Understanding the efficacy of drugs in animal models is cost and labor intensive. Our protocol which uses an ex vivo tissue explant model is significantly lower both in terms of cost and scientific personnel required to conduct the study. The main advantage of this technique is the use of porcine eyes, which have anatomy and physiology very similar to human eyes as opposed to some other small animal models.
When we test drugs in vitro, they might show efficacy which might be absent in animal or human models. Ex vivo tissue systems are the best way to understand whether or not our in vitro systems can be translated into animal and human trials. The porcine cornea system, although less labor intensive, requires trainable skills to isolate, maintain, infect and treat with the drugs, especially the process of isolation of cornea from the entire porcine eye is tricky to express in words, but requires visual demonstration.
Upon receiving porcine eyes from a suitable vendor, store the tissues on ice until processing and put on the appropriate personal protection equipment. Disinfect the work area with 70%ethanol and secure a bench cover onto the workspace. When the work area is ready, place the eyes onto a 50 millimeter piece of gauze and use the gauze to grasp the posterior section with one hand.
Using a 30 gauge needle, carefully make a single puncture at approximately the center of the epithelial surface of the eye, taking care not to damage the stroma, and use a sharp sterilized blade to make a small incision on the sclera at a one millimeter distance from the cornea. Using a swift and smooth rotating action of the hand and taking care that the vitreous humor does not leak, cut the edge of the cornea and holding the cornea at the cornea-sclera edge with a flat tweezer, use the blade to cut off the remaining tethering membranes. As each cornea is isolated, place the tissues face up into individual wells of a 12-well plate containing two milliliters of cornea medium per well and add five microliters of a five times 10 to the fifth plaque-forming unit of 17 GFP virus solution to the debrided site on each corneal surface.
When all of the corneas have been collected, place the plate into at 37 degree Celsius, 5%carbon dioxide incubator for 72 hours and spray any additional eyes not used in the experiment with 10%bleach for their secure disposal in biohazard bags. Every day before the addition of drugs, place the plate of corneas under a stereo microscope and select the GFP filter and a 500 microsecond exposure time. Capture the images at the lowest magnification before imaging the corneas at a series of increasing magnifications until all of the viral spread and dendrite formation can be visualized clearly.
When all of the corneas have been imaged, return the plate to the tissue culture incubator. The day before the infection, wash a confluent vero cell culture two times with 10 milliliters of PBS per wash, and treat the cells with one milliliter of 0.05%trypsin per well for five to 10 minutes at 37 degrees Celsius. When the cells have detached, use nine milliliters of whole medium to ensure that the cells dislodge completely from the flask surface and transfer the resulting cell suspension to a 15 milliliter centrifuge tube.
Add 300 microliters of cells to each well of a new six well-plate, followed by the addition of two milliliters of medium to each well, then place the plate in the cell culture incubator overnight. The next morning, to collect the virus from each cornea for quantification, soak sterile cotton swab tips in 500 microliters of serum-free medium per swab in multiple microcentrifuge tubes in a biosafety cabinet for at least five minutes. At the end of the incubation, carefully place the infected porcine cornea cultures into the cabinet.
Using the wet cotton swabs, make three revolutions clockwise and three revolutions anti-clockwise five millimeters from the center of each infected porcine cornea without applying excessive pressure. At the end of the series of revolutions, rotate each swab in its serum-free medium filled microcentrifuge tube clockwise and anticlockwise five times before cutting the swabs so that the tips fit within each tube with the lid closed. When all of the corneas have been swabbed, vortex the tubes at high speed for one minute.
To quantify the amount of virus collected from each cornea, prepare a log10 one-fold dilution of the virus in serum-free medium in microcentrifuge tubes until a dilution of one times 10 to the negative eight is reached. After aspirating the growth medium from each well of cells, transfer one milliliter of each virus dilution starting at one times 10 to the negative three to the plated cell monolayers and place the infected cells in the cell culture incubator for two hours. At the end of the incubation, gently wash each well two times with PBS before adding two milliliters of methylcellulose-laden medium to each well for a 72-hour incubation or until the formation of plaques can be observed.
Upon plaque observation, slowly add one milliliter of methanol to the corner of each well for a 15-minute incubation at room temperature. At the end of the incubation, slowly aspirate the contents from each well without disturbing the cell monolayer. Next, label each well with one milliliter of crystal violet working solution, taking care that all of the cells are covered for a 30-minute incubation protected from light.
At the end of the incubation, discard the solution and dry the wells on a sheet of absorbent paper, then count the number of plaques at the highest dilution well to quantify the total virus content in the starting solution three times. As observed in this stereoscopic fluorescence imaging analysis, the antiviral efficacy of BX795 is similar to that of TFT in controlling viral spread, while corneas treated with vehicle only demonstrate spread of the virus from the central infection zone to the periphery by six days post-initial viral inoculation. Similarly, the ocular swabs taken on days two and four post-infection exhibit a complete inhibition of virus in the positive control and BX795-treated samples, while a sharp increase in infectious virus titer is observed in the negative control samples.
The isolation of the porcine cornea from the whole porcine eye is the most important step. Steps 2.3 to 2.6 are the most crucial in this process. Our model can be used to understand the dendritic spread of the virus in porcine corneas.
This method can be extended towards bacterial and fungal infections as well. This method has helped in visualizing dendritic spread of the virus in corneas outside a human model. This model is helpful in testing the efficacy of drugs prior to moving towards animal and human trials.
