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.
