U.S. patent application number 12/568443 was filed with the patent office on 2010-03-25 for novel cellular compositions and methods for their preparation.
Invention is credited to Daniel Dryden, James Hardy.
Application Number | 20100075295 12/568443 |
Document ID | / |
Family ID | 35136896 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100075295 |
Kind Code |
A1 |
Dryden; Daniel ; et
al. |
March 25, 2010 |
Novel Cellular Compositions and Methods for Their Preparation
Abstract
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.
Inventors: |
Dryden; Daniel;
(Westminster, MD) ; Hardy; James; (Ijamsville,
MD) |
Correspondence
Address: |
LOEB & LOEB, LLP
321 NORTH CLARK, SUITE 2300
CHICAGO
IL
60654-4746
US
|
Family ID: |
35136896 |
Appl. No.: |
12/568443 |
Filed: |
September 28, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11110879 |
Apr 21, 2005 |
7604929 |
|
|
12568443 |
|
|
|
|
Current U.S.
Class: |
435/1.3 ;
435/29 |
Current CPC
Class: |
C12N 5/067 20130101 |
Class at
Publication: |
435/1.3 ;
435/29 |
International
Class: |
A01N 1/02 20060101
A01N001/02; C12Q 1/02 20060101 C12Q001/02 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
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(.beta.)-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.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/110,879, filed Apr. 21, 2005, which
application is herein incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] 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.
BACKGROUND OF THE INVENTION
[0003] Hepatocytes are parenchymal liver cells, and make up 60-80%
of the cytoplasmic mass of the liver. Hepatocytes play a key role
in the detoxification, modification and excretion of exogenous and
endogenous substances (Ponsoda, X. et al. (2004) "Drug Metabolism
By Cultured Human Hepatocytes: How Far Are We From The In Vivo
Reality?" Altern. Lab Anim. 32(2):101-110). One of the detoxifying
functions of hepatocytes is to modify ammonia to urea for
excretion. They are also important in protein synthesis and
storage, in the transformation of carbohydrates and in the
synthesis of cholesterol, bile salts and phospholipids (Postic, C.
et al. (2004) "Role Of The Liver In The Control Of Carbohydrate And
Lipid Homeostasis," Diabetes Metab. 30(5):398-408). The hepatocyte
is the only cell in the body that manufactures albumin, fibrinogen,
and the prothrombin group of clotting factors. It is the main site
for the synthesis of lipoproteins, ceruloplasmin, transferrin, and
glycoproteins. Hepatocytes manufactures their own structural
proteins and intracellular enzymes. Hepatocytes are also important
depots for vitamin B12 and iron.
[0004] Due to these attributes, isolated and cultured hepatocytes
have become very attractive as models systems for the study of
liver functions (Chesne, C. et al. (1993) "Viability And Function
In Primary Culture Of Adult Hepatocytes From Various Animal Species
And Human Beings After Cryopreservation," Hepatology 18(2):406-414;
Guillouzo, A. et al. (1986) "Isolated and Cultured Hepatocytes,"
Paris: les Editions INSERM and London: John Libbey Eurotext);
Ponsoda. X. et al. (2004) "Drug Metabolism By Cultured Human
Hepatocytes: How Far Are We From The In Vivo Reality?" Altern. Lab
Anim. 32(2):101-110; Gomez-Lechon, M. J. et al. (2004) "Human
Hepatocytes In Primary Culture: The Choice To Investigate Drug
Metabolism In Man," Curr. Drug Metab. 5(5):443-462; Lemaigre, F. et
al. (2004) "Liver Development Update: New Embryo Models, Cell
Lineage Control, And Morphogenesis," Curr Opin Genet Dev.
14(5):582-590; Nanji, A. A. (2004) "Animal Models Of Nonalcoholic
Fatty Liver Disease And Steatohepatitis," Clin Liver Dis.
8(3):559-574; Hewitt, N. J. et al. (2004) Cryopreserved Rat, Dog
And Monkey Hepatocytes: Measurement Of Drug Metabolizing Enzymes In
Suspensions And Cultures." Hum Exp Toxicol. 23(6):307-316).
[0005] In addition to their use in liver models, hepatocytes have
the potential of being used to produce Bioartificial Livers (BALs)
or in hepatocyte transplantation that can provide liver functions
for individuals suffering from liver disease or liver failure.
Bioartificial Livers (BALs) are described by Anand, A. C. (1996)
"Bioartificial Livers: The State Of The Art," Trop Gastroenterol.
17(4):197-198, 202-211; Gan. J. H. et al. (2005) "Hybrid Artificial
Liver Support System For Treatment Of Severe Liver Failure," World
J. Gastroenterol. 11(6):890-894; Fukuda, J. et al. (2004)
"Hepatocyte Organoid Culture In Elliptic Hollow Fibers To Develop A
Hybrid Artificial Liver," hit J Artif Organs. 27(12):1091-1099;
Meng, Q. et al. (2004) "Hepatocyte Culture In Bioartificial Livers
With Different Membrane Characteristics," Biotechnol Lett.
26(18):1407-1412; Sekido, H. et al. (2004) "Usefulness Of
Artificial Liver Support For Pretransplant Patients With Fulminant
Hepatic Failure," Transplant Proc. 36(8):2355-2356; WO 03/105663A2,
WO05/000376A2, and U.S. Pat. No. 6,759,245. Hepatocyte
transplantation is described by Chan, C. et al. (2004) "Hepatic
Tissue Engineering For Adjunct And Temporary Liver Support:
Critical Technologies," Liver Transpl. 10(11):1331-1342; Lee, S. W.
et al. (2004) "Hepatocyte Transplantation: State Of The Art And
Strategies For Overcoming Existing Hurdles," Ann. Hepatol.
3(2):48-53; Horslen, S. P. (2004) "Hepatocyte Transplantation,"
Transplantation 77(10): 1481-1486; Burlina, A. B. (2004)
"Hepatocyte Transplantation For Inborn Errors Of Metabolism," J.
Inherit. Metab. Dis. 27(3):373-83; and Fox, I. J. et al. (2004)
"Hepatocyte Transplantation," Am. J. Transplant. 4 Suppl.
6:7-13.
[0006] A limiting factor in the development of such model systems
and to the development of Bioartificial Livers (BALs) has been the
erratic source and limited availability of hepatocytes, especially
human hepatocytes. Fresh hepatocytes are obtainable only from liver
resections or non-transplantable livers of multi-organ donors
(Lloyd, T. D. R. et al. (2003) Cryopreservation Of Hepatocytes: A
Review Of Current Methods For Banking," Cell and Tissue Culture
Banking 4:3-15). The supply of such tissue is inconsistent and
often geographically inconvenient in light of the limited
functional lifespan of liver tissue (Smrzova, J. et al. (2001)
"Optimization Of Porcine Hepatocytes Cryopreservation By Comparison
Of Viability And Enzymatic Activity Of Fresh And Cryopreserved
Cells," Acta Veterinaria Brunensis 70:141-147).
[0007] One approach to addressing this problem has involved the
development of hepatocyte storage conditions that allow hepatocytes
to be maintained over time with their cellular functions preserved.
