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Jemin J. Chae, Qiaozhi Lu, Joseph Choi, Qiongyu Guo, Oliver D. Schein, Jennifer H. Elisseeff; Preparation and Evaluation of Decellularized Porcine Cornea. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6036.
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© ARVO (1962-2015); The Authors (2016-present)
To assess and investigate the potential of decellularized porcine cornea regarding its clarity, stability and biocompatibility for use in corneal lamellar xenotransplantation.
To optimize the decellularizing method for porcine cornea, various methods of treatment including freeze-thawing, hypertonic saline, Dextran hyperosmolarity solution, Sodium dodecyl sulfate (SDS) solution, Triton-X solution and DNase solution were performed. The potency of each method was evaluated using H&E staining and DNA assay. Following evaluation, the effective methods were combined to maximize the effect of decellularization. The live / dead cell analysis and cell proliferation assay were performed to evaluate toxicity and properties of the decellularized cornea. In addition, decellularized corneal tissue was transplanted into a rabbit corneal stromal pocket to assess the possibility of lamellar xenotransplantation. To improve clarity and increase mechanical properties, three procedures were performed. First, the cornea was dehydrated at 4°C. Then, the decellularized cornea was treated with 15% Dextran solution and 0.1% Riboflavin solution. Finally, it was cross-linked using UV radiation for 3 hours on each side.
Freeze-thawing, hypertonic saline and hyperosmolarity methods neither removed cells nor significantly decreased the DNA content. However, SDS, Triton-X and DNase methods decreased cell concentration and DNA content by 12%, 51% and 14%, respectively. Moreover, the combination of SDS, Triton-X and DNase methods eliminated all cells and decreased DNA content by approximately 97%. In cell based tests, while mostly survived cells in the decellularized matrix initially proliferated at a similar rate as that in the normal tissue culture plates, the maximum concentration of cells was much higher in the decellularized matrix. In the rabbit xenotransplantation study, there was substantial integration of the decellularized porcine cornea with the recipient cornea, demonstrated by repopulation of the original host keratocyte population into the implanted cornea. After treating with dehydration, hyperosmolarity solution and riboflavin cross-linking, the challenges of post-treatment in the combined decellularization method, decreased transparency and increased swelling, were overcome.
We developed an effective method of decellularizing the porcine cornea with the SDS, triton-X and DNase combination. Also, modification of decellularized cornea could improve clarity and mechanical properties. These findings suggest that a decellularized cornea may have the potential to someday substitute conventional human allograft transplantation.
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