U.S. patent application number 11/353453 was filed with the patent office on 2006-06-29 for cell culture media for mammalian cells.
This patent application is currently assigned to University of Pittsburgh. Invention is credited to Geoffrey D. Block.
Application Number | 20060141447 11/353453 |
Document ID | / |
Family ID | 24473190 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060141447 |
Kind Code |
A1 |
Block; Geoffrey D. |
June 29, 2006 |
Cell culture media for mammalian cells
Abstract
A chemically defined mammalian cell culture medium is provided
that supports maintenance and long term clonal growth of mammalian
hepatocytes and other cells.
Inventors: |
Block; Geoffrey D.;
(Pittsburgh, PA) |
Correspondence
Address: |
THE WEBB LAW FIRM, P.C.
700 KOPPERS BUILDING
436 SEVENTH AVENUE
PITTSBURGH
PA
15219
US
|
Assignee: |
University of Pittsburgh
|
Family ID: |
24473190 |
Appl. No.: |
11/353453 |
Filed: |
February 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10722378 |
Nov 25, 2003 |
7022520 |
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11353453 |
Feb 14, 2006 |
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10075048 |
Feb 12, 2002 |
6670180 |
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10722378 |
Nov 25, 2003 |
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09267551 |
Mar 12, 1999 |
6413772 |
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10075048 |
Feb 12, 2002 |
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08617325 |
Mar 18, 1996 |
6043092 |
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09267551 |
Mar 12, 1999 |
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Current U.S.
Class: |
435/4 ;
435/366 |
Current CPC
Class: |
C12N 5/067 20130101;
G01N 33/5017 20130101; C12N 2500/38 20130101; G01N 33/5067
20130101; C12N 2500/34 20130101; G01N 33/74 20130101; C12N 2501/148
20130101; C12N 2500/25 20130101; G01N 33/5008 20130101; C12N
2501/12 20130101; C12N 2500/90 20130101; C12N 2500/32 20130101;
C12N 2500/20 20130101; C12N 2501/11 20130101 |
Class at
Publication: |
435/004 ;
435/366 |
International
Class: |
C12Q 1/00 20060101
C12Q001/00; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method of producing recombinant pancreatic islet cells
expressing a heterologous gene comprising: producing pancreatic
islet cells in vitro without serum by introducing pancreatic islet
cells into a cell culture medium in vitro, said culture medium
comprising 1-150 mg/L arginine; 1-120 mg/L proline; 1-3050 mg/L
nicotinamide; 0.1-100 mg/L transferrin chelated with iron; greater
than 10.sup.11 M insulin or insulin-like growth factors; 10.sup.-12
M-10.sup.-3 M glucocorticoid steroid; 1-6000 .mu.g/L zinc salt;
1-250 .mu.g/L manganese salt; 1-1000 .mu.g/L copper salt; 1-150
.mu.g/L selenium salt; 2.0-10.0 mM L-glutamine; 0.01-5.0 g/L
D-galactose or 0.01-5.0 g/L D-glucose, or when both D-galactose and
D-glucose are included together, 0.01-8.0 g/L, and culturing said
introduced cells in said medium; and transforming said cultured
pancreatic islet cells with a nucleic acid capable of expressing
said gene in said cells.
2. A method of using pancreatic islet cells produced according to
claim 1 comprising infusing said pancreatic islet cells into a
patient and allowing said gene to be expressed.
3. A method for pancreatic islet cell transplantation comprising
introducing recombinant pancreatic islet cells produced according
to claim 1 into a patient.
4. A method for manufacturing a gene product comprising culturing
recombinant pancreatic islet cells produced according to claim 1
and recovering the gene product.
5. A method for testing a drug comprising introducing said drug to
recombinant pancreatic islet cells produced according to claim 1
and assaying for the effect of the drug.
6. A method of producing recombinant hepatocytes expressing a
heterologous gene comprising: producing hepatocytes in vitro
without serum by introducing hepatocytes into a cell culture medium
in vitro, said culture medium comprising 1-150 mg/L arginine; 1-120
mg/L proline; 1-3050 mg/L nicotinamide; 0.1-100 mg/L transferrin
chelated with iron; greater than 10.sup.-11 M insulin or
insulin-like growth factors; 10.sup.-12 M-10.sup.-3 M
glucocorticoid steroid; 1-6000 .mu.g/L zinc salt; 1-250 .mu.g/L
manganese salt; 1-1000 .mu.g/L copper salt; 1-150 .mu.g/L selenium
salt; 2.0-10.0 mM L-glutamine; 0.01-5.0 g/L D-galactose or 0.01-5.0
g/L D-glucose, or when both D-galactose and D-glucose are included
together, 0.01-8.0 g/L, and culturing said introduced cells in said
medium; and transforming said cultured hepatocytes with a nucleic
acid capable of expressing said gene in said cells.
7. A method of using hepatocytes produced according to claim 6
comprising infusing said hepatocytes into a patient and allowing
said gene to be expressed.
8. A method for hepatocytes transplantation comprising introducing
recombinant hepatocytes produced according to claim 6 into a
patient.
9. A method for manufacturing a gene product comprising culturing
recombinant hepatocytes produced according to claim 6 and
recovering the gene product.
10. A method for testing a drug comprising introducing said drug to
recombinant hepatocytes produced according to claim 6 and assaying
for the effect of the drug.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/722,378 filed Nov. 25, 2003, which is a continuation of U.S.
application Ser. No. 10/075,048 filed Feb. 12, 2002, now U.S. Pat.
No. 6,670,180, which is a divisional of U.S. application Ser. No.
09/267,551 filed Mar. 12, 1999, now U.S. Pat. No. 6,413,772, which
is a divisional of U.S. application Ser. No. 08/617,325 filed Mar.
18, 1996, now U.S. Pat. No. 6,043,092, all of which are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0002] The present invention relates generally to cell culture
media for mammalian cells. In particular, the invention relates to
cell culture media that allow long-term expansion and maintenance
of a cell population of mammalian hepatocytes, hepatocyte-derived
cell lines, hepatocyte-derived malignant cells, and other
cells.
[0003] It is well-known that specific cell lines can be grown
in-vitro in optimally formulated culture or nutrient media.
Examples of some of the culture media developed for special
purposes are: RPMI Media 1640 medium for growth of human B-lymphoid
cells and malignant cells, Changs medium for growth of amniotic
fluid cells, Medium 199 for growth of mouse fibroblast cells,
Minimal Essential Medium (MEM) medium, a "minimal" medium for
growth of attached mammalian cells, and Leibovitz medium for growth
in absence of CO.sub.2. Such various media are distinguished from
one another in that they contain critically different components in
precise amino acids, vitamins, organic salts, trace elements, and
other organic compounds which promote the maximum growth of the
cultured cells.
[0004] For the growth of mammalian cells chemically defined media
are usually supplemented with various sera, preferably fetal calf
or newborn calf serum, and other incompletely defined growth
factors. A major drawback to serum, however, is that its
constituents may vary widely, thereby introducing undefined
biological components into the nutrient medium which contributes to
the variability of biochemical and cellular events. Additionally,
serum is expensive and may result in critical immune reactions in
patients if the cells are used for clinical purposes.
[0005] The present invention is primarily directed to culturing
mammalian hepatocyte cells using chemically defined media that
allow long-term expansion of the cell population. The term
"chemically defined media" is used in tissue culture to refer to
culture media of known chemical composition, both quantitatively
and qualitatively, as contrasted with media which contain natural
products such as animal serum.
