The purpose of this video is to describe a human corneal organ culture model of Descemet's Stripping Only with accelerated healing, stimulated by engineered fibroblast growth factor one. Fuch's Endothelial Corneal Dystrophy, or FECD, is a disease characterized by lost pump function in corneal endothelial cells and the excessive buildup of collagen and other extracellular matrix proteins on the surface of Descemet's membrane, forming corneal guttae. The only known treatment for FECD is endothelial keratoplasty in varying forms, all of which come with risk of rejection and endothelial cell loss.
While advancements in ophthalmic surgery have enabled these procedures to become less invasive over time, any form of transplantation comes with the risk of rejection and possibility of lifetime steroid use. Descemet's Stripping Only, or DSO, is an experimental alternative in which FECD patients with guttae localized to the center of the cornea have the central four millimeter circle of Descemet's membrane removed without graft replacement. The removal of guttae encourages healthy peripheral cells to migrate inwards and reform the endothelial monolayer, eventually reversing stromal edema and improving vision.
Though there are many advantages to this method, the healing process is lengthy and inconsistent, and some patients require rescue transplantation if no healing is seen in the month's following surgery. A treatment that stimulates faster wound healing following DSO, may make the procedure a more feasible option for patients with FECD. This protocol models the clinical DSO procedure in an organ culture format using human donor corneas, with the goal of testing treatments to improve wound healing following DSO, to make DSO a more feasible option for patients with FECD.
Our lab uses this model to test wound healing in corneas treated with an engineered fibroblast growth factor for which we will show representative results later in this video. In this part of the procedure, a biopsy punch will be used to mark the four millimeter wound area from which Descemet's membrane will be stripped. Using sterile forceps, remove the cornea from media and rinse in 1X PBS to remove residual media and cell debris.
After rinsing, place the cornea endothelial side up on the lid of a Petri dish. Dip a new four millimeter biopsy punch in trypan blue in a weld dish and tap off excess. Using both hands position the punch above the center of the cornea and lower it straight down onto the endothelial surface, applying minimal pressure.
Without changing the position or pressure on the cornea shift the punch to one hand and reach for forceps with the newly free hand. Use the forceps to hold the cornea in place while gently twisting the biopsy punch about 90 degrees back and forth several times. Lift the biopsy punch straight up from the cornea and set aside.
Rinse once more in 1X PBS to remove excess trypan blue. Transfer cornea on Petri dish lid to a dissecting scope. Holding the cornea in place with curved forceps score Descemet's membrane by lightly dragging the tip of a sharp 30 gauge needle along the ring of trypan blue left by the biopsy punch.
Use minimal pressure to avoid disrupting the underlying stroma. With a sinskey hook use gentle scooping motions to lift and peel back Descemet's membrane around the wound edge. Working toward the center of the lesion.
Descemet's membrane should come up with very little resistance. If you are experiencing difficulty you're likely pulling up stroma. If this happens, start again, lifting from a new point along the wound edge.
Once the majority of Descemet's membrane has been separated from the stroma use Gorovoy forceps to remove the membrane and set aside. Examine the stripped area for any remaining pieces of membrane and remove with Gorovoy forceps. Following Descemet's stripping stain the cornea with trypan blue to visualize the wound area.
Rinse the cornea in 1X PBS containing 0.01%calcium chloride and magnesium chloride to remove cell debris that could interact with trypan blue and facilitate tight adherence of the remaining CECs to the intact Descemet's membrane. Place the cornea in a welded dish and pipette 30 microliters of trypan blue onto the endothelial layer for 30 seconds. Use forceps to gently rock the cornea to ensure that the entire endothelial surface is covered.
Rinse off excess trypan blue in PDS with calcium and magnesium, and image the stained cornea under a dissecting microscope for the day zero time point. After this procedure is completed, culture corneas in a six well plate containing low serum media consisting of the following listed components. Culture the left cornea in eight mills of low serum media alone, and the right cornea in low serum media supplemented with engineered FGF-1 or other desired test treatments.
Incubate the corneas at 37 degrees Celsius with 6%CO2 for 14 days with daily media changes. Repeat the trypan staining procedure on days, three, six, nine, 12 and 14, and image each cornea immediately after staining. Be sure to keep camera settings consistent across all time points.
Following the culture period additional stains can be performed on the corneas based on research interests, particular to the experiment. Additional stains relevant to our lab's research interests include Alizarin staining, as well as fluorescent staining for EDU incorporation and ZO-1 tight junction proteins. Alizarin staining is performed to visualize cell borders to confirm trypan staining results and visualize cell morphology in the healed area.
ZO-1 immunofluorescent staining allows for visualization of tight junction formation between CECs which is of particular interest in and around the healed area. EdU labeling is performed as a qualitative measure to confirm that proliferation contributes to healing and can also be used to quantify proliferating cells in some contexts. More detailed instructions on how to perform these specific staining procedures is included in the article corresponding to this video.
Here are representative images of a pair of corneas judged dystrophic by the Eye-Bank that were incubated with or without the engineered FGF-1 over a period of 14 days. As seen in the images, the engineered FGF-1 treated cornea demonstrated accelerated healing compared to the untreated control cornea. Additional Alizarin staining at the end of the culture period, served to delineate cell borders and confirmed with these findings.
This observation was replicated across 11 pairs of dystrophic corneas to validate the model in a sample of cornea's most relevant to the clinical DSO procedure. Image processing software, such as ImageJ, can be used to measure the stained area in these images to quantify wound healing progress, as can be seen here. All dystrophic corneas treated with engineered FGF-1 showed greater healing at day 14, compared to the untreated mate resulting in an average of 91%healing as opposed to 38%in control corneas.
And this difference was statistically significant. Additional immunohistochemistry visualized by confocal imaging revealed semi-organized expression of the ZO-1 tight junction protein, reflecting previous Alizarin staining patterns. EDU labeling confirmed the presence of proliferating cells in and around the wound area and was visible in higher levels in treated corneas compared to untreated controls.
In some cornea pairs, CEC death may be visible peripheral to the wound area, particularly in dystrophic corneas. Here are example images of trypan stained corneas exhibiting substantial peripheral cell death. As seen in the images, peripheral cell death occurs primarily at earlier time points and gradually reverses over the culture period.
This peripheral staining can complicate quantitation of the wound area when it connects to the wound as it can be difficult to ascertain the location of the wound area edge. However, in all cases, the peripheral trypan staining cleared enough to produce measurable images at the final day 14 time point. We believe this phenomenon is unique to donor corneas cultured ex vivo, and is not relevant to human corneas in vivo.
As damage to the peripheral endothelium has not been reported in any clinical case studies of DSO to our knowledge. A second staining pattern referred to as, far peripheral staining, was captured in the Alizarin Red images, but was also apparent in trypan blue images throughout the culture period. This observation is characterized by a ring of dark staining around the limbus indicating compromised endothelium.
Despite the term, this pattern was so common in both normal and dystrophic corneas that they were not counted as positive for peripheral staining. This protocol allowed us to demonstrate improved healing outcomes in cultured corneas treated with engineered FGF-1 following Descemet's stripping. Additionally, this stripping protocol serves as a valuable method for other researchers studying DSO and can be utilized for a wide variety of applications including, testing other wound healing therapies, evaluating modifications to surgical technique, and comparing healing across different donor populations or stages of disease.
We hope that this research and other research using this protocol encourages clinicians to consider DSO as a valuable treatment option for their eligible FECD patients in the future.
