Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board

1. Preparation of an inoculum

1. Pour 10 mL of LB broth into a 50 mL conical flask with a foam stopper.
2. Transfer a colony of P. aeruginosa strain PAO1 or strain PA14 from a fresh agar plate and incubate at 37 °C for 3-4 h until the bacteria are in mid-log phase.
3. Transfer the culture of bacteria to a 50 mL tube and centrifuge at 3,000 x g for 5 min. Remove the supernatant and re-suspend the cell pellet in PBS.
4. Repeat step 1.3 two more times to wash the cells. Resuspend the cell pellet in PBS and adjust the optical density at 600 nm to approximately 0.6 using sterile PBS as a blank.

2. Infecting the corneoscleral button

1. Remove media from the Petri dish and rinse corneas twice with 1 mL of sterile PBS.
2. Gently squeeze forceps while holding the cornea in-between. Use a 10A scalpel to make four cuts–two vertical, two horizontal-in the central section of the corneoscleral button through the epithelial layer to the underlying stroma.
3. Place a sterile glass mold in a 6-well plate with the wide part up and place the cornea in the middle of the glass mold, epithelium side facing down. Make the cut right in the center of the bottom part of the glass mold.
4. CRITICAL STEP: Pour 1 mL of 1% (w/v) low melting point agar dissolved in DMEM to fill the glass mold with cornea completely.
5. Allow the agar to set and then invert the glass mold so that the corneal epithelium is facing upwards.
6. Pipette 15 μL of the bacterial culture with OD_600nm = 0.6 (for P. aeruginosa this equates to approximately 1 x 10⁷ colony forming units (CFU) in 15 μL) directly into a cut area and then add 85 μL of PBS to the top to keep the corneal epithelium moist. Dilute the remaining bacterial culture and plate on agar to count colony forming units for the inoculum.
7. Add 1 mL of DMEM without antibiotics to the bottom of each well with the glass mold. Incubate the 6-well plate with the infected corneoscleral buttons in a humidified incubator at 37 °C with 5% CO₂ for up to 24 h.
8. Set up uninfected control cornea alongside every experiment. To set up uninfected control, replace the 15 μL of bacterial culture in step 2.6 with sterile PBS.