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

Overview

This video describes a protocol to set up an ex vivo model for bacterial keratitis using a corneoscleral button dissected from the porcine eye. Inoculated bacteria traverse the corneal epithelial incisions to invade the underlying stroma and establish an infection within the corneal tissues that mimic the in vivo infection.

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
50 mL Falcon tube	SLS	352070
Amphotericin B	Sigma	A2942
Cellstar 12 well plate	Greiner Bio-One	665180
Dextran	Sigma	31425-100mg-F
Distel	Fisher Scientific	12899357
DMEM + glutamax	SLS	D0819
Dual Oven Incubator	SLS	OVe1020	Sterilising oven
Epidermal growth factor	SLS	E5036-200UG
F12 HAM	Sigma	N4888
Foetal calf serum	Labtech International	CA-115/500
Forceps	Fisher Scientific	15307805
Heracell VIOS 160i	Thermo Scientific	15373212	Tissue culture incubator
Insulin, recombinant Human	SLS	91077C-1G
LB agar	Sigma	L2897
Multitron	Infors	Not appplicable	Bacterial incubator
PBS	SLS	P4417
Penicillin-Streptomycin	SLS	P0781
Petri dish	Fisher Scientific	12664785
Petri dish 35x10mm CytoOne	Starlab	CC7672-3340
Povidone iodine	Weldricks pharmacy	2122828
Safe 2020	Fisher Scientific	1284804	Class II microbiology safety cabinet
Scalpel blade number 15	Fisher Scientific	O305
Scalpel Swann Morton	Fisher Scientific	11849002