Decellularized Human Cornea for Reconstructing the Corneal Epithelium and Anterior Stroma

In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. The elastic modulus between the NaCl decellularized cornea and the native cornea (control) showed no significant difference compared by the Student’s t-test. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro. For in vivo study, NaCl decellularized graft was transplanted on a rat cornea ablated with excimer laser showed graft epithelialization as demonstrated by a reduction in flourescein staining over time. In summary, this is the first study on decellularization of human cornea with which recellularization with both corneal epithelial cells and fibroblasts was demonstrated. The novel method for decellularization has potential applications in corneal and limbal tissue engineering, where a biopsy may be used to grow both epithelial cells and stromal cells to reconstruct the corneal epithelium with a stromal niche.