[0006] Liver regeneration is achieved primarily by cell division of
mature adult hepatocytes as reported by Grisham, J. W., et al.,
Cancer Res. 22:842 (1962), the disclosure of which is incorporated
herein by reference. These cells, or a fraction thereof, have a
high capacity for clonal growth, as shown by hepatocyte
transplantation experiments in ectopic sites (Jirtle, R. L., et
al., Cancer Res. 42:3000 (1982), the disclosure of which is
incorporated herein by reference) and in transgenic mouse models
(Rhim, J. A., et al., Science 263:1149 (1994), the disclosure of
which is incorporated herein by reference). It has been shown in
several studies, however, that when liver is stimulated to
regenerate while proliferation of mature hepatocytes is suppressed,
faculative stem cells emerge and proliferate. See, for example,
Thorgeirsson, S. S., et al., Proc. Soc. Exp. Biol. Med. 204:253
(1993), the disclosure of which is incorporated herein by
reference. Such cells, sometimes referred to as "oval cells", can
mature into hepatocytes in defined animal models or ductular
structures composed of cells ("ductular hepatocytes") with mixed
hepatocyte and bile duct epithelial markers. See, Gerber, M. A., et
al., Amer. J. Path. 110:70 (1983) and Vandersteenhoven, A. M., et
al., Arch. Pathol. Lab. Med. 114:403 (1991), the disclosures of
which are incorporated herein by reference. Little is known,
however, about their origin and about the controls that regulate
their phenotypic transitions to hepatocytes or ductular cells.
[0007] Despite the high capacity of hepatocytes to proliferate in
vivo, directly or via faculative stem cell growth, the conditions
that determine their growth potential and their phenotypic
transitions are not thoroughly understood because of only limited
success in hepatocyte growth in primary culture. It is typically
the case that hepatocytes in primary culture under the influence of
primary mitogens enter into one or two divisions and then the cells
degenerate and die. Heretofore various investigators have failed to
develop a medium that allows hepatocytes to both proliferate and
survive.
[0008] For example, Berry, N. M., et al., J. Cell. Biol. 43:506
(1969), the disclosure of which is incorporated herein by
reference, taught the collagenase perfusion technique which allows
liver tissue to dissociate into its component cellular elements,
based on size. Later, Bissell, D. M., et al., J. Cell. Biol.
59(3):722 (1973) and Bonney, R. J., et al., In vitro 9:399 (1974),
the disclosures of which are incorporated herein by reference,
described the first methods for culturing isolated hepatocytes
which perhaps survived for one or two days. Long term culture of
hepatocytes on collagen gels for a maximum of 7 to 10 days was
reported by Michalopoulos, G., et al., Exp. Cell. Res. 94(1):70
(1975), the disclosure of which is incorporated herein by
reference. The common characteristic of all of the above-referenced
systems is that the hepatocytes in those systems were maintained in
culture without there being any evidence of cell proliferation.
These systems were, instead, only maintenance cultures of
non-proliferating cells for a brief period.
[0009] The first successful attempt to initiate DNA synthesis in
hepatocytes used the then newly-discovered epidermal growth factor
(EGF) as reported by Richman, R. A., et al., Proc. Nat. Acad. Sci.
USA 73:3589 (1976), the disclosure of which is incorporated herein
by reference. Over the ensuing years several other groups of
researchers used EGF as a mitogen for hepatocytes and reported on
the mitogenic effects of EGF and their modulation by other factors
such as, for example, matrices such as collagen Type I, zinc and
proline.
[0010] Hepatocyte growth factor, also known as scatter factor
(hereinafter referred to as "HGF" or "HGF/SF") was discovered,
cloned and ultimately sequenced by the late 1980's. See
Michalopoulos, G., et al., Federation Proceedings 42:1023 (1983);
Michalopoulos, G., et al., AACR Proceedings 24:58 (1983);
Michalopoulos, G., et al., Cancer Res. 44(10):4414 (1984); and
Miyazawa, K., et al., Biochem. Biophys. Res. Comun. 163:967 (1989),
the disclosures of which are incorporated herein by reference.
HGF/SF was found to be a mitogen for many hepatocytes as well as
for epithelial cells. HGF/SF's importance for the liver is due to
the fact that it is the trigger for liver regeneration through an
endocrine mechanism.
[0011] Recently, several studies have shown that HGF/SF, epidermal
growth factor ("EGF"), and transforming growth factor a
("TGF.alpha.") are the primary mitogens for hepatocytes in culture
by stimulating limited hepatocyte DNA synthesis in chemically
defined media. See, for example, Michalopoulos, G. K., Fed. Am.
Soc. Exp. Biochem J. 4:176 (1990), the disclosure of which is
incorporated herein by reference. These growth factors were later
found to additionally play a role in vivo in liver regeneration
after partial hepatectomy. Injection of HGF/SF, TGF.alpha., or EGF
in rats induces DNA synthesis in hepatocytes. See, for example,
Liu, M. L., et al., Hepatology 19:1521 (1994), the disclosure of
which is incorporated herein by reference.
[0012] In all of these systems, however, it was reported that
hepatocytes entered into DNA synthesis and mitosis for only a
limited time, typically 1-3 days. After 1-2 rounds of DNA synthesis
and cell division, the cultures degenerated and all the cells were
dead in about 7-10 days. Until now, there has also been no
documented expansion of the number of hepatocytes in cell culture
by adding either EGF or HGF alone, or in combination. The cell
replication in cultures containing these growth factors is instead
self-limited and the number of hepatocytes that die exceeds the
number of hepatocytes that are newly generated. The cell
replication in cultures containing other hepatocyte mitogens such
as transforming growth factors, such as TGF.alpha., and acidic
fibroblast growth factor is similarly self-limited.
[0013] More recently, Mitaka, T., et al., Hepatology 13(1):21
(1991); Mitaka, T., et al., Hepatology 16(2):440 (1992); Mitaka,
T., et al., Virchows Arch. B. Cell Pathol. Incl. Mol. Pathol-62:329
(1992); and Mitaka, T., et al., Cancer Res. 53: 3145 (1993), the
disclosures of which have been incorporated herein by reference,
have reported that adding nicotinamide, dexamethasone and EGF to a
conventional culture medium resulted in the formation of colonies
of small hepatocytes arising in a dense culture of standard size
parenchymal hepatocytes. In a further study, Mitaka, T., et al., J.
Cell. Physiol. 157:461 (1993), the disclosure of which is
incorporated herein by reference, have reported that the numbers of
colonies induced by the combinations of EGF+HGF, EGF+TGF-.alpha.,
and HGF+TGF-.alpha. were not different from those of colonies
induced by each mitogen alone. In these studies, however, there was
no significant expansion of the total cell population, no evidence
of clonal growth, and there was loss of differentiation.
[0014] Until now there has been no chemically defined medium,
supplemented or not, which is able to support long term
proliferation, differentiation, and viability of hepatocytes. While
for many purposes the use of an undefined supplement is
satisfactory, in cases where studies are made of growth,
metabolism, and/or differentiation of cells in culture, it is most
desirable to have a supplement that is defined. The introduction of
undefined components to a cell culture can contribute to
variability, unpredictability, and contamination in study results
and applications of cell cultures. The use of defined media is
particularly important and advantageous in areas of drug
metabolism, artificial organ development, cell transplantation,
gene therapy, and basic investigational cell studies.