Cryopreservation methods for the storage of hepatocytes have been
developed to address this need (see, Lloyd, T. D. R. et al. (2003)
Cryopreservation Of Hepatocytes: A Review Of Current Methods For
Banking," Cell and Tissue Culture Banking 4:3-15; Loretz, L. J. et
al. (1989) "Optimization Of Cryopreservation Procedures For Rat And
Human Hepatocytes," Xenobiotica. 19(5):489-498; Shaddock, J. G. et
al. (1993) "Cryopreservation And Long-Term Storage Of Primary Rat
Hepatocytes: Effects On Substrate-Specific Cytochrome
P450-Dependent Activities And Unscheduled DNA Synthesis," Cell Biol
Toxicol. 9(4):345-357; Novicki, D. L. et al. (1982)
"Cryopreservation Of structural proteins and intracellular enzymes.
Hepatocytes are also important depots for vitamin B12 and iron.
Isolated Rat Hepatocytes," In Vitro. 18(4):393-399; Zaleski, J. et
al. (1993) "Preservation Of The Rate And Profile Of Xenobiotic
Metabolism In Rat Hepatocytes Stored In Liquid Nitrogen," Biochem
Pharmacol. 46(1):111-116). Typically, such measures comprise
storage in liquid nitrogen (-196.degree. C.) or in frozen nitrogen
gas (-150.degree. C.). The ability to recover viable thawed cells
has been found to depend on multiple factors such as the rate of
freezing, the concentration of hepatocytes, the type of
cryoprotectant employed, and the final cooling temperature. Cell
concentrations of 10.sup.6-10.sup.7 cells/ml have been typically
employed. The isolated hepatocytes are typically incubated in
suspension for a period (e.g., 4-48 hours) to allow them to recover
from the isolation process. Thereafter, a cryoprotectant (such as
glycerol, DMSO, polyvinylpyrrolidone, or dextrin) is added, and the
hepatocytes are frozen. The art has developed various freezing
procedures, all designed to minimize or prevent the occurrence of
intracellular ice. The freezing rates typically vary from
-0.05.degree. C./min to -50.degree. C./min (see, Lloyd, T. D. R. et
al. (2003) Cryopreservation Of Hepatocytes: A Review Of Current
Methods For Banking," Cell and Tissue Culture Banking 4:3-15).
[0008] While the development of cryopreservation methods for the
storage of hepatocytes has significantly facilitated the
availability of human hepatocytes, cryopreservation has been found
to cause a significant decrease in cellular viability (e.g.,
25-35%) (Dou, M. et al. (1992) "Thawed Human Hepatocytes In Primary
Culture," Cryobiology 29:454-469; Alexandre, E. et al. (2002)
"Cryopreservation Of Adult Human Hepatocytes Obtained From Resected
Liver Biopsies," Cryobiology 44:103-113). Coundouris, J. A. et al.
(1993) reported viability of 67% after 24 hours, declining to 49%
after 14 days (Coundouris, J. A. et al. (1993) "Cryopreservation Of
Human Adult Hepatocytes For Use In Drug Metabolism And Toxicity
Studies." Xenobiotica. 23(12):1399-1409). Adams, R. M. et al. have
reported that the viability of hepatocytes may be enhanced to
greater than 90% using specialized cyropreservation fluids,
however, only 16% of cells were found to be capable of replication
(Adams, R. M. et al. (1995) "Effective.sub.--Cryopreservation And
Long-Terms Storage Of Primary Human Hepatocytes With Recovery Of
Viability, Differentiation, And Replicative Potential," Cell
Transplant. 4(6):579-586). Methods of cryopreservation are
disclosed in U.S. Pat. Nos. 5,795,711, 6,136,525, 5,895,745;
International Patent Publications WO04/009766, WO92/12722,
WO/0153462, European Patent No. EP0834252B, and United States
Patent Applications Publication Nos. US20020039786A1,
US20030134418A1. The poor recovery of cells when cryopreserved
continues to limit the use of hepatocytes in in vitro liver
models.
[0009] A second major problem affecting the use of both fresh and
cryopreserved hepatocytes is the variation of liver enzyme
expression that is observed in tissue from different donors (Li. A.
P. et al. (1999) "Present Status Of The Application Of
Cryopreserved Hepatocytes In The Evaluation Of Xenobiotics:
Consensus Of An International Expert Panel," Chem Biol Interact.
121(1):117-123; Li, A. P. et al. (1999) "Cryopreserved Human
Hepatocytes: Characterization Of Drug-Metabolizing Enzyme
Activities And Applications In Higher Throughput Screening Assays
For Hepatotoxicity, Metabolic Stability, And Drug-Drug Interaction
Potential," Chem Biol Interact. 121(1):17-35; O'Brien, Z. Z. et al.
(undated) "The Construction Of A Representative Human Cryopreserved
Hepatocyte Pool For Metabolism Study." One solution to this sample
variation involves pooling samples from different sources to
produce a "composite" hepatocyte preparation having the
characteristics of "average" liver cells. However, the frequency of
receipt of fresh tissue and the need to cryopreserve hepatocytes
immediately after isolation has been cited as preventing the
preparation of hepatocyte pools. Thus, multiple companies (e.g.,
Xenotech, LLC; BD Biosciences) refrain from selling pooled
hepatocytes thus forcing the end user to thaw and pool hepatocytes
from several different donors. This difficulty remains even though
pooled cryopreserved human hepatocytes are a valid model for
metabolic studies (Zhang, J. G. et al. (undated) "Validation Of
Pooled Cryopreserved Human Hepatocytes As A Model for Metabolic
Studies."
[0010] Thus, despite all prior advances, a need remains for
processes that would enable the availability of hepatocytes for
medical research and other purposes. A need further exists for a
stable and reproducible source of human hepatocytes. The present
invention permits the production and availability of hepatocyte
preparations that may be repeatedly cryopreserved and thawed
without unacceptable loss of viability. The invention thus permits
multiple hepatocyte samples to be pooled to produce pooled
hepatocyte preparations, especially pooled cryopreserved human
hepatocyte preparations. Using such advance, pooled cryopreserved
human hepatocytes are now commercially available from In Vitro
Technologies (Baltimore, Md.).
SUMMARY OF THE INVENTION
[0011] The present invention relates to novel cell (e.g.,
hepatocyte) compositions and methods for their preparation and use.
In particular, the invention concerns methods of processing
preparations of cells, especially hepatocytes, 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.
[0012] In detail, the invention particularly concerns a
multi-cryopreserved hepatocyte preparation comprising hepatocytes
that have been frozen and thawed at least two times, wherein
greater than 50% and more preferably 70% or more of the hepatocytes
of the preparation are viable.
[0013] The invention further concerns the embodiment of such a
multi-cryopreserved hepatocyte preparation wherein the 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.
[0014] The invention further concerns the embodiment of such a
multi-cryopreserved hepatocyte preparation wherein the preparation
comprises a pooled preparation of hepatocytes of multiple sources,
which may be of the same or different gender, race, or health
state, or which provide the pooled preparation with a desired level
of a metabolic activity (especially wherein the metabolic activity
is selected from the group consisting of COUM, DEX, ECOD, 7-HCG,
7-HCS, MEPH, TEST, PHEN and CZX).
[0015] The invention further concerns a method of producing a
desired preparation of multi-cryopreserved hepatocytes, the
hepatocytes being capable of being frozen and thawed at least two
times, and in which greater than 50% and more preferably 70% or
more of the hepatocytes of the preparation are viable, the method
comprising:
[0016] (A) subjecting hepatocytes that have been frozen and thawed
to density gradient fractionation (especially PERCOLL.RTM. density
centrifugation) to separate viable hepatocytes from non-viable
hepatocytes,
[0017] (B) recovering the separated viable hepatocytes, and
[0018] (C) cryopreserving the recovered viable hepatocytes to
thereby form the desired preparation of hepatocytes.
[0019] The invention further concerns the embodiment of such a
method in which the 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.
