BASEMENT-MEMBRANE SYNTHESIS BY HUMAN CORNEAL EPITHELIAL-CELLS IN-VITRO

Purpose. Collagen gels may prove to be potential carriers for transpla ntation of cultured corneal epithelial cells. The purpose of this stud y was to evaluate the suitability of collagen gels in comparison with corneal stromal blocks as the substrate to support the growth of human corneal epithelial cells in culture and the synthesis and deposition of the basement membrane components by these cells. Methods. Corneal e pithelial sheets, freed from the culture dishes using Dispase II (Boeh ringer Mannheim, Indianapolis, IN), were cultured on corneal stromal b locks. Deposition of laminin, type IV collagen, type VII collagen, and perlecan (heparan sulfate proteoglycan) were evaluated immunohistoche mically after 4 days, 7 days, 2 weeks, and 3 weeks. Human limbal expla nt cultures were established on collagen gels prepared from bovine typ e I collagen with or without addition of cultured human corneal fibrob lasts. After 1, 2, 3, and 4 weeks, the deposition of the basement memb rane components was evaluated immunohistochemically. Results. Corneal epithelial cells, cultured on corneal stromal blocks as well as on col lagen gels with or without fibroblasts, deposited laminin, type IV col lagen, perlecan, and type VII collagen at the interface of the cells a nd the substrates. However, different substrates differentially influe nced the temporal pattern of the deposition of various basement membra ne components. On the stromal blocks, deposition of laminin, type IV c ollagen, and perlecan by the epithelial cells was evident at 1 week. T ype VII collagen was detected at 2 weeks. On the collagen gels with fi broblasts, deposition of laminin, type IV collagen and perlecan was de tectable at 1 week. In the epithelial cultures on the collagen gels wi thout fibroblasts, only perlecan was detectable at 1 week. At 2 weeks, all of the basement membrane components, including type VII collagen were detectable on the collagen gels, either with or without fibroblas ts. Conclusion. Human corneal epithelium cultured on collagen gels or on corneal stromal blocks can synthesize and deposit basement membrane components, including laminin, type IV collagen, type VII collagen, a nd perlecan within 2 weeks in culture. Therefore, collagen gels may se rve as potential carriers for human corneal epithelial transplantation .