[0015] The above-described limited capacity of hepatocytes to
proliferate in primary culture has hindered long-term studies or
uses that required long-term viability or proliferation.
Applications of hepatocyte cultures to cellular transplantation and
gene therapy have thus been hindered. Consequently, there remains a
need for a chemically defined medium that will allow hepatocytes to
proliferate and survive long term. Among the potential applications
for such a medium and the hepatocytes and other cells so cultured
are gene therapy, bioartificial organs, cell transplants, drug
production, and drug and chemical testing.
[0016] As stated above, the current state of the art does not
provide a hepatocyte culture system in which hepatocytes expand as
a cell population by sustained proliferation, and there is a need
for such a system. The present invention provides a fully defined
culture medium which allows sustained proliferation and long-term
expansion of hepatocytes.
SUMMARY OF THE INVENTION
[0017] According to the present invention, a new chemically defined
cell culture medium is provided. This medium supports sustained
clonal growth of primary hepatocytes and hepatocyte cell lines,
genetically transformed hepatocytes, and hepatocytes obtained from
neoplastic sources, resulting in expansion of the cell population.
This medium further allows complete differentiation of metabolic,
structural, and secretory functions of the cells grown therein.
Under these conditions, hepatocytes undergo multiple proliferative
cycles. Once confluency is reached, or in the presence of specific
matrix components, nutrients, and/or growth factors, these
proliferating cells stop dividing and maintain a mature hepatocyte
phenotype for many months or longer.
[0018] Accordingly it is a primary object of the present invention
to provide a culture medium for sustained proliferation and
viability of hepatocytes.
[0019] Another object of the present invention to provide a culture
medium for sustained differentiation and viability of
hepatocytes.
[0020] Yet another object of the present invention is to provide a
culture medium for sustained proliferation of hepatocytes that
revert to complete differentiation as growth ceases.
[0021] Another object of the present invention is to provide a
culture medium for long-term expansion of hepatocytes that contains
no serum such that the medium is fully defined.
[0022] Yet another object of the present invention is to provide a
culture medium for sustained proliferation, differentiation, and
viability of hepatocytes on a variety of matrix substrates.
[0023] These and other objects of the present invention are
achieved by one or more of the following embodiments.
[0024] In one aspect, the invention features a chemically defined
HBM culture medium for maintenance, differentiation, and long-term
growth of mammalian hepatocytes, comprising:
[0025] (a) a synthetic stock basal medium designed for mammalian
cell culture; and
[0026] (b) a hepatocyte cell growth promoting amount of components
selected from among nicotinamide, amino acids, transferrin,
hormones, dexamethasone, trace metals, and simple carbohydrate
selected from the group consisting of D-glucose and D-galactose and
any combination thereof.
[0027] In a preferred embodiment the invention features HBM culture
medium further comprising buffer, antibiotics, and albumin.
[0028] In another aspect, the invention features a mammalian cell
culture medium comprising the composition of HGM as defined in
Tables I and II, wherein the stock basal media of Table I comprises
a blended DMEM such that the final concentration of D-glucose is
preferably about 2.0 g/L and the amount of D-galactose is
preferably about 2.0 g/L.
[0029] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiment, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIGS. 1A-lC are graphs showing results of studies in which
rat hepatocytes were cultured in HBM medium as described herein
supplemented with growth factors, as indicated. All points
represent the mean and standard error of at least three separate
cultures.
[0031] FIG. 1A shows the percent labeled nuclei (BRdU) of
proliferating cell cultures at different times after hepatocyte
isolation. The cells were grown in HBM supplemented with HGF/SF and
EGF.
[0032] FIG. 1B shows the incorporation of [.sup.3H] thymidine
(disintegrations per minute) into DNA in cultures at different
times after hepatocyte isolation. Cells were exposed to HGF/SF in
HBM (.diamond.); EGF in HBM (O); HGF/SF+EGF in HBM (.DELTA.); and a
control, HBM alone (.quadrature.).
[0033] FIG. 1C shows the amount of DNA per plate at different days
of cells grown in HBM medium alone (control) (.box-solid.); HGF/SF
in HBM (.DELTA.); EGF in HBM (.gradient.); and HGF/SF+EGF in HBM
(.circle-solid.).
[0034] FIG. 2 is a graph showing the day 15 DNA per plate of rat
hepatocytes grown in HBM with the indicated growth factors. Cells
represented by control (day t=0) and day 15 were grown in HBM
without any growth factors.
[0035] FIG. 3 is a graph showing the amount of DNA synthesis per
plate (.mu.g/culture) of rat hepatocytes at day 15 in HBM
supplemented with HGF/SF and EGF grown on different matrices. "CC"
means collagen coated; "ECL" is a commercial matrix; "m COLL IV"
means mouse collagen IV; "CC VITROGEN" means bovine skin collagen
type I; "POLY D LYS" means poly D-Lysine; "m LAMININ" means mouse
laminin; "h FIBRONECT" means human fibronectin; and "m COL I" means
mouse collagen I.
[0036] FIGS. 4A-4G are photographs of rat hepatocytes cultured in
HBM as described herein under various conditions.
[0037] FIG. 4A shows hepatocytes in HEM medium with HGF/SF and EGF
at day 1 after isolation showing typical subconfluent
non-proliferating hepatocytes.
[0038] FIG. 4B shows hepatocytes at day 4 in HBM media induced with
HGF/SF showing typical scattered morphology.
[0039] FIG. 4C shows hepatocyte cultures having reached confluency
at day 15 showing typical morphology.
[0040] FIGS. 4D and 4E are election photomicrographs of the cells
in FIGS. 4A and 4C, respectively.
[0041] FIGS. 4F and 4G are photomicrographs of stained hepatocytes
that were transfected at day 3 with a lac-Z-containing replication
defective retrovirus and stained for .beta.-galactosidase
expression as described below and then photographed at day 1 (FIG.
4F) and day 10 (FIG. 4G) after transfection. The cells were
cultured in HBM medium supplemented with HGF/SF and EGF as
described below.
[0042] FIGS. 5A and 5B are photographs of Northern blots showing
expression of specific genes at different days in rat hepatocyte
cultures maintained in HBM in the presence of HGF/SF and EGF as
described herein. GAPDH expression and the intensity of the 28S RNA
after ethidium bromide staining were used as internal controls.
[0043] FIGS. 6A-6F are photographs of proliferating rat hepatocytes
cultured in HBM medium in the presence of HGF/SF and EGF as
described herein and Northern blots thereof.
[0044] FIG. 6A is a phase contrast photomicrograph of cultures of
proliferating hepatocytes overlaid with Matrigel at day 8 and
photographed at day 18. The granular cytoplasm and the appearance
of typical bile canaliculi appear as bright lines between
cells.
[0045] FIGS. 6B and 6C are low and high power electron
photomicrographs, respectively, of cells at day 18 in culture, 10
days after overlay with Matrigel. Typical features of hepatocyte
cytoplasm are shown such as lamellae of endoplasmic reticulum
wrapping around mitochondria, microbodies with crystalline center,
bile canaliculi ("c") (FIG. 6B), abundant mitochondria ("M"), and
glycogen ("G") (both in FIG. 6C).
[0046] FIG. 6D is a photograph of a Northern blot showing increased
expression of albumin mRNA after addition of Matrigel. Albumin mRNA
is expressed in control cultures, immediately after isolation from
liver by collagenase perfusion and before culture. Expression is
minimal at day 8 in culture. At days 3 and 7 after Matrigel
addition (lanes marked with "+"), there was an increase in
expression of albumin mRNA. GADPH expression was used as an
internal control.
