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 three dimensional cell culture comprising cells derived from an adherent stromal cell culture on a substrate, said substrate forming a physiologically acceptable three-dimensional network of fibers, wherein said cells are capable of supporting expansion of undifferenciated hemopoietic stem cells, whereby said cells derived from said stromal cells culture are from a first individual and further whereby said undifferenciated hemopoietic stem cells are from a different individual. 
   
   
       19 . The three dimensional cell culture of  claim 18 , wherein said undifferentiated hemopoietic stem cells are cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone-marrow. 
   
   
       20 . The three dimensional cell culture of  claim 18 , wherein said first individual and said second individual are of different species. 
   
   
       21 . The three dimensional cell culture of  claim 18 , wherein said cells derived from said adherent stromal cell culture are grown to a density of at least 5×106 cells per a cubic centimeter of said substrate. 
   
   
       22 . The three dimensional cell culture of  claim 18 , wherein said cells derived from said adherent stromal cell culture are grown to a density of at least 107 cells per a cubic centimeter of said substrate. 
   
   
       23 . The three dimensional cell culture 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. 
   
   
       24 . The three dimensional cell culture of  claim 18 , wherein said substrate is made of fibers 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. 
   
   
       25 . The three dimensional cell culture of  claim 18 , wherein said substrate 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. 
   
   
       26 . The three dimensional cell culture of  claim 18 , wherein said substrate has a pore volume as a percentage of total volume of from 60 to 95%. 
   
   
       27 . The three dimensional cell culture of  claim 18 , wherein the substrate has a height of 50-1000 mm. 
   
   
       28 . The three dimensional cell culture of  claim 18 , wherein the material of the substrate is selected from the group consisting of polyesters, polyalkylenes, polyfluorochloroethylenes, polyvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibers, and inert metal fibers. 
   
   
       29 . The three dimensional cell culture of  claim 18 , wherein the substrate is in a shape selected from the group consisting of squares, rings, discs, and cruciforms. 
   
   
       30 . The three dimensional cell culture of  claim 18 , wherein the substrate is in the form of a disc. 
   
   
       31 . The three dimensional cell culture of  claim 18 , wherein the substrate is coated with poly-D-lysine.