[0020] The invention further concerns the embodiment of such a
method in which the preparation comprises a pooled preparation of
hepatocytes of multiple sources, which may be of the same or
different gender, race, or health state, or which provide the
pooled preparation with a desired level of a metabolic activity
(especially wherein the metabolic activity is selected from the
group consisting of COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH, TEST, PHEN
and CZX).
[0021] The invention also concerns 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%
and more preferably 70% or more of the hepatocytes of the
preparation are viable.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0022] The present invention relates to novel cell compositions and
methods for their preparation and use. In particular, the invention
concerns methods of processing preparations of cells so as to
permit their repeated cryopreservation and thawing while retaining
substantial viability. The methods of the present invention are
generally applicable to a wide variety of cell types, including
hepatocytes, kidney cells, spleen cells, thymus cells, bone marrow
cells, stem cells, muscle cells (including cardiac muscle cells),
endocrine cells (including pancreatic cells, adrenal cells, thyroid
cells, etc.) epidermal cells, endodermal cells, etc. The methods of
the present invention are illustrated below with respect to a
preferred cell type: hepatocytes.
[0023] The invention also concerns preparations of cells, e.g.,
hepatocytes, that have been repeatedly cryopreserved and thawed to
obtain a high viability preparation useful for a variety of
experimental, diagnostic and therapeutic purposes. The present
invention extends the ability of hepatocytes to be cryopreserved
and thawed for later use, so as to permit hepatocyte preparations
to be repeatedly cryopreserved and thawed without an unacceptable
loss of viability.
[0024] As used herein, the term "cell preparation" denotes a liquid
or frozen composition of cells from one or more sources (e.g.,
"hepatocyte preparation", denotes a composition of liver cells from
one or more sources). The sources may be primary cells that have
been dissociated from or isolated from tissue as by resection,
biopsy, or from donor organs, or they may be secondary,
immortalized or transformed cell cultures. The cells may be derived
from any mammalian source, including human, porcine, simian,
canine, feline, bovine, equine, ovine or rodent sources. The use of
human, porcine or rodent (especially rat) cells is preferred.
Preferably, greater than 50% and more preferably 70% or more of the
hepatocytes of such preparations will be viable.
[0025] As used herein, the term "multi-cryopreserved cell
preparation" denotes a cell preparation that has been frozen and
then thawed at least two times (e.g., a "multi-cryopreserved
hepatocyte preparation" denotes a hepatocyte preparation that has
been frozen and then thawed at least two times). Such preparations
may have been frozen and thawed three, four, five, or more
times.
[0026] The term "pooled preparation" denotes a cell (e.g.,
hepatocyte) preparation in which the cells (e.g., hepatocytes) are
derived from two, three, four, five, or more different sources,
such as different donors, biopsies, tissue resections from
different tissue samples or different tissue sources, or different
primary, secondary, immortalized or transformed cell (e.g.,
hepatocyte) cultures. The cells of such pooled preparations may be
randomly selected cell, or may have been selected to provide the
pooled preparation with a desired level of one or more metabolic
activities (such as for example, a preparation of hepatocytes
having a desired level of COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH,
TEST, PHEN and/or CZX activity), or a desired cell characteristic
(such as, for example, a preparation of hepatocytes derived from
sources of the same gender, age, race (e.g., Caucasian, etc.), or
health state (e.g., hepatocytes of hepatitis virus-infected liver,
hepatocytes of HIV-1 infected liver, hepatocytes of healthy liver,
hepatocytes of cigarette smokers, hepatocytes of individuals
suffering from cirrhosis of the liver, or from other diseases or
conditions). For example, to obtain a pooled hepatocyte preparation
with minimal DEX activity, a pooled preparation could be prepared
from Lot. Nos. 067, CEK, ETR, PFM, VTA, or WWM (see, Table
III).
[0027] In a preferred embodiment, illustrated with respect to
hepatocyte cells, the practice of the invention comprises some or
all of the following steps: the isolation of hepatocytes, a first
cryopreservation of the isolated primary hepatocytes to obtain a
first cryopreserved hepatocyte preparation, the thawing of the
first cryopreserved hepatocyte preparation to obtain viable
hepatocytes, and the reformulation of the thawed viable hepatocytes
to permit their further storage and use through repeated
cryopreservation and thawing to obtain viable hepatocytes.
The Isolation Of Hepatocytes
[0028] Any of a wide variety of methods may be employed or adapted
to permit the isolation of the primary hepatocytes used in the
present invention. For 30 example, suitable techniques for the
isolation of hepatocytes are outlined in Morsiani et al, (1995)
"Automated Liver Cell Processing Facilitates Large Scale Isolation
And Purification Of Porcine Hepatocytes," ASAIO Journal 41:155-161
and in Seglen, P. O. (1976) "Preparation Of Isolated Rat Liver
Cells," Meth. Cell Biol. 13:29-83). Specific reference is made to
the two-step collagenase digestion procedure described in Li, A. P.
et al. (1992) "Isolation And Culturing Of Hepatocytes From Human
Liver," J. Tissue Cult. Meth. 14:139-146.
[0029] The hepatocytes may be cultured in any suitable hepatocyte
culture medium. By way of illustration and not limitation mention
may be made of the following culture media Chee's Essential Media
(Hamilton, G. A. et al. (2001) "Effects Of Medium Composition On
The Morphology And Function Of Rat Hepatocytes Cultured As
Spheroids And Monolayers," In Vitro Cell Dev Biol Anim.
37(10):656-667; Zurlo, J. et al. (1996) "Characterization Of A
Primary Hepatocyte Culture System For Toxicological Studies," In
Vitro Cell Dev Biol Anim. 32(4):211-220; Arterburn, L. M. et al.
(1995) "A Morphological Study Of Differentiated Hepatocytes In
Vitro," Hepatology 22(1):175-187), Modified Eagle Medium (or
Dulbecco's Modified Eagle Medium) (Arikura, J. et al. (2002) "UW
Solution: A Promising Tool For Cryopreservation Of Primarily
Isolated Rat Hepatocytes," J Hepatobiliary Pancreat Surg.
9(6):742-749; Washizu, J. et al. (2000) "Amino Acid Supplementation
Improves Cell-Specific Functions Of The Rat Hepatocytes Exposed To
Human Plasma," Tissue Eng. 6(5):497-504; Iwata, H. et al. (1999)
"In Vitro Evaluation Of Metabolic Functions Of A Bioartificial
Liver," ASAID J. 45(4):299-306; Stutenkemper, R et al. (1992) "The
Hepatocyte-Specific Phenotype Of Murine Liver Cells Correlates With
High Expression Of Connexin32 And Connexin26 But Very Low
Expression Of Connexin43," Exp Cell Res. 201(1):43-54), Leibowitz
medium (Coundouris, J. A. et al. (1993) "Cryopreservation Of Human
Adult Hepatocytes For Use In Drug Metabolism And Toxicity Studies,"
Xenobiotics: 23(12):1399-1409), Waymouth (Vind, C. et al. (1992)
"Regulation By Growth Hormone And Glucocorticoid Of Testosterone
Metabolism In Long-Term Cultures Of Hepatocytes From Male And
Female Rats," Biochem Pharmacol. 44(8):1523-1528; Nemoto, N. et al.
(1991) "Proline Is Required For Transcriptional Control Of The
Aromatic Hydrocarbon Inducible P(1) 450 Gene In C57BL16 Mouse
Monolayer-Cultured Hepatocytes," Jpn J Cancer Res. 82(8):901-908;
Dich, J. et al. (1988) "Long-Term Culture Of Hepatocytes: Effect Of
Hormones On Enzyme Activities And Metabolic Capacity," Hepatology.
