The overall goal of this procedure is to grow pseudomonas aerogen biofilms at the apical surface of live human airway epithelial cells. This is accomplished by first establishing a confluent monolayer of airway epithelial cells on a plastic surface or a glass cover slip. The second step of the procedure is to grow a culture of p aerogen bacteria constitutively, expressing the green fluorescent protein.
The next step is to place the bacteria and airway cells in contact with each other in either a static setting or in a flow chamber. The final step of the procedure is to let bacterial biofilms develop over time while assessing the viability of the airway cells. Results can be obtained that show biofilm formation on live airway cells through fluorescence microscopy.
The implication of this technique extend toward therapy of cystic fibrosis patients because these co culture models can be used to develop and validate new antibiotic regimens, able to eradicate biofilms responsible for chronic colonization of the airways. In cystic fibrosis patients, The static co-culture biofilm model uses CFBE cells, which are immortalized human airway. Epithelial cells originally developed from an individual with cystic fibrosis seven to 10 days prior to bacterial inoculation seed the CFBE cells in a 24 well tissue culture plate.
We seed cells at a concentration of two times 10 to the fifth cells per well in 0.5 milliliters of minimal essential medium or MEM supplemented with 10%fetal bovine serum two millimolar L-glutamine, 50 units per milliliter, penicillin and 50 micrograms per milliliter. Streptomycin grow the cells at 37 degrees Celsius and 5%carbon dioxide. 95%air for seven to 10 days change the medium every two to three days.
These conditions will lead to formation of a confluent, monolayer, and tight junctions one day prior to the experiment. Inoculate a five milliliter LB culture with pierro gen from a frozen stock and grow 18 hours at 37 degrees Celsius on a rotator. At 200 RPM we use p aerogen carrying the PSMC 21 plasmid for constitutive expression of GFP on the day of bacterial inoculation.
Remove the medium from the CFPE cells and add an equal volume of microscopy medium, which is MEM without phenol red, supplemented with two millimolar L-glutamine inoculate confluence, CFBE monolayers with posa at a multiplicity of infection of approximately 30 to one relative to the number of CFBE cells originally seeded for a 24 well plate. This equates to 1.2 times 10 of the seventh CFU per milliliter in 0.5 milliliters of MEM per. Well incubate the plate at 37 degrees Celsius and 5%carbon dioxide, 95%air for one hour.
Following the one hour incubation, remove the snat and replace with fresh microscopy. Medium supplemented with 0.4%arginine. The addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFPE cells.
Incubate the plate at 37 degrees Celsius and 5%carbon dioxide, 95%air for various time points up to approximately eight hours every couple of hours. Analyze the integrity of the CFBE monolayer and the growth of the biofilm using microscopy. The flow cell co-culture biofilm model requires the formation of a confluent monolayer of CFBE cells on a 40 millimeter diameter glass cover slip.
To accomplish this first place a sterile cover slip into a sterile 60 millimeter diameter plastic dish. Then add three milliliters of prewarm cell growth, medium pressing on the cover slip with the tip of the pipette to remove any bubbles trapped underneath, and to force the cover slip to the bottom of the plastic dish seed, two times 10 to the sixth CFBE cells per dish. Shake the dish gently back and forth, but avoid swirling to prevent centrifuging the cells against the sides of the dish.
Place the dish in a 5%carbon dioxide, 95%air incubator at 37 degrees Celsius for eight to 10 days. Feed the cells every other day with three milliliters of fresh growth medium. Under these conditions, the cells will form a confluent monolayer on the glass cover slip.
The next step is to prepare the P aerogen bacterial strain one day prior to the experiment. Grow p aerogen in five milliliters of LB for 18 hours at 37 degrees Celsius on a rotator. At 200 RPM, we use P aerogen strain, PO one, carrying the PSM C 21 plasmid for constitutive expression of GFP on the day of the experiment.
