Ll 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 1326631 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cells after only 2 passages (GS Peh and JS Mehta, unpublished observation).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 TA-02 conservatively, this could be equated to 20 RAFT preparations, 10 mm diameter in size, meaning that one donor cornea 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 such 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+ –Pentagastrin site 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.Ll 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 1326631 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 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 such 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.
