Patent ID: 8609408

Claim:
A method for reconstruction of a tissue-engineered human corneal endothelium, the method comprising: a) human corneal endothelial cells are cultured and proliferated in 20% calf bovine serum-containing DMEM/F12 medium; b) after digestion with 0.25% trypsin, human corneal endothelial cells are harvested with centrifugation at 270-300 g for 10-15 minutes; c) the cells are suspended in a DMEM/F12 medium supplemented with 20% calf bovine serum and 0.05%-0.1% type-IV collagen, to produce human corneal endothelial cell suspension; d) a freeze-dried human amniotic membrane is placed up-side down on a Petri dish with an epithelial layer of the freeze-dried human amniotic membrane underneath; e) the epithelial layer of the amniotic membrane is digested with 0.25% trypsin solution using an “inverted digestion method” to produce denuded human amniotic membrane, which is subsequently punched into discs having the same diameter as culture plate wells with a puncher; f) the amniotic membrane discs are tiled onto the bottom of wells of a 48-well cell culture plate with said denuded human amniotic membrane discs on top, the plate being kept in a 37° C. incubator till the discs are dried and completely adhered to a bottom of the plate, to obtain amniotic membrane scaffold carriers; g) the human corneal endothelial cell suspension is inoculated into the culture plate tiled with the amniotic membrane scaffolds and inoculation amount varies between 0.1 mL and 0.2 mL; h) when the cells are completely adhered to the scaffold carriers, additional specific medium is added to lift liquid surface of culture medium to ¾ wells height; and i) when the human corneal endothelial cells expand to form a single layer, a tissue-engineered human corneal endothelium is reconstructed.