This protocol presents the first research model allowing organotypical long-term cultivation of corneal endothelial cells achieved with the preparation of split corneal buttons. By partially removing the outer corneal layers, the endothelial cell loss is significantly reduced, allowing the cultivation of corneal endothelial cells for at least 15 days. Therefore, this model is valuable for investigating the effects of specific substances or techniques of interest such as irrigation solutions or ophthalmic viscoelastic devices on the corneal endothelium.
After receiving pig eyes that were removed shortly post-mortem, use eye scissors and Colibri forceps to remove all of the orbital adnexes. Place five to six eyes into five percent iodine PBS solution for a total of five minutes to properly disinfect the eye surfaces, with careful stirring once a minute to ensure that the surfaces are fully submerged to achieve a complete disinfection. After five minutes, transfer the disinfected eyes to a cup of pure PBS, and carefully stir it to thoroughly wash the iodine PBS solution from the eye surfaces.
Before beginning the dissection, use a blunt catheter to fill a five-milliliter syringe with two milliliters of freshly thawed fetal calf serum, and strain the serum through a 0.22 micrometer filter into 80 milliliters of dextran-free culture medium. Gently agitate the mixture to achieve a homogenous distribution of the serum, and add three milliliters of the medium to each well of a 12-well cell culture plate. When the plate is ready, transfer an eye bulb into an eye bulb holder under an ophthalmic surgical microscope with the cornea facing up, and use a syringe filled with 0.9%sodium chloride to apply slight suction to the eye over the eye bulb holder to fix the eye in place.
Use a trephine containing a standardized inlay to cut superficially into the central cornea, no deeper than 300 micrometers, and use Colibri forceps and a single-use scalpel with a triangular blade to cut and remove the partially trephined fragment of the cornea horizontally through the stroma. Superficially place a 10-0 suture into the stroma to allow differentiation of the endothelial from the stromal side during the experiments, without penetrating the corneal endothelium, and use a trephine without an inlay to advance the trephine cut to the full depth until the anterior eye chamber is reached. A distinct drop in resistance and fluid leaking from the anterior eye chamber can be perceived after complete penetration of the cornea.
Place the obtained split corneal button containing part of the stroma, the Descemet's membrane, and the corneal endothelium into one well of the 12-well plate with the tagged side facing down. Then label each well on the culture plate with an individual number for every split corneal button for identification during follow-ups. When all of the corneal buttons have been collected, place the 12-well plate in a cell culture incubator at 37 degrees celsius, five percent carbon dioxide, and 95%humidity, replacing the supernatant on day eight if a seven-day incubation period is exceeded.
To perform an unstained examination, add three milliliters of hypotonic balanced salt solution to each well of a 12-well plate, and carefully place a single split corneal button into each well to induce swelling of the corneal endothelial cells, and to improve the visibility of the cells for counting. After one to two minutes, use a microscope camera to acquire at least three images of three different areas of the endothelium to obtain a representative impression of the actual condition of the corneal endothelium, and add a scale bar with the true-to-scale length of 100 micrometers for later analysis. To prevent osmotic damage, transfer the split corneal button from the solution after a maximum of five minutes back into the culture medium.
To perform a stained examination, place the split corneal buttons in individual Petri dishes, endothelial side up, and use a pipette to slowly drop 0.25%Trypam blue solution onto each corneal endothelium for 90 seconds per specimen. Next, carefully rinse the buttons three times in 0.9%sodium chloride in a small glass beaker before using a pipette to slowly drop 0.2%Alizarin Red S solution onto the endothelia for 90 seconds. Then image the samples as just demonstrated.
To prepare the images for cell density and morphology assessment, open the images in an appropriate graphics editing software program and project a square with a true-to-scale side length of 100 micrometers on the image. To crop the scale bar, use the rectangular marquee tool to border the scale bar and select edit and crop. Click file and new, and select clipboard.
Note the corresponding pixels for the other pictures and click OK.Click file and new again, and insert the width and length of the counting square in pixels in the pop-up window according to the length of the scale bar. Select white as a background color, and click OK to select all, and copy the selected image. Select the image of the corneal endothelium and paste the square on the corneal endothelium.
Select the square layer and set the opacity to 30%Then click OK and save the image with the projected square with the true-to-scale slide length of 100 micrometers. To assess the endothelial cell density, open the images with the projected squares in ImageJ, and open the cell counter plugin. To count the cells within the square, click initialize to count the endothelial cells on two sides of the square, and record the result.
To assess the morphological parameters, count the joint meeting of at least four cells per cell border, the characteristic rosette-shaped appearance, and the Alizarin Red areas within the projected squares. Over a period of 15 days, split corneal buttons demonstrate a steady decline in the endothelial cell density, with an average weekly percentage endothelial cell loss of 4.90%Staining of the endothelial cell layer on day 15 allows morphological analysis of the corneal cells, with reformation figures making up approximately seven percent of the merging cell borders, a median of approximately one rosette formation per millimeter squared per sample, and about 13 punctual cell losses per millimeter squared. Here, representative images of the corneal endothelium during microscopic evaluation in hypotonic balanced salt solution, and after staining with Trypan blue and Alizarin Red S, can be observed.
If the split corneal buttons are cultivated with the endothelial side facing down, extensive endothelial cell damage is to be expected. Non-split corneal buttons suffer significantly increased endothelial cell loss due to stromal swelling, causing Descemet's membrane folding over 15 days of cultivation, whereas split corneal buttons exhibit a largely preserved corneal endothelium after 15 days of cultivation, as evidenced by scattered punctual Alizarin Red-stained areas, indicative of single destroyed cells. To minimize cell loss, care should be taken to not touch the corneal endothelium throughout any steps of this protocol.
