This paper described a simple method for assaying wounding in a three-dimensional, multi-cellular organ culture model system. We do this by using pig eyes, but even if you are not familiar with eyes, you can do this assay. As an overview, we received porcine eyes, cut out the globes, make a circular wound in the cornea that goes through the epithelium and about one third of the stroma.
We cut out the cornea, and mount it on an Agar base, and put this construct in the incubator. The tissue will fill in creating a scar in the cornea. You don't need to add any growth factors, or even serum.
This is done in serum-free media. Although this is performed in the cornea, it can be used as a model system for fibrotic healing in general. As many of the cellular pathways that are activated during scarring are similar between systems.
In a biosafety hood, use a straight edge surgical blade to remove the globe from the lid on an ethanol-cleaned chopping board, and remove the excess fatty tissue from each eye. Holding the cleaned globe posteriorly with forceps immediately dip the eye in PBS followed by three quick dips in 10%iodine, and two quick dips in PBS. Then wrap the eye circumferentially with a clean lab tissue to wrap with enough pressure to achieve a taut corneal surface without contacting the cornea.
Using a six millimeter trephine, penetrate the epithelium in the anterior stroma at the center of the cornea without making a full thickness wound through the entire cornea, and rotate the trephine 180 degrees clockwise and counterclockwise five times while applying light pressure to deepen the wound. When the wound is deep enough to allow a tissue flap to be lifted with forceps, use a surgical blade to cut the flap parallel to the globe while continuing to lift the anterior cornea within the wound margin. When the entire flap has been removed, a circular wound should be located at the center of the cornea.
To harvest the cornea grasp the eye with the lab tissue, and use a surgical blade to make a small incision one millimeter away from the edge of the cornea to include the limbus and tissue collection. Using small, sharp scissors continue the incision around the globe keeping a millimeter margin across the cornea to keep the limbus in tact. Then place the cornea, wound-side down, in a 60 millimeter, or 100 millimeter dish containing one milliliter of PBS.
To mount the cornea, use two pairs of forceps to hold the cornea epithelial-side up to create a cup, and use a sterile transfer pipette to add warmed agar solution into the cornea until the cup is full. After the agar hardens, carefully place the cornea in a new 60 millimeter plate agar-side down and cover the plate with a lid. Then add four milliliters of supplemented serum-free medium to each plate of corneas maintaining the corneas at an air-liquid interface at the limbal border in a cell culture incubator at 37 degrees celsius in 5%carbon dioxide, and wetting the corneal surfaces once daily with one drop of supplemented serum-free medium from the conditioned medium in the dish to keep the tissues hydrated.
For gene knockdown, mix five microliters of small interfering or siRNa with 50 microliters of reduced serum minimum essential medium and two microliters of transfection reagent. With 50 microliters of reduced serum medium per cornea. After five minutes, mix the two mixtures together, and add 200 microliters of reduced serum minimum medium to each siRNA mixture.
Then pipette the siRNA solutions drop wise onto each wound. Then put the corneas into the incubator. After three hours, use the medium in each dish to wash the siRNA from the corneal surfaces, and replace the conditioned medium in each dish with fresh supplemented serum free medium plus antibiotics.
Then return the corneal cultures to the cell culture incubator, and incubate for two weeks. Six hours after wounding, the corneal epithelium is absent. Six days after wounding, the epithelium regrows.
Fluorescence amino staining with alpha smooth muscle actin reveals a gradient of active myofibroblasts along the wound margin from the anterior to posterior stroma. Immuno-histochemical staining for alpha smooth muscle actin reveals a dramatic increase in alpha smooth muscle actin protein expression in wounded plus control siRNA corneas. Compared to unwounded and target protein siRNA treated, wounded corneas.
Similarly, fibronectin extra domain A is upregulated in siRNA treated wounded corneas compared to unwounded and target protein siRNA treated wounded corneas. Demonstrating a successful knock down of the target protein. Further, treatment of the wounded corneas with the toxin that nonspecifically targets the target protein prevents re-epithelialization and results in qualitative cell death, a disorganized matrix, and stromal vacuoles suggesting that the toxin does not promote healing at the concentration assayed.
In conclusion, while attempting this procedure it's important to remember to keep the blade parallel to the tissue while cutting, so that it doesn't penetrate the globe. Different wounding techniques can be employed with this assay, including chemical burn or epithelial scrape. It can also be used for drug toxicity assays to determine if the epithelium heals and at what rate.
This is a cost-savings ex vivo, organ culture model system that can precede your in vivo wound healing studies.
