Establishing a Co-Culture Model to Study Host-Pathogen Interactions at the Air-Liquid Interface

Abstract

Source: Montefusco-Pereira, C. V. et al. P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives. J. Vis. Exp.(2020)

This video demonstrates a co-culture model of bronchial epithelial cells and macrophage-like cells at the air-liquid interface to study host-pathogen interactions. Pathogenic bacterial infection compromises epithelial membrane integrity, triggering macrophage-like cell activation and promoting pathogen clearance.

Protocollo

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 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 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 atmosphere.
      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, resuspend macrophage cells in 3 mL of THP-1 medium in a 15 mL conical tube, count the cells as described in 1.1.2. and incubate for a maximum of 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 at 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 Pseudomonas 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 colony-forming unit (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.

Materiali

Elenco dei materiali utilizzati in questo articolo

Nome	Azienda	Numero di catalogo	Commenti
Accutase	Accutase	AT104
Ampicillin	Carl Roth, Germany	HP62.1
CASY TT cell counter and analyzer	OLS Omni Life Sciences	-
CellTrace Far Red	Thermo Fischer	C34564
Centrifuge Universal 320R	Hettich, Germany	1406
CFBE41o- cells	1. Gruenert Cell Line Distribution Program2. Sigma-Aldrich	1. gift from Dr. Dieter C. Gruenert2. SCC151
Chopstick electrode set for EVOM2, 4mm	World Precision Instruments, Sarasota, USA	STX2
D-(+) Glucose	Merck	47829
Epithelial voltohmmeter	World Precision Instruments, Sarasota, USA	EVOM2
Falcon permeable support for 12 Well Plate with 3.0μm transparent PET membrane, sterile	Corning, Amsterdam, Netherlands	353181
Fetal calf serum	Lonza, Basel, Switzerland	DE14-801F
LB broth	Sigma-Aldrich, Germany	L2897-1KG
MEM (Minimum essential medium)	Gibco Thermo Fisher Scientific Inc.	11095072
Non-essential amino acids solution (100X)	Gibco Thermo Fisher Scientific Inc.	11140050
P. aeruginosa strain PAO1	American Type Culture Collection	47085
P. aeruginosa strain PAO1-GFP	American Type Culture Collection	10145GFP
Phosphate buffer solution buffer	Thermo Fischer	10010023
Petri dishes	Greiner	664102
Phorbol 12-myristate 13-acetate (PMA)	Sigma, Germany	P8139-1MG
Roswell Park Memorial Institute (RPMI) 1640 medium	Gibco by Lifetechnologies, Paisley, UK	11875093
T75 culture flasks	Thermo Fischer	156499
THP-1 cells	Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany)	No. ACC-16
Trypsin-EDTA 0.05%	Thermo Fischer	25300054