8(1):39-45; Goethals, F. et al. (1984) "Critical Biochemical
Functions Of Isolated Hepatocytes As Sensitive Indicators Of
Chemical Toxicity," Fundam Appl Toxicol. 4 (3 Pt 1):441-450),
Kreb's medium (House, J. D. (2001) "Threonine Metabolism In
Isolated Rat Hepatocytes," Am J Physiol Endocrinol Metab.
281(6):E1300-1307; Irvine, F. et al. (1993) "Extracellular Calcium
Modulates Insulin's Action On Enzymes Controlling Cyclic AMP
Metabolism In Intact Hepatocytes," Biochem J. 293 (Pt 1):249-253;
Marsh, D. C. et al. (1991) "Hypothermic Preservation Of
Hepatocytes. III. Effects Of Resuspension Media On Viability After
Up To 7 Days Of Storage," Hepatology 13(3):500-508), etc.
[0030] In a preferred embodiment, hepatocytes are cryopreserved in
a medium containing approximately 10% DMSO and approximately 90%
fetal bovine serum (Loretz, L. J. et al. (1989) "Optimization Of
Cryopreservation Procedures For Rat And Human Hepatocytes,"
Xenobiotics 19:489-498; Ruegg, C. E. et al. (1997) "Cytochrome-P450
Induction and Conjugated Metabolism In Primary Human Hepatocytes
After Cryopreservation," In Vitro Toxicol. 10:217-222).
[0031] The viability of the isolated hepatocytes may be determined
using any of a variety or methods. Preferable, such viability will
be determined using the Trypan blue exclusion method (see, e.g.,
Berry, M. N. et al. (1992) "Techniques For Pharmacological And
Toxicological Studies With Isolated Hepatocyte Suspensions," Life
Sci. 51(1):I-16). Thus the phrases "viable hepatocytes" or "percent
viability", as used herein, refers to hepatocyte viability as
assessed using the method of Trypan Blue exclusion.
Cryopreservation of the Isolated Primary Hepatocytes
[0032] The hepatocytes of the present invention are preferably
cryopreserved using liquid nitrogen, and most preferably within 36
hours of their isolation. Considerations for the cryopreservation
of human hepatocytes are discussed in Lloyd, T. D. R. et al. (2003)
Cryopreservation Of Hepatocytes: A Review Of Current Methods For
Banking," Cell and Tissue Culture Banking 4:3-15. Suitable
procedures for the cryopreservation of hepatocytes may also be
found in the following documents: Adams, R. M. et al. (1995)
"Effective Cryopreservation And Long-Term Storage Of Primary Human
Hepatocytes With Recovery Of Viability, Differentiation, And
Replicative Potential," Cell Transplant. 4(6):579-586; Chesne, C.
et al. (1993) "Viability And Function In Primary Culture Of Adult
Hepatocytes From Various Animal Species And Human Beings After
Cryopreservation," Hepatology 18(2):406-414; Coundouris, J. A. et
al. (1993) "Cryopreservation Of Human Adult Hepatocytes For Use In
Drug Metabolism And Toxicity Studies," Xenobiotica.
23(12):1399-1409; Hewitt, N. J. et al. (2004) Cryopreserved Rat Dog
And Monkey Hepatocytes: Measurement Of Drug Metabolizing Enzymes In
Suspensions And Cultures," Hum Exp Toxicol. 23(6):307-316; Novicki,
D. L. et al. (1982) "Cryopreservation Of Isolated Rat Hepatocytes,"
In Vitro. 18(4):393-399; Shaddock, J. G. et al. (1993)
"Cryopreservation And Long-Term Storage Of Primary Rat Hepatocytes:
Effects On Substrate-Specific Cytochrome P450-Dependent Activities
And Unscheduled DNA Synthesis," Cell Biol Toxicol.9(4):345-357;
Zaleski, J. et al. (1993) "Preservation Of The Rate And Profile Of
Xenobiotic Metabolism In Rat Hepatocytes Stored In Liquid
Nitrogen," Biochem Pharmacol. 46(1):111-116.
[0033] Preferably, isolated hepatocytes are suspended in a
cryoprotective medium, and the suspended cells are dispensed into
freezer-safe containers. A cryoprotective medium typically
comprises a hepatocyte culture medium that contains at least one
cryoprotectant that minimizes the deleterious effects of
cryopreservation such as the formation of intracellular ice during
freezing. By way of illustration and not limitation, the following
commonly used cryoprotectants are listed: dimethylsulfoxide (DMSD),
polyethylene glycol, amino acids, propanediol, and glycerol. A
preferred cryoprotectant of the present invention is DMSD. Suitable
cryoprotectants and methods for their use in hepatocyte
cryopreservation can be found, for example, in: Lore, L. J. et al.
(1989) "Optimization Of Cryopreservation Procedures For Rat And
Human Hepatocytes," Xenobiotica. 19(5):489-498; Chesne, C. et al.
(1993) "Viability And Function In Primary Culture Of Adult
Hepatocytes From Various Animal Species And Human Beings After
Cryopreservation," Hepatology 18(2):406-414; Diener, B. et al.
(1993) "A Method For The Cryopreservation Of Liver Parenchymal
Cells For Studies Of Xenobiotics," Cryobiology 30(2):116-127;
Lawrence, J. N. et al. (1991) "Development Of An Optimal Method For
The Cryopreservation Of Hepatocytes And Their Subsequent Monolayer
Culture. Toxicology In Vitro," 5(1):39-51; Houle, R. et al. (2003)
"Retention of Transporter Activities in Cryopreserved, Isolated Rat
Hepatocytes," Drug Metab. Disposit. 31(4):447-451; Silva, J. M. et
al. (1999) "Induction Of Cytochrome-P450 In Cryopreserved Rat And
Human Hepatocytes," Chem-Biol Interact 121:49-63.
[0034] The isolated hepatocytes are preferably suspended in a
cryoprotective medium in preparation for freezing. The suspended
cells are preferably dispensed into freezer resistant containers at
a cell density of from about 105 cells/ml to about 4.times.10'
cells/ml. Preferred freezing volumes range from 0.1-10.0 ml. The
preferred freezing volume is 1.0 ml.
[0035] The dispensed hepatocytes are then preferably cryopreserved
using a controlled rate freezing process, most preferably at a
freezing rate of between about -1.degree. C./min to about
-25.degree. C./min until a final temperature of about -90.degree.
C. is reached. During the initial phase of the cryopreservation
process, seeding may be employed to induce controlled
crystallization or ice formation in cell suspensions that have
already been cooled to below the freezing point of the culture
medium. Such seeding serves to minimize ice formation-related
damage and therefore may be beneficial to cell viability. Suitable
seeding methods include inserting a cold metal rod into the
freezing containers, and introducing a blast of liquid nitrogen
into the freezing containers.
[0036] Once the desired final temperature has been reached, the
frozen cell samples may be transferred to liquid nitrogen freezers
for prolonged storage. The frozen samples may be stored in either
the liquid nitrogen phase or the gas phase of liquid nitrogen.
Preferably storage is accomplished in the gas phase of liquid
nitrogen. The frozen samples may be stored in this manner for days,
months, or years, with the length of storage in the gas phase of
liquid nitrogen having little effect on the post-thaw viability and
function.
