Cryopreservation of cells and subcellular fractions

Abstract

The invention provides cryopreserved compositions of cells, wherein the compositions are advantageously in the form of self-sustaining bodies that can be individually handled and combined independently of a container, allowing for easy customization of the eventual pooled preparation. The invention also provides pre-pooled stacks of the self-sustaining cryopreserved compositions for eventual thawing to produce pooled preparations of cells. A mold and methods for forming the self-sustaining bodies are also provided. The invention is also concerned with methods of forming pooled preparations of cells using single-cryopreserved compositions of cells.

1 . A cryopreserved composition of cells, said composition being in the form of a self-sustaining body. 
     
     
         2 . The composition of  claim 1 , said self-sustaining body being in the form of a cryopreserved pellet having a volume of from about 10 μL to about 2 mL. 
     
     
         3 . The composition of  claim 2 , said pellet having a thickness of from about 2 mm to about 15 mm. 
     
     
         4 . The composition of  claim 2 , said pellet having a width of from about 6 mm to about 20 mm. 
     
     
         5 . The composition of  claim 1 , said cryopreserved composition being formed from a suspension of cells dispersed in a solution comprising a culture medium and a cryoprotectant. 
     
     
         6 . The composition of  claim 5 , wherein said cryoprotectant is selected from the group consisting of fetal bovine serum, dimethyl sulfoxide, polyethylene glycol, amino acids, propanediol, glycerol, and mixtures thereof. 
     
     
         7 . The composition of  claim 1 , wherein said cells are hepatocytes. 
     
     
         8 . The composition of  claim 7 , wherein said suspension comprises about from about 1 million viable cells/mL to about 20 million viable cells/mL. 
     
     
         9 . The composition of  claim 7 , wherein said hepatocytes are from a single source or from multiple sources. 
     
     
         10 . The composition of  claim 7 , wherein said hepatocytes are human hepatocytes. 
     
     
         11 . The composition of  claim 1 , wherein said cells are cellular or subcellular fractions selected from the group consisting of mitochondria, cytosol, S9, and microsomes. 
     
     
         12 . The composition of  claim 1 , wherein said composition is a single-cryopreserved composition. 
     
     
         13 . The combination of:
 a first cryopreserved composition of cells, said first composition being in the form of a first self-sustaining body; and   a second cryopreserved composition of cells, said second composition being in the form of a second self-sustaining body,   wherein said first self-sustaining body and said second self-sustaining body are in physical contact with each other.   
     
     
         14 . The combination of  claim 13 , wherein said first self-sustaining body and said second self-sustaining body are stacked adjacent one another in a vial, said first self-sustaining body and said second self-sustaining body remaining discrete bodies. 
     
     
         15 . The combination of  claim 13 , wherein said first composition comprises cells obtained from a first source and said second composition comprises cells obtained from a second source, said second source being different from said first source. 
     
     
         16 . A method of forming a first cryopreserved self-sustaining body formed from a composition of cells, said method comprising:
 providing a first receptacle comprising a plurality of wells configured to contain said cells;   adding a quantity of a first composition of cells from a first source to each of said wells; and   cryopreserving said first composition to yield discrete cryopreserved self-sustaining bodies formed from said first composition in each of said wells.   
     
     
         17 . The method of  claim 16 , wherein said first source comprises cells from a single source or multiple sources. 
     
     
         18 . The method of  claim 16 , further comprising:
 removing said self-sustaining bodies from said wells; and   transferring said self-sustaining bodies to a second receptacle.   
     
     
         19 . The method of  claim 18 , wherein said transferring is carried out in the vapor phase of liquid nitrogen. 
     
     
         20 . The method of  claim 18 , wherein said second receptacle is a container comprising at least a second cryopreserved self-sustaining body formed from a second composition of cells, said second composition of cells being from a second source different from said first source, wherein said transferring comprises forming a pre-pooled stack of said first and second cryopreserved self-sustaining bodies in said second receptacle. 
     
     
         21 . The method of  claim 18 , wherein said second receptacle comprises an about 1 mL to about 50 mL vial. 
     
     
         22 . The method of  claim 16 , wherein said wells of said first receptacle each have a volume of from about 20 μL to about 2 mL. 
     
     
         23 . The method of  claim 16 , wherein from about 10 μL to about 2 mL of said first composition is added to each well. 
     
     
         24 . The method of  claim 16 , wherein said cryopreserving comprises subjecting said first composition to controlled rate freezing of from about −1° C./min. to about −25° C./min., until a temperature of about −90° C. is reached. 
     
     
         25 . A method of forming a pooled preparation of cells, said method comprising:
 providing a pre-pooled stack comprising a plurality of discrete, cryopreserved self-sustaining bodies in a container, said self-sustaining bodies formed from respective compositions of cells, each of said respective compositions comprising cells from a different source; and   thawing said stack, wherein said respective compositions, upon thawing, form a single composition comprising said cells from different sources, thereby creating said pooled preparation in situ in said container.   
     
     
         26 . The method of  claim 25 , wherein said thawing comprises placing said container in a water bath at a temperature of from about 35° C. to about 40° C., for about 0.1 to about 4 minutes. 
     
     
         27 . The method of  claim 25 , wherein said cryopreserved self-sustaining bodies are single-cryopreserved compositions of cells. 
     
     
         28 . The method of  claim 25 , wherein said cells are hepatocytes. 
     
     
         29 . The method of  claim 28 , wherein at least about 70% of said hepatocytes in said pooled preparation are viable, based upon the total recovered hepatocytes in the preparation taken as 100%. 
     
     
         30 . The method of  claim 28 , further comprising subjecting said pooled preparation to density gradient centrifugation to separate viable and non-viable hepatocytes, wherein at least about 70% of said hepatocytes in said pooled preparation are viable after said density gradient centrifugation, based upon the total recovered hepatocytes in the preparation taken as 100%. 
     
     
         31 . The method of  claim 28 , wherein said hepatocytes are human hepatocytes. 
     
     
         32 . The method of  claim 28 , further comprising:
 incubating said pooled preparation with a xenobiotic; and   determining the metabolic fate of the xenobiotic, or the affect of the xenobiotic on the hepatocytes in said pooled preparation or on an enzyme or metabolic activity thereof.   
     
     
         33 . The method of  claim 25 , wherein said cells are cellular or subcellular fractions selected from the group consisting of mitochondria, cytosol, S9, and microsomes. 
     
     
         34 . The method of  claim 25 , wherein said pre-pooled stack consists of from about 10 to about 20 of said self-sustaining bodies. 
     
     
         35 . The method of  claim 25 , wherein each of said cryopreserved self-sustaining bodies is in the form of a pellet having a volume of from about 10 μL to about 2 mL. 
     
     
         36 . The method of  claim 25 , wherein said pooled preparation has a volume of from about 100 μL to about 40 mL. 
     
     
         37 .- 39 . (canceled)