Culture system and method for maintenance and proliferation of undifferentiated human embryonic stem cells

Abstract

The present invention discloses an improved culture system for proliferative and undifferentiated growth of human embryonic stem cells, comprising an extracellular matrix prepared from feeder cells and a conditioned medium preconditioned by feeder cells. This invention also relates to a culture method utilizing the foregoing culture system.

1 . A culture system for proliferation and undifferentiated growth of human embryonic stem cells comprising: 
 a culture matrix comprising extracellular matrix prepared from feeder cells; and    a conditioned medium being preconditioned by feeder cells;    wherein said feeder cells are selected from primary mouse embryonic fibroblasts (PMEF), a mouse embryonic fibroblast cell line (MEF), murine fetal fibroblasts (MFF), human embryonic fibroblasts (HEF), human fetal muscle (HFM), human fetal skin cells (HFS), human adult skin cells, human foreskin fibroblasts (HFF), human adult fallopian tubal epithelial cells (HAFT) or human marrow stromal cells (hMSCs).    
     
     
         2 . The culture system according to  claim 1 , wherein said feeder cells are mouse embryonic fibroblasts or human foreskin fibroblasts.  
     
     
         3 . The culture system according to  claim 1 , wherein said culture matrix is prepared by removing intracellular substances by means of treating said feeder cells with NaOH or trinitrotoluene to obtain extracellular matrix as culture matrix.  
     
     
         4 . The culture system according to  claim 3 , wherein the intracellular substances are removed by washing with water or buffered solution.  
     
     
         5 . The culture system according to  claim 4 , wherein said buffered solution is phosphate buffered saline.  
     
     
         6 . The culture system according to  claim 1 , wherein said conditioned medium is prepared by the steps of: 
 (a) inactivating the feeder cells;    (b) placing the cells obtained in step (a) in a culture solution; and    (c) collecting the cell culture solution as conditioned medium.    
     
     
         7 . The culture system according to  claim 6 , wherein said feeder cells are inactivated by irradiation with gamma ray or treatment with mitomycin C.  
     
     
         8 . The culture system according to  claim 7 , wherein said feeder cells are inactivated by treating with mitomycin C.  
     
     
         9 . A method for culturing human embryonic stem cells, comprising the steps of: 
 obtaining human embryonic stem cells;    culturing the human embryonic stem cells in the culture system described according to claims  1 ; and    continuing the culture to maintain human embryonic stem cells in a substantially proliferative and undifferentiated state.    
     
     
         10 . The method according to  claim 9 , wherein said human embryonic stem cells are maintained in a substantially undifferentiated and proliferative state over the course of at least five passages in said culture system.