Development of a three-dimensional organ culture model for corneal wound healing and corneal transplantation. Invest Ophthalmol Vis Sci 47

PURPOSE. To develop and evaluate a three-dimensional organ culture system of the cornea anterior chamber that could replicate the in vivo processes occurring during corneal wound healing and corneal transplantation. METHODS. Bovine corneoscleral buttons were clamped in a specially designed chamber through the sclera outside the limbus. The epithelium was exposed to air, and its anterior surface was automatically irrigated. The endothelial layer was perfused separately with media under normal intraocular pressure. Wound healing and corneal transplantation were observed using light, scanning, and transmission electron microscopy. RESULTS. The organ culture system maintained the epithelium, the putative epithelial stem cells in the limbus, the stroma, and the endothelium in good condition for the 10-day period during which the system was evaluated. The authors observed that the processes of wound healing and corneal transplantation in the model appeared similar to those occurring in vivo. CONCLUSIONS. In vitro model closely replicated the in vivo processes of wound healing and corneal transplantation. The authors believe this model will be useful for basic investigations into the cornea, such as study of the response of the cornea to surgery, wound healing, toxins, and therapeutic agents. (Invest Ophthalmol Vis Sci. 2006;47:2840 -2846) DOI: 10.1167/iovs.05-1367 T he role of in vitro models is becoming increasingly important in the field of ophthalmology. The use of primary and immortalized cell lines has meant that much work that previously could only be carried out on animals can now be performed more quickly and at greatly reduced cost in the cell culture laboratory. The use of corneal cell lines is particularly valuable in the early stages of investigation, such as when screening potential pharmacologic agents or novel biomaterials. In addition to these practical considerations, there are ethical issues to consider, and there is increasing pressure from the public and government, particularly in Europe, to find ways to minimize animal experimentation. Although the use of cell lines is helpful, it has clear limitations, and cell lines cannot come close to replicating the complex interactions that occur between the different cell types in the cornea. This has prompted investigators to attempt to develop an in vitro culture model for the whole cornea or anterior chamber. Several research groups have published various versions of this concept. One of the earliest models 1 involved the use of human material and clamping of the corneoscleral button into a special holder where the posterior chamber could be perfused with medium. This system was successful for the endothelium, though no evaluation was made of the epithelium or stroma. Another group 2 cultured human corneoscleral buttons in a simple system using Petri dishes and reported excellent results for the endothelium, epithelium, and stroma. Yet another group 3 working with rabbit cornea showed that an in vitro model could be used to evaluate epithelial wound healing over a 72-hour period. Other workers 4 developed a novel system with an agar-collagen gel to maintain the anterior chamber and demonstrated excellent epithelial wound healing in human and bovine cornea. Most recently, a sophisticated in vitro system was developed for pig corneas with separate epithelial irrigation and endothelial perfusion. 5 This produced excellent results, but, because the epithelial irrigation was performed manually, it was only evaluated for 14 hours. In addition to these corneal models, an advanced and successful in vitro model exists of the trabecular meshwork. In this article we describe how we have attempted to develop a model that meets these objectives. We describe how we have evaluated all the corneal cell types, including the putative epithelial stem cell population, in our model at the ultrastructural level and how we have assessed the usefulness of our model by monitoring epithelial wound healing and the model's response to penetrating keratoplasty