The Thawing of Cryopreserved Hepatocytes
[0037] Frozen samples may be thawed for further processing by
removing them from the presence of liquid nitrogen or liquid
nitrogen vapor. Frozen samples are preferably thawed by placing the
samples immediately into a prewarmed water bath having a
temperature of between about 37.degree. C. to about 42.degree. C.
Preferably, cells are thawed to at least the stage in which ice
chunks can be dislodged when the sample container is inverted. The
thawed cells are then preferably rapidly processed to remove the
cells from contact with DMSO, for example by PERCOLL.RTM.
(colloidal silica particles of 15-30 nm diameter (23% w/w in water)
which have been coated with polyvinylpyrrolidone (PVP)) gradient
centrifugation (as described below) or by sequential washings.
[0038] In a preferred embodiment, the cells are thawed into
Complete INVITROGRO.TM. CP medium (In Vitro Technologies,
Baltimore, Md.; Roymans. U. et al. (2004) "Determination Of
Cytochrome P450 IA2 And Cytochrome P4503a4 Induction In
Cryopreserved Human Hepatocytes," Biochem. Pharmacol.
67(3):427-437) (hepatocyte plating medium, which contains water,
Dulbecco's Modified Eagle Medium, sodium bicarbonate, HEPES,
fructose, bovine serum albumin, sodium hydroxide, MEM non-essential
amino acids, insulin, hydrocortisone, and newborn calf serum). The
medium is prepared by thawing TORPEDO.TM. Antibiotic Mix (In Vitro
Technologies, Baltimore, Md.) (a mixture of antibiotics selected to
inhibit bacterial growth in hepatocyte cell cultures that contains
penicillin, streptomycin, gentamicin, amikacin and fungizone) to
37.degree. C. in a water bath until thawed, and then removed from
the water bath. 1.0 ml of TORPEDO.TM. Antibiotic Mix is then mixed
with 45 ml INVITROGRO.TM. CP medium. Following the addition of
TORPEDO.TM. Antibiotic Mix, the shelf life for the complete medium
is 7 days. When thawing a single vial, the INVITROGRO.TM. CP medium
is prewarmed to approximately 37.degree. C. 5 ml of warmed
INVITROGRO.TM. CP medium is added to a sterile 50 ml conical tube.
The vial of frozen hepatocytes is carefully removed from the
freezer. If the vial was stored in the liquid phase, its cap is
carefully removed, any liquid nitrogen present in the vial is
decanted, and the cap is reclosed before placing the vial into the
water bath. It is preferred to then immediately immerse the vial
into a 37.degree. C. water bath, and to shake the vial gently until
the ice is entirely melted, but no longer than it takes to
completely thaw the vial. It may be helpful to remove any labels
from the vial so that it will be easier to view the vial contents.
The thawed contents are then emptied into the pre-warmed
INVITROGRO.TM. CP medium. 1.0 ml of pre-warmed INVITROGRO.TM. CP
medium then is added to each vial to resuspend any remaining cells.
The contents of the vial are then decanted or pipetted into the
hepatocyte suspension. The hepatocytes are preferably resuspended
by gently inverting the receiving container (e.g., vial, test rube,
etc.) several (e.g., three) times.
[0039] When thawing multiple vials, it is preferred that all of the
vials be thawed in the water bath simultaneously. As before, the
medium (preferably, INVITROGRO.TM. CP medium) should be warmed to
37.degree. C. It is desirable to ensure that there is enough medium
to permit 5 ml of pre-warmed INVITROGRO.TM. CP medium to he used
for each vial of cryopreserved hepatocytes. After vials have
thawed, their caps should be quickly removed and their contents
poured into a sterile tube or beaker that contains at least 5 ml of
pre-warmed INVITROGRO.TM. CP medium per vial thawed. For example,
25 ml of media is preferably employed for 5 vials in a container
that can hold a volume of 50 ml.
[0040] If desired, the total cell count and the number of viable
cells may be determined using the Trypan Blue exclusion method.
Cells may he diluted to 0.70.times.10.sup.6 viable cells/ml with
INVITROGRO.TM. CP medium.
The Reformulation of Thawed Hepatocytes to Permit Further
Cryopreservation and Thawing
[0041] One aspect of the present invention concerns the ability to
reformulate the thawed cells so that they may be refrozen and
rethawed on one or more subsequent occasions. Such
multi-cryopreserved hepatocyte preparations have multiple uses.
They may be used in bioartificial livers, liver cell transplants,
liver assist devices, hepatocyte transplantations, and in vitro
applications. In particular, multi-cryopreserved hepatocyte
preparations may be used in in vitro drug metabolism studies (for
example, in identifying hepatocytes with unique characteristics
(e.g., metabolic polymorphisms, genetic polymorphisms, etc.), in
studies on the metabolic fate of the xenobiotic and studies on the
affect of the xenobiotic in altering the drug-metabolizing enzyme
profile of the hepatocytes, in inhibition studies to determine the
IC50 of xenobiotics on liver enzymes and functions (e.g.
cholesterol metabolism), in gene induction studies with
xenobiotics, in protein induction studies with xenobiotics, in
toxicity assessment of xenobiotics on hepatocytes, transport
studies with xenobiotics (e.g. studies on P-glycoprotein transport
systems, organic ion transporters, organic cation transporters,
etc.), in metabolic clearance studies with xenobiotics, and in
efficacy assays (e.g. lipoprotein processing, gluconeogenesis,
protein secretion etc.). Multi-cryopreserved hepatocyte
preparations may also be used to study or propagate hepatitis
viruses and other infectious viruses and agents. Recovered cells
may be reformulated for use in DNA, mRNA or proteomic studies or in
studies of metabolic polymorphisms. Multi-cryopreserved hepatocyte
preparations may also be used in metabolic clearance studies and
efficacy assays (e.g., lipoprotein processing, gluconeogenesis,
protein secretion, etc.). Cells may be reformulated for use in
seeding bioreactors for large scale incubations or as models for
gene regulation via micro RNA, or for use in combination systems
with other cell types (e.g. non-parenchymal cells from liver or
cells from other sources, e.g. Caco-2 cells).
[0042] In a preferred embodiment of the invention, such
reformulation comprises separating viable and non-viable cells
prior to a subsequent refreezing. Density gradient centrifugation
is preferably employed for this purpose. For example, a 30%
PERCOLL.RTM. gradient centrifugation procedure may be employed
(Madan. A. et al. (1999) "Effect of Cryopreservation on Cytochrome
P-450 Enzyme Induction in Cultured Rat Hepatocytes, Drug Metab.
Dispos. 27(3):327-335; Sun, E. L. et al. (1990) "Cryopreservation
Of Cynomologus Monkey (Macaca fascicularis) Hepatocytes For
Subsequent Culture And Protein Synthesis Studies," In Vitro Cell
Development and Biology 25:147-150; Lawrence, J. N. et al. (1991)
"Development Of An Optimal Method For The Cryopreservation Of
Hepatocytes And Their Subsequent Monolayer Culture," Toxicology In
Vitro, 5(1):39-51; Dou, M. et al. (1992) "Thawed Human Hepatocytes
In Primary Culture," Cryobiology 29(4):454-469; Utesch, D. et al.
(1992) "Characterization Of Cryopreserved Rat Liver Parenchymal
Cells By Metabolism Of Diagnostic Substrates And Activities Of
Related Enzymes," Biochemical Pharmacology 44:309-315). For
example, the thawed cells may be resuspended in a prewarmed
(approximately 37.degree. C.) 30% PERCOLL.RTM. isotonic
fractionation buffer and then centrifuged at 100.times.g at room
temperature for twenty minutes to pellet viable cells. The
supernatant is discarded and the cells are resuspended in media for
a subsequent cryopreservation step directly or for further
processing prior to cryopreservation.
