Method and apparatus for maintenance and expansion of hemopoietic stem cells and/or progenitor cells

Abstract

A method of preparing a stromal cell conditioned medium useful in expanding undifferentiated hemopoietic stem cells to increase the number of the hemopoietic stem cells is provided. The method comprising: (a) establishing a stromal cell culture in a stationary phase plug-flow bioreactor under continuous flow on a substrate in the form of a sheet, the substrate including a non-woven fibrous matrix forming a physiologically acceptable three-dimensional network of fibers, thereby expanding undifferentiated hemopoietic stem cells ; and (b) when a desired stromal cell density has been achieved, collecting medium from the stationary phase plug-flow bioreactor, thereby obtaining the stromal cell conditioned medium useful in expanding the undifferentiated hemopoietic stem cells.

1 - 17 . (canceled) 
     
     
         18 . A bioreactor plug comprising a container having an outlet and an inlet and containing therein a substrate in the form of a sheet, said substrate including a non-woven fibrous matrix forming a physiologically acceptable three-dimensional network of fibers, said substrate supporting at least 5×10 6  stromal cells per cubic centimeter of said substrate. 
     
     
         19 . The bioreactor of  claim 18 , wherein said fibers form a pore volume as a percentage of total volume of from 40 to 95% and a pore size of from 10 microns to 100 microns. 
     
     
         20 . The bioreactor of  claim 18 , wherein said matrix is made of fiber selected from the group consisting of flat, non-round, and hollow fibers and mixtures thereof, said fibers being of from 0.5 microns to 50 microns in diameter or width. 
     
     
         21 . The bioreactor of  claim 18 , wherein said matrix is composed of ribbon formed fibers having a width of from 2 microns to 20 microns, and wherein the ratio of width to thickness of the fibers is at least 2:1. 
     
     
         22 . The bioreactor of  claim 18 , wherein said matrix having a pore volume as a percentage of total volume of from 60 to 95%. 
     
     
         23 . The bioreactor of  claim 18 , wherein the matrix has a height of 50-1000 μm. 
     
     
         24 . The bioreactor of  claim 18 , wherein the material of the matrix is selected from the group consisting of polyesters, polyalkylenes, polyfluorochloroethylenes, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers, and inert metal fibers. 
     
     
         25 . The bioreactor of  claim 18 , wherein the matrix is in a shape selected from the group consisting of squares, rings, discs, and cruciforms. 
     
     
         26 . The bioreactor of  claim 18 , wherein the matrix is in the form of a disc. 
     
     
         27 . The bioreactor of  claim 18 , wherein the matrix is coated with poly-D-lysine. 
     
     
         28 . A plug-flow bioreactor comprising the bioreactor plug of  claim 18 . 
     
     
         29 . A bioreactor comprising a container having an outlet and an inlet, the container comprising a substrate forming a physiologically acceptable three-dimensional network of fibers, said substrate comprising at least 5×10 6  stromal cells per cubic centimeter of said substrate. 
     
     
         30 . The bioreactor of  claim 29 , wherein the stromal cells are at a density of at least 10 7  cells per cubic centimeter of substrate. 
     
     
         31 . The bioreactor of  claim 29 , wherein the stromal cells comprise stromal cells of a primary culture. 
     
     
         32 . The bioreactor of  claim 29 , wherein the stromal cells comprise stromal cells of a cell line. 
     
     
         33 . The bioreactor of  claim 29 , wherein the bioreactor is a plug flow bioreactor. 
     
     
         34 . The bioreactor of  claim 29 , wherein the stromal cells support expansion of undifferentiated hematopoietic stem cells. 
     
     
         35 . A cell culture comprising adherent stromal cells, wherein the adherent stromal cells are grown to confluence in a bioreactor on a matrix comprising a three dimensional substrate. 
     
     
         36 . The cell culture of  claim 35 , wherein the adherent stromal cells support expansion of undifferentiated hematopoietic stem cells. 
     
     
         37 . The cell culture of  claim 35 , wherein the bioreactor is a plug flow bioreactor. 
     
     
         38 . The cell culture of  claim 35 , wherein the three dimensional substrate is a non-woven fibrous matrix forming a physiologically acceptable three-dimensional network of fibers. 
     
     
         39 . The cell culture of  claim 35 , wherein the stromal cell culture is at a density of at least 10 7  cells per cubic centimeter of substrate.