ULTRASTRUCTURE AND IMMUNOCHEMISTRY OF 3-D IMENSIONAL CULTURES OF HUMAN KERATOCYTES IN COLLAGEN GEL MATRIX

Purpose To investigate the ultrastructural and immunochemical features of three-dimensional cultures of human keratocytes in collagen gel ma trix. Methods Human keratocytes were obtained from primary cultures of stromal explants. They were cultured in bovine type I collagen gel ma trix for 6 weeks. Keratocyte-populated gels were analyzed by means of immunochemistry and transmission electron microscopy. Results Human ke ratocytes cultured in collagen gel matrix developed processes and form ed networks of connecting cells. They showed positive staining for vim entin, collagen I, V, and VI, and connexin. Electron microscopy showed elongated cells with processes and gap junctions. Keratocytes synthes ized collagen fibrils and filaments. No fibrils' organization similar to that observed in the normal human collagen stroma (i.e. pai allel b undles of collagen fibrils) was observed. Conclusion Ultrastructure an d immunochemical phenotype of three-dimensional cultures of human kera tocytes in collagen gel matrix are similar to those observed in situ. These cultures represent a useful in vitro model to study the differen t corneal stroma components.