Er and include Descemet’s stripping (automated) endothelial keratoplasty (DSEK (or

Er and include Descemet’s stripping (automated) endothelial keratoplasty (DSEK (or DSAEK)) and Descemet’s membrane endothelial keratoplasty (DMEK). There are many advantages to the lamellar techniques over the PK procedure because the corneal surface is not compromised allowing for faster visual recovery, suture related problems are eliminated as endothelial keratoplasty requires no corneal sutures and wound healing complications are rare as the procedure can be performed through a self-sealing limbal or scleral tunnel incision at the periphery of the cornea [2,3]. Although these new techniques are an improvement on the classic PK method, the Epigenetics worldwide donor cornea shortage is increasingly becoming an issue [4], compounded by the fact that demand for corneal transplantation is expected to increase due to a rise 25033180 in the aging population globally [5]. This has led to considerable interest in the development of a strategy to treatPC Collagen for Endothelial Transplantationendothelial disorders using cell replacement therapy as an alternative to one donor ?one recipient tissue transplants. The considerable challenge here is that corneal endothelial cells are maintained in a G1 cell cycle phase arrested state and do not proliferate in vivo [6]. However, these cells do retain their proliferative capacity and many research groups have successfully stimulated cell division in order to expand endothelial cell numbers in vitro [7?0]. Expanded cell therapy could potentially allow many patients to be treated using one donor cornea and may alleviate some of the current donor shortage problems. However, this approach requires a supporting material with properties enabling easy transfer of the propagated cells to the recipient while at the same time exhibiting no detrimental effect on the functionality of the endothelial cell population [11]. We have developed a process of plastic compression of type 1 collagen hydrogels to produce a thin (60?00 mm) collagen membrane-like construct with enhanced mechanical properties, which we have termed Real Architecture For 3D Tissues (RAFT). If required, RAFT can be seeded directly with cells in the collagen before compression and we have previously shown it to be suitable for the generation of a human corneal epithelial cell surface layer [12]. The major advantage of RAFT is its rapid, simple and reproducible method of production and ability to be easily manipulated, particularly important if they are to be handled by surgeons for transplantation. Here we describe the first use of plastic compressed RAFT as a highly effective, novel carrier for cultured human corneal endothelial cells for transplantation.at 4uC in Optisol GS (Chiron Ophthalmics Inc., Irvine, California) and used within 13 days of enucleation.Isolation and Culture of Human Corneal Endothelial CellsHuman corneal endothelial cells (hCECs) were isolated using a 2-step peel and digest method as previously described [7]. Briefly, corneas were incubated in three washes of antibiotic/antimycotic solution in phosphate Autophagy buffered saline (PBS; Life Technologies, Ltd., Paisley, UK) and then endothelial layer and Descemet’s membrane were removed for incubation with collagenase A (Sigma-Aldrich Ltd., Dorset, UK) for up to 6 hours at 37uC. Cell clusters were then subjected to TrypLE Express (Life Technologies, Ltd., Paisley, UK) treatment for 5 min at 37uC to disperse the cell clumps. Cells were then seeded onto fibronectin/collagen (FNC) coating mix (US Biologica.Er and include Descemet’s stripping (automated) endothelial keratoplasty (DSEK (or DSAEK)) and Descemet’s membrane endothelial keratoplasty (DMEK). There are many advantages to the lamellar techniques over the PK procedure because the corneal surface is not compromised allowing for faster visual recovery, suture related problems are eliminated as endothelial keratoplasty requires no corneal sutures and wound healing complications are rare as the procedure can be performed through a self-sealing limbal or scleral tunnel incision at the periphery of the cornea [2,3]. Although these new techniques are an improvement on the classic PK method, the worldwide donor cornea shortage is increasingly becoming an issue [4], compounded by the fact that demand for corneal transplantation is expected to increase due to a rise 25033180 in the aging population globally [5]. This has led to considerable interest in the development of a strategy to treatPC Collagen for Endothelial Transplantationendothelial disorders using cell replacement therapy as an alternative to one donor ?one recipient tissue transplants. The considerable challenge here is that corneal endothelial cells are maintained in a G1 cell cycle phase arrested state and do not proliferate in vivo [6]. However, these cells do retain their proliferative capacity and many research groups have successfully stimulated cell division in order to expand endothelial cell numbers in vitro [7?0]. Expanded cell therapy could potentially allow many patients to be treated using one donor cornea and may alleviate some of the current donor shortage problems. However, this approach requires a supporting material with properties enabling easy transfer of the propagated cells to the recipient while at the same time exhibiting no detrimental effect on the functionality of the endothelial cell population [11]. We have developed a process of plastic compression of type 1 collagen hydrogels to produce a thin (60?00 mm) collagen membrane-like construct with enhanced mechanical properties, which we have termed Real Architecture For 3D Tissues (RAFT). If required, RAFT can be seeded directly with cells in the collagen before compression and we have previously shown it to be suitable for the generation of a human corneal epithelial cell surface layer [12]. The major advantage of RAFT is its rapid, simple and reproducible method of production and ability to be easily manipulated, particularly important if they are to be handled by surgeons for transplantation. Here we describe the first use of plastic compressed RAFT as a highly effective, novel carrier for cultured human corneal endothelial cells for transplantation.at 4uC in Optisol GS (Chiron Ophthalmics Inc., Irvine, California) and used within 13 days of enucleation.Isolation and Culture of Human Corneal Endothelial CellsHuman corneal endothelial cells (hCECs) were isolated using a 2-step peel and digest method as previously described [7]. Briefly, corneas were incubated in three washes of antibiotic/antimycotic solution in phosphate buffered saline (PBS; Life Technologies, Ltd., Paisley, UK) and then endothelial layer and Descemet’s membrane were removed for incubation with collagenase A (Sigma-Aldrich Ltd., Dorset, UK) for up to 6 hours at 37uC. Cell clusters were then subjected to TrypLE Express (Life Technologies, Ltd., Paisley, UK) treatment for 5 min at 37uC to disperse the cell clumps. Cells were then seeded onto fibronectin/collagen (FNC) coating mix (US Biologica.