We would like to thank G. O Toole for guidance and suggestions in developing these models. This work was supported by the Cystic Fibrosis Foundation (ANDERS06F0 to G.G.A., STANTO07RO and STANTO08GA to B.A.S.), the National Institutes of Health (T32A107363 to G.G.A. and R01-HL074175 to B.A.S.), and the National Center for Research Resources Centers for Biomedical Research Excellence (COBRE P20-RR018787 to B.A.S.).

1. Static Co-culture Biofilm Model
<ol>
 <li>The Static Co-culture Biofilm Model1 uses CFBE41o- cells (CFBE cells), which are immortalized cells originally developed from an individual with CF homozygous for the &Delta;F508-CFTR mutation 2,3,4. CFBE cells should be seeded at a concentration of 106 cells/well in a 6-well tissue culture plate or 2 X 105 in a 24-well tissue culture plate in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine, 50 U/mL penicillin, and 50 &mu;g/mL streptomycin. We use 1.5 mL medium per well in 6-well plates and 0.5 mL medium per well in 24-well plates.</li>
 <li>Cells should be grown at 37&deg;C and 5% CO2-95% air for 7-10 days to form a confluent monolayer before inoculation with bacteria. The medium must be changed every 2-3 days. These conditions have been shown to lead to formation of a confluent monolayer and tight junctions.</li>
 <li>Grow P. aeruginosa in 5 mL LB for 18 hours at 37&deg;C on an incubator shaker at 200 rpm. Under these conditions, P. aeruginosa cultures will typically reach a density of 5x109 CFU/ mL.</li>
 <li>For bacterial inoculation, remove the medium from CFBE cells and add an equal volume of MEM without phenol red, supplemented with 2 mM L-glutamine (Microscopy medium). Confluent CFBE monolayers are inoculated with P. aeruginosa at a multiplicity of infection of approximately 30:1 relative to the number of CFBE cells originally seeded. This equates to 2 X 107 CFU/mL in 1.5 mL MEM/well for 6-well plates and 1.2 X 107 CFU/mL in 0.5 mL MEM/well for 24-well plates.</li>
 <li>Incubate plates for 1 hour at 37&deg;C and 5% CO2-95% air.</li>
 <li>Following the 1 hour incubation, the supernatant should be removed and replaced with fresh Microscopy medium supplemented with 0.4% arginine.</li>
 <li>Incubate at 37&deg;C and 5% CO2-95% air for various time points (up to approximately 8 hours) and analyze the integrity of the CFBE monolayer using phase contrast microscopy. If airway cells are non-confluent, P. aeruginosa will rapidly (within minutes) gain access to the basolateral surface of the cells and destroy cellular integrity. Inoculation with P. aeruginosa strains that constitutively express GFP results in fluorescent biofilm microcolonies which can be visualized by epifluorescence or confocal microscopy.</li>
 <li>The bacterial CFU in the biofilm can be determined by washing the co-culture 2-3 times with phosphate-buffered saline (PBS) to eliminate planktonic bacteria. Following the wash, treat with 0.1% Triton X-100 in PBS or MEM for 10-15 minutes to lyse the epithelial cells and disperse the biofilm.</li>
 <li>Vortex for 3 minutes and prepare serial dilutions of the lysate. Plate these dilutions onto LB agar and incubate overnight at 37&deg;C. Count the colonies the next day to determine the CFU.</li>
</ol>
2. Flow Cell Co-culture Biofilm Model
<ol>
 <li>The Flow Cell Co-culture Biofilm Model (flow assay) involves modification of the standard biofilm flow cell apparatus to accommodate CFBE cells5.</li>
 <li>Place several 40-mm diameter glass coverslips into a clean 100-mL beaker. Cover tightly with 2 layers of aluminum foil and autoclave on a dry cycle for 20 min.</li>
 <li>Working in a cell culture hood, grab one sterile coverslip from the beaker using ethanol washed forceps and place into a sterile 60-mm diameter plastic dish.</li>
 <li>Add 3 mL of pre-warmed cell growth medium, pressing on the coverslip with the tip of the pipette to remove any bubble trapped underneath and to force the coverslip to the bottom of the plastic dish.</li>
 <li>Seed 2x106 cells per dish and shake the dish gently back and forth. Avoid swirling to prevent centrifuging the cells against the sides of the dish.</li>
 <li>Place the dish in a 5% CO2-95% air incubator at 37&deg;C and feed the cells every other day with 3 mL fresh growth medium for 8 to 10 days. The cells will form a confluent monolayer on the glass coverslip.</li>
 <li>Grow P. aeruginosa in 5 mL LB for 18 hours at 37&deg;C on an incubator shaker at 200 rpm. Under these conditions, P. aeruginosa cultures will typically reach a density of 5x109 CFU/mL. We use P. aeruginosa strain PAO1 carrying the pSMC21 plasmid for constitutive expression of GFP6.</li>
 <li>Add 1 mL of bacterial culture into a sterile microcentrifuge tube. Centrifuge at 6000 rpm for 3 min, and wash the bacterial pellet twice in 1 mL of Microscopy medium.</li>
