P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

1. Growth and differentiation of cells in permeable support inserts

1. Cultivate CFBE41o^– in a T75 flask with 13 mL of minimum essential medium (MEM) containing 10% fetal calf serum (FCS), 1% non-essential amino acids and 600 mg/L glucose at 37 °C with 5 % CO₂ atmosphere. Add fresh medium to the cells every 2–3 days.
   1. Detach the cells after reaching 70% confluence in the flask with 3 mL of trypsin- Ethylenediaminetetraacetic acid (EDTA) at 37°C for 15 min. Add 7 mL of fresh MEM and centrifuge the cells at 300 x g for 4 min at room temperature (RT). Discard the supernatant and add new 10 mL of MEM while disrupting the clumps by gently pipetting up and down.
   2. Count the cells with an automated cell counter or hemocytometer chamber. Seed cells with a density of 2 x 10⁵ cells/well in 12-well plates with permeable supports (pore size of 3 μm, see Table of Materials).
      NOTE: The automated cell counter determines cell number, size distribution, and viability of the cells (see Table of Materials). Permeable supports with a pore size of 0.4 μm could be used here; however, the macrophages, in this condition, should be added directly to the apical side, and their transcellular migration will not be assessed in this case.
   3. Seed cells at liquid-liquid condition (LLC) by adding 500 µL of the cell suspension on the apical side of the permeable support and 1.5 mL of fresh medium in the basolateral side. Then incubate cells at 37°C under 5% CO₂, for 72 h.
   4. To shift to the air-liquid interface (ALI) culture, on the third day after seeding, remove the medium from the basolateral side first, then from the apical side. To the basolateral side, add 500 µL of fresh MEM and change the medium every second day until cells form a confluent monolayer.
      NOTE: For the conditions used in this protocol, the CFBE41o^– cells usually are confluent after 3-7 days in culture.
   5. Assess the epithelial barrier properties on day 7 by incubating CFBE41o^– cells with 500 μL cell medium in the apical side and 1.5 mL in the basolateral side for 1 h, at 37°C under 5% CO₂.
   6. Measure barrier properties via transepithelial electrical resistance (TEER), with an STX2 chopstick electrode and an epithelial volt-ohmmeter; after 7 days this is higher than 300 Ω×cm².
      NOTE: Eventually, in some membrane inserts, the cells have low TEER. Therefore permeable inserts with TEER < 300 Ω×cm² are not used.
2. To cultivate THP-1 cells, grow them in a T75 flask using 13 mL of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FCS, and incubate at 37°C under 5% CO₂. Split cells every second day by seeding 2 x 10⁶ cells/mL cells in a new T75 flask.
   NOTE: Non-differentiated THP-1 cells are grown as monocytes in suspension.
   1. Differentiate the THP-1 cells as follows. Centrifuge contents of a T75 at 300 x g for 4 min. Discard the supernatant, resuspend the pellet in fresh medium and put in a new T75. Add 10 ng/mL Phorbol 12-myristate 13-acetate (PMA) incubatig the cells in RPMI for 48 h at 37 °C and 5% CO₂ atmosphere¹⁵.
      NOTE: After the differentiation with PMA, cells do not proliferate anymore and attach to the flask.
   2. To detach THP-1 macrophage-like cells, wash once with phosphate-buffered saline (PBS) at 37 °C and incubate with 3 mL of cell detachment solution (e.g. accutase) containing 0.5 mM EDTA for 10 min at room temperature.
   3. Inspect the cells under an inverted microscope to look for cell detachment. Add 7 mL of fresh medium and centrifuge at 300 x g for 4 min at RT.
      NOTE: Macrophages can also be detached with trypsin-EDTA, 37 °C for 20 min. However, trypsin is harsher to macrophages than the chosen cell detachment solution (see Table of Materials).
   4. After removing the supernatant, re-suspend macrophage cells in 3 mL of THP-1 medium into a 15 mL conical tube, count the cells as described in 1.1.2. and incubate for a maximum 1 h at 37 °C under 5% CO₂ before setting up the co-culture.
      NOTE: THP-1 cells in suspension can be stained with viability dyes to image the co-culture further. At this step, use the procedure below (step 1.2.5).
   5. Stain macrophages with 10 µM of a cell viability dye (based on the conversion of acetate moieties by intracellular esterases, see Table of Materials) in which 3 µL of the cell viability dye is applied to the cell suspension. Incubate cells for 20 min at 37 °C, 5% CO₂, then wash 1x with PBS 37°C to remove the dye.
      NOTE: Centrifuge the cells to remove the dye at 300 x g for 4 min at room temperature (RT).

