Expansion Medium for CD34-Negative Stem Cells

Abstract

This invention provides a cell growth medium comprising (a) a human platelet lysate free of solid matter greater than 0.22 μm in diameter; (b) a human fresh frozen plasma (FFP) filtrate free of solid matter greater than 0.22 μm in diameter; (c) heparin; (d) L-glutamine; and (e) a serum-free, low glucose medium suitable for mammalian cell growth, wherein the cell growth medium permits the expansion of human CD34 − stem cells and wherein the resulting expanded CD34 − stem cells retain the ability to differentiate. This invention also provides related cell growth medium supplements, a sterile human platelet lysate and human fresh frozen plasma (FFP) filtrate, kits, CD34 − stem cell-containing compositions, and related production and cell expansion methods.

1 .- 26 . (canceled) 
     
     
         27 . A composition of matter comprising (a) human CD34 −  stem cells and (b) a cell growth medium comprising (a) a human platelet lysate free of solid matter greater than 0.22 μm in diameter, wherein the lysate constitutes from 2% to 15% of the total volume of the cell growth medium; (b) a human fresh frozen plasma (FFP) filtrate free of solid matter greater than 0.22 μm in diameter, wherein the FFP filtrate constitutes from 1% to 10% of the total volume of the cell growth medium; (c) heparin at a concentration of from 0 U/ml to 10 U/ml of the cell growth medium; (d) L-glutamine at a concentration of from 0.5 mM to 10 mM; and (e) a serum-free, low glucose medium suitable for mammalian cell growth, wherein the serum-free, low glucose medium constitutes from 75% to 97% of the total volume of the cell growth medium, and may contain the L-glutamine of part (d); (i) wherein the FFP is the fluid portion of human blood that has been centrifuged, separated, and frozen solid within six hours of collection, (ii) wherein the cell growth medium permits the expansion of human CD34 −  stem cells, and (iii) wherein the resulting expanded CD34 −  stem cells retain the ability to differentiate. 
     
     
         28 . The composition of  claim 27 , wherein the platelet lysate of part (a) is prepared according to the following steps: freezing human platelet concentrate, thereby lysing the platelets therein; (ii) thawing the resulting lysed platelets; (iii) centrifuging the thawed lysed platelets at a speed and for a duration suitable to pellet solid matter therein; (iv) re-centrifuging the supernatant from step (iii) at a speed and for a duration suitable to pellet solid matter therein; and (v) filtering the supernatant from step (iv) at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         29 . The composition of  claim 28 , wherein the human platelet concentrate of step (i) is prepared by: centrifuging platelets and thereby pelleting them; separating the pelleted platelets from the liquid phase; and reconstituting the resulting platelets in FFP. 
     
     
         30 . The composition of  claim 27 , wherein the FFP filtrate of part (b) is prepared by: thawing human FFP; centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof and filtering the resulting liquid portion at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         31 . The composition of  claim 27 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof. 
     
     
         32 . The composition of  claim 31 , wherein the thawed FFP from step (iii) is frozen and thawed at least once again before the centrifugation step (iv). 
     
     
         33 . The composition of  claim 27 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof, wherein the liquid portion resulting from step (iv) is not filtered. 
     
     
         34 . The composition of  claim 27 , wherein the cell growth medium is free of animal serum. 
     
     
         35 . The composition of  claim 27 , wherein the platelet lysate and FFP filtrate are matched with respect to blood group antigens ABO and Rh. 
     
     
         36 . A method for expanding a population of human CD34 −  stem cells comprising the step of incubating the stem cells at a suitable temperature in a cell growth medium comprising (a) a human platelet lysate free of solid matter greater than 0.22 μm in diameter, wherein the lysate constitutes from 2% to 15% of the total volume of the cell growth medium; (b) a human fresh frozen plasma (FFP) filtrate free of solid matter greater than 0.22 μm in diameter, wherein the FFP filtrate constitutes from 1% to 10% of the total volume of the cell growth medium; (c) heparin at a concentration of from 0 U/ml to 10 U/ml of the cell growth medium; (d) L-glutamine at a concentration of from 0.5 mM to 10 mM; and (e) a serum-free, low glucose medium suitable for mammalian cell growth, wherein the serum-free, low glucose medium constitutes from 75% to 97% of the total volume of the cell growth medium, and may contain the L-glutamine of part (d); (i) wherein the FFP is the fluid portion of human blood that has been centrifuged, separated, and frozen solid within six hours of collection, (ii) wherein the cell growth medium permits the expansion of human CD34 −  stem cells, and (iii) wherein the resulting expanded CD34 −  stem cells retain the ability to differentiate. 
     
