Human Corneal Organotypic Cultures

Abstract

Purpose. In order to test irritancy levels of commercial products, the Draize animal test has been the universally accepted choice. However, since this test uses live animals, many researchers have been trying to develop an alternative organotypic culture. These organotypic cultures, however, utilize animal cells, and it is our belief that a model using human cells would be more predictive for determining human irritancy levels. Methods. Primary human corneal epithelial cells and fibroblasts were separately isolated from a limbal rim and grown in culture. SV40-transformed mouse corneal endothelial cells were seeded onto a membrane and grown to confluence. A homogenous fibroblast/collagen mixture was then added and allowed to gel. After about a week, the epithelial cells were seeded on top of the gel and the constructs were kept submerged in culture for 3-7 days. The constructs were then airlifted to allow for the stratification of the epithelial cells. Following this, the constructs were either fixed and processed for methacrylate sectioning to study morphology, or they were frozen and sectioned for indirect immunofluorescence. Indirect immunofluorescence was performed with ZO-1, a marker of tight junctions; keratins 3 and 12, markers for differentiation; and laminin, a marker of basement membrane components. Initially, a SV40-transformed human endothelial cell line was to be used; however, it did not grow well in this culture system. Results. In these experiments, we tested two methods of isolating epithelial cells. One method, the explant technique-a method we previously used to isolate rabbit epithelial cells-could not be maintained beyond two passages, and when added to the construct only stratified to 2-3 layers of flattened cells. However, with the second method, the dispase technique, the epithelial cells obtained grew rapidly and could be maintained beyond two passages. These cells, when added to the construct, stratified to 5-7 layers and exhibited a more epithelium-like morphology. Staining with ZO-1 indicated that tight junctions in the superficial cells were formed. Staining with keratins 3 and 12 indicated that the epithelial cells were differentiating, and staining with laminin indicated that basement membrane components were being synthesized. Conclusion. Constructing an organotypic culture with human corneal cells is possible, and the morphology of the construct appears to be equivalent to a construct already created with bovine cells that has been used for irritancy studies.