 <li>Dilute 0.5 mL of washed and resuspended bacteria into 4.5 mL Microscopy medium to achieve a concentration of ~5x108 CFU/mL.</li>
 <li>Observation of live cells in real time requires an imaging chamber coupled to a peristaltic pump (to ensure a flow of nutrients for extended lengths of time) and a temperature controller. We use the Bioptechs FCS2 (Focht Live-Cell) chamber connected to a low-flow micro-perfusion pump with 1/16th C-flex tubing cut to appropriate lengths and autoclaved for sterility, and temperature-regulated via the FCS2 chamber controller. We keep the input source of medium in a 37&deg;C water bath located right next to the microscope.</li>
 <li>Assemble the flow chamber. The FCS2 chamber includes a self-locking base (which sits on the stage adapter during imaging) and an upper half connected to the perfusion tubes and the chamber controller. The chamber is assembled upside-down before being flipped over and placed onto the stage adapter. Hold the upper half of the chamber upside down so that the perfusion tubes are visible, and align the clearance holes of a 0.75-mm thick rubber gasket onto the perfusion tubes. Stack the microaqueduct slide provided with the chamber on top of the rubber gasket, making sure the grooved side is up, and place another rubber gasket on top of the slide. The thickness and internal geometry of this second gasket will determine the volume of the chamber. We typically work with a 0.75-mm thick rubber gasket with a 30-mm round internal geometry.</li>
 <li>Add 1 mL of pre-warmed (37&deg;C) Microscopy medium at the center of the slide.</li>
 <li>Retrieve a 60-mm dish from the cell culture incubator, remove the spent medium and wash the cells once with 3 mL of pre-warmed Microscopy medium. This step ensures the elimination of the phenol red and antibiotics present in the cell growth medium. Phenol red is mildly fluorescent and can interfere with the imaging, while antibiotics can eradicate P. aeruginosa bacteria before they can establish biofilms.</li>
 <li>Using ethanol-washed forceps, retrieve the coverslip from the dish and lower it upside down onto the bead of Microscopy medium placed onto the chamber. The coverslip is now resting on the second rubber gasket and the monolayer of airway cells is facing downward.</li>
 <li>Holding the assembled components in one hand, place the base of the chamber on top of the stack, and turn the chamber over swiftly so that everything is right side up. Lock the base into place by turning the ring.</li>
 <li>Connect the inlet tube to the low-flow micro-perfusion pump and start the flow at a rate of 20 mL/h; this flow rate is within the swimming speed capability of P. aeruginosa. A second piece of tubing links the pump to a flask of Microscopy medium placed in the 37&deg;C water bath located right next to the microscope.</li>
 <li>Attach sterile pre-cut 1/16th C-Flex tubing to the inlet and outlet perfusion tubes of the chamber, connect the temperature controller, then place the assembled chamber onto the microscope stage of an inverted fluorescence microscope.</li>
 <li>Using a 1-mL disposable syringe, inject the previously prepared bacterial suspension into the chamber, using a two-way valve placed in-line between the pump and the chamber. In our particular setting, it takes a 0.6 mL volume of bacterial suspension to reach the chamber. Adjust this volume according to your particular tubing length. We also find it useful to place an in-line disposable 0.22 &mu;m filter between the pump and the two-way valve to prevent bacteria from swimming upstream and contaminating the input flask.</li>
 <li>To allow bacteria to attach to the airway cells, stop the pump for 2h. The flow can then be reinitiated and maintained at 20 mL/h for the rest of the experiment.</li>
 <li>Monitor the integrity of the airway cells by differential interference contrast (DIC) microscopy throughout the experiment to check for signs of damage to the monolayer. Simultaneously, follow the development of GFP-labeled P. aeruginosa biofilms at the apical surface of airway cells by acquiring images with an inverted confocal or wide-field fluorescence microscope.</li>
</ol>
3. Representative Results
We use a strain of P. aeruginosa containing the pSMC21 plasmid which allows for constitutive expression of GFP6. For this reason, GFP-labeled biofilms growing on a CFBE monolayer can be visualized by epifluorescence microscopy. Alternatively, visualization of the biofilm can be achieved by staining unlabeled P. aeruginosa with a 1% calcofluor white solution for 1 hour at 37&deg;C1.
