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

In the vertebrate eye, the cornea - the outermost protective covering- is infected by Pseudomonas aeruginosa, resulting in a corneal inflammatory condition - keratitis. 

To establish an ex vivo corneal keratitis model, place a porcine eye corneoscleral button in a Petri dish. This tissue segment consists of the cornea surrounded by remnants of sclera - the fibrous eye covering. Supplement the Petri dish with antibiotic-containing media to eliminate contaminating microbes from the corneal epithelial surface.

Locate the central portion of the cornea. Make superficial horizontal and vertical incisions in the desired pattern piercing the outermost corneal epithelial layer. Transfer the incised tissue, cornea side down, into a specialized hollow mold. 

Fill the corneal cavity with liquified agar and allow to solidify, restraining the tissue inside the mold. Invert the mold to expose the wounded surface of the corneal epithelium. Pipette the Pseudomonas suspension into the incised areas of the cornea. Overlay the mold with more growth medium for optimal bacterial growth. Supplement the culture dish with media and incubate. 

Bacteria traverse the epithelial incision to invade the stroma. Inside the stroma, bacteria cause the infiltration of immune cells, proteins, and fluid in the cornea resulting in an inflammatory response. The fluid build-up increases the corneal opacity, confirming bacterial keratitis.

Remove the media from the Petri dish, and rinse the corneas twice with one milliliter of sterile PBS. Hold the cornea with forceps and squeeze gently. 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.

Place a sterile glass mold in a six-well plate with the wide part up. Then, place the cornea in the middle of the glass mold, with the epithelium side facing down, and the wounded part of the cornea centered in the glass mold.

Fill the glass mold completely by adding one milliliter of agar solution. After allowing the agar to set, invert the glass mold so that the corneal epithelium is facing upwards.

The glass mold has to be sealed with agar up to the brim to prevent leaking of the inoculum or drug solution.

Pipette 200 microliters of the bacterial culture directly into a cut area. Also, for each experiment, set up a control by adding 200 microliters of sterile PBS to one cornea, instead of adding the bacterial culture.

Next, add 185 microliters of PBS to the top of each cornea to keep the epithelium moist. Then, add one milliliter of DMEM without antibiotics to the bottom of each well. Incubate the six-welled plate at 37 degrees Celsius with humidity and 5% carbon dioxide, for up to 24 hours.

Discard the DMEM from the six-welled plate and add one milliliter of sterile PBS to each well to rinse it. Remove the PBS gently, without touching the central part of the corneoscleral button. Remove the glass mold using sterile forceps, and place it in 5% Distel. Gently rinse the top of the corneoscleral button twice with one milliliter of PBS.

Use fine-tip forceps to lift the edge of the corneoscleral button, detach it from the agar underneath, and transfer it to a 50-milliliter tube filled with one to two milliliters of ice-cold PBS. To help detach bacteria from the corneal epithelium and the cut area, add PBS to the tube and use a fine-tip homogenizer to shear the top of the infected cornea. The tissue does not have to be completely liquidized.

Vortex to resuspend settled bacteria, and add 20 microliters of the homogenized cornea to 180 microliters of PBS. Then, perform serial dilutions of the homogenate in a 96-well plate.

Pipette 10 microliters of the diluted homogenate onto a blood agar plate.

After incubating the plate for 16 hours, count the number of colony-forming units.