Decellularizing corneal stroma using N(2) gas

PURPOSE: To examine the efficacy of a novel method of decellularizing porcine corneal stroma using N(2) gas from liquid N(2) and the feasibility of using decellularized porcine corneal stroma in a corneal transplantation model in rabbits. METHODS: Porcine corneas were placed in a tube, and N(2) gas from liquid N(2) was poured into the tube to freeze the corneas and make the inside of the tube hypoxic. After fastening the cap firmly, the tube was kept at room temperature for seven days, and the porcine corneas were examined histologically. A porcine corneal stromal disk treated with the aforementioned method was inserted into a pocket of rabbit corneal stroma and observed for six months. RESULTS: Hoechst 33342 and hematoxylin and eosin staining both showed few cellular nuclei in the porcine corneal stroma incubated in N(2) gas for one week. A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay showed many positively stained nuclei in the porcine corneal stroma incubated in N(2) gas for three days. The porcine corneal stroma that was decellularized and transplanted into a rabbit corneal stromal pocket remained clear for six months after transplantation. CONCLUSIONS: This method using N(2) gas decellularizes corneal stroma without reducing corneal transparency.