The overall goal of the following experiment is to use the vertical diffusion chamber or VDC model to investigate bacterial interactions with an invasion of intestinal epithelial cells. This is achieved first by using a special filter seated with a polarized monolayer of caco two intestinal epithelial cells to create separate apical and basal lateral compartments in the VDC. In the second step, the bacterial inoculum of interest is added to the apical compartment.
Under micro aerobic conditions and cell culture, medium is added to the basolateral compartment under aerobic conditions, then at the desired time points during the co-culture, the filter is removed and the numbers of interacting or intracellular bacteria are enumerated. Ultimately, an increase in the number of interacting and intracellular bacteria can be observed using the VDC model compared to standard aerobic culture conditions. The main advantage of this technique over existing methods, such as the standard tissue culture adhesion and invasion assays, is that with this technique, the co-culture of bacteria and intestinal epithelial cells is performed under in vitro conditions that more closely mimic the in vivo conditions of the human intestine.
Demonstrating the procedure will be N Vida nas, a PhD student from my laboratory and Dominic Mills, a postdoc who developed the vertical diffusion chamber model as part of his PhD in my laboratory. Begin by seeding four by 10 to the fifth caco, two intestinal epithelial cells per 0.4 micron filter in culture medium, then grow the cells to polarization for 21 days, changing the media every two to three days to confirm the polarization status of the monolayer. Use a volt ohm resistance meter to measure the trans epithelial electrical resistance.
24 hours before beginning the VDC co-culture assay incubate jejuni on a fresh blood agar plate under micro aerobic conditions in a variable atmosphere incubator. At the same time, pre incubate 30 milliliters of Bruce broth under the same conditions on the day of the experiment, immerse the VDC half chambers as well as the O-rings and plugs in sterilization solution. Then use a sterile 20 milliliter syringe to flush the solution through the gas inlets in both half chambers After an hour, rinse the VDC components with fresh sterile water using another sterile 20 milliliter syringe to rinse the gas inlets.
Then harvest half of the CJ jui blood agar plate culture into one milliliter of the pre incubated barella broth to yield approximately 10 to the 10th CFU per milliliter. Measure the optical density of the bacterial suspension at 600 nanometers to semi quantify the bacterial colony forming units or CFUs, and then adjust the bacterial suspension to the desired inoculum level in four milliliters total of the pre incubated Bruce broth. Perform a serial dilution from this final bacterial suspension, then plate the appropriate dilution on blood agar plates and triplicate and incubate the plates in the variable atmosphere Incubator for 48 hours.
Now, place the lower half chamber of the VDC flat onto the bench and fit an O-ring onto it. Then detach one filter carrying the intestinal epithelial cells from the carrier and wash it three times with 400 microliters of sterile PBS. Place the filter onto the lower half chamber, making sure the O-ring remains in place, and then gently lower the upper half chamber into place.
Once the two half chambers are assembled, clamp them together using the ring clamps and place the VDC horizontally on the benchtop with the two openings facing upwards. Add the bacterial inoculum into the apical half chamber and then add four milliliters of cell culture medium into the basal lateral half chamber. Place the end pieces into the openings on both half chambers, and then place the VDC into the variable atmosphere incubator.
In close proximity to the gas manifold, open the gas supply regulator connected to the gas manifold. Attach the tube from the manifold into the gas inlet on the basal lateral half chamber of the VDC and then attach the gas outlet pipe leading out of the variable atmosphere incubator to one of the outlets in the end piece on the basal lateral half chamber. Close off the other outlet in the end piece on the basal lateral half chamber.
Then open the gas flow on the gas manifold very slowly to avoid excess gas pressure and to achieve a gas flow of one bubble every two to five seconds after the appropriate co-culture period. Close the gas supply regulator connected to the gas manifold. Then blows off the gas flow into the VDC at the manifold and disconnect the gas inlet, the gas outlet, and the stopper from the VDC.
Next, remove the VDC from the variable atmosphere incubator. Then remove the apical and basal lateral supernatants and store them at negative 80 degrees Celsius for subsequent analysis. Remove the ring clamps and gently take apart the VDC.
Remove the filter from the half chamber and transfer it to a sterile six well cell culture dish. Then wash the VDC with sterile water and store it for the next round of experiments for enumeration of the total number of interacting bacteria. Wash the intestinal epithelial cells three to times with 400 microliters of sterile PBS and then lyce them in 400 microliters of sterile PBS containing Triton X 100 for 20 minutes at room temperature, perform a serial dilution from the cell lysate, followed by plating of the appropriate dilutions on blood agar plates in triplicate.
Then incubate the plates at 37 degrees Celsius in the variable atmosphere incubator for 48 hours for enumeration of the total number of invading bacteria. Incubate the intestinal epithelial cells with 400 microliters of cell culture medium containing 150 micrograms per milliliter of Gentamycin or two hours in a standard tissue culture incubator to kill the extracellular bacteria. Then quantify the colony forming units after cell lysis, serial dilution, and culture as just demonstrated.
The following data illustrate a comparison between the number of CJ jui interacting with or invading caco two cells after three, six, or 24 hours of co-culture under standard cell culture assay conditions versus using the VDC model. After 24 hours of co-culture, for example, less than 10 to the seventh, CFUs of CJ Juna can be observed interacting with CACO two cells under standard cell culture conditions. However, after 24 hours of co-culture in the VDC model, approximately 10 to the eighth, CFU of CJ jui can be observed interacting with CACO two cells, an increase in CJ jui IEC interactions of almost tenfold.
When comparing the number of invading CJ jui under standard versus VDC culture conditions, approximately 10 to the fifth intracellular CFUs of CGE jui can be isolated from the CACO two cells after 24 hours under standard cell culture conditions. Whereas after 24 hours of co-culture in the VDC model, approximately 10 to the seventh intracellular CFUs can be isolated from the CACO two cells and increase in intracellular CGE of almost 100 fold. While attempting this procedure, it's important to monitor the gas flow in the basal lateral chamber After its development.
This technique paved the way for researchers in the field of abuc pathogenesis to investigate both the genetic basis of bacterial interaction with intestinal epithelial cells, as well as the host in innate immune response to bacterial infection. After watching this video, you should have a good understanding of how to use the vertical diffusion chamber model to study the interactions of other enteric bacteria with intestinal epithelial cells and the conditions that more closely represent those in the human intestine.