At one milliliter of bacterial culture into a sterile micro centrifuge tube and centrifuge at 6, 000 RPM for three minutes. Wash the bacterial pellet twice in one milliliter of microscopy. Medium dilute 0.5 milliliters of washed and resuspended bacteria into 4.5 milliliters of microscopy medium to achieve a concentration of about five times 10 of the eighth CFUs per milliliter.
Now we are ready to observe the CFPE cells in real time, which 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 a standard biofilm flow cell apparatus. The biotechs FCS two chamber modified to accommodate CFPE cells.
One of the most critical steps in the flow cell co-culture biofilm model is assembling the chamber without damaging the cell monitor To assemble the chamber. 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 millimeter thick rubber gasket onto the perfusion tubes. Stack the micro aqueduct slide provided with the chamber on top of the rubber gasket, making sure the grooved side is up.
Then 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. Next, add one milliliter of prewarm microscopy medium At the center of the slide.
Place the imaging chamber on a sterile surface while you retrieve the CFPE cells from the cell culture incubator. Remove the spent medium from the dish and wash the CFPE cells once with three milliliters of prewarm microscopy medium. Using ethanol washed forceps.
Retrieve the cover slip from the dish and lower it upside down onto the bead of microscopy medium placed onto the chamber. The cover slip is now resting on the second rubber gasket and the monolayer of airway cells is facing downward. 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. Connect the inlet tube to the low flow micro perfusion pump. A second piece of tubing, links the pump to a flas of microscopy medium placed in the 37 degree Celsius water bath.
Located right next to the microscope. Start the flow at a rate of 20 milliliters per hour. This flow rate is within the swimming speed capability of posa.
Attach sterile precut one 16th CFL tubing to the inlet and outlet perfusion tubes of the chamber, and then connect the temperature controller. Place the assembled chamber onto the microscope stage of an inverted fluorescence microscope. Using a one milliliter 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 to allow bacteria to attach to the airway cells. Stop the pump for two hours. After two hours, the flow can be reinitiated and maintained at 20 milliliters per hour.
For the rest of the experiment, monitor the integrity of the airway cells by differential interference. Contrast microscopy throughout the experiment to check for signs of damage to the monolayer. Simultaneously follow the development of GFP labeled p aerogen biofilms at the apical surface of airway cells.
By acquiring images with an inverted confocal or widefield fluorescence microscope in both the static and the flow cell assays, it was found that the CFPE monolayer could withstand the presence of p aerogen for up to eight hours after inoculation without any sign of alteration. Epithelial monolayer integrity can be assessed by phase contrast microscopy using an inverted as shown by this example of a confluent monolayer of CFPE cells grown on tissue culture plates. Over time, p aerogen will produce toxins and virulence factors that can damage the epithelial cell monolayer fully or in sections.
In this example of a compromised CFPE monolayer, P aerogen bacteria shown in green 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. This image shows an overgrown p aerogen biofilm observed 24 hours post inoculation after successfully supporting biofilm formation.
The CFPE 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 cover slip. When the integrity of the airway monolayer is not compromised.
P aerogen biofilms can successfully form and develop at the apical surface of airway cells in both co-culture models. Shown here is a representative image of A GFP expressing p aerogen biofilm grown on a confluent monolayer of CFPE cells using the static co-culture biofilm model assessed by epi fluorescence microscopy. The image is an overlay of the face contrast channel and the fluorescence channel.
This next image shows A GFP labeled p aerogen. Biofilm grown for six hours on a confluent mono layer of CFBE cells using the flow cell co-culture biofilm model to facilitate the visualization of the airway. Monolayer nuclei were stained with HEXT 3 33 42 prior to inoculation with posa and appear blue in color.
Films presenting as green clumps attached to the apical surface of the CFPE cells are dispersed across the airway cells. The typical mushroom like structures of six hour old aerogen biofilms forming on A-C-F-P-E cell monolayer after 3D reconstruction are shown here. After watching this video, you should have a good understanding of how to successfully grow and visualize bacterial biofilms on an established monolayer of airway cells using the co-culture models described here, and coupled to standard microbiological methods and fluorescence microscopy.
