Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The overall goal of this procedure is to create a clinically relevant model where wound repair can be followed in its native microenvironment. This is accomplished by first removing the lens from the eye of a chicken embryo. The second step is to make a small incision in the anterior of the lens.

Then hydro elute the lens fiber cell mass. Next, the lens capsule with associated epithelium is flattened by making additional cuts in the anterior capsule. The final step is to secure the wounded culture to the tissue culture dish in order to follow wound healing as it occurs.

Ultimately, time-lapse microscopy, confocal microscopy, and biochemical analysis are used to show mechanisms of wound repair in a model that closely recapitulates the in vivo environment. This method can help answer key questions in the wound healing field, such as how cells coordinately move together to close the wound. Though this method can give insight into the process of wound repair.

It can also be applied to studies of ABER wound healing, particularly fibrosis and scarring. Start by placing three 100 millimeter Petri dishes in a sterile laminar flow hood. Fill two of the Petri dishes halfway with tris dextrose buffer at room temperature, leaving the third empty.

Then remove a fertile embryonic day 15 white leghorn chick egg from the 37.7 degrees Celsius incubator and clean the outside of the shell with 70%ethanol in order to sterilize it. Next carefully crack the egg and place its contents into the empty 100 millimeter Petri dish. Using standard forceps and fine scissors, decapitate the embryo and place the head into one of the dishes containing the TST dextrose buffer.

Within 15 minutes, move the chick embryo head onto a Petri dish lid. Use high precision forceps to pinch the back of the eye, creating a small opening in the back of the eye. Then grasp the vitreous humor with forceps and gently tug on the vitreous with the rolling motion.

The vitreous with the lens attached will dislodge from the eye. Place the lens and vitreous in the remaining Petri dish containing Tex extras Buffer. Allow the lenses to remain in the buffer for no longer than 30 minutes before continuing with the dissection.

Next, move the lens to a new Petri dish lid under a dissecting microscope with high precision forceps, carefully brush away any ciliary body cells that were dislodged with the lens using the edge of the forceps. Then separate the lens from the vitreous humor by pinching off the vitreous body from its association with the posterior lens capsule. Once separated, transfer the lens into a 200 microliter drop of tris dextrose buffer in a 35 millimeter tissue culture dish, and orient it with the anterior aspect of the lens facing up.

The anterior of the lens is easily identifiable by the presence of a dense ring in the tissue that notes the border between the interior and equatorial region of the lens epithelium. Using two high precision forceps, make a small incision approximately 850 microns in length in the center of the anterior lens capsule by grasping the tissue with one forceps in each hand and gently tugging in opposing directions. Next, take a one milliliter syringe with the 27.5 gauge needle tip and fill it with 300 microliters of TST dextrose buffer.

Insert the needle tip into the incision made in the anterior lens capsule and about halfway into the lens. Then gently depress the syringe, injecting the TST dextrose buffer into the lens. Fiber cell mass.

Inject between 50 and 200 microliters of buffer to loosen the fiber cell mass from the surrounding epithelium and lens capsule. Never inject more than 300 microliters. Using high precision forceps, remove the loosened fiber cell mass from the lens through the anterior incision site.

The removal of the fiber cell mass from its attachment on the posterior capsule creates a highly reproducible wound area on the basement membrane surrounded by the wounded epithelium. Starting with the capsular bag, make five cuts into the anterior aspect of the lens and through to the equator. Flatten the resultant five flaps of lens capsule with the attached epithelium on the culture dish capsule side down and cell side up.

Press down softly with the forceps at each point of the star to secure the capsule to the dish. This will make a small indentation at the five most outside tips of the x explan and result in sustained attachment to the dish. Then remove the tris dextrose buffer from the 35 millimeter dish and replace it with 1.5 milliliters of pre-war M1 99.

Media supplemented with 1%of both L-glutamine and penicillin. Streptomycin covered the dish with its lid and place in the incubator. Place the culture dish under the dissecting microscope and observe the demarcation between the central migration zone and the original attachment zone of the epithelial cells.

Then use two high precision forceps to grasp the edge of both sides of the demarcation line between the two zones. Gently pull the original attachment zone along the line to easily separate the two zones. Continue to separate the two zones around the entire culture until the two regions are completely separated.

Study the two fractions using standard molecular or biochemical analytical techniques. Immediately following removal of the fiber cell mass, a subpopulation of ment enrich mesenchymal repair cells are activated and migrate to the wound edge of the epithelium. These cells rapidly move onto the cell-free region of the endogenous basement membrane capsule, the central migration zone to begin healing.

The wound, the technique described here perfectly prepares this model system for viewing this process with a microscope or video camera wound healing progresses quickly covering a significant area of the wound by day one in culture. By day three, the wound healing process is typically completed following separation of the central migration and original attachment zones. Molecular differences between these distinct zones were analyzed here.

The levels of focal adhesion kinase were measured in the two zones and the amount of phosphorylated, focal adhesion kinase was found to be increased in the migration specific central migration zone. Once mastered, this mock cataract surgery technique can be completed in three minutes if it is performed properly. After watching this video, you should have a good understanding of how to create a mock cataract surgery model that closely mimics wound repair in a natural microenvironment.