[0047] FIG. 6E is a photograph of a Northern blot showing induction
of cytochrome IIB1 mRNA in cultures treated with Matrigel at day 8
and exposed to phenobarbitol ("PB") 2 days later (day 10 of
culture). Cells were harvested at day 15 of culture. GAPDH
expression was used as an internal control for mRNA loading.
[0048] FIG. 6F is a photograph of a Northern blot of cells cultured
additionally with Matrigel (added at day 8 in culture). Cytokeratin
19, a bile duct marker expressed by the proliferating hepatocytes,
is suppressed by addition of Matrigel and is not suppressed in
control cultures not receiving Matrigel. 28S rRNA stained by
ethidium bromide was used as an internal control.
[0049] FIGS. 7A-7E show the results of studies showing formation of
ductular/acinar structures in cultures of rat hepatocytes kept from
the beginning of culture between two type I collagen gel layers in
HBM in the presence of HGF/SF. The FIGS. 7A-7D photographs are of
15 day cultures.
[0050] FIG. 7A is a phase contrast photomicrograph (100.times.)
showing the appearance of the ductular structures surrounded by
collagen fibrils.
[0051] FIG. 7B is a photomicrograph (100.times.) of parrafin
sections of the FIG. 7A type I collagen gels stained with
hematoxylin and eosin.
[0052] FIGS. 7C and 7D are electron micrographs of cells
surrounding the same lumen but in different locations around the
lumen of one of the ductular structures seen in FIG. 7B. FIG. 7C
shows cells with a morphology similar to bile duct epithelium, with
long parallel contacts joined by many desmosomes and with abundant
keratin intermediate filaments. FIG. 7D shows cells resembling more
of the hepatocyte phenotype, with rough endoplasmic reticulum and
mitochondria, densely stained secondary lysosomes and fewer
filaments.
[0053] FIG. 7E is a photograph of a Northern blot showing that
expression of cytokeratins 18 and 19 increases in cultures with
ductular/acinar structures whereas albumin is only slightly
expressed.
[0054] FIG. 8 shows photographs of stained human hepatocyte
cultures grown in HBM supplemented with HGF/SF and EGF as described
herein taken at day 1, 3, 5, 7, 10, 12, and 19.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The present invention provides a hepatocyte basal medium,
designated HBM herein, which allows long-term expansion,
differentiation and survival of the cell population of hepatocytes,
hepatocyte-derived cell lines such as HepG2, hepatic fetal
epithelial cells, and hepatic primary hepatocarcinoma cells in
vitro. Additionally, the media of the present invention can be used
for culturing pancreatic islet cells, renal tubular cells, Ito
cells, small intestine epithelial cells, and a variety of cell
lines, including MRC5, CaCo, and 3T3 cells, as well as others. The
HBM of the present invention maintains metabolic pathways and
synthesis functions, i.e., differentiation of mature adult
hepatocytes. Additionally, the HBM of the present invention, when
combined with specific mitogens and extracellular matrix
compositions, hepatocytes can be driven to transdifferentiate into
bile duct-like structures. The HBM of the present invention can be
used for culture of mammalian hepatocytes, and other cells,
including, but not limited to those of human, rat, dog, pig, mouse,
and baboon origin.
[0056] The HBM medium comprises appropriate levels of essential and
non-essential amino acids and bulk ions and trace elements,
buffers, vitamins, carbohydrates, lipids, proteins, and hormones to
function as a nutrient medium for in vitro mammalian cell
culture.
[0057] In its broadest aspect the invention features a chemically
defined basal media, HBM, that by itself allows for long-term
survival, differentiation, and growth of mammalian hepatocytes and
other cells. Additionally, in the presence of growth factors such
as HGF/SF, EGF, or TGF.alpha., as well as other mitogens, cells
growing in the supplemented HBM have a more rapid population
expansion and clonal growth. As further demonstrated below, the HBM
of the present invention when supplemented with HGF/SF causes
formation of bile duct-like structures in conjunction with certain
matrix constructs. As stated above, however, such growth factors
are not required for specific differentiation patterns in primary
cultures, but do accelerate growth and population expansion if this
is desired for a specific purpose.
[0058] The HBM medium of the present invention comprises a
chemically defined stock basal medium (hereinafter referred to as
"SM") designed for mammalian cell culture. The stock basal medium
can be preferably constructed using Dulbecco's Modified Eagle
Medium ("DMEM") as one ingredient, but the present invention is not
to be so limited as long as the formulations are within the
guidelines set forth herein. Examples of other defined basal medias
which may be used in accordance with the present invention include,
but are not limited to: Basal Media Eagle (BME), DMEM/F-12 (1:1
DMEM and F-12 vol:vol); Medium 199; F-12 (Ham) Nutrient Mixture;
F-10 (Ham) Nutrient Mixture; Minimal Essential Media (MEM),
Williams' Media E; and RPMI 1640, all of which are available from
Gibco-BRL/Life Technologies, Inc., Gaithersburg, Md., among others.
Several versions of many of these media are available, and those
that are particularly useful to construct HBM include, but are not
limited to: DMEM 11966, DMEM 10314, MEM 11095, Williams' Media E
12251, Ham F12 11059, MEM-alpha 12561, and Medium-199 11151 (all
available from Gibco-BRL/Life Technologies (1995-1996 catalog)).
Therefore, for example, if L-arginine and/or D-glucose are already
in the stock basal media in the necessary amounts, then little or
no additional ingredient will have to be added as a supplement.
[0059] Depending on the particular composition of the stock basal
medium, the SM is then supplemented, as described more fully
herein, with D-glucose and/or D-galactose, nicotinamide, other
micronutrients comprising amino acids and trace metals not already
present in the SM such as L-proline, L-glutamine, L-arginine,
L-ornithine, zinc, manganese, copper, and selenium, purified
transferrin to which is bound elemental iron or apo-transferrin in
combination with iron gluconate, hormones such as dexamethasone and
insulin and a pH buffer such as HEPES
(N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]).
Antibiotics such as penicillin and streptomycin, a pH indicator,
albumin and/or dextran, essential fatty acids, alternative buffers,
vitamins, osmotic agents, and other forms of trace metals may also
be optionally added. Typically, a basal medium will have a pH in
the range of 6.5-8.2, preferably 7.0-7.7, and most preferably
7.2-7.5. Phenol red is a typical indicator added to aid in the
control of pH. The SM and supplements thereto comprise the HBM
medium of the present invention.
[0060] The HBM medium may then be optionally supplemented with one
or more growth factors such as, for example, HGF/SF, EGF and
TGF.alpha. if accelerated growth is desired.
[0061] Simple carbohydrates D-glucose and/or D-galactose are added
to the stock basal media to comprise HBM. If both D-glucose and
D-galactose are used, the sum of their total concentrations is
preferably 8.0 g/L or less but greater than 0.01 g/L, taking into
account the amount of D-glucose present in the stock basal media,
if any. When only one of D-glucose or D-galactose is used, the
concentration is preferably 5.0-0.1 g/L. For example, in the
Example herein, the blended DMEM stock basal medium that was used
contained 2.0 g/L D-glucose and no further D-glucose was added. 2.0
g/L of D-galactose was then added as a supplement.