2. Establishment of an epithelial-macrophage co-culture on permeable supports

1. Use CFBE41o^– monolayers at ALI with TEER ≥ 300 Ω×cm² (step 1.1.6.). Remove the medium from the lower chamber, carefully invert the support inside a sterile glass Petri dish (50 mm x 200 mm), and remove the cells overgrown through the membrane pores on the bottom side of the membrane using a cell scraper.
   NOTE: Due to the pore size of 3 µm, epithelial cells tend to grow through the pores toward the basolateral side. Therefore, one needs to remove them before adding the macrophages on this side. CFBE41o^– lung epithelial cells can be stained at this step. The procedure in step 1.2.5 can be used; however, instead of a cell suspension, the dye solution in MEM is applied (500 µL apical side only) on the adhered cells on the permeable support.
2. Use 2 x 10⁵ cells/well (in 200 µL of RPMI) from the cell suspension of PMA-differentiated THP-1 macrophages and place the cells on the basolateral side of the inverted inserts.
3. Close the Petri dishes carefully and incubate for 2 h at 37 °C under 5% CO₂.
4. Place the inserts back into the 12-well microplates and add 500 µL of MEM medium in the basolateral side of the permeable insert to maintain ALI conditions. The cells are now ready for infection.

3. Infection by P. aeruginosa

NOTE: All following steps from here must be done in a biosafety level 2 (BSL2) laboratory.

1. Inoculate 15 mL of lysogeny broth (LB) supplemented with 300 μg/mL ampicillin in an Erlenmeyer flask (50 mL) with a single colony of P. aeruginosa PAO1-GFP.
   NOTE: Other strains of P. aeruginosa could also be used here, for instance, PAO1 wild type, PA14, or clinical strains, following their own cultivation protocols.
2. Incubate the bacteria for 18 h at 37°C, shaking at 180 rpm.
3. Transfer the contents after the 18 h to a 50 mL conical tube and centrifuge at 3850 x g for 5 min. Discard the supernatant and add 10 mL of sterile PBS at 37°C.
4. Measure optical density on a spectrophotometer at wavelength 600 nm and adjust the concentration of bacteria using the cell culture medium to a final concentration of 2 x 10⁵ CFU/mL. This corresponds to a multiplicity of infection (MOI) of one bacterium per epithelial cell.
5. Add 100 μL of bacterial suspension to the apical side of the permeable support (step 2.4.) and incubate at 37 °C under 5% CO₂ for 1 h, to allow bacteria attachment to the cells. Then, remove apical liquid carefully with a pipette to restore ALI conditions. Keep some samples uninfected as a control.
   NOTE: At this stage, the bacteria attached should be plated in LB agar (see steps 5.4/5.5) to determine the initial bacteria inoculum.
6. Incubate the drug of interest after bacterial adhesion in the cells. For treatment experiments, add 500 μL of a drug solution diluted in cell medium (in this protocol tobramycin 6 μg/mL was used) to the apical side. Add 1,500 µL of cell medium without drug on the basolateral side.
   NOTE: Instead of instilling the drugs as a solution, the model can also be adapted to aerosol deposition. For such purposes, the cells at ALI are fed from the basolateral side with 500 µL of cell medium. The drug is then first nebulized and allowed to deposit in the apical compartment by an appropriate device (not described here). The infected and treated sample can be checked for the endpoints outlined in the sections 4–7. From this step on, permeable supports can be used to create either images (section 4) or to get results of bacteria growth and mammalian cell viability, amongst others (sections 5–7).

