The overall goal of this experimental protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy, in organ-cultured corneas. This method can help answer key questions in the diabetic corneal field, such as what molecular alterations underline delayed diabetic epithelial wound healing, and abnormal expression of epithelial SEM SEM molecules. The main advantage of this technique is that it allows testing of gene therapy approaches targeting various mediators of corneal epithelial wound healing and stem cell function in organ-culture corneas.
This implications of this technique extend toward therapy of other corneal diseases related to stem cell dysfunction, because we have demonstrated the validity of the method for normalizing ultradiabetic corneal stem cells. Place the corneas epithelial-side down in sterile 60-millimeter dishes, and fill the concavity with 0.5 milliliters of agar-collagen mixture. The mixture solidifies on the corneas within two minutes.
Once solidified, place the corneas agar-side down in sterile 60-millimeter dishes containing enough full supplemented medium to reach the level of the limbus for air-liquid interface culture. Then place the dishes containing the corneas into a 5 percent carbon dioxide incubator humidified with a water pan at 35 degrees Celsius. To perform an epithelial debridement of the central cornea, first soak a 5-millimeter filter paper disc in N-heptanol, and place on the central corneal anterior surface for 75 seconds.
Remove the filter, and wash the corneas in full medium. After debridement, the epithelial cells usually die and slough off, leaving behind microscopically intact basement membrane seen on the left. Add 100 microliters of medium to the epithelium daily to moisten the corneas.
Monitor the corneal healing microscopically. Take photographs at 4X and 10X every day, until the epithelial defect is completely healed. During the healing process, black spider-like cells are observed at the top focal plane.
These cells are apoptotic stromal keratocytes that died after epithelial removal. Healing is complete when these dead cells are overgrown by the healing epithelium and are no longer visible. Prepare transduction medium by supplementing full medium with 75 micrograms per milliliter sildenafil citrate.
Add adenovirus expressing the c-met gene, or vector, at 0.8 to 1.25 times 10 to the 8th platforming units per cornea. Transfer the corneas to a 24-well plate. Add the appropriate volume of virus-containing media, and ensure that the corneas are under the medium surface.
Incubate for 48 hours at 37 degrees Celsius. To transduce only the limbal cells, incubate the corneas with virus at the air-liquid interface with medium at the level of the limbus. As sildenafil has a short half life in aqueous solutions, four hours later, replenish, by adding sildenafil citrate to the medium.
Following the incubation, use a round-end sterile spatula to transfer corneas to new dishes containing medium without adenovirus, and culture, keeping the medium level at the limbus. GFP-containing transduced cells are seen at the limbus under fluorescent light. During culture, routinely moisten the corneas by adding 100 microliters of medium on top of the cornea.
After an additional four to eight days of incubation, process the adenovirus-treated corneas for various analyses. Or test for epithelial wound healing. If whole corneas or globes are used, first isolate the limbal areas.
To do this, place the cornea on a sterile plastic dish with the epithelial side up, and use a 9-millimeter trephine to remove the central area and discard. Then excise the limbal zone using a 13-millimeter trephine or surgical scissors, and discard the outer conjunctival part. Prepare 1.5 milliliters of keratinocyte theram-free medium containing 2.4 units per milliliter of dispace-two per corneal schleral rim.
Immerse the corneal schleral rims in this solution, and incubate at 37 degrees Celsius for two hours. Then, under a dissecting stereo microscope, use forceps to gently ease the limbal epithelial cell sheet off the rim. Dissociate the cells in 1 milliliter of 0.25 percent trypsin, 0.02 percent EDTA solution for 30 minutes at room temperature.
After dissociation, wash the cells in 10 milliliters of medium, and pellet them at 300 g in a tabletop centrifuge for five minutes at room temperature. Re-suspend the cells in supplemented culture medium, and seat cells in KSFM medium at 10, 000 cells per milliliter into coded 60 millimeter dishes. Culture the cells in a humidified carbon dioxide incubator at 37 degree Celsius until they form fully confluent monolayers.
This may take one to two weeks. Passage the confluent cells according to standard techniques and plate the cells onto a 24-well plate at 2 times 10 to the 4th cells per milliliter. Prepare transduction medium containing 2 nanograms per milliliter of EGF, and a polycation enhancer to facilitate adenovirus binding to the cell surface.
Add the required volume of adenovirus, expressing a specific gene, or shRNA inhibitor, or scrambled shRNA as control, to transduce cells at a multiplicity of infection of 1 to 300 plaque-forming units per cell. Add the virus-containing media to the cells, and incubate in the humidified incubator for 24 hours at 37 degrees Celsius. After 24 hours, replace the adenovirus-containing media with fresh media without adenovirus.
Return to the incubator for four days. After four days, evaluate the GFP expression level using an inverted fluorescent microscope. Then make scratch wounds in the monolayer, by scratching the cells in the straight linear motion with a 200-microliter pipette tip.
After wounding, change the medium to remove the detached cells. Photograph the wounds every day with a digital camera attached to an inverted microscope at 4X magnification. Record the time when the wound edges come into contact along the entire wound, and the healing is complete.
This series of images shows healing of 8.5-millimeter epithelial wounds in a pair of diabetic corneas. The arrows show the wound edge, and the asterisk, the non-healed part. For a vector-transduced cornea, healing is complete in eight days, whereas for the adenovirus c-met-transduced cornea, healing is complete in five days.
As seen here, c-met gene transduction leads to a significant decrease of corneal epithelial wound healing time. Moreover, combined gene therapy with adenovirus harboring the c-met gene and shRNAs to MMP10 and cathepsin-F genes completely normalizes epithelial wound healing time. Significance was established by paired student's t-test in comparison with respective vector treatments.
The bars represent the standard error of the mean. The following images show immunostaining of diabetic corneal sections for various markers after limbal gene therapy. c-met expression and combination treatments result in markedly increase limbal staining for the diabetic markers integrin alpha-3-beta-1 and nitogen-1, and the putative stem cell markers keratin 15 and Delta-Np-63-alpha.
The level of GFP expression in transduced limbal epithelial cells depends on the multiplicity of infection of adenovirus GFP. Here, cells were transduced with 30 plaque-forming units per cell for three days. This is the result with 120 PFU per cell, and finally with 300 PFU per cell at three days of transduction.
It is worth noting that limbal cell migration is adversely affected by increasing concentration of adenovirus, as revealed by the scratch wound test. For this reason, transduction dose must be optimized to avoid virus toxicity. This image shows live untransduced cells.
Here, cells are transduced with 20 PFU per cell, 80 PFU per cell, and finally 120 PFU per cell. While attempting this procedure, it's important to remember to keep corneas sterile to optimize the viral tighters, to moisten the corneal surface after gene transduction at least once a day, and to document the healing process daily. After watching this video, you should have a good understanding of how to prepare corneal organ cultures, corneal stem cell cultures, make wounds, and apply viral gene therapy to accelerate wound healing in diseased corneal tissue in an in-vitro setting.