[0062] Nicotinamide, another HBM component, has been shown to
maintain hepatocyte differentiation, enhance expression of
cytochrome P450, and prolong hepatocyte survival in conventional
culture to 10-14 days. See, Rosenberg, M.., et al., In Vitro 18:
775 (1982) and Inoue, C., et al., Biol. Chem. 264(9): 4747 (1989),
the disclosures of which are incorporated herein by reference.
[0063] Transferrin is an iron binding protein that interacts with a
transferrin receptor on the cell membrane. It serves to both
chelate and transport iron ions. The transferrin preferably used in
the present invention is either holo-transferrin 30% saturated with
iron or is completely unsaturated (apo-transferrin) and is combined
with iron gluconate.
[0064] Dexamethasone, a synthetic corticosteroid, has been shown to
enhance EGF-induced DNA synthesis as reported by Sand, T.-F., et
al., Acta. Endocrinol. 109:369 5 (1985), the disclosure of which is
incorporated herein by reference. As used in the present invention,
dexamethasone can be any cortisol derivative such as, prednisone,
prednisilone, cortisol, hydrocortisone, and other derivatives.
[0065] Insulin and insulin-like growth factors are required for
glucose uptake, amino acid transport, and maintenance of multiple
intermediary metabolic pathways. These effects help to maintain
differentiation and support proliferation.
[0066] The inclusion of L-arginine in HBM, in the stock basal
medium, or supplemental thereto, appears to be important because
hepatocytes in culture tend to lose their capacity to synthesize
arginine through the urea cycle. In the absence of L-arginine,
hepatocytes in culture cannot live for very long because they
become incapable of synthesizing L-arginine, thereby blocking their
protein synthesis. The use of D-galactose in addition to D-glucose
is advantageous for HBM because the combination gives the greatest
growth potential over either substance alone.
[0067] HGF/SF, EGF, and TGF.alpha., as discussed above, are
mitogens which as shown below exhibit an enhanced proliferative
effect when used together or alone in accordance with the present
invention. The foregoing list of mitogens that can be used to
supplement the HBM of the present invention is not exhaustive. It
is to be noted, however, that these mitogens are not required for
survival or differentiation of hepatocytes when grown in HBM.
Hepatocytes grown in HBM will proliferate at a slower rate than if
mitogens are added.
[0068] The insulin, EGF, HGF, and TGF.alpha. used in the presently
claimed media can either be recombinantly produced, genetically
engineered, or purified from natural sources. The species can be,
for example, human, bovine, equine, murine, porcine, or rat.
[0069] Several preferred stock basal media--DMEM 11966, DMEM 10314,
MEM 11095, and Williams' Medium E 12251 contain the following
components per 1000 ml of sterile deionized water as shown below in
Table I: TABLE-US-00001 TABLE I Compositions of Stock Basal Media
(mg/L) WILLIAMS' DMEM DMEM MEM MEDIUM E COMPONENT 11966 10314 11095
12251 INORGANIC SALTS: CaCl.sub.2 (anhyd.) 200.00 200.00 200.00
200.00 CuSO.sub.4.5H.sub.2O -- -- -- 0.0001
Fe(NO.sub.3).sub.3.9H.sub.20 0.10 0.10 -- 0.0001 KCl 400.00 400.00
400.00 400.00 MnCl.sub.2.4H.sub.2O -- -- -- 0.0001
MgSO.sub.4.(anhyd.) 97.67 97.67 97.67 97.67 NaCl 6,400.00 6,400.00
6,800.00 6,800.00 NaHCO.sub.3 3,700.00 3,700.00 2,200.00 2,200.00
NaH.sub.2PO.sub.4.H.sub.2O 125.00 125.00 140.00 --
NaH.sub.2PO.sub.4 -- -- -- 140.00 ZnSO.sub.4.7H.sub.2O -- -- --
0.0002 OTHER COMPONENTS: D-Glucose -- 4,500.00 1,000.00 2,000.00
Glutathione -- -- -- 0.05 Methyl Linoleate -- -- -- 0.03 Phenol red
15.00 15.00 10.00 10.00 Sodium Pyruvate -- -- -- 25.00 AMINO ACIDS:
L-Alanine -- -- -- 90.00 L-Arginine.HCl 84.00 84.00 126.00 --
L-Arginine -- -- -- 50.00 L-Asparagine.H.sub.2O -- -- -- 20.00
L-Aspartic Acid -- -- -- 30.00 L-Cysteine -- -- -- 40.00 L-Cystine
-- 48.00 -- -- L-Cystine.2HCL 63.00 63.00 31.00 26.10 L-Glutamine
584.00 584.00 292.00 -- L-Glutamic Acid -- -- -- 50.00 Glycine
30.00 30.00 -- 50.00 L-Histidine.HCl.H.sub.2O 42.00 42.00 42.00 --
L-Histidine -- -- -- 15.00 L-Isoleucine 105.00 105.00 52.00 50.00
L-Leucine 105.00 105.00 52.00 75.00 L-Lysine.HCl 146.00 146.00
73.00 87.50 L-Methionine 30.00 30.00 15.00 15.00 L-Phenylalanine
66.00 66.00 32.00 25.00 L-Proline -- -- -- 30.00 L-Serine 42.00
42.00 -- 10.00 L-Threonine 95.00 95.00 48.00 40.00 L-Tryptophan
16.00 16.00 10.00 10.00 L-Tyrosine.2Na.2H.sub.2O 104.00 104.00
52.00 50.70 L-Valine 94.00 94.00 46.00 50.00 VITAMINS: Ascorbic
Acid -- -- -- 2.00 Biotin -- -- -- 0.50 D-Ca pantothenate 4.00 4.00
1.00 1.00 Choline chloride 4.00 4.00 1.00 1.50 Ergocalciferol -- --
-- 0.10 Folic acid 4.00 4.00 1.00 1.00 i-inositol 7.20 7.20 2.00
2.00 Menadione -- -- -- 0.01 Na Bisulfate Niacinamide 4.00 4.00
1.00 1.00 Pyridoxine HCl -- 4.00 -- -- Pyridoxal HCl 4.00 -- 1.00
1.00 Riboflavin 0.40 0.40 0.10 0.10 .alpha.-Tocopherol -- -- --
0.01 Phosphate Disodium Thiamine.HCl 4.00 4.00 1.00 1.00 Vitamin A
Acetate -- -- -- 0.10 Vitamine B.sub.12 -- -- -- 0.20
[0070] In accordance with the preferred embodiment of present
invention, the following supplemental components in Table II are
added to the stock basal media of Table I in a total volume of 1000
ml. The preferred amounts listed coincide with those described
below in the Example for which the basal stock medium was
constructed from 445 ml of DMEM 10314 and 555 ml 11966 to provide a
D-glucose concentration of 2.0 g/L such that no additional
D-glucose was added. It is to be understood that when using other
basal stock media the optimal amounts of these components that are
added may vary. TABLE-US-00002 TABLE II Additions to SM for HBM
Preferred Concentration Amount Range units/L units/L D-Galactose
2.0 g 0.01-5.0 g* D-Glucose 2.0 g** 0.01-5.0 g* Nicotinamide 610.0
mg 1-3050 mg L-Proline 30.0 mg** 1-120 mg L-Arginine 84.0 mg**
1-150 mg L-Ornithine 100 mg 1-500 mg human-holo-Transferrin 5.0 mg
0.1-100 mg (30% Fe saturated) h-Insulin 5.0 mg greater than
10.sup.-11 M Dexamethasone 1 .times. 10.sup.-7 M
10.sup.-12-10.sup.-3M ZnCl.sub.2 .544 mg** 1-3000 .mu.g MnSO.sub.4
0.025 mg** 1-250 .mu.g ZnSO.sub.4.7H.sub.2O .750 mg** 1-3000 .mu.g
CuSO.sub.4.5H2O 0.20 mg** 1-1000 .mu.g Selenium 5.0 .mu.g 1-150
.mu.g L-Glutamine*** 5.0 mM 2.0-10.0 mM (additional to SBM) HEPES
20.0 mM 5-50 mM *If both D-galactose and D-glucose are used, the
upper limit is about 8.0 g/L or less, taking into consideration the
amounts present in the particular stock basal medium used, if any.
