Cryopreservation of human red blood cells

Abstract

A red blood cell storage composition includes a composition of red blood cells and biochemistry altering reagents, the biochemistry altering reagents being present at a concentration so as to reduce the percent hemolysis of the red blood cells during the freeze-thaw cycle below that of the percent hemolysis of the red blood cells in the absence the biochemistry altering reagents. The red blood cell storage composition preferably includes reagents selected from: modifiers of glycolytic/metabolic components, modifiers of antioxidant potential, effectors of intracellular ionic distribution, modifiers of membrane fluidity, modifiers of cytoskeletal structure, effectors of the cyclooxygenase second messenger pathway, effectors of the lipoxygenase second messenger pathway, effectors of the hexose monophosphate second messenger pathway, effectors of the phosphorylation second messenger pathway, modifiers of specific messenger molecules, and combinations thereof.

1 .- 7 . (canceled)  
   
   
       8 . A red blood cell composition comprising: 
 (a) red blood cells;    (b) a combination of biochemistry altering reagents selected from the group consisting of pentoxyfylline, flurbiprofen, nicotinamide, nikethamide, nifedipine, amiloride, taxol, cytochalasin B, trehalose, ribose, a protease inhibitor, a bioflavonoid, and bioflavonoid derivative; and    (c) one or more cryoprotective agents.    
   
   
       9 . The composition of  claim 8 , further comprising a storage composition.  
   
   
       10 . The composition of  claim 8 , wherein the protease inhibitor is leupeptin.  
   
   
       11 . The composition of  claim 8 , wherein the bioflavinoid derivative is selected from the group consisting of rutin, quercetin, and curcumin.  
   
   
       12 . The composition of  claim 8 , wherein the one or more cryoprotective agents is selected from the group consisting of glycerol, dimethyl sulfoxide, 2,3 butanediol, 1,2-propanediol, 1,3-propanediol, polypropylene glycol, N,N-dimethylacetamide, and 1,3,5-trimethyl pentanetriol.  
   
   
       13 . The composition of  claim 8 , wherein the one or more cryoprotective agents is selected from the group consisting of hydroxyethyl starch, dextran, polyvinyl pyrrolidone, polyethylene, and polyethylene glycol.  
   
   
       14 . The composition of  claim 8 , wherein the one or more cryoprotective agents comprise a combination of one or more cell membrane penetrating cryoprotectants and one or more cryoprotectants that do not penetrate cell membranes.  
   
   
       15 . The composition of  claim 14 , wherein the one or more cryoprotectants that do not penetrate cell membranes have molecular weights of at least 5,000 MW.  
   
   
       16 . The composition of  claim 8 , comprising pentoxyfylline, flurbiprofen, nikethamide, nifedipine, taxol, cytochalasin B, trehalose, and ribose.  
   
   
       17 . The composition of  claim 8 , wherein the nifedipine has a concentration of about 500 μM, the cytochalasin B has a concentration of about 20 μM, the pentoxifylline has a concentration of about 5 mM, the flurbiprofen has a concentration of about 25 μg/ml, the nikethamide has a concentration of about 1% (v/v), the ribose has a concentration of about 50 mM, and the trehalose has a concentration of about 50 mM.  
   
   
       18 . The composition of  claim 8 , wherein the biochemistry altering reagents are present in a concentration so that upon in vitro preservation of the red blood cells at a temperature at or below the red blood cells' physiological temperature for a period of about 2-7 days, the red blood cells exhibit decreased hemolysis during a freeze-thaw cycle and increased in vitro functional activity when compared to red blood cells preserved under the same conditions but in the absence of the biochemistry altering reagents.  
   
   
       19 . A method for the prolonged storage of red blood cells comprising creating the composition of  claim 8  and storing the composition at a temperature below the red blood cells' physiological temperature.  
   
   
       20 . The method of  claim 19 , wherein the temperature below the red blood cells' physiological temperature is from about −10° C. to about −193° C.  
   
   
       21 . The method of  claim 19 , wherein the temperature below the red blood cells' physiological temperature is about −80° C.  
   
   
       22 . The method of  claim 12 , wherein the red blood cells are isolated from human blood prior to creating the composition.