Corneoscleral Button Isolation: A Surgical Procedure to Obtain Corneoscleral Button from Porcine Eye

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

1. Sterilization

1. CRITICAL STEP: Disinfect all forceps and scissors by soaking for 1 h in 5% (v/v) solution of Distel in distilled water, clean with a brush, rinse with tap water and sterilize in an oven at 185 °C for a minimum of 2 h.
2. Sterilize all other glassware and reagents by autoclaving at 121 °C for 15 minutes or prepare reagents according to the manufacturer's instructions. Carry out the following procedures in a class II microbiology safety cabinet.

2. Sample collection

1. Collection of porcine eyes
   1. Large white landrace sows, a cross with a Hampshire boar was used. The animals were stunned with an electric current and the eyes were enucleated 2 h later in the abattoir.
   2. CRITICAL STEP: Once enucleated, transfer the eyes to the lab in a sterile phosphate buffered saline (PBS) solution to prevent them from drying out and process them immediately upon arrival.

3. Preparation of the corneoscleral button

1. Use sterile forceps to hold the tissue surrounding the eyeball and transfer it to a Petri dish. Remove the conjunctiva and muscle tissue around the eyeball on a Petri dish using scalpel blade no. 15 and forceps.
2. Gently lift the eyeball while holding the optic nerve with forceps and transfer to a 0.5 L jar filled with sterile PBS.
3. Once all eyes are cleared of surrounding tissue, move them using sterile forceps to another 0.5 L jar filled with 3% (v/v) povidone iodine in PBS and leave for 1 min.
4. Transfer eyeballs to another 0.5 L jar with sterile PBS.
5. Use forceps to hold the eye still on a Petri dish and make a cut near the cornea with a scalpel blade no 10A.
6. CRITICAL STEP: Hold the edge of the cut and use scissors to excise the cornea leaving about 3 mm of sclera surrounding the cornea. Ensure the sharp end of scissors does not pierce the iris or the choroidal tissue and is in the supra-choroidal space.
7. Hold the corneoscleral button with forceps and use another pair of pointed end forceps to gently separate the uveal tissue.
8. Lift the corneoscleral button from remaining globe and briefly rinse it in 1.5% (v/v) povidone iodine solution in PBS in a 12 well plate.
9. Place the corneoscleral button into another 12 well plate filled with sterile PBS.