For imaging biofilm formation on CFBE cells in the flow assay, we use a custom-made stage adapter, but several companies can now provide specially fitted stage adapters. We work with an Olympus IX70 inverted microscope equipped with an ORCA-AG deep cooling CCD camera and an x60 oil-immersion objective (numerical aperture 1.40). The filter wheel is equipped with a 480/40 nm band pass excitation filter and a 535/30 nm band pass emission filter. Digital images were acquired with the OpenLab 4.0.3 software package (Improvision) and volumes were deconvolved by iterative restoration using the Volocity 3.5.1 software (Improvision). Quantitative analysis of 3D biofilm structures was achieved with the COMSTAT image analysis software package7,8.
For any co-culture model considered, one must pay particular attention to the cytotoxicity developing between the components of the model. In both the static and the flow cell assays, we found that the CFBE monolayer could withstand the presence of P. aeruginosa for up to 8 hours after inoculation without any sign of alteration. Epithelial monolayer integrity can be assessed by phase-contrast microscopy using an inverted microscope1 (Figure 1A) or by differential interference contrast (DIC) microscopy throughout the experiment5. Over time, P. aeruginosa will produce toxins and virulence factors which accumulate and can damage the epithelial cell monolayer fully or in sections (Figure 1B-1C). Cytotoxicity can be quantitatively assessed using various kits to measure the release of lactate dehydrogenase (LDH) from the epithelial cells. LDH is a stable cytosolic enzyme released in the extracellular milieu upon cell lysis or cell death.
When the integrity of the airway monolayer is not compromised (typically for ~8 h following inoculation), P. aeruginosa biofilms can successfully form and develop at the apical surface of airway cells in both co-culture models described (Figure 2A-2B). Following 3D reconstruction (Figure 2C) and quantification, biomass accumulation can be accurately determined. We have also used these co-culture biofilms in several phenotypic assays. For instance, as mentioned above, biofilm CFU can be easily determined by lysing the epithelial cells, serially diluting the lysate, and plating on agar plates. We have found this technique advantageous in determining the resistance of different strains to antibiotic treatment. Furthermore, biofilm toxicity toward the epithelial cells can be measured using commercially available LDH detection kits. In this manner, the role of virulence factors in toxicity of biofilms can be assessed. These model systems also support a number of gene expression applications, including promoter fusion studies, RT-PCR, and microarray analysis1,5,9.
<img src="/files/ftp_upload/2186/2186fig1.jpg" alt="Figure 1" /> Figure 1. Monolayer of CFBE cells and representative images of compromised and damaged airway cell monolayers due to P. aeruginosa biofilm growth. (A) Representative image of a confluent monolayer of CFBE cells grown in tissue culture plates, assessed by phase-contrast microscopy. Scale bar, 120 &mu;m. (B) Example of a compromised CFBE monolayer. Even though the monolayer does not display obvious visible signs of damage yet, P. aeruginosa bacteria are seen spreading between the tight junctions of the epithelial cells and gaining access to the basolateral membranes. Biofilm formation is typically not achieved under these conditions due to the monolayer deteriorating. Scale bar, 20 &mu;m. (C) Example of an overgrown P. aeruginosa biofilm observed 24 h post-inoculation. After successfully supporting biofilm formation, the CFBE monolayer was damaged beyond repair and is now virtually absent. Residual biofilm, growing as a flat layer of bacteria, is shown attaching to the glass coverslip. Scale bar, 20 &mu;m.
<img src="/files/ftp_upload/2186/2186fig2.jpg" alt="Figure 2" /> Figure 2. P. aeruginosa biofilms grown at the apical surface of confluent CFBE cells using the Static Co-culture Biofilm Model and the Flow Cell Co-culture Biofilm Model. (A) Representative image of a GFP-expressing P. aeruginosa biofilm grown on a confluent monolayer of CFBE cells using the Static Co-culture Biofilm Model, assessed by epifluorescence microscopy. Image is an overlay of the phase contrast channel and the fluorescence channel. Scale bar, 35 &mu;m. (B) Representative image of a GFP-labeled P. aeruginosa biofilm grown for 6 h on a confluent monolayer of CFBE cells using the Flow Cell Co-culture Biofilm Model. To facilitate the visualization of the airway monolayer, nuclei were stained with 10 &mu;g/mL Hoechst 33342 (Molecular Probes) for 30 min prior to inoculation with P. aeruginosa. Merged and pseudocolored images were viewed by differential interference contrast (DIC), and the corresponding fluorescent images are shown. Biofilms, presenting as green clumps attached to the apical surface of the CFBE cells, are dispersed across the airway cells. Scale bar, 20 &mu;m. (C) Three-dimensional reconstruction of z-series image stacks showing the typical mushroom-like structures of 6 h-old P. aeruginosa biofilms forming on a CFBE cell monolayer. Scale bar, 10 &mu;m.

<ol>
	<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> Infect Immun. 76, 1423-1433 (2008).</li><li>Bruscia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CF+cell+lines+corrected+at+DeltaF508-CFTR+locus+by+SFHR-mediated+targeting.">Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting.</a> Gene Ther. 9, 683-685 (2002).</li><li>Cozens, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+expression+and+chloride+secretion+in+polarized+immortal+human+bronchial+epithelial+cells.">CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.</a> Am J Respir Cell Mol Biol. 10, 38-47 (1994).</li><li>Hentchel-Franks, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+airway+cl-+secretion+in+human+subjects+by+adenosine.">Activation of airway cl- secretion in human subjects by adenosine.</a> Am J Respir Cell Mol Biol. 31, 140-146 (2004).</li><li>Moreau-Marquis, S. <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> Am J Physiol Lung Cell Mol Physiol. 295, 25-37 (2008).</li><li>Kuchma, S. L., Connolly, J. P., 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=A+three-component+regulatory+system+regulates+biofilm+maturation+and+type+III+secretion+in+Pseudomonas+aeruginosa.">A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa.</a> J Bacteriol. 187, (2005).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+reproducibility+in+flow-chamber+biofilms.">Experimental reproducibility in flow-chamber biofilms.</a> Microbiology. 146 (Pt 10), 2409-2415 (2000).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Anderson, G. G., Yahr, T. L., Lovewell, R. 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=The+Pseudomonas+aeruginosa+magnesium+transporter+MgtE+inhibits+transcription+of+the+type+III+secretion+system.">The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system.</a> Infect Immun. 78, (2010).</li><li>Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+microbial+biofilms.">Overview of microbial biofilms.</a> J Ind Microbiol. 15, 137-140 (1995).</li><li>Hoiby, N., Frederiksen, B., Pressler, T. <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+early+Pseudomonas+aeruginosa+infection.">Eradication of early Pseudomonas aeruginosa infection.</a> J Cyst Fibros. 4, 49-54 (2005).</li><li>Ko, Y. H., Pedersen, P. L. <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:+a+brief+look+at+some+highlights+of+a+decade+of+research+focused+on+elucidating+and+correcting+the+molecular+basis+of+the+disease.">Cystic fibrosis: a brief look at some highlights of a decade of research focused on elucidating and correcting the molecular basis of the disease.</a> J Bioenerg Biomembr. 33, 513-521 (2001).</li><li>Gibson, R. L., Burns, J. L., Ramsey, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+management+of+pulmonary+infections+in+cystic+fibrosis.">Pathophysiology and management of pulmonary infections in cystic fibrosis.</a> Am J Respir Crit Care Med. 168, 918-951 (2003).</li><li>Furukawa, S., Kuchma, S. L., 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=Keeping+their+options+open:+acute+versus+persistent+infections.">Keeping their options open: acute versus persistent infections.</a> J Bacteriol. 188, 1211-1217 (2006).</li><li>Merritt, J. H., Kadouri, D. E., 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=Growing+and+analyzing+static+biofilms.">Growing and analyzing static biofilms.</a> Curr Protoc Microbiol. Chapter 1, Unit 1B 1-Unit 1B 1 (2005).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signalling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis.</a> Mol Microbiol. 28, 449-461 (1998).</li><li>Gomez, M. I., Prince, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunistic+infections+in+lung+disease:+Pseudomonas+infections+in+cystic+fibrosis.">Opportunistic infections in lung disease: Pseudomonas infections in cystic fibrosis.</a> Curr Opin Pharmacol. 7, 244-251 (2007).</li></ol>

No conflicts of interest declared.

Biofilms are communities of bacteria that form in response to environmental stimuli. These environmental signals lead to global regulatory changes within each bacterium, resulting in binding to a surface, aggregation, production of exopolysaccharides, and other phenotypes such as increased antibiotic resistance10. Over the last couple of decades, much evidence has supported the hypothesis that biofilms play a large role in the pathogenesis of chronic infections. For instance, it is well accepted that P. ae...

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<td>FCS2 (Focht Live-Cell) chamber</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
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<td>FCS2 chamber controller</td>
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<td>40 mm glass coverslips</td>
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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.

Watch this Scientific Journal Video about Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells at JoVE.com

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.

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 ...

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Immunology and Infection

22.1K ビュー.  Dartmouth College. 本論文では、成長の異なる方法を説明します緑膿菌培養ヒト気道上皮細胞上のバイオフィルム。これらのプロトコルは、バイオフィルムの可視化を含むバイオフィルム形成の異なる側面を、研究するために適応、バイオフィルムの染色、バイオフィルムのコロニー形成単位（CFU）を測定し、バイオフィルム細胞毒性を勉強することができます?...

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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 ...

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

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

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

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

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

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

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.

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.

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