Novel Cellular Compositions and Methods for Their Preparation
The present invention relates to novel cell (e.g., hepatocyte, etc.) compositions and methods for their preparation and use. In particular, the invention concerns methods of processing preparations of such cells so as to permit their repeated cryopreservation and thawing while retaining substantial viability. The invention also concerns preparations of cells (e.g., hepatocytes) that have been repeatedly cryopreserved and thawed.
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10 . A method of producing a desired preparation of multi-cryopreserved hepatocytes, said hepatocytes being capable of being frozen and thawed at least two times, and in which greater than 50% of the hepatocytes of said preparation are viable after the final thaw, said method comprising:
(A) subjecting hepatocytes that have been frozen and thawed to density gradient fractionation to separate viable hepatocytes from non-viable hepatocytes,
(B) recovering the separated viable hepatocytes, and
(C) cryopreserving the recovered viable hepatocytes to thereby form said desired preparation of hepatocytes without requiring a density gradient step after thawing the hepatocytes for the second time, wherein the hepatocytes are not plated between the first and second cryopreservations, and wherein greater than 50% of the hepatocytes of said preparation are viable after the final thaw.
11 . The method of claim 10 , wherein said density gradient fractionation comprises density centrifugation through polyvinylpyrrolidone-coated colloidal silica particles.
12 . The method of claim 10 , wherein said hepatocytes are selected from the group consisting of human hepatocytes, porcine hepatocytes, simian hepatocytes, canine hepatocytes, feline hepatocytes, bovine hepatocytes, equine hepatocytes, ovine hepatocytes and rodent hepatocytes.
13 . The method of claim 12 , wherein said hepatocytes are human hepatocytes.
14 . The method of claim 10 , wherein said preparation comprises a pooled preparation of hepatocytes of multiple sources.
15 . The method of claim 14 , wherein said multiple sources are of the same gender, race, or health state.
16 . The method of claim 14 , wherein the hepatocytes of said pooled preparation of hepatocytes provide said pooled preparation with a desired level of a metabolic activity.
17 . The method of claim 16 , wherein said metabolic activity is selected from the group consisting of coumarin 7-hydroxylase (COUM), dextromethorphan O-demethylase (DEX), 7-ethoxycourmarin O-deethylase (ECOD), activities responsible for the phase II metabolism of 7-hydroxycoumarin (7-HCG and 7-HCS), mephenyloin 4-hydroxylase (MEPH), testosterone 6(β)-hydroxylase (TEST), tolbutamide 4-hydroxylase (TOLB), phenacetin O-deethylase (PHEN), and chlorzoxazone 6-hydroxylase (CZX).
18 . The method of claim 10 , wherein greater than about 70% of the hepatocytes of said preparation are viable.
19 . The method of claim 10 , wherein greater than 80% of the hepatocytes of said preparation are viable.
20 . A method of investigating in vitro drug metabolism comprising incubating hepatocytes of a multi-cryopreserved hepatocyte preparation in the presence of a xenobiotic, and determining the metabolic fate of the xenobiotic, or the effect of the xenobiotic on the hepatocytes or on an enzyme or metabolic activity thereof, wherein the hepatocytes have been frozen and thawed at least two times, and wherein greater than 50% of the hepatocytes of said preparation are viable after a final thaw and without requiring a density gradient step after thawing the hepatocytes for the second time, wherein the hepatocytes are not plated between the first and second cryopreservations.
21 . The method of claim 20 , wherein greater than about 70% of the hepatocytes of said preparation are viable.
22 . The method of claim 14 , wherein said multiple sources are of different gender, race or health state.
23 . The method of claim 16 , wherein said multiple sources are selected based upon metabolic activity, and wherein the pooled preparation exhibits a desired level of one or more metabolic activities.