CRYOPRESERVATION OF CELLS AND SUBCELLULAR FRACTIONS
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)