Cultured Cell Sheet, Production Method and Tissue Repair Method Using Thereof

Abstract

It is intended to provide a cultured cell sheet with excellent tissue adherence and flexibility. The above object can be achieved by culturing cells on a support for cell culture in which a surface of a substrate is coated with a temperature-responsive polymer whose lower or upper critical solution temperature against water is in the range of 0 and 80° C. along with a surfactant protein or a crosslinking inhibitor and producing a cultured cell sheet by detaching it by setting the temperature of the culture to the upper critical solution temperature or higher or to the lower critical solution temperature or lower.

1 . A cultured cell sheet, having superior tissue adhesiveness and excellent flexibility. 
     
     
         2 . The cultured cell sheet according to  claim 1 , wherein the cells are selected from the group consisting of: fibroblasts, cells of alveolar tissue, cells of myocardial tissue, cells of hepatic tissue, cells of vascular tissue, mesenchymal stem cells, and adipose derived cells. 
     
     
         3 . The cultured cell sheet according  claim 1  or  2 , wherein the cultured cell sheet is a layered cultured cell sheet. 
     
     
         4 . The cultured cell sheet according to  claim 3 , wherein the layered cell sheet is prepared by layering of the cell sheet of  claim 2 . 
     
     
         5 . The cultured cell sheet according to any one of  claims 1  to  4 , wherein the superior adhesiveness allows the cultured cell sheet to cover a site of air leakage, blood leakage or bodily fluid leakage on tissue, and thereby to suppress such leakage. 
     
     
         6 . The cultured cell sheet according to any one of  claims 1  to  5 , wherein the cultured cell sheet has a flexibility such that the cultured cell sheet obstructs performance of the tissue not more than 20% after tissue has been covered by the cultured cell sheet. 
     
     
         7 . The cultured cell sheet according to any one of  claims 1  to  6 , wherein the flexibility results from a surfactant protein. 
     
     
         8 . The cultured cell sheet according to  claim 7 , wherein the surfactant protein is produced by the cultured cell sheet. 
     
     
         9 . The cultured cell sheet according to any one of  claims 1  to  8 , wherein the flexibility results from a cross-linking inhibitor. 
     
     
         10 . The cultured cell sheet according to  claim 9 , wherein a cross-linking inhibitor is a collagen cross-linking inhibitor, β-aminopropylnitrile. 
     
     
         11 . The cultured cell sheet according to any one of  claims 1  to  10 , wherein the cultured cell sheet is used for a treatment for suppressing air leakage, blood leakage or bodily fluid leakage from a surface layer of an organ. 
     
     
         12 . The cultured cell sheet according to  claim 11 , wherein the treatment comprises covering the surface of an affected portion of an organ with the cultured cell sheet. 
     
     
         13 . The cultured cell sheet according to either  claim 11  or  12 , wherein the subject to be treated is air leakage from lung tissue, bleeding from liver tissue or bleeding from vascular tissue. 
     
     
         14 . The cultured cell sheet according to any one of  claims 11  to  13 , wherein the cultured cell sheet is cut according to the size and shape of the affected area, before covering of the surface of the affected area. 
     
     
         15 . A method for preparing a cultured cell sheet, wherein cells are cultured on a cell culture support having a surface coated with a temperature responsive polymer having an upper or lower critical solution temperature of 0° C. to 80° C. in water, and, thereafter, comprising steps of:
 (1) adjusting the temperature of the culture medium to a temperature above the upper critical solution temperature or below the lower critical solution temperature; and   (2) detaching the cultured cell sheet.   
     
     
         16 . The method according to  claim 15 , wherein the temperature responsive polymer is poly(N-isopropyl acrylamide). 
     
     
         17 . The method according to either  claim 15  or  16 , wherein the cell sheet is not treated with a proteolytic enzyme upon being detached. 
     
     
         18 . The method according to any one of  claims 15  to  17 , wherein β-aminopropylnitrile is added to the culture medium. 
     
     
         19 . A treatment method comprising transplanting the cultured cell sheet of any one of  claims 1  to  14  to the affected area of the surface layer of the organ from which air, blood or bodily fluid is leaking. 
     
     
         20 . The method according to  claim 19 , wherein the transplantation is conducted by covering of the surface layer of the affected area of the organ. 
     
     
         21 . The method according to  claim 19  or  20 , wherein the cultured cell sheet is cut according to the size and shape of the affected portion, before covering of the surface layer of the affected area of the organ. 
     
     
         22 . The method according to any one of  claims 19  or  21 , wherein the subject to be treated is air leakage from alveolar tissue, bleeding from liver tissue, or bleeding from vascular tissue.