Li on the cell BI 78D3 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 POR8 site 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 cell loss or damage leads to stromal oedema, loss of transparency, and will eventually lead to blindness. The transplantation of a healthy endothelial layer is generally required to reverse the oedema. The worldwide shortage of donor corneal tissue has led to increased pressure to optimise protocols for the reproducible expansion of endothelial cells in vitro to enable development of tissue engineered endothelial constructs. Published methods for isolation and expansion of endothelial cells vary greatly between research groups [5], but success is difficult to evaluate when donor variability is not taken into consideration. Colleagues have evaluated the effectiveness of published culture methods comparing the efficiency in supporting endothelial cell growth from a single donor to eliminate the differences caused by donor sample variability [7]. They compared four different cell culture media after isolating cells using a two-step peel and digest method and were able to determine the optimal culture medium for expansion of cells. Endothelial cells were cultured for this study using this method and it was found that cells could be expanded from an initial starting number of approximately 36105 per donor pair to between 6?56106 cells after only 2 passages (GS Peh and JS Mehta, unpublished observation). In this study we were able to seed human corneal endothelial cells at a concentration that produced a final density of, on average, 1941.2 cells/mm2. This figure is within range of the 2000 cells/mm2 minimum for transplantation which is employed by 70 of European eye banks, according to a 2010 European Eye Bank Association Directoryreport [14]. Seeding at these densities with the number of cells obtained from one cornea and estimating conservatively, this could be equated to 20 RAFT preparations, 10 mm diameter in size, meaning that one donor cornea 1379592 pair could potentially treat 20 eyes, a dramatic improvement on the one to one approach with whole tissue transplant. The endothelial layer is at the boundary of the fluid filled anterior chamber and a major function of the corneal endothelium is to maintain corneal transparency by regulating corneal hydration. The “leaky” barrier formed by the endothelial cells allows aqueous humour to flow into the cornea to supply nutrients to the avascular stroma and tight junctional proteins su.Li 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 cell loss or damage leads to stromal oedema, loss of transparency, and will eventually lead to blindness. The transplantation of a healthy endothelial layer is generally required to reverse the oedema. The worldwide shortage of donor corneal tissue has led to increased pressure to optimise protocols for the reproducible expansion of endothelial cells in vitro to enable development of tissue engineered endothelial constructs. Published methods for isolation and expansion of endothelial cells vary greatly between research groups [5], but success is difficult to evaluate when donor variability is not taken into consideration. Colleagues have evaluated the effectiveness of published culture methods comparing the efficiency in supporting endothelial cell growth from a single donor to eliminate the differences caused by donor sample variability [7]. They compared four different cell culture media after isolating cells using a two-step peel and digest method and were able to determine the optimal culture medium for expansion of cells. Endothelial cells were cultured for this study using this method and it was found that cells could be expanded from an initial starting number of approximately 36105 per donor pair to between 6?56106 cells after only 2 passages (GS Peh and JS Mehta, unpublished observation). In this study we were able to seed human corneal endothelial cells at a concentration that produced a final density of, on average, 1941.2 cells/mm2. This figure is within range of the 2000 cells/mm2 minimum for transplantation which is employed by 70 of European eye banks, according to a 2010 European Eye Bank Association Directoryreport [14]. Seeding at these densities with the number of cells obtained from one cornea and estimating conservatively, this could be equated to 20 RAFT preparations, 10 mm diameter in size, meaning that one donor cornea 1379592 pair could potentially treat 20 eyes, a dramatic improvement on the one to one approach with whole tissue transplant. The endothelial layer is at the boundary of the fluid filled anterior chamber and a major function of the corneal endothelium is to maintain corneal transparency by regulating corneal hydration. The “leaky” barrier formed by the endothelial cells allows aqueous humour to flow into the cornea to supply nutrients to the avascular stroma and tight junctional proteins su.