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Matthew Giegengack, Faraaz Khan, Keith Walter, Jin San Choi, Min Jeong Kim, Guoguang Niu, Shay Soker; Bioengineered Corneal Endothelium for Transplantation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3636.
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The objectives of this study are to fabricate gelatin-based scaffold as a carrier of human corneal endothelial cells (HCEC) and to characterize gelatin-based bioengineered cornea, containing HCECs, for endothelium transplantation. We hypothesize that we may overcome the supply issue in Descemet’s stripping endothelail keratoplasty (DSEK) by creating a process whereby a single high quality donor can be used to create many high quality corneal donor grafts.
Human CEC were obtained from discarded corneas of eye donors by digestion in collagenase II (2 mg/mL D-PBS) for 40 min at 37°C. The cells were cultured in EGM-2 with 10% FBS and thoroughly characterized. Corneal scaffolds were made using a 10% gelatin solution that was subsequently stablized through N-ethyl-N'-[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) cross-linking. The EDC/NHS was then washed out for 2 days using de-ionized water. Approximately 130, 600, and 3000 cells/mm2 were seeded on the gelatin-based scaffolds. The constructs were placed in EGM-2 + 10% FBS for 7 days, then evaluated cell-coverage of surface on the film by alizarin red S.
The modulus of the gelatin gel is around 3.0 Mpa and the tensile strain is about 70%. The gelatin gel has good transparency with visible light scope (>90%); this is higher than natural corneal stroma, transparency (80-90%). There was no signficant difference in gelatin gel permeability with or without cells. A 1:5 molar ratio between gelatin’s amine and EDC/NHS, rather than 1:10, provided higher rates of diffusion and permeability. Initially cells grew better in TCP; however, after 7 days cell growth was similar between TCP and the gelatin-based scaffold. Alizarin red staining shows good cell integrity with confluent HCECs growth on gelatin-based scaffold.
We obtained a highly transparent gelatin gel with proper mechanical properties. The gelatin gel is easy to handle and can withstand manipulation. The porous structure of the gelatin gel enables efficient diffusion of nutrients through the gelatin-based scaffold. HCECs can be seeded successfully on the gelatin-based scaffolds and show high levels of cell proliferation. Currently, a rabbit model is being used as an in vivo test for the gelatin-based scaffold seeded with HCECs. The gelatin-based scaffold is a potential scaffolding candidate for endothelial cell transplantation.
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