[0043] Cryopreserved preparations that result from the freezing of
a previously frozen-thawed preparation will preferably have a
post-thaw cell viability of greater than 50% and more preferably
70% or more. Such high viabilities enable the present invention to
accomplish the repeated freezing and thawing of hepatocytes without
unacceptable losses of cells or the need for ever greater samples
sources.
Pooled Hepatocyte Preparations
[0044] The capacity of the present invention to enable the repeated
freezing and thawing of hepatocytes additionally facilitates the
production of pooled hepatocyte preparations, especially pooled
human hepatocyte preparations. As discussed above, individual liver
samples yield hepatocytes having differing metabolic capabilities.
In order to facilitate the reproducible use or study of
hepatocytes, it is desirable to minimize hepatocyte differences
attributable to such sample variation by pooling hepatocytes from
different sources to obtain a composite or "average" hepatocyte
preparation. Such composite hepatocyte preparations may thus be
formulated so as to provide a preparation having the metabolic
activities of an "average" hepatocyte sample or a preparation whose
hepatocyte enzyme functions approximate the hepatocyte enzyme
functions of freshly isolated hepatocytes. Such metabolic
activities may include, for example, some or all of the following
enzymatic activities: coumarin 7-hydroxylase (COUM),
dextromethorphan O-demethylase (DEX), 7-ethoxycoumarin O-deethylase
(ECOD), activities responsible for the phase II metabolism of
7-hydroxycoumarin (7-HCG and 7-HCS), mephenyloin 4-hydroxylase
(MEPH), testosterone 6(B)-hydroxylase (TEST), tolbutamide
4-hydroxylase (TOLB), phenacetin O-deethylase (PHEN), or
chlorzoxazone 6-hydroxylase (CZX). The substrates, methods of
measurements and assay units for assays of such metabolic
activities are provided in Table 1.
TABLE-US-00001 TABLE I Hepatocyte Metabolic Activities Abbreviation
Substrate/Assay Method of Measurement Units 7-HCG 7- Phase II
metabolism of 7- Pmol/min/10.sup.6 cells hydroxycoumarin
hydroxycoumarin glucuronide 7-HCS 7- Phase II metabolism
Pmol/min/10.sup.6 culls hydroxycoumarin of 7-hydroxycoumarin
sulfate NAT1 p-aminobenzoic acid N-acetylation of p- nmol/mg/min
aminobenzoic acid NAT2 Sulfamethazine N-acetylation of nmol/mg/min
Sulfamethazine VBTY viability Trypan Blue .TM. percentage Exclusion
AP Alkaline Phosphatase Sigma kit units/mg protein GGT
gamma-glutamyl GGT stain positive transpeptidase UGT1
7-hydroxycoumarin Phase I1 metabolism 169 pmol/mg/min of
7-hydroxycoumarin P450 cytochrome p450 carbon monoxide Not
determined for content difference spectrum cryo hepatocytes nmol/mg
protein CZX chlorzoxazone chlorzoxazone 6- 31.1 pmol/mg/min*
hydroxylation COUM coumarin coumarin 7- 50.0 pmol/mg/min*
hydroxylation DEX dextromethorphan dextromethorphan O- 21.4
pmol/mg/min* demethylation MEPH mephenytoin mephenytoin 4- 24.1
pmol/mg/min* hydroxylation PHEN phenacetin phenacetin O- 28.9
pmol/mg/min* deethylation TEST testosterone testosterone 6(beta)-
96.8 pmol/mg/min* hydroxylation TOLB tolbutamide tolbutamide 4-
30.6 pmol/mg/min* hydroxylation PROT protein content Pierce protein
kit Not determined for cryo hepatocytes mg/mL ECOD ethoxycoumarin
7-ethoxycoumarin O- 37.3 pmol/mg/min* demethylation
[0045] For example, preferred preparations of pooled hepatocytes
will yield assay values within the ranges identified in Table II.
Alternatively, the hepatocytes samples used to form the pooled
preparation may be selected so as to maximize, minimize, or
emphasize certain hepatocyte functions over other functions so as
to yield a pooled preparation that exhibits a user desired profile
of liver cell function(s).
[0046] The pooled hepatocyte preparations of the present invention
may comprise hepatocytes obtained from the same source at differing
times, or from two or more different sources. Preferably, pooled
hepatocyte preparations will result from the pooling of hepatocytes
obtained from three, four, five, six or more different sources.
[0047] Most preferably, the pooled hepatocyte preparations of the
invention will comprise at least one population of hepatocytes that
were cryopreserved prior to pooling. For example, a pooled
hepatocyte preparation may comprise one or more hepatocyte
specimens that were cryopreserved prior to pooling with one or more
freshly isolated hepatocyte specimens. Alternatively, a pooled
hepatocyte preparation may comprise only hepatocyte specimens that
were previously cryopreserved. Table II provides the normal range
(i.e., the range between Assay Minimum and Assay Maximum for each
Assay). Table II values are derived data of the last 150+ lots of
human cryopreserved hepatocytes.
TABLE-US-00002 TABLE II Hepatocyte Assays Normal Range Assay
(pmol/min/10.sup.6 cells) coumarin 7-hydroxylation 1 to 154
Dextromethorphan O-demethylation 0.5 to 96 7-ethoxycoumarin
O-deethylation 1 to 154 Phase I metabolism of 7-hydroxycoumarin 2
to 545 Phase II metabolism of 7-hydroxycoumarin 0 to 110
mephenytoin 4-hydroxylation 0.2 to 442 testosterone
6(beta)-hydroxylation 2 to 675 tolbutamide 4-hydroxylation 1.8 to
82 phenacetin O-deethylation 1 to 125 chlorzoxazone 6-hydroxylation
2 to 215
[0048] In certain embodiments of the invention, hepatocyte
preparations will have assay values in the above stated ranges for
at least three of, and preferably for at least four of, still more
preferably for at least six of, and most preferably for at least
eight of the following assays: the COUM assay; the DEX assay; the
ECOD assay; the 7-HCG assay; the 7-HCS assay; the MEPH assay; the
TEST assay; the TOLB assay; the PHEN assay; the CZX assay.
[0049] If desired, the cryopreserved hepatocytes may be plated on
to collagen-coated tissue culture plates, or tissue culture plates
coated with other extracellular matrix proteins including but not
limited to laminin, fibronectin, entactin, poly-L-lysine, gelatin,
or any combination thereof. Preferably, this is accomplished by
diluting an appropriate volume (e.g., 0.2 ml to 2.5 ml) of diluted
cells (e.g., cells having a concentration of approximately
0.7.times.106 cells/ml) onto the plates. For plating on a 96-well
microliter plate, it is desirable to further dilute the cell
suspension to a concentration of 0.35.times.106 cells/ml with
INVITROGRO.TM. Medium, and to add 100 .mu.l of the cell suspension
to each well. It is preferred to even distribute the cells in the
wells. This can be accomplished by gently shaking the plates in a
back-and-forth and side-to-side manner; the use of a circular
motion will cause the cells to evenly pool in the center of the
wells. Human hepatocytes handled in this manner will attach to the
plates in 2-4 hours, however, if minimal handling is desired, the
cells can be allowed to attach overnight.
[0050] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples, which are provided by way of illustration and are not
intended to be limiting of the present invention unless
specified.
Example 1
Refreezing of Thawed Hepatocyte Preparations
[0051] Cryopreserved hepatocytes are thawed and refrozen as
indicated below.