     
         37 . The method of  claim 36 , wherein the platelet lysate of part (a) is prepared according to the following steps: freezing human platelet concentrate, thereby lysing the platelets therein; (ii) thawing the resulting lysed platelets; (iii) centrifuging the thawed lysed platelets at a speed and for a duration suitable to pellet solid matter therein; (iv) re-centrifuging the supernatant from step (iii) at a speed and for a duration suitable to pellet solid matter therein; and (v) filtering the supernatant from step (iv) at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         38 . The method of  claim 37 , wherein the human platelet concentrate of step (i) is prepared by: centrifuging platelets and thereby pelleting them; separating the pelleted platelets from the liquid phase; and reconstituting the resulting platelets in FFP. 
     
     
         39 . The method of  claim 36 , wherein the FFP filtrate of part (b) is prepared by: thawing human FFP; centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof; and filtering the resulting liquid portion at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         40 . The method of  claim 36 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof. 
     
     
         41 . The method of  claim 40 , wherein the thawed FFP from step (iii) is frozen and thawed at least once again before the centrifugation step (iv). 
     
     
         42 . The method of  claim 36 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof, wherein the liquid portion resulting from step (iv) is not filtered. 
     
     
         43 . The method of  claim 36 , wherein the cell growth medium is free of animal serum. 
     
     
         44 . The method of  claim 36 , wherein the platelet lysate and FFP filtrate are matched with respect to blood group antigens ABO and Rh. 
     
     
         45 . A human CD34 −  stem cell (i) having the ability to differentiate, and (ii) resulting from an expansion of a population of human CD34 −  stem cells, wherein the expansion comprises incubating the population of stem cells at a suitable temperature in a cell growth medium comprising (a) a human platelet lysate free of solid matter greater than 0.22 μm in diameter, wherein the lysate constitutes from 2% to 15% of the total volume of the cell growth medium; (b) a human fresh frozen plasma (FFP) filtrate free of solid matter greater than 0.22 μm in diameter, wherein the FFP filtrate constitutes from 1% to 10% of the total volume of the cell growth medium; (c) heparin at a concentration of from 0 U/ml to 10 U/ml of the cell growth medium; (d) L-glutamine at a concentration of from 0.5 mM to 10 mM; and (e) a serum-free, low glucose medium suitable for mammalian cell growth, wherein the serum-free, low glucose medium constitutes from 75% to 97% of the total volume of the cell growth medium, and may contain the L-glutamine of part (d); (i) wherein the FFP is the fluid portion of human blood that has been centrifuged, separated, and frozen solid within six hours of collection, (ii) wherein the cell growth medium permits the expansion of human CD34 −  stem cells, and (iii) wherein the resulting expanded CD34 −  stem cells retain the ability to differentiate. 
     
     
         46 . The stem cell of  claim 45 , wherein the platelet lysate of part (a) is prepared according to the following steps: freezing human platelet concentrate, thereby lysing the platelets therein; (ii) thawing the resulting lysed platelets; (iii) centrifuging the thawed lysed platelets at a speed and for a duration suitable to pellet solid matter therein; (iv) re-centrifuging the supernatant from step (iii) at a speed and for a duration suitable to pellet solid matter therein; and (v) filtering the supernatant from step (iv) at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         47 . The stem cell of  claim 46 , wherein the human platelet concentrate of step (i) is prepared by: centrifuging platelets and thereby pelleting them; separating the pelleted platelets from the liquid phase; and reconstituting the resulting platelets in FFP. 
     