The lower limit for when both or only one of D-glucose or
D-galactose is used is about 0.01 g/L or greater. For example, in
the Example herein, the blended DMEM medium used contained 2.0 g/L
D-glucose with no additional amount added, and 2.0 g/L D-galactose
was added. **If not already in SM stock basal medium.
***L-glutamine is a required nutrient in the present invention.
Many stock basal medias are provided with L-glutamine included.
However, it is well known by practitioners of the art of cell
culture that L-glutamine will degrade by oxidation, such that all
L-glutamine will be degraded over a period of a few weeks following
manufacture. Therefore, additional L-glutamine is added to the HBM
medium of the present invention as described to compensate for
this.
[0071] Table III below lists substances which may be optionally
added to HBM medium to optimize cell growth for specific purposes
and for specific species origin of hepatocytes and specific cell
lines or malignant cells. Again, these amounts will vary depending
on whether a particular component is already present in the stock
basal medium being used. TABLE-US-00003 TABLE III Optional
Additives for HBM Preferred Concentration Amount Range units/L
units/L Albumin* 2.0 g 0-10.0 g Penicillin** 100 U 0-2500 U
Streptomycin** 100 .mu.g 0-2500 .mu.g Sodium Pyruvate 0.15 g 0-2.0
g Gamma Tocopherol 0.35 mg 0-3.5 mg Alpha Tocopherol 0.15 mg 0-2.5
mg Vitamin D.sub.3 0.20 mg 0-1.8 mg Dextran* 2.0 g 0-5.0 g Iron
gluconate*** 25.0 .mu.g 0-100 .mu.g Linolenic Acid 1.0 g 0-5.0 g
Apo-transferrin*** 5.0 mg 0-20.0 mg Retinol 0.05 mg 0-2.0 mg
Vitamin B.sub.12 0.15 mg 0-2.0 mg Ascorbic acid 3.0 mg 0-10.0 mg
Choline Chloride 1.5 mg 0.2-12.5 mg Biotin 0.75 mg 0.2-15.0 mg
*Dextran can be substituted for albumin. **Other antibiotics may be
used, such as for example, gentamycin. ***Iron gluconate and
apo-transferrin can be substituted for iron-saturated
holo-transferrin.
[0072] In instances where accelerated growth is desired various
growth factors can be added to the HBM of the present invention.
While HGF/SF, EGF, and TGF.alpha. are presently preferred, it is to
be understood that other growth factors may also be used. The
preferred amount of such growth factors typically vary with the
particular source used. The amounts listed herein are therefore
necessarily limited to the particular sources used herein. In the
Example below HGF/SF was preferably added to HBM at 40 ng/ml; EGF
was preferably added at 20 ng/ml; and TGF.alpha. was preferably
added at 20 ng/ml.
[0073] The following examples are included for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLE
Materials and Methods
Materials
[0074] Male Fischer 344 rats from Charles River, (Pa.) were used
for all the experiments involving rat hepatocyte isolation. EGF and
Matrigel (a mixture of matrix components derived from EHS mouse
tumor) were obtained from Collaborative Research (Waltham, Mass.).
[.sup.3H] Thymidine was obtained from ICN Radiochemicals (Irvine,
Calif.). Collagenase for hepatocyte isolation was obtained from
Boehringer Mannheim (Indiannapolis, Ind.). Vitrogen (Celtrix Labs,
Palo Alto, Calif.) was used for the construction of the collagen
gels. General reagents were obtained from Sigma Chemical Co. (St.
Louis, Mo.). HGF/SF used for these studies was the .DELTA.5
variant. ECL matrix was purchased from Upstate Biotechnology (Lake
Placid, N.Y.).
Isolation and Culture of Hepatocytes
[0075] Rat hepatocytes were isolated by an adaptation of the
calcium two step collagenase perfusion technique as taught by Kost,
D. P., et al, J. Cell. Physiol. 147:274 (1991), the disclosure of
which is incorporated herein by reference. All such preparations
were designed to obtain a population of pure hepatocytes. Unlike
many other hepatocyte culture systems, the present invention does
not require or use a feeder cell co-culture or feeder-cell
conditioned media. After isolation of hepatocytes, the cells were
suspended in a media used for cell attachment to the culture
plates. This media was MEM (GIBCO 12570) with NEAA (GIBCO 11140),
insulin 5 mg/L, and gentamycin 5 .mu.g/ml. Hepatocytes were plated
on a single layer of collagen coating as described below and left
to attach for 2 hours. Six-well cluster plates (9.8 square
centimeters per plate) from Corning were used. For experiments
where hepatocytes were not going to be induced to proliferate, or
where assessments of differentiated function were performed, the
cells were plated at a density of 80,000 hepatocytes per square
centimeter of surface area. For experiments intended to induce
proliferation or genetic transduction with foreign DNA, cells were
plated at an initial density of 1,000 or 10,000 hepatocytes per
square centimeter of surface area. The plating medium was replaced
with the invention HBM medium at 2 hours after the cells were
plated and every 48 hours thereafter. Thymidine, growth factors,
and other ingredients, were added at the time of medium change as
required.
[0076] Human hepatocytes were isolated by an adaptation of the
collagenase perfusion technique as described by Strom, S. C., et
al., Journal of National Cancer Institute 65(5):771-8 (1982), the
disclosure of which is incorporated herein by reference. Cells were
cultured as described above for the rat hepatocytes.
[0077] Collagen gels were prepared as described by Michalopoulos,
G. K., et al., Exp. Cell. Res. 94:70 (1975), the disclosure of
which is incorporated herein by reference. Dry coating of plates
with collagen and Matrigel was also performed as specified by the
manufacturer. Matrigel gels were made by adding 50 .mu.l of
Matrigel solution into 0.5 ml of medium directly on the top of
attached cells.
[0078] DNA synthesis was measured by the uptake of tritiated
thymidine into trichloroacetic acid (TCA) precipitable material as
described by Kost, D. P., et al. (1991), cited above. Collagen
gels, where necessary, were digested with 2 mg of collagenase per
ml of MEM medium used. Incubation was then carried out for 30
minutes at 37.degree. C. The digested gels were treated with NaOH
followed by TCA to precipitate DNA, RNA, and proteins as described
by Kost, D. P., et al. (1991), cited above.