[0052] Materials: 30 cc syringe, two couplers, PERCOLL.RTM. in a 1
L bag, INVITROGRO.TM. CP medium in a 2 L bag, 1-2 L autoclaved
beaker, heater, waterbath, centrifuge tube rack.
[0053] Procedure:
[0054] 1) Set up a recirculating waterbath heater at 37-42.degree.
C.
[0055] 2) Add approximately 200-400 mls of INVITROGRO.TM. CP medium
to a 1-2 L beaker. Equip Biological Safety Cabinet with a manual
pipet or a liquid handling robot.
[0056] 3) Remove approximately 50 cryovials from dewar receptacle
and quickly place them in 2 test tube racks. When possible, space
vials apart.
[0057] 4) Submerge solid cell suspensions into heated waterbath
until the ice chunks can be dislodged when the vial is
inverted.
[0058] Pour cell suspension from each vial into the beaker. Add 1
ml of INVITROGRO.TM. CP medium from the small beaker into each vial
to rinse and pour contents into the beaker. Transfer thawed cell
suspension into a 1 L sterile bag.
[0059] 6) Attach bag, INVITROGRO.TM. CP medium in a 2 L bag and
PERCOLL.RTM. bagged at 30% onto COBE.RTM. automated cell processor
(a centrifugal system incorporating a flexible membrane that allows
the removal of fluids while spinning through use of a rotating
seal, hydraulic pump and flexible membrane) and process according
to standard practices.
[0060] 7) Perform a cell count.
[0061] 8) Cryopreserve cell suspension.
[0062] PERCOLL.RTM./REDIGRAD.TM. (Amersham Biosciences) is employed
for the PERCOLL.RTM. density centrifugation. PERCOLL.RTM. is
composed of colloidal silica coated with polyvinylpyrrolidone
(PVP). The REDIGRAD.TM. formulation is also composed of colloidal
silica but is covalently coated with silane. These coatings are
thought to render the material non-toxic and ideal for use with
biological materials. Both particles have a density of 1.13 g/ml.
Centrifugation of samples in the presence of
PERCOLL.RTM./REDIGRAD.TM. results in the spontaneous formation of a
density gradient due to the heterogeneity of particle sizes in the
medium.
[0063] PERCOLL.RTM./REDIGRAD.TM. is best used in balanced salt
solutions, such as physiological saline (0.15M NaCl), although 0.25
M sucrose may be employed. The addition of 9 parts (v/v) of
PERCOLL.RTM./REDIGRAD.TM. to 1 part (v/v) of either 1.5 M NaCl,
10.times. concentrated cell culture medium, or 2.5 M sucrose will
result in a solution adjusted to about 340 mOs/kg H.sub.2O,
Solutions of different osmotic pressure can be produced by
adjusting the relative volumes of PERCOLL.RTM./REDIGRAD.TM. and
salt or sucrose solution. (Vincent, R. et al. (1984) "Adjustment Of
The Osmolality Of Percoll For The Isopycnic Separation Of Cells And
Cell Organelles," Anal. Biochem. 141(2):322-328). The final
adjustment to the required osmolality can be carried out by the
addition of salts or distilled water. Concentrations other than
10.times. physiological saline may also be used satisfactorily.
[0064] PERCOLL.RTM./REDIGRAD.TM. will form self-generated gradients
by centrifugation in fixed-angle rotor heads after 15 minutes.
Hepatocytes can be separated by centrifugation at 50-100 g.sub.av
in fixed-angle or swinging bucket rotor heads after 10-30
minutes.
Example 2
Variation of Primary Hepatocyte Samples
[0065] To illustrate the sample-to-sample variation of different
sources of individual (unpooled) hepatocytes, hepatocytes are
isolated from 82 different donors and analyzed for cell viability
and enzyme function. The following metabolic activities are
evaluated: COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH, TEST, TOLB, PHEN,
and CZX. The results are shown in Table III.
TABLE-US-00003 TABLE III Variation of Hepatocyte Samples 7- 7- Lot
No. Sex % V COUM DEX ECOD HCG HCS MEPH TEST TOLB PHEN CZX 067 M 62%
67 1 70 231 24 2 44 35 27 24 086 F 74% 51 23 10 50 9 1 38 13 BQL 18
089 F 77% 25 21 8 23 6 1 11 13 BQL 29 090 F 74% 30 25 7 13 BQL 2 19
15 BQL 16 091 F 73% 13 29 66 44 10 12 252 36 27 36 094 F 67% 41 12
37 24 4 21 126 40 19 87 099 F 86% 21 15 7 4 BQL 1 60 10 BQL 22 104
M 81% 63 21 44 247 25 2 58 37 8 20 105 M 67% 59 15 24 38 14 1 29 27
12 27 110 F 77% 45 24 35 23 4 6 206 11 54 36 111 F 71% 4 10 9 2 3 3
147 2 19 19 114 F 75% 39 23 21 10 5 5 59 TBD 3 45 122 M 79% 26 30
29 80 5 1 42 23 4 25 129 F 90% 4 24 27 67 18 1 16 33 10 51 ACU F
81% 53 8 25 74 18 8 80 16 4 23 AIT F 83% 45 29 13 118 14 BQL 82 15
11 7 AOK M 73% 60 21 58 283 64 5 86 30 24 21 ATR M 73% 7 11 1 39 11
BQL 11 8 3 4 AVF M 70% 59 13 50 210 29 BQL 54 37 11 24 BTP M 88% 66
29 36 214 25 3 50 45 11 12 CEC M 86% 47 26 17 105 27 21 32 57 38 36
CEK F 80% 55 2 39 141 5 16 302 41 7 16 CHD F 77% 30 14 53 471 42 4
28 23 13 26 CPN M 81% 28 6 40 100 13 2 168 14 37 21 ECM M 85% 8 11
10 55 18 6 81 18 21 9 EFA M 69% 9 9 35 47 5 18 66 12 47 67 EHI F
90% 88 3 56 291 45 1 248 21 45 43 EJR F 75% 89 14 32 288 43 8 62 41
1 41 ENR M 73% 69 28 27 124 31 107 77 36 38 32 EOB M 88% 14 11 18
65 9 31 49 14 21 35 ETR F 88% 30 1 34 13 13 7 13 13 37 69 EVY M 80%
2 20 23 218 77 33 24 25 17 38 FNL M 85% 17 27 62 282 32 6 6 50 14
24 FRW M 75% 46 22 16 106 17 48 25 54 44 8 GBE F 77% 5 49 31 165 20
2 16 19 5 54 GNG F 74% 57 17 33 54 8 5 95 22 16 22 GTV F 71% 32 6 8
47 7 BQL 40 28 16 11 GUY M 92% 65 12 11 73 12 20 90 13 5 8 HHG M
83% 2 8 14 251 29 BQL 28 12 4 40 HRU M 90% 43 28 39 175 15 4 69 40
57 44 ICJ M 74% 134 20 60 287 17 BQL 129 82 28 7 IEM M 88% 34 17 23
129 34 72 48 23 19 34 IHR F 76% 17 43 8 84 9 9 95 46 41 7 LID M 86%
36 31 50 307 54 1 142 49 21 51 IRX F 73% 57 5 40 172 24 12 113 43 6
18 JUL M 82% 7 11 3 41 9 7 23 12 2 3 KK5 M 83% 1 8 27 319 38 BQL 61
17 17 42 KPT F 83% 9 12 32 248 30 55 65 26 56 32 KRJ F 76% 6 40 76
359 37 1 11 61 23 20 KRM F 78% 126 36 55 83 17 103 98 46 74 44 K5E
M 73% 65 27 52 206 74 21 123 42 93 16 KZO F 82% 38 16 38 262 8 6 75
32 30 20 LAE M 76% 58 15 50 294 22 14 67 63 125 21 MOF F 91% 79 17
29 10 12 2 85 5 7 46 MRS M 72% 119 21 110 450 50 2 675 54 68 28 MTR
F 69% 2 33 23 218 3 5 38 67 39 8 MYO F 94% 40 24 9 24 BQL BQL 12 7
