Cryogenic preservation of biological material by circulating a cooling fluid

Abstract

Viable biological material is cryogenically preserved (cryopreservation) by chemically preparing the material for freezing, immersing the material in a tank of cooling fluid, and circulating the cooling fluid past the material at a substantially constant predetermined velocity and temperature to freeze the material. A method according to the present invention freezes the biologic material quickly enough to avoid the formation of ice crystals within cell structures (vitrification). The temperature of the cooling fluid is preferably between −20° C. and −30° C., which is warm enough to minimize the formation of stress fractures in cell membranes due to thermal changes. Cells frozen using a method according to the present invention have been shown to have approximately an 80 percent survival rate, which is significantly higher than other cryopreservation methods.

What is claimed is:  
     
         1 . A method of cryopreservation comprising: 
 immersing biologically active material in cooling fluid; and    circulating the cooling fluid past the biologically active material at a substantially constant predetermined velocity and temperature to freeze the biologically active material such that the biologically active material is vitrified, and the formation of stress fractures in cell membranes is minimized.    
     
     
         2 . The method as in  claim 1 , wherein the biologically active material comprises viable single cells.  
     
     
         3 . The method as in  claim 1 , wherein the biologically active material comprises viable organs.  
     
     
         4 . The method as in  claim 1 , wherein the biologically active material comprises viable nucleic acids.  
     
     
         5 . The method as in  claim 1 , wherein the biologically active material comprises viable ribonucleic acids.  
     
     
         6 . The method as in  claim 1 , wherein the biologically active material comprises viable amino acid based compounds.  
     
     
         7 . The method as in  claim 1 , wherein the biologically active material comprises viable lipid based compounds.  
     
     
         8 . A method of cryopreservation comprising; 
 immersing biologically active material in cooling fluid; and    freezing the biologically active material directly to a temperature higher than about −30 degrees centigrade by circulating the cooling fluid past the biologically active material at a substantially constant predetermined velocity and temperature such that the biologically active material is vitrified.    
     
     
         9 . The method as in  claim 8 , wherein the biologically active material comprises viable single cells.  
     
     
         10 . The method as in  claim 8 , wherein the biologically active material comprises viable organs.  
     
     
         11 . The method as in  claim 8 , wherein the biologically active material comprises viable nucleic acids.  
     
     
         12 . The method as in  claim 8 , wherein the biologically active material comprises viable ribonucleic acids.  
     
     
         13 . The method as in  claim 8 , wherein the biologically active material comprises viable amino acid based compounds.  
     
     
         14 . The method as in  claim 8 , wherein the biologically active material comprises viable lipid based compounds.  
     
     
         15 . A biological material having been subjected to a cryopreservation process, the cryopreservation process comprising: 
 immersing the biological material in cooling fluid; and    circulating the cooling fluid past the biological material at a substantially constant predetermined velocity and temperature to freeze the biological material such that the biological material is vitrified, and the formation of stress fractures in cell membranes is minimized.    
     
     
         16 . The biological material as in  claim 15 , wherein said biological material comprises viable single cells.  
     
     
         17 . The biological material as in  claim 15 , wherein said biological material comprises viable organs.  
     
     
         18 . The biological material as in  claim 15 , wherein said biological material comprises viable nucleic acids.  
     
     
         19 . The biological material as in  claim 15 , wherein the biological material comprises viable ribonucleic acids.  
     
     
         20 . The biological material as in  claim 15 , wherein the biological material comprises viable amino acid based compounds.  
     
     
         21 . The biological material as in  claim 15 , wherein the biological material comprises viable lipid based compounds.