Hematopoietic cell culture nutrient supplement

Abstract

The present invention provides a serum-free supplement which supports the growth of hematopoietic cells in culture. Also provided are a medium comprising a basal medium supplemented with the serum-free supplement of the present invention. The present invention also provides methods for culturing and for differentiating hematopoietic cells.

1 - 54 . (canceled) 
     
     
         55 . A serum-free, eukaryotic cell culture medium comprising the ingredients N-acetyl-L-cysteine, 2-mercaptoethanol, and D,L-tocopherol acetate, or derivatives or mixtures thereof,
 wherein each of said ingredients is present in said medium at a concentration that supports the culture of a hematopoietic stem cell, a CD34 +  hematopoietic cell, or a myeloid lineage cell, in suspension culture.   
     
     
         56 . The serum-free, eukaryotic cell culture medium of  claim 55  further comprising transferring and/or insulin substitutes. 
     
     
         57 . The serum-free, eukaryotic cell culture medium of  claim 55  further comprising iron and/or zinc. 
     
     
         58 . The serum-free, eukaryotic cell culture medium of  claim 55  wherein said hematopoietic stem cell or CD34 +  hematopoietic cell is derived from bone marrow, peripheral blood or neonatal cord blood. 
     
     
         59 . A method of cultivating a hematopoietic stem cell, a CD34 +  hematopoietic cell, or a myeloid lineage cell, the method comprising:
 a. contacting the hematopoietic stem cell, the CD34 +  hematopoietic cell, or the myeloid lineage cell with a serum-free medium of  claim 55 ; and   b. culturing the myeloid lineage cell in serum-free suspension culture, in the absence of stromal cells, under conditions that facilitates its growth and cultivation.   
     
     
         60 . The method of  claim 59 , wherein the hematopoietic stem cell, the CD34 +  hematopoietic cell, or the myeloid lineage cell is a mammalian or an avian cell. 
     
     
         61 . The method of  claim 60 , wherein the mammalian cell is human. 
     
     
         62 . A method of differentiating a hematopoietic stem cell, a CD34 +  hematopoietic cell, or a myeloid lineage, the method comprising:
 a. contacting the hematopoietic stem cell, the CD34 +  hematopoietic cell, or the myeloid lineage cell with a serum-free medium of  claim 55 ; and   b. culturing the hematopoietic stem cell, the CD34 +  hematopoietic cell, or the myeloid lineage cell in serum-free suspension culture, in the absence of stromal cells, under conditions that facilitate the differentiation of said hematopoietic stem cell, said CD34 +  hematopoietic cell, or said myeloid lineage cell to a desired differentiated line.   
     
     
         63 . The method of  claim 62 , wherein the hematopoietic stem cell, the CD34 +  hematopoietic cell, or the myeloid lineage cell is a mammalian or an avian cell. 
     
     
         64 . The method of  claim 63 , wherein the mammalian cell is human. 
     
     
         65 . A method of enriching a CD34 +  marker carrying cell from CD34 −  bone marrow cells, the method comprising:
 a. contacting bone marrow cells with a serum-free medium of  claim 55 ; and   b. culturing the bone marrow cells in serum-free suspension culture, in the absence of stromal cells, under conditions that facilitate the expansion of the CD34 +  hematopoietic cells.   
     
     
         66 . The method of  claim 55 , wherein the serum-free culture medium further comprises at least one component selected from the group consisting of human serum albumin, soluble human lipids, ethanolamine, human zinc insulin, iron-saturated transferrin, Se 4+ , hydrocortisone, Ca 2+ , K + , Mg 2+ , Na + , CO 3   2− , PO 4   3− , D-glucose, HEPES, sodium pyruvate, phenol red, glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine HCL, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, D-calcium pantothenate, choline chloride, folic acid, i-inositol, niacinamide, pyridoxal HCl, riboflavin, thiamine HCl, and vitamin B 12 . 
     
     
         67 . The method of  claim 55 , wherein the serum-free medium comprises at least one component selected from the group consisting of a trace element, a glucocorticoid, an inorganic salt, an energy source, a buffering agent, a pyruvate salt, a pH indicator, an amino acid, and a vitamin. 
     
     
         68 . The method of  claim 55 , wherein the serum-free medium comprises at least one cytokine or at least one growth factor. 
     
     
         69 . The method of  claim 55 , wherein the serum-free medium comprises at least one glucocorticoid. 
     
     
         70 . The method of  claim 55 , wherein the cell is a hematopoietic progenitor cell. 
     
     
         71 . A method of providing CD34 +  hematopoietic cells to a mammal comprising:
 (a) cultivating CD34 +  hematopoietic cells according to the method of  claim 59 ; and   (b) introducing said recombinant cells into said mammal.