Establishment of fundamental technology for clinically applicable regenerative medicine in corneal endothelium and stroma.

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Long-term efficacy of transplantation of cultured human corneal endothelial cells(HCEC) with Descemet's stripping automated endothelial keratoplasty(DSAEK) was investigated. DSAEK grafts with cultured HCEC had normal stromal lamellar structure and endothelial cell layer with intercellular adhesion structure. The corneas in which cultured HCEC were thinner than those in the control group and were transparent at 1 year after the surgery. These results indicated that DSAEK using cultured HCEC is effective and clinically feasible.The other method of seeding cultured HCEC on the posterior corneal stroma which was obtained after a thick corneal flap was made using a microkeratome, was confirmed to be effective and safe.A new temperature-responsive culture dish was developed and evaluated. The results indicated that the temperature-responsive culture dish can produce a sheet of corneal epithelium containing a lot of progenitor cells after culture with non-serum and non-feeder cells. We plan to use this new temperature-responsive culture dish to produce a cell sheet of corneal endothelial cells.A new culture method of HCEC using bi-phosphate ascorbic acid(Asc-2P) was investigated. The results indicated that the primary culture of HCEC was successful in all cases under the culture condition with athello-collagen as a carrier and Asc-2P and bGFGF in the media. Even after several passage, HCEC showed a high proliferative capacity and a cobble-stone like appearance. The expression level and intrecellular distribution pattern of ZO-1 and NA+/K+-ATPase in cultured HCEC were similar to those in in vivo HCEC. 8-OHdG and MDA increased in HCEC cultured without Asc-2P as passage increased whereas they were suppressed in HCEC cultured with Asc-2P. HCEC cultured with Asc-2P showed high proliferative capacity and appearance similar to in vivo HCEC. These results suggested that Asc-2P elongated the lifespan of HCEC by reducing intracellular oxidative stress.