Suspension Culture of Human Embryonic Stem Cells

Abstract

This disclosure provides an improved system for culturing human embryonic stem cells. The cells are cultured in suspension so as to maximize the production capacity of the culture environment. The new culture system of this invention allows for bulk proliferation of hES cells in a more cost-effective manner, which facilitates commercial production of important products for use in human therapy.

The invention claimed is: 
     
         1 . A culture of human embryonic stem (hES) cells in suspension, wherein the hES cells are substantially undifferentiated. 
     
     
         2 . The culture of  claim 1 , wherein the cells are cultured in a medium containing fibroblast growth factor at a concentration of at least about 40 ng/mL. 
     
     
         3 . The culture of  claim 2 , wherein the medium also contains transforming growth factor beta (TGFβ), stem cell factor (SCF), or Flt3 ligand (Flt3L). 
     
     
         4 . The culture of any preceding claim, wherein the cells are cultured in a medium containing one or more soluble or suspended extracellular matrix components. 
     
     
         5 . The culture of  claim 4 , wherein the extracellular matrix component(s) include human laminin and/or human fibronectin. 
     
     
         6 . The culture of any preceding claim, wherein the cells are cultured in suspension with solid microparticle. 
     
     
         7 . The culture of  claim 7 , wherein the microparticle are coated with one or more soluble or suspended extracellular matrix components. 
     
     
         8 . A method for culturing hES cells, comprising:
 a) suspending the cells in a nutrient medium;   b) maintaining the cells in suspension while culturing;   c) changing the medium periodically;   d) optionally splitting the culture from time to time so as to reduce cell density; and finally   e) harvesting cells from the culture.   
     
     
         9 . The method of  claim 8 , wherein the cells are cultured in a medium containing fibroblast growth factor at a concentration of at least about 40 ng/mL. 
     
     
         10 . The method of  claim 9 , wherein the medium also contains transforming growth factor beta (TGFβ), stem cell factor (SCF), or Flt3 ligand (Flt3L). 
     
     
         11 . The method of any of  claims 8 - 10 , wherein the cells are cultured in a medium containing one or more soluble or suspended extracellular matrix components. 
     
     
         12 . The method of  claim 11 , wherein the extracellular matrix component(s) include human laminin and/or human fibronectin. 
     
     
         13 . The method of any of  claims 8 - 12 , wherein the cells are cultured in suspension with solid microparticle. 
     
     
         14 . The method of  claim 13 , wherein the microparticle are coated with one or more soluble or suspended extracellular matrix components. 
     
     
         15 . The method of any of  claims 8 - 14 , wherein the cells are cultured in suspension for at least two months. 
     
     
         16 . The method of any of  claims 8 - 15 , wherein the cells undergo at least a 3-fold expansion while in suspension culture. 
     
     
         17 . The method of any of  claims 8 - 16 , further comprising plating the harvested cells back onto a solid surface and continuing to culture the cells so as to maintain a substantially undifferentiated cell population. 
     
     
         18 . The method of any of  claims 8 - 17 , further comprising differentiating the harvested cells. 
     
     
         19 . A system or kit for culturing hES cells in suspension, comprising a nutrient medium that contains at least 40 ng/mL fibroblast growth factor. 
     
     
         20 . The system or kit of  claim 19 , also comprising an extracellular matrix component or microparticle for adding to the medium.