Cultured cell sheet for tissue regeneration, production method thereof, and application method thereof

Abstract

Provided is a cell sheet suitable for cartilage repair. The present invention provides a cell sheet for cartilage repair, formed from a culture of cells derived from a cartilage tissue, and the cell sheet is negative for immunostaining using an antibody against type II collagen. The present invention also provides a method for producing a cell sheet for cartilage repair, formed from a culture of cells derived from a cartilage tissue, and the method includes culturing cells derived from a cartilage tissue on a surface of a membrane, where a temperature-responsive polymer is immobilized on the surface, to give the cell sheet. The culturing is stopped before the cell sheet becomes positive for immunostaining using an antibody against type II collagen.

1 . A cell sheet for cartilage repair, the cell sheet being formed from a culture of cells derived from a cartilage tissue,
 the cell sheet being negative for immunostaining using an antibody against type II collagen.   
     
     
         2 . The cell sheet according to  claim 1 , wherein the cell sheet is positive for immunostaining using an antibody against type I collagen. 
     
     
         3 . The cell sheet according to  claim 1  or  2 , wherein the cell sheet is negative for safranin O staining. 
     
     
         4 . The cell sheet according to  claim 1 , wherein the cell sheet is positive for immunostaining using an antibody against aggrecan. 
     
     
         5 . The cell sheet according to  claim 1 , wherein the cells are derived from a cartilage tissue of a polydactyly animal. 
     
     
         6 . The cell sheet according to  claim 1 , wherein the cells comprise a mesenchymal stem cell. 
     
     
         7 . A method for producing a cell sheet for cartilage repair, the cell sheet being formed from a culture of cells derived from a cartilage tissue, the method comprising:
 culturing cells derived from a cartilage tissue on a surface of a membrane, a temperature-responsive polymer being immobilized on the surface, to give the cell sheet,   wherein the culturing is stopped before the cell sheet becomes positive for immunostaining using an antibody against type II collagen.   
     
     
         8 . The production method according to  claim 7 , wherein the cell sheet is positive for immunostaining using an antibody against type I collagen when the culturing is stopped. 
     
     
         9 . The production method according to  claim 7  or  8 , wherein the culturing is continued until the cell sheet becomes positive for immunostaining using an antibody against aggrecan. 
     
     
         10 . The production method according to  claim 7 , wherein the culturing is stopped before the cell sheet becomes positive for safranin O staining. 
     
     
         11 . The production method according to  claim 7 , wherein an amount of the polymer immobilized on the surface is 0.3 to 5.0 μg/cm 2 . 
     
     
         12 . The production method according to  claim 7 , wherein the membrane is a pore membrane, and in the culturing, the cells are in contact with a culture medium on an upper side of the pore membrane and is in contact with a culture medium on a lower side of the pore membrane through pores in the pore membrane. 
     
     
         13 . The production method according to  claim 7 , wherein the culturing is performed by using a cell culture insert.