     
         48 . The stem cell of  claim 45 , wherein the FFP filtrate of part (b) is prepared by: thawing human FFP; centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof; and filtering the resulting liquid portion at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         49 . The stem cell of  claim 45 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof. 
     
     
         50 . The stem cell of  claim 49 , wherein the thawed FFP from step (iii) is frozen and thawed at least once again before the centrifugation step (iv). 
     
     
         51 . The stem cell of  claim 45 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof, wherein the liquid portion resulting from step (iv) is not filtered. 
     
     
         52 . The stem cell of  claim 45 , wherein the cell growth medium is free of animal serum. 
     
     
         53 . The stem cell of  claim 45 , wherein the platelet lysate and FFP filtrate are matched with respect to blood group antigens ABO and Rh. 
     
     
         54 . A population of human CD34 −  stem cells (i) having the ability to differentiate, and (ii) resulting from an expansion of a population of human CD34 −  stem cells, wherein the expansion comprises incubating the population of stem cells at a suitable temperature in a cell growth medium comprising (a) a human platelet lysate free of solid matter greater than 0.22 μm in diameter, wherein the lysate constitutes from 2% to 15% of the total volume of the cell growth medium; (b) a human fresh frozen plasma (FFP) filtrate free of solid matter greater than 0.22 μm in diameter, wherein the FFP filtrate constitutes from 1% to 10% of the total volume of the cell growth medium; (c) heparin at a concentration of from 0 U/ml to 10 U/ml of the cell growth medium; (d) L-glutamine at a concentration of from 0.5 mM to 10 mM; and (e) a serum-free, low glucose medium suitable for mammalian cell growth, wherein the serum-free, low glucose medium constitutes from 75% to 97% of the total volume of the cell growth medium, and may contain the L-glutamine of part (d); (i) wherein the FFP is the fluid portion of human blood that has been centrifuged, separated, and frozen solid within six hours of collection, (ii) wherein the cell growth medium permits the expansion of human CD34 −  stem cells, and (iii) wherein the resulting expanded CD34 −  stem cells retain the ability to differentiate. 
     
     
         55 . The stem cell population of  claim 54 , wherein the platelet lysate of part (a) is prepared according to the following steps: freezing human platelet concentrate, thereby lysing the platelets therein; (ii) thawing the resulting lysed platelets; (iii) centrifuging the thawed lysed platelets at a speed and for a duration suitable to pellet solid matter therein; (iv) re-centrifuging the supernatant from step (iii) at a speed and for a duration suitable to pellet solid matter therein; and (v) filtering the supernatant from step (iv) at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         56 . The stem cell population of  claim 55 , wherein the human platelet concentrate of step (i) is prepared by: centrifuging platelets and thereby pelleting them; separating the pelleted platelets from the liquid phase; and reconstituting the resulting platelets in FFP. 
     
     
         57 . The stem cell population of  claim 54 , wherein the FFP filtrate of part (b) is prepared by: thawing human FFP; centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof; and filtering the resulting liquid portion at least twice using filters of decreasing pore size, wherein the first filtration is performed using a filter having pores of at least 5 μm, and the last filtration is performed using a filter having pores no larger than 0.22 μm. 
     
     
         58 . The stem cell population of  claim 54 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof. 
     
     
         59 . The stem cell population of  claim 58 , wherein the thawed FFP from step (iii) is frozen and thawed at least once again before the centrifugation step (iv). 
     
     
         60 . The stem cell population of  claim 54 , wherein the FFP filtrate of part (b) is prepared by: (i) thawing human FFP; (ii) freezing the thawed human FFP; (iii) thawing the resulting human FFP; and (iv) centrifuging the resulting thawed FFP at a speed and for a duration suitable to separate the liquid and solid portions thereof, wherein the liquid portion resulting from step (iv) is not filtered. 
     
     
         61 . The stem cell population of  claim 54 , wherein the cell growth medium is free of animal serum. 
     
     
         62 . The stem cell population of  claim 54 , wherein the platelet lysate and FFP filtrate are matched with respect to blood group antigens ABO and Rh.