BQL 11 NPX F 79% 36 32 13 130 6 15 76 25 20 10 NQT M 85% 76 12 39
80 23 2 151 14 20 34 OAU F 81% 47 26 24 86 8 6 85 46 53 13 OZL M
76% 16 15 61 300 109 3 165 29 17 43 PFM F 87% 21 1 11 67 10 3 116 8
15 33 PXK M 80% 86 35 63 433 78 2 109 62 32 60 QWG F 77% 16 32 29
300 21 9 50 10 BQL 15 REL F 77% 40 20 15 109 9 65 100 33 75 10 RFA
F 78% 130 42 49 444 52 6 195 30 28 17 RKB F 95% 42 16 16 100 8 3 36
17 20 19 RML F 76% BQL 6 45 129 31 14 152 24 42 29 RNG F 91% 119 14
97 298 27 177 207 34 71 41 ROE F 82% 73 24 36 302 37 2 55 17 2 51
SEO F 72% 36 25 18 106 9 66 102 50 81 11 SQJ F 74% 115 12 100 285
19 175 210 30 81 42 SRA M 79% 50 6 71 409 84 10 23 28 18 44 TPZ F
83% 120 13 101 301 26 171 204 31 82 41 TSR F 62% 47 66 58 175 20
BQL 6 77 16 34 VCM M 82% 42 28 79 415 110 0.2 94 16 4 215 VEN F 70%
79 69 89 328 53 2 32 58 48 81 VTA M 78% 32 1 25 84 12 5 120 21 40
15 WWM M 84% 42 1 27 127 12 6 58 21 16 37 ZAG M 85% 35 28 39 96 73
1 11 42 17 18 ZCR M 80% 84 11 39 160 22 38 14 57 20 18 ZIJ M 72% 6
33 31 320 29 13 25 34 3 13 BQL (Below Quantization Limit) TBD (To
Be Determined)
Example 3
Characterization of Pooled Hepatocytes
[0066] Cryopreserved pooled lots of hepatocytes are prepared and
analyzed for post-thaw viability and enzyme function. The following
metabolic activities are evaluated: COUM, DEX, ECOD, 7-HCG, 7-HCS,
MEPH, TEST, TOLB, PHEN, and CZX.
[0067] Six lots of pooled hepatocytes, comprising either five-donor
pools or ten-donor pools, are prepared as described above.
Hepatocytes are harvested from individual donors and then
cryopreserved as individual lots using liquid nitrogen as freezing
agent. Cryopreservation is accomplished by suspending the
hepatocytes into freezer-safe vials containing a medium having
approximately 10% DMSD and approximately 90% Cryopreservation
Medium. The dispensed hepatocytes are then frozen in a controlled
rate freezer until a final temperature of approximately -80.degree.
C. is reached.
[0068] To form the pooled hepatocyte preparations, individual lots
are thawed, and the viable cells are isolated by PERCOLL.RTM.
gradient centrifugation. Vials of individual donor cryopreserved
hepatocytes were thawed in a 37.degree. C. waterbath (perhaps it
would be better to give a range such as 30-40.degree. C.
waterbath?) for 60-90 seconds. The thawed cells are decanted into
37.degree. C. media containing 30% Isotonic PERCOLL.RTM. and 70%
CP-2 media. The cell suspension is centrifuged at 100 g for 20
minutes. The viable cells are recovered in cryopreservation media
and counted. The viable cells are diluted to 20 million cells per
mL. A second solution containing 20% DMS4 and 80% cryopreservation
media (equal volume to the cell suspension listed above) is
prepared. The 20% DMSO and 80% cryopreservation media is slowly
added to the cells suspension mixture. The addition takes 5-10
minutes. The resulting mixture is 10% DMSO, 90% cryopreservation
media with cells at 10 million cells per mL. This solution is
aliquoted into cryovials at 1.0 mL per vial. The cells are then
cryopreserved. Viable cells from individual lots are then pooled to
form pooled hepatocyte preparations whose cells have functional
assay values within desired ranges.
[0069] The pooled lots are then cryopreserved. Table IV below shows
the results of the post-thaw viability ("% V") and enzyme function
analysis of the pooled lots. As indicated in Table III, pools had
an average viability of 79% (S.D. .+-.6%).
TABLE-US-00004 TABLE IV Pool % V COUM DEX ECOD 7-HCG 7-HCS MEPH
TEST TOLB PHEN CZX MJI.sup.a 89 63 28 66 301 44 12 70 61 50 62
YDJ.sup.a 72 66 30 80 470 41 1 165 31 27 71 APO 79 79 21 55 276 43
2 112 22 26 30 HMB.sup.b 76 61 18 70 231 46 3 151 23 20 83
IJU.sup.a 75 32 19 35 232 44 2 124 32 11 42 RKS.sup.b 81 84 20 73
336 53 2 131 28 23 55 .sup.a5-donor lots .sup.b10-donor lots
[0070] For comparison, Table V below shows summary data for a
post-thaw viability and enzyme function analysis of eighty-one
individual lots that are cryopreserved (i.e., subjected to one
cycle of cryopreservation). This data confirms that the lot-to-lot
variability of enzyme function found in individual hepatocyte
sources is very high. The data confirms the desirability of
employing pooled hepatocyte preparations for providing
cryopreserved cells that approximate the enzyme function of
"average" hepatocytes for a wide variety of enzymes.
TABLE-US-00005 TABLE V Summary of Pooled Hepatocyte Lot Data % V
COUM DEX ECO 7-HCG 7-HCS MEPH TEST TOLB PHEN CZX Avg 79 37 17 51
276 27 8 35 35 15 19 High 95 134 69 110 471 110 177 675 82 125 215
Low 62 6 1 31 231 24 2 25 34 3 13
Example 4
[0071] Characterization of the Viability of Pooled Hepatocytes
After Thawing
[0072] A common use of for cryopreserved hepatocytes is to thaw the
hepatocytes and then incubate them with various xenobiotics. For
this purpose, it is preferred that the hepatocytes maintain their
viability for at least several hours. To examine the post-thaw
viability over time for one lot of pooled cryopreserved
hepatocytes, the cells were thawed, aliquoted into the wells of a
12-well plate, and incubated at 37.degree. C. with 5% CO.sub.2. The
viability of the hepatocytes is then measured at time-points for up
to six hours. Table VI shows the results of this analysis, wherein,
at six hours, 39% of the hepatocytes remained viable
TABLE-US-00006 TABLE VI Post-Thaw Viability Analysis of a Pooled
Hepatocyte Lot Timepoint % Viability.sup.a T = 0 88% 0.5 hrs 79%
1.0 hrs 84% 2.0 hrs 79% 3.0 hrs 73% 4.0 hrs 67% 6.0 hrs 69%
.sup.aviability determined by Trypan Blue
[0073] All publications and patents mentioned in this specification
are herein incorporated by reference to the same extent as if each
individual publication or patent application was specifically and
individually indicated to be incorporated by reference.
[0074] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth
* * * * *