Ch as ZO-1, expressed at lateral cell borders, are thought to regulate paracellular permeability. The tendency for stromal swelling is counteracted by the removal of excess fluid via Na+ K+ -ATPase ionic pumps located on the lateral plasma membranes. Hence, the expression of ZO-1 and Na+ K+ -ATPase, detected by antibodies, in primary endothelial cells and endothelial cell lines in get Linolenic acid methyl ester culture supports the assumption that cells are exhibiting proper pump function as seen in native tissue. Tissue engineering an endothelial sheet ex vivo relies upon the fact that the cells can be expanded in vitro, but this approach would fail if cells did not survive or lost functional phenotype, i.e. expression of ZO-1 or Na+ K+ -ATPase, during the time taken to expand to confluence or after deposition on a carrier substrate. The culture of primary endothelial cells in vitro is a considerable challenge, requiring specific knowledge and skills and donor material of a particular specification. For initial exploratory experiments to determine the suitability of RAFT as a carrier for endothelial cells, a human corneal endothelial cell line was used before use of primary endothelial cells. We have shown that both the hCECL cells and primary hCECs seeded onto RAFT attach and mature to form a stable confluent monolayer after only 4 days in culture. Cells retained the typical characteristics of endothelial cells including cobblestone morphology and ultrastructural features of apical microvilli and tight junctions between neighbouring cells and even after 14 days were shown to retain expression of ZO-1 and Na+ K+ -ATPase. This suggests that RAFT is a suitable substrate for long-term culture of human endothelial cells for subsequent transplantation. Additionally, this validates the use of the endothelial cell line as an experimental alternative when it is not possible to culture primary cells due to lack of suitable donor material or knowledge of the complex culture protocols. A simple corneal endothelial tissue 3-Amino-1-propanesulfonic acid web equivalent suitable for many in vitro testing applications can be rapidly created using the endothelial cell line with RAFT as the stromal portion. A number of different cell carriers have been trialled for the purpose of endothelial layer construction but the possibilities are limited by the specific requirements of a substrate in this context. The required properties include; cytocompatibility, reproducibility, ease of production/supply, transparency, ability to be handled easily by surgeons ideally with tuneable properties such as thickness. Amongst the materials tested by others are bioengineered materials such as collagen vitrigels [15], atellocollagen and gelatin hydrogel sheets [16], silk fibroin [17], and tissues such as the xenogeneic substrate of bovine corneal posterior lamellae [18], human anterior lens capsule [19] and amniotic membrane [20]. Tissues such as amniotic membrane are beneficial, as they have been widely used in ocular surgery and have already been proven to successfully support the culture of other ocular cells such as limbal epithelial cells ([21?4] and reviewed in [25]). However, the donor variability between biological materials such as these renders them unreliable and amniotic membrane in particular displays sub-optimal transparency limiting its use in this context.PC Collagen for Endothelial TransplantationAn in vivo study using RAFT would provide important information regarding degradation time in the presence of cells and ante.Ch as ZO-1, expressed at lateral cell borders, are thought to regulate paracellular permeability. The tendency for stromal swelling is counteracted by the removal of excess fluid via Na+ K+ -ATPase ionic pumps located on the lateral plasma membranes. Hence, the expression of ZO-1 and Na+ K+ -ATPase, detected by antibodies, in primary endothelial cells and endothelial cell lines in culture supports the assumption that cells are exhibiting proper pump function as seen in native tissue. Tissue engineering an endothelial sheet ex vivo relies upon the fact that the cells can be expanded in vitro, but this approach would fail if cells did not survive or lost functional phenotype, i.e. expression of ZO-1 or Na+ K+ -ATPase, during the time taken to expand to confluence or after deposition on a carrier substrate. The culture of primary endothelial cells in vitro is a considerable challenge, requiring specific knowledge and skills and donor material of a particular specification. For initial exploratory experiments to determine the suitability of RAFT as a carrier for endothelial cells, a human corneal endothelial cell line was used before use of primary endothelial cells. We have shown that both the hCECL cells and primary hCECs seeded onto RAFT attach and mature to form a stable confluent monolayer after only 4 days in culture. Cells retained the typical characteristics of endothelial cells including cobblestone morphology and ultrastructural features of apical microvilli and tight junctions between neighbouring cells and even after 14 days were shown to retain expression of ZO-1 and Na+ K+ -ATPase. This suggests that RAFT is a suitable substrate for long-term culture of human endothelial cells for subsequent transplantation. Additionally, this validates the use of the endothelial cell line as an experimental alternative when it is not possible to culture primary cells due to lack of suitable donor material or knowledge of the complex culture protocols. A simple corneal endothelial tissue equivalent suitable for many in vitro testing applications can be rapidly created using the endothelial cell line with RAFT as the stromal portion. A number of different cell carriers have been trialled for the purpose of endothelial layer construction but the possibilities are limited by the specific requirements of a substrate in this context. The required properties include; cytocompatibility, reproducibility, ease of production/supply, transparency, ability to be handled easily by surgeons ideally with tuneable properties such as thickness. Amongst the materials tested by others are bioengineered materials such as collagen vitrigels [15], atellocollagen and gelatin hydrogel sheets [16], silk fibroin [17], and tissues such as the xenogeneic substrate of bovine corneal posterior lamellae [18], human anterior lens capsule [19] and amniotic membrane [20]. Tissues such as amniotic membrane are beneficial, as they have been widely used in ocular surgery and have already been proven to successfully support the culture of other ocular cells such as limbal epithelial cells ([21?4] and reviewed in [25]). However, the donor variability between biological materials such as these renders them unreliable and amniotic membrane in particular displays sub-optimal transparency limiting its use in this context.PC Collagen for Endothelial TransplantationAn in vivo study using RAFT would provide important information regarding degradation time in the presence of cells and ante.