petri dish, use a cell scraper to remove the cells overgrown through the membrane pores.Place 200, 000 THP1 macrophages in 200 microliters of RPMI on the basal lateral side of the inverted insert in each well, close the Petri dishes, and incubate them for two hours. Then place the inserts back into the 12 well micro plates and add 500 microliters of MEM medium to the basal lateral side of the permeable insert. Inoculate 15 milliliters of lb supplemented with 300 micrograms per milliliter ampicillin with a single colony of P aeruginosa, PAO1GFP, incubate the bacteria for 18 hours at 37 degrees Celsius, while shaking at 180 RPM.After the incubation, transfer the bacteria to a 50 milliliter conical tube, and centrifuges at 3, 850 times G for five minutes. Discard the supernatant and add 10 milliliters of sterile PBS that has been preheated to 37 degrees Celsius. Measure optical density at 600 nanometers and adjust the concentration of bacteria with the cell culture medium to a final concentration of 200, 000 colony forming units per milliliter, which corresponds to a multiplicity of infection of one bacterium per epithelial cell.Add 100 microliters of bacterial suspension to the apical side of the permeable support and incubate the plate at 37 degrees Celsius and 5%carbon dioxide for one hour to allow bacteria to attach to the cells, then carefully remove apical liquid with a pipette to restore ALI conditions. For experiments with drug treatment, add 500 microliters of a drug solution diluted in cell medium to the apical side. Then add 1.5 milliliters of cell medium to the basal lateral side.Collect 500 microliters of the atypical and basal lateral medium and pool them to assess CFU of non-attached bacteria. To assess survival of attached or internalized bacteria, add 500 microliters of sterile deionized cold water to each compartment of the permeable support, and incubate the cells for 30 minutes at room temperature. If assessing CFU from frozen samples, thaw them at 37 degrees Celsius for 10 minutes.Then use pipette tips to scrape the membrane surface taking care to not put too much pressure on the wells. Pipette up and down to remove all adhered content. With the bacterial suspension from both fractions, make a one to 10 serial dilution with PBS tween and plate the bacteria on lb agar plates.Incubate the agar plates at 30 degrees Celsius for 16 to 72 hours, then count the colonies and calculate CFU. The morphology of the resulting co-culture of human bronchial epithelial cells and macrophages on the apical and basil lateral side of the permeable supports are shown here. A higher TEER measurement and immunostaining for the tight junction proteins ZO1, proved epithelial barrier integrity.To model a bacterial infection, P aeruginosa was inoculated on CFBE41 o minus cells. Macrophages were observed on the apical side of the co-culture six hours after infection. The TEER dropped to 250 ohm per centimeter squared, indicating a compromised epithelial barrier, which was also suggested by ZO1 staining.Macrophage trans migration through the permeable filter pores and bacteria uptake by THP-1 cells on the apical side is shown here. In the THP-1 monocultures, macrophage migration as early as one hour post-infection. Meanwhile the migration was seen after three hours post-infection in the co-culture.Infected co-cultures treated with tobramycin for six or 20 hours were imaged with confocal scanning laser microscopy. Without treatment, either the epithelial cells or macrophages died after 20 hours of infection. Despite being seen after six hours of treatment in the microscopy pictures, a CFU assay demonstrated that the bacteria did not proliferate.Nevertheless, the bacteria recovered their proliferation capability after the 20 hour treatment. TEER of monocultures and co cultures were also measured. The co-culture of CFBE41 o minus cells with THP-1 did not induce any changes in the epithelial barrier integrity compared to the monoculture.Upon infection, the TEER value dropped. when performing this procedure, it's crucial to ensure that the cells are seated properly and the filter membrane is not disrupted. Therefore the cell seeding steps need to be performed carefully.In addition to this protocol, nebulization of antibiotics on the inserts would make it possible to see if bacteria survival changes compared to submersed conditions.

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

Research

JoVE Journal

Immunology and Infection

10.5K Views.  Helmholtz Institute for Pharmaceutical Research Saarland (HIPS). So when we are evaluating novel anti-infectives, we have to address two things. One side is the bacteria. If they are planktonic or informed biofilms, the other side is the presence of host cells, especially epithelial host cells, which form tight biological barriers.If these barriers are effected, how the monological response might be, and with this new model, we can actually monitor both parameters at the same time. When treating bacterial infections, we have to realize what organ is...

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