4. Sample preparation for confocal laser-scanning microscopy (CLSM)

1. After the establishment of the co-culture, infection and drug treatment, remove all medium from the apical and basolateral side. Wash 1x with PBS at 37°C and then fix the cells with 3% paraformaldehyde (PFA) for 1 h at RT (300 µL on apical/600 µL on basolateral). Cell nuclei are stained with 5 µg/mL of DAPI-PBS for 30 min at room temperature.
   CAUTION: PFA is hazardous.
2. Cut the membranes using a scalpel and place them between two 12 mm microscopy cover slides using a mounting medium (see Table of Materials). Let it dry inside the flow bench for 30 min before storage at 4 °C. Visualize by confocal scanning microscopy.
   NOTE: After the co-culture and before the mounting, tight junctions immunostaining can be performed. For that, cells are fixed with paraformaldehyde 3% for 30 min, washed again with PBS, and permeabilized with saponin 0.05%/BSA 1% in PBS. In this protocol, the zonula occludens protein (ZO-1) was detected via mouse anti-human ZO-1 antibody (1:400, incubation at 4 °C overnight). The samples were then incubated for 2 h at RT with goat anti-mouse IgG antibody Alexa Fluor 633 (1:2000 in red). Nuclei were stained with DAPI (1 µg/mL) and mounted with mounting medium on coverslips.
3. Use a confocal microscope for imaging the stored membranes. Choose 25x or 63x water-immersion objectives and lasers at 405, 488, 505 or 633 nm for detection. Images should have a 1024 x 1024 pixel resolution.
   NOTE: The lasers are chosen according to the stain used.
4. Acquire apical and cross-section views, and use zeta-stack mode (10–15 stacks) for the construction of a three-dimensional model using imaging software.

5. Measurement of bacterial proliferation via colony-forming units (CFU)

1. Collect the apical and basolateral medium (containing bacteria) to assess CFU of non-attached bacteria. Collect 500 µL from the apical and basolateral sides and pool them.
   NOTE: Use this suspension directly to count bacteria (step 5.4) or centrifuge at 21,250 x g for 10 min to evaluate lactate dehydrogenase (LDH) from the supernatant (section 6) and/or re-suspended in PBS to count extracellular bacteria (step 5.4).
2. Assess survival of bacteria attached and/or internalized in the cells by adding 500 µL of sterile deionized cold water in each compartment of the permeable support. Incubate cells for 30 min at room temperature.
   NOTE: The samples can either be plated on LB agar (see step 5.4) or frozen (as whole insert plate) at -20 °C for plating later on.
3. For assessing CFU of adherent/internalized bacteria, thaw samples at 37°C for 10 min (if frozen). Using pipette tips for each well, scrape the membrane surface and pipette up and down to remove all adhered content.
   NOTE: At this step, all the epithelial cells are lysed and adherent/internalized bacteria are available as a suspension to be plated.
4. With the bacterial suspension from both fractions, perform a 1/10 serial dilution using Tween-80 0.05% in PBS and plate the bacteria on LB agar plates.
   NOTE: Dilutions in between 1 to 10 are recommended. The bacteria should be counted in the highest dilution, where single colonies are first identified.
5. Incubate agar plates at 30°C for 16–72 h to count colonies, and calculate CFU accordingly.
   NOTE: A temperature of 30°C at the time of plate incubation is essential for treated-samples and to observe delayed-growth of colonies.

6. Evaluation of cell cytotoxicity via lactate dehydrogenase assay

1. Use the supernatant of infected cells containing bacteria (from step 5.1) for cell viability assessment for LDH assay¹⁶. Centrifuge the supernatant at 21,250 x g for 10 min to pellet the bacteria and eventually rest of the cells. Use the bacteria-free supernatant to measure LDH release.
   NOTE: The supernatant should not be frozen before measuring LDH by this assay.
2. Transfer 100 µL of the supernatant to a 96-well plate, and add 100 µL of the LDH assay solution (see the Table of Materials). Incubate at room temperature for 5 min in the dark, then read absorbance at 492 nm.

7. Assessing the release of human cytokines

1. For cytokine quantification, use either ELISA or cytometric bead array immunoassay¹⁷(see the Table of Materials). For this, centrifuge supernatant from step 5.1 at 21,250 x g for 10 min and measure either immediately or store -80 °C for up to 15 days until analysis.
2. Evaluate supernatants with a commercially available ELISA kit.
   NOTE: The procedure follows the manufacture instructions, which include the coating of plates with the capture antibody, addition of the samples (100 µL) and cytokine standards, incubation, washing, and addition of detection antibody to provide a colorimetric measurement of cytokine presence. Alternatively, flow cytometry can be used to measure cytokines via commercially available kits (see Table of Materials).