Sheila MacNeil

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 micros-copy. 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 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 biomateri-als. 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 per-fused with medium. This system was successful for the endo-thelium, 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. 6,7 All these models must be considered valuable and important contributions to the ultimate objective of producing an in vitro model for the cornea. However, it is clear that considerable scope remains for improvement. Ideally, an in vitro corneal model should include an epithelium exposed to the air, as occurs in vivo, and an automatic irrigation system that attempts to replicate the tear film and that assists in epithelial desqua-mation. The anterior chamber should be perfused separately to allow the anterior chamber to be maintained at normal intraoc-ular pressure. It should be possible to maintain the cornea long enough for it to be useful for epithelial and endothelial wound healing studies, and the stroma and keratocytes should remain healthy and transparent during this period. Finally, the species of cornea used should be cheaply and readily available but should avoid the deliberate sacrifice of animals. 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.

Poly(N-vinylpyrrolidinone) (PNVP) has been used in various biomedical applications for many years. This study explores two PNVP hydrogels for their biocompatibility with skin cells and their ability to support the growth of skin cells in direct and indirect contact with the cells. Two crosslinked PNVP's were investigated, one crosslinked with ethylene glycol dimethacrylate (EGDMA) and the other crosslinked with diethylene glycol bisallylcarbonate (DEGBAC). The different crosslinkers lead to hydrogels with different mechanical and slightly different biological properties. While neither hydrogel proved to be a suitable substrate for culturing cells (based on fibroblasts and a range of other cells), indirect contact with both showed them to be biocompatible and even stimulatory to fibroblasts. The P(NVP-co-DEGBAC) hydrogel stimulated fibroblast viability more reliably than the P(NVP-co-EGDMA) hydrogel when in indirect contact with cells. This effect was shown to be independent of the presence of foetal calf serum in the culture media, and could not be explained by any hydrogel breakdown products during the course of these experiments. Rather the phenomenon was observed to be the result of a dynamic interaction between the hydrogels and the cells.