Cell sheets for ectocornea formation, method of producing the same and method of using the same

Abstract

The present invention has as its objective providing acorneal epithelium forming cell sheet that will adhere well to an anterior segment tissue. To attain this objective, a corneal epithelium forming cell sheet is produced by a process comprising the steps of cultivating under specified conditions corneal epithelium forming cells on a cell culture support comprising a substrate having its surface covered with a temperature responsive polymer of which the hydrating force varies in a temperature range of 0-80° C., optionally stratifying the layer of cultured cells, and thereafter, (1) adjusting the temperature of the culture solution to either above an upper critical dissolution temperature or below a lower critical dissolution temperature, (2) bringing the cultured corneal epithelium forming cells into close contact with a carrier, and (3) detaching the sheet together with the carrier under specified conditions.

1 . A corneal epithelium forming cell sheet in close contact with a carrier.  
     
     
         2 . The corneal epithelium forming cell sheet according to  claim 1  which has been detached from a support without being subjected to any treatment with a proteinase, the detached cell sheet having a shrinkage not greater than 20%.  
     
     
         3 . The cell sheet according to  claim 1  which has at least 80% of a basement membrane-like protein left intact.  
     
     
         4 . The cell sheet according to  claim 1  which has at least 80% of a desmosome structure left intact.  
     
     
         5 . The corneal epithelium forming cell sheet according to  claim 1  for treating a diseased site where an anterior segment tissue is partly or entirely damaged or deficient.  
     
     
         6 . The corneal epithelium forming cell sheet according to  claim 5 , wherein the anterior segment tissue is a corneal epithelial tissue, Bowman's membrane or a corneal stromal tissue.  
     
     
         7 . The corneal epithelium forming cell sheet according to  claim 1 , which is detached within a period of 21 days after the cells became confluent on a support's surface.  
     
     
         8 . The corneal epithelium forming cell sheet according to  claim 1 , which is a regenerated corneal epithelial cell sheet which is single-layered or stratified.  
     
     
         9 . The corneal epithelium forming cell sheet according to  claim 8 , which is a stratified sheet obtained by cultivating corneal epithelial cells in layers.  
     
     
         10 . The corneal epithelium forming cell sheet according to  claim 1 , which is a corneal epithelium substitute cell sheet which is single-layered or stratified.  
     
     
         11 . The corneal epithelium forming cell sheet according to  claim 10 , wherein the corneal epithelium substitute cell sheet comprises oral mucosa cells, hair root cells or conjunctival epithelial cells, as taken individually or in admixture of two or more kinds, or in admixture thereof with corneal epithelial cells.  
     
     
         12 . The corneal epithelium forming cell sheet according to  claim 10 , which is a stratified sheet obtained by cultivating in layers oral mucosa cells, hair root cells or conjunctival epithelial cells, as taken individually or in admixture of two or more kinds, or in admixture thereof with corneal epithelial cells.  
     
     
         13 . The corneal epithelium forming cell sheet according to  claim 11 , wherein the oral mucosa cells originate from the buccal membrane.  
     
     
         14 . The corneal epithelium forming cell sheet according to  claim 1 , which is applied to a diseased site to be treated such that it covers the diseased site without suturing.  
     
     
         15 . The corneal epithelium forming cell sheet according to  claim 1 , which is cut to the size and shape of the diseased site before it is applied to cover the diseased site.  
     
     
         16 . A process for producing a corneal epithelium forming cell sheet, comprising the steps of cultivating corneal epithelium forming cells on a cell culture support comprising a substrate having its surface covered with a temperature responsive polymer of which the hydrating force varies in a temperature range of 0-80° C. with respect to water, optionally stratifying the layer of cultured cells by the usual method, and thereafter, 
 (1) adjusting the temperature of the culture solution to either above an upper critical dissolution temperature or below a lower critical dissolution temperature,    (2) bringing the cultured corneal epithelium forming cells into close contact with a carrier, and    (3) detaching the sheet together with the carrier.    
     
     
         17 . The process according to  claim 16 , wherein the corneal epithelium forming cell sheet is a regenerated corneal epithelial cell sheet or a corneal epithelium substitute cell sheet, each of which is single-layered or stratified.  
     
     
         18 . The process according to  claim 16 , wherein the cell culture support is a membrane-based cell insert.  
     
     
         19 . The process according to  claim 16 , wherein the temperature responsive polymer is poly(N-isopropylacrylamide).  
     
     
         20 . The process according to  claim 16 , wherein the carrier is of annular shape having a cutout in the center.  
     
     
         21 . The process according to  claim 16 , wherein the sheet is detached without treatment with a proteinase.  
     
     
         22 . A method of treating a diseased site where an anterior segment tissue is partly or entirely damaged or deficient by grafting to the diseased site the corneal epithelium forming cell sheet according to  claim 1 .  
     
     
         23 . The method of treatment according to  claim 22 , wherein the grafted sheet covers the diseased site without suturing.  
     
     
         24 . The method of treatment according to  claim 23 , wherein the corneal epithelium forming cell sheet is cut to the size and shape of the diseased site before it is applied to cover the diseased site.  
     
     
         25 . The method of treatment according to  claim 22 , wherein the disease to be treated is corneal erosion, corneal ulceration, or a binocular refractory conjunctival disease.  
     
     
         26 . The method of treatment according to  claim 22 , wherein the treatment involves refractive correction by the RK method, the PRK method, the LASIK method, or the LASEK method.