Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies

After obtaining freshly enucleated porcine eyes from the local abattoir, place the samples in 4 degrees Celsius, full medium. Use scissors to remove the extracellular tissues, and soak the eyes in 5% povidone-iodine ophthalmic solution for five minutes. Next, place the disinfected samples in sterile PBS at room temperature.

Using a spectral-domain optical coherence tomography device, screen the eyes for major anterior segment pathologies, such as corneal scarring, edema, and other opacities. After screening, position the eyes in front of a slit-lamp unit equipped with a Nd:YAG laser with a wavelength of 1064 nanometers, and a focal spot diameter of 10 micrometers in air. Select the 12X magnification, and deflect the illumination to visualize the individual corneal layers.

After setting the pulse energy and focus point to the appropriate parameters for the selective ablation of the corneal endothelial cells, apply several laser shots to the tissue. Then, under a dissecting microscope, place a clear cornea paracentesis close to the limbus, and inject viscoelastic to stabilize the interior chamber. Then, use an 8-millimeter trephine to excise the laser-treated central cornea, and place the isolated cornea into one well of a 12-well plate, endothelial side up. When all of the corneas have been collected, add 3 milliliters of full medium to each sample well, and incubate the specimens for up to three days at 37 degrees Celsius.