Ern of high expression at cell borders of cells on RAFT also matched that of cells on permanox slides coated with FNC coating (Fig. 5F), whereas representative negative isotype controls showed no staining in these areas (Fig. 5E). The average cell area of hCECs on RAFT was 614.86157.6 mm2 compared with 185.1614.5 mm2 for hCECL cells, which was a significant difference (p,0.05). The large variability in cell size observed with hCECs is fairly typical of cells in primary cultures compared to the often homogenous cell lines. In both cases hCECs expressed ZO-1 (Fig. 6A and C) and Na+/K+-ATPase (Fig. 6B and D) in abundance whether on glassConformation of Endothelial Cell Layers on RAFTTissue sections of human cornea and endothelial cells on RAFT were stained with haematoxylin and eosin or toluidine blue and basic fuschin to 
reveal similarities in conformation of the cells on RAFT compared to native tissue. Human endothelial cells line the posterior portion of the cornea in a single monolayer separated from the collagen stroma by the Descemet’s membrane (Fig. 4A). hCECL on RAFT form a confluent monolayer on the collagen surface (Fig. 4B) that is very similar to the formation seen in corneal specimens. The same can be said for hCECs seeded onto RAFT where an endothelial monolayer can be seen (Fig. 4C).PC Collagen for Endothelial Transplantation(Fig. 6A and B) or RAFT (Fig. 6C and D) after 4 days in culture. This expression pattern was also seen after 14 days (Fig. 6E and F), indicating that cells remain viable and maintain their typical endothelial phenotype even after longer- term culture. Additionally, the expression pattern of ZO-1 confirmed the typical cobblestone appearance of hCECs on RAFT by labelling cell junctions and borders. The isotype matched controls were negative as expected (data not shown).Ultrastuctural Properties of Endothelial Cells on RAFTScanning electron microscopy revealed the randomly orientated collagen fibre structure of the seeding surface of RAFT (Fig. 7A). A low magnification image of hCECs on the collagen surface showed a confluent monolayer of polygonal cells (Fig. 7B) and at 
higher magnification the tightly opposed borders were apparent (Fig. 7C), additional evidence of typical endothelial morphology. Further magnification reveals the highly interdigitated apical flaps of cell borders and microvilli on the cell surface (Fig. 7D). Transmission electron microscopy confirmed apical microvilli on the cell surface, which leads to significant enlargement of cell surface area (Fig. 8A) and tight junctions between adjacent cells (Fig. 8B and C). Anchoring filaments protruding into RAFT were also identified, which presumably aid attachment and security of the endothelial layer to the underlying collagen substrate (Fig. 8D).DiscussionThe SPDP chemical information application of plastic compressed collagen for the culture of human corneal endothelial cell layers offers an attractive alternative for surgical restoration of corneal endothelium using a simple and rapidly produced tissue engineered substrate. The present study demonstrates that RAFT is a suitable substrate for culture of corneal endothelial cells and additionally indicates that this carrier has sufficient AKT inhibitor 2 site mechanical strength for transplantation using current clinical delivery techniques. This feasibility study provides encouraging evidence for further development using an in vivo model to confirm endothelial functionality and RAFT suitability in a living system. Corneal endothelial ce.Ern of high expression at cell borders of cells on RAFT also matched that of cells on permanox slides coated with FNC coating (Fig. 5F), whereas representative negative isotype controls showed no staining in these areas (Fig. 5E). The average cell area of hCECs on RAFT was 614.86157.6 mm2 compared with 185.1614.5 mm2 for hCECL cells, which was a significant difference (p,0.05). The large variability in cell size observed with hCECs is fairly typical of cells in primary cultures compared to the often homogenous cell lines. In both cases hCECs expressed ZO-1 (Fig. 6A and C) and Na+/K+-ATPase (Fig. 6B and D) in abundance whether on glassConformation of Endothelial Cell Layers on RAFTTissue sections of human cornea and endothelial cells on RAFT were stained with haematoxylin and eosin or toluidine blue and basic fuschin to reveal similarities in conformation of the cells on RAFT compared to native tissue. Human endothelial cells line the posterior portion of the cornea in a single monolayer separated from the collagen stroma by the Descemet’s membrane (Fig. 4A). hCECL on RAFT form a confluent monolayer on the collagen surface (Fig. 4B) that is very similar to the formation seen in corneal specimens. The same can be said for hCECs seeded onto RAFT where an endothelial monolayer can be seen (Fig. 4C).PC Collagen for Endothelial Transplantation(Fig. 6A and B) or RAFT (Fig. 6C and D) after 4 days in culture. This expression pattern was also seen after 14 days (Fig. 6E and F), indicating that cells remain viable and maintain their typical endothelial phenotype even after longer- term culture. Additionally, the expression pattern of ZO-1 confirmed the typical cobblestone appearance of hCECs on RAFT by labelling cell junctions and borders. The isotype matched controls were negative as expected (data not shown).Ultrastuctural Properties of Endothelial Cells on RAFTScanning electron microscopy revealed the randomly orientated collagen fibre structure of the seeding surface of RAFT (Fig. 7A). A low magnification image of hCECs on the collagen surface showed a confluent monolayer of polygonal cells (Fig. 7B) and at higher magnification the tightly opposed borders were apparent (Fig. 7C), additional evidence of typical endothelial morphology. Further magnification reveals the highly interdigitated apical flaps of cell borders and microvilli on the cell surface (Fig. 7D). Transmission electron microscopy confirmed apical microvilli on the cell surface, which leads to significant enlargement of cell surface area (Fig. 8A) and tight junctions between adjacent cells (Fig. 8B and C). Anchoring filaments protruding into RAFT were also identified, which presumably aid attachment and security of the endothelial layer to the underlying collagen substrate (Fig. 8D).DiscussionThe application of plastic compressed collagen for the culture of human corneal endothelial cell layers offers an attractive alternative for surgical restoration of corneal endothelium using a simple and rapidly produced tissue engineered substrate. The present study demonstrates that RAFT is a suitable substrate for culture of corneal endothelial cells and additionally indicates that this carrier has sufficient mechanical strength for transplantation using current clinical delivery techniques. This feasibility study provides encouraging evidence for further development using an in vivo model to confirm endothelial functionality and RAFT suitability in a living system. Corneal endothelial ce.
