Method of manufacturing or differentiating mammalian pluripotent stem cellsor progenitor cells using a hollow fiber bioreactor

Abstract

The present disclosure provides a method of manufacturing and differentiating mammalian stem cells, and in one embodiment human induced pluripotent stem cells (iPSc), e.g., manufacturing neuron progenitors, e.g., derived from iPSC, on a large scale by the use of an automated hollow fiber reactor system. In one embodiment, human iPSc that can be differentiated into cardiomyocytes or neuron progenitors are provided. The method comprises seeding a hollow fiber reactor with stem cells such as iPSc, or differentiated iPSc, growing and expanding the seeded cells using appropriate growth factors and nutrients, and harvesting the cells after expansion from the hollow fiber reactor walls, e.g., with the use of an enzyme. The method can produce billions of cells per week from seeding the reactor with a minimum number of starting stem cells or neuron progenitor cells.

1 . A large-scale method to culture and differentiate stem cells, comprising: providing a system comprising a hollow fiber bioreactor comprising a plurality of fibers that is part of a sterile closed-loop circuit for media and gas exchange; a gas exchange module; a waste bag; and a cell harvest bag;
 introducing induced pluripotent stem cells to the hollow fibers;   incubating the cells under conditions that allow for cell growth and differentiation; and   harvesting the cells from the bioreactor.   
     
     
         2 . The method of  claim 1  wherein the fibers comprise polysulfone or cellulose derivatives. 
     
     
         3 . The method of  claim 2  wherein molecular weight cutoff of the fibers is about 5 kDa. 
     
     
         4 . The method of claim I wherein the fibers are coated with an adhesion promoter. 
     
     
         5 . The method of claim I wherein the conditions allow for differentiation to cardiomyocytes. 
     
     
         6 . The method of claim I wherein the conditions include control of gas exchange, control of temperature, control of metabolite concentration, control of growth factor concentration, control of differentiation factor concentration, or any combination thereof. 
     
     
         7 . A large-scale method to culture neuron progenitor cells, comprising: providing a system comprising a hollow fiber bioreactor comprising a plurality of fibers that is part of a sterile closed-loop circuit for media and gas exchange; a gas exchange module; a waste bag; and a cell harvest bag, wherein the fibers are coated with a glycoprotein and a molecule or mixture that promotes cell attachment;
 introducing stern cells or neuron progenitor cells to the hollow fibers;   incubating the cells under conditions that allow for cell expansion; and   harvesting the cells from the bioreactor.   
     
     
         8 . The method of  claim 7  wherein the cells are stem cells. 
     
     
         9 . The method of  claim 7  wherein the fibers comprise polysulfone or cellulose derivatives. 
     
     
         10 . The method of  claim 7  wherein molecular weight cutoff of the fibers is about 5 kDa. 
     
     
         11 . The method of  claim 7  wherein the glycoprotein enhances attachment and spreading of cells. 
     
     
         12 . The method of  claim 7  wherein the glycoprotein comprises laminin, fibronectin, vitronectin, or a combination thereof. 
     
     
         13 . The method of  claim 7  wherein the molecule or mixture comprises poly-L ornithine, poly-lysine, Matrigel or a plant derived protein. 
     
     
         14 . The method of  claim 7  wherein the neuron progenitors are derived from iPSc. 
     
     
         15 . The method of  claim 7  wherein the conditions include control of gas exchange, control of temperature, control of metabolite concentration, control of growth factor concentration, or time, or any combination t