Composition of the HBM Medium
[0079] DMEM, HEPES, L-glutamine, and antibiotics were purchased
from GIBCO/BRL (Gaithersburg, Md.). ITS mixture (Insulin,
Transferrin, Selenium) was purchased from Boehringer Mannheim. All
other additives were cell culture grade (Sigma). Unless otherwise
indicated for specific experiments, the stock basal medium
consisted of DMEM 11966 and DMEM 10314 blended to achieve a final
D-glucose concentration of 2.0 g/L. In this case, 445 ml of DMEM
10314 was blended with 555 ml of DMEM 11966 to achieve this
concentration. The formulas for these stock basal media are set
forth in Table I above. The resulting blended medium was then
supplemented with: purified bovine albumin 2.0 g/L, D-galactose 2.0
g/L, L-ornithine 0.1 g/L, L-proline 0.030 g/L, nicotinamide 0.610
g/L, ZnCl.sub.2 0.544 mg/L, ZnSO.sub.4.7H.sub.2O 0.750 mg/L,
CuSO.sub.4.5H.sub.2O 0.20 mg/L, MnSO.sub.4 0.025 mg/L, glutamine
5.0 mM, ITS (rh-insulin 5.0 mg/L, human transferrin 5.0 mg/L (30%
diferric iron saturated], selenium 5.0 .mu.g/L), dexamethasone
10.sup.-7 M, and HEPES buffer 20.0 mM. Penicillin and streptomycin
were added at 100 U/L and 100 .mu.g/L, respectively. The mixed
basal HBM was sterilized by filtration through a 0.22-.mu.m low
protein-binding filter system (Corning), stored at 4.degree. C.,
and used within 4 weeks. The growth factors, as required, were
added to HBM fresh at the specified concentrations every time the
medium was changed.
Retroviral Transfection and Assessment of Clonal Expansion
[0080] Hepatocytes were initially plated at 10.sup.4/cm.sup.2 and
grown in HBM supplemented with HGF/SF (40 ng/ml) and EGF (20
ng/ml). After 68 hours the media was replaced with supernatant from
CR.PSI.P-packaged, replication-deficient, amphotropic retrovirus
(MFG-5.times.10.sup.5 units per ml) containing the E. coli
.beta.-galactosidase gene under an LTR promoter as described by
Zitvogel, L. H., et al., Hum. Gene. Ther. 5:1493 (1994), the
disclosure of which is incorporated herein by reference. Polybrene
was added at 2 .mu.g/ml. The supernatant was replaced after 18
hours with HBM that was supplemented with EGF at 20 ng/ml and
HGF/SF at 40 ng/ml. The brief exposure with the virus-containing
supernatant did not have an adverse effect on hepatocyte survival
or proliferation. At indicated times, cells were fixed with 0.5%
glutaraldehyde in PBS for 10 min and developed with X-Gal substrate
at 37.degree. C. for 16 hours. Transduced cells expressing the E.
coli gene stained positive as shown in FIGS. 4G and 4F. Appropriate
controls for each component were negative for X-Gal staining.
Transmission Electron Microscopy
[0081] Samples for transmission electron microscopy (TEM) were
fixed on the culture plates for 1-1.5 hours in 0.1 M sodium
cacodylate buffer (pH 7.4) that contained 2.5% glutaraldehyde and
2% formuldehyde. The plates were then rinsed 2 times with 0.1 M
sodium cacodylate buffer (pH 7.4) and 2 times with 0.1 M sodium
cacodylate buffer containing 5% sucrose (pH 7.4). They were held in
the sucrose buffer for 1-7 days, rinsed 2 times with 0.1 M sodium
cacodylate buffer (pH 7.4), and then postfixed for 1 hour in
OsO.sub.4 in 0.1 M sodium cacodylate buffer. The plates were then
rinsed again in buffer, and the fixed and processed collagen gels
were then cut in strips with a razor blade. The strips were then
transferred to glass specimen vials, dehydrated through a graded
series of ethanol (25-100%) and two propylene oxide changes, and
infiltrated with Epon-Araldite resin (BioTec, Tex.). Several
changes of resin were made over 2 days, as the collagen gels tended
to hold the propylene oxide. The collagen strips were flat-embedded
and cured overnight at 60.degree. C.
Analysis of Gene Expression by Northern Blots
[0082] Extraction of Total RNA and mRNA from Cultures.
[0083] Total RNA was extracted from unwashed cell cultures using
2.0 ml of RNAzol B (BioTec) per well and purified per the
manufacturer's guidelines. RNA concentration and purity were
determined by routine spectrophotometry. Size separation of 20
.mu.g RNA per lane was completed on denaturing 1% agarose gels and
transferred to nylon membranes (Amersham, Arlington Heights, Ill.)
by the capillary method. After cross-linking under UV light, the
membranes were hybridized overnight with specific cDNAs (as
indicated in the figures) that had been labeled with
[.alpha.-.sup.32P] dCTP using an Amersham random primer kit.
Membranes were subsequently washed under high stringency conditions
and exposed to XAR film (Eastman Kodak, Rochester, N.Y.) for 1-3
days. Quantification of the RNA hybridization bands were performed
by laser densitometry.
Sources of cDNA Probes
[0084] cDNA probes used to study gene expression were obtained as
gifts, and are available upon request from the following sources:
Cytokeratin 8 from Dr. Norman Marceau (Laval University);
Cytokeratin 14 from Dr. Dennis Roop (Baylor College of Medicine);
Cytokeratin 18 from Dr. Robert Oshima (LaJolla Cancer Research
Foundation); Cytokeratin 19 from Dr. Andre Royal (University of
Montreal); TGF.alpha. (rat) originated from Dr. David Lee
(University of North Carolina at Chapel Hill); EGF-R (rat)
originated from Dr. Sheldon Earp (University of North Carolina at
Chapel Hill); aFGF from American Type Culture Collection (ATCC)
(catalog No. 78222); aFGF-R from ATCC (catalog No. 65796); uPA
originated from Dr. Jay Degen (University of Cincinnati);
cytochrome 11B1 from Dr. Steve Strom (University of Pittsburgh);
cDNAs for albumin, .alpha. fetoprotein, and transcription factor
analysis were generated by Dr. Joe Locker (University of
Pittsburgh).
RESULTS OBTAINED
Role of Media Components and Matrix Substrates on Cell
Proliferation
[0085] The full description of the HBM medium is given above. To
evaluate the relative importance of different media components
several experiments were performed whose results are shown in Table
IV (A, B, and C) below. The components D-glucose, albumin,
dexamethasone, transferrin and selenium, nicotinamide, and trace
elements were individually subtracted from the full HBM medium
composition as shown in Table IV A. The total DNA per culture after
14 days of growth is shown therein. As can be readily seen, removal
of dexamethasone had the most dramatic effect, followed by removal
of nicotinamide. In Table VI B, growth of cells achieved by day 14
is compared between HBM medium containing diferric transferrin
(iron saturated) versus iron unsaturated transferrin. Addition of
iron containing diferric transferrin (30% saturation) was found to
be much more effective in promoting growth. The addition of
elemental iron (FeSO.sub.04, 0.1 .mu.M) to the unsaturated
transferrin failed to overcome the difference. Table VI C provides
information on the relative effects of D-glucose, D-galactose, and
L-ornithine in HBM medium. All three of these components are
potential sources of energy to the cells. Complete cessation of
growth was noticed when all three components were removed. Addition
of D-glucose alone restored most of the response whereas the
addition of D-galactose alone was less effective. Ornithine alone
had minimal effect. Concentrations of 2 g per liter each of albumin
and D-glucose were found to be optimal though the effects were not
statistically different than 1 or 3 g per liter in each case. The
effect of complete removal of these components is shown in Table IV
A. TABLE-US-00004 TABLE IV .mu.g/well A. Effect of Removal of
Specific HBM Components on Hepatocyte Growth Zero time DNA 13.90
.+-. 3.60 DNA at day 14 in HBM (with HGF/SF and EGF) supplemented
with: +All components 84.90 .+-. 0.50 -Glucose 69.80 .+-. 2.90
-Albumin 68.70 .+-. 0.50 -Dexamethasone 13.70 .+-. 0.20
-(Transferrin and selenium) 63.10 .+-. 2.00 -Nicotinamide 35.20
.+-. 1.70 -Trace elements 65.00 .+-. 2.40 -All components 20.20
.+-. 4.30 B. Effects of Iron and Transferrin on Hepatocyte Growth
Zero time DNA 9.21 .+-. 1.01 Diferric transferrin 70.15 .+-. 1.21
(iron saturated) Diferric transferrin plus 1.83 .+-. 0.41 added
iron (5 .mu.M) Iron poor transferrin 24.84 .+-. 4.30 Iron poor
transferrin with 1.10 .+-. 0.90 added iron C. Effect of Removal of
Glucose, Ornithine, or Galactose Zero time 11.0 .+-. 0.4 Control
(Gluc.+, Orn.+, Gal+) 59.0 .+-. 2.5 Gluc.-, Orn.-, Gal.- 10.6 .+-.
0.8 Gluc.-, Orn.+, Gal.- 14.4 .+-. 0.9 Gluc.-, Orn.-, Gal.+ 44.2
.+-. 6.0 Gluc.+, Orn.-, Gal.- 59.0 .+-. 4.0 Gluc.+, Orn.+, Gal.-
62.7 .+-. 1.4 Gluc.+, Orn.-, Gal.+ 63.6 .+-. 0.3 The indicated HBM
components were removed and hepatocyte were grown in the modified
media for 14 days. The total DNA per culture in micrograms was
measured at day 14 to evaluate growth cell growth. Time zero was
the sample of hepatocyte suspension that was inoculated into the
plate immediately after cell isolation. The data are expressed as
mean .+-. standard error of three separate plates.
Hepatocytes Enter Diffusely into Proliferation under the Influence
of HGF/SF, EGF, and TGF.alpha.
[0086] FIGS. 1A and 1B show the uptake of thymidine per .mu.g DNA,
as well as the BRdU nuclear labeling index at different days in
culture in cells growing in the presence of HGF/SF and EGF (40, 20
ng/ml respectively) as described above. As can be seen, most of the
proliferation occurs at days 5-12. By day is the cultures were
confluent and DNA synthesis slowed down. The high nuclear labeling
index during the times of sustained proliferation indicates that
the proliferating cells derive directly from the mature
hepatocytes.
[0087] The growth factors HGF/SF, EGF, TGF.alpha., KGF
(keratinocyte growth factor), SCF (stem cell factor), and aFGF
(acidic fibroblast growth factor) were added individually to HBM
medium. Of the growth factors added, HGF/SF, EGF, and TGF.alpha.
(shown in FIG. 2 as "TGFa") caused significant cell proliferation,
as shown by the total amount of DNA per culture at day 15. KGF,
aFGF, and SCF when added alone or in combination had no
proliferative effect as shown in FIG. 2. TGF.alpha. had a stronger
proliferative effect than any of the other mitogens when added
alone to HBM medium. HGF/SF and EGF together had the strongest
proliferative effect for a given time interval of 15 days together
than did any other single mitogens or combinations. Addition of all
the growth factors together had no more effect than the combined
HGF/SF and EGF as seen in FIG. 2. The detailed cell kinetics
induced by HGF/SF and EGF alone or in combination are shown in FIG.
1C. The total DNA per culture is shown as a function of the time in
culture. The largest amount of accumulated DNA at day 15 was seen
with the combination of HGF/SF and EGF. The DNA per culture at day
15 was 12 times that at time 0, reflecting the increase in cell
number. HGF/SF and EGF were about equally potent. TGF.alpha. alone
was more mitogenic than either HGF/SF or EGF alone.
Effect of Matrix Substrates
[0088] Several matrix substrates promoted cell growth in this
system. Rat hepatocytes were cultured for 15 days in HBM
supplemented with HGF/SF (40 ng/ml) and EGF (20 ng/ml) grown on
various matrices as seen in FIG. 3. The cells were cultured as
described above. Dry coating with collagens type IV (mouse), type I
(bovine), fibronectin, and laminin were equally effective in
promoting cell growth as assayed by measurement of total DNA per
culture at day 15. Dry coating with ECL (a commercial derivative of
EHS gel, UBI) had superior effects. Coating with type I collagen
(Vitrogen commercial preparation) was the standard method used for
the experiments unless otherwise specified. The effect of matrix
gels in promoting specific phenotypic conversions in these cultures
is further discussed below.
Phenotypic Chances of Hepatocytes During Proliferation
[0089] The morphology of the proliferating cells (cultured in HBM
as described above supplemented with 40 ng/ml HGF/SF and 20 ng/ml
EGF) varied at different times after the stimulation of cell
proliferation. From a normal hepatocyte morphology as seen in FIG.
4A, the proliferating cells in the first 4 days acquired long
projections assuming the phenotype typically described as due to
the "scattering" effect of HGF/SF on hepatocytes as seen in FIG. 4B
as described by Michalopoulos, G. K., et al, J. Cell. Physiol.
156:443 (1993), the disclosure of which is incorporated herein by
reference. Between days 6 and 8, the proliferating cells lost most
of their cytoplasmic granules, the nuclei became less prominent,
the projections diminished and the cells began to grow as monolayer
patches. Eventually these patches merged as the cells continued to
grow to form a continuous monolayer as seen in FIG. 4C. Examination
by electron microscopy seen in FIGS. 4D and 4E showed that most of
the features typical of mature hepatocytes were missing. By day 15
there were no lamallae of endoplasmic reticulum wrapping around
mitochondria and there are no glycogen rosettes or peroxisomes.
Bile canaliculi were absent. There was a prominent increase in
bundles of keratin intermediate filaments. The nuclei were angular
with very prominent nucleoli. After confluency the morphology
gradually revert back to mature differentiated hepatocytes with
endoplasmic reticulum lamallae, mitochondria, glycogen,
peroxisomes, bile canaliculi, etc., similar to that in FIGS. 6B and
6C.
[0090] Clonal growth of the proliferating rat hepatocytes is
demonstrated in FIGS. 4F and 4G. Hepatocytes were transfected at
day three in culture with a replication-deficient retrovirus
containing the lac-Z gene under the influence of a viral LTR and
were stained for expression of .beta.-galactosidase. Mostly single
cells were stained positive at day four in culture (1 day after
transfection) as seen in FIG. 4F. On the other hand, staining at
day 10 and continuing through day 28 showed patches of
positive-stained, hepatocytes, consistent with clonal growth of the
original transfected hepatocytes as seen in FIG. 4G. The percentage
of lac-Z-positive cells (-20%) did not appear to change during
culture.
Proliferating Hepatocytes Express Altered Levels of Various
Genes
[0091] The expression of several specific genes was assayed in
proliferating hepatocytes. These included mRNA genes associated
with hepatocyte differentiation (albumin, ctyochrome IIB1 (labeled
as P450 in FIG. 5A)), genes encoding cytokeratin markers
(cytokeratins 14, 18, and 19) or related to hepatocyte growth
(urokinase (uPA), HGF/SF and its receptor c-met (labeled as MET in
FIG. 5A), EGF (labeled as EGFR) and TGF.alpha. and their receptor,
acidic FGF and its receptor, and TGF.beta.1). These genes were
studied by Northern blot analysis of RNA from cