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-glut...

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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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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>FCS2 (Focht Live-Cell) chamber</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319131616</td>
<td>&nbsp;</td>
<tr>
<td>FCS2 chamber controller</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319-2-0303</td>
<td>&nbsp;</td>
<tr>
<td>40 mm glass coverslips</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>PH 40-1313-0319</td>
<td>&nbsp;</td>
<tr>
<td>MEM</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>#10-010-CV</td>
<td>&nbsp;</td>
<tr>
<td>MEM without phenol red</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>Mediatech, Manassass, VA</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 ...

co-culture-models-pseudomonas-aeruginosa-biofilms-grown-on-live-human

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Immunology and Infection

22.0K vistas.  Dartmouth College. Este documento describe los diferentes métodos de cultivo Pseudomonas aeruginosa Biofilms en cultivos de células epiteliales de las vías humanos. Estos protocolos pueden ser adaptados para estudiar diferentes aspectos de la formación de biopelículas, incluyendo la visualización del biofilm, la tinción de la biopelícula, la medición de unidades formadoras de colonias (UFC) de la película biológica, y el estudio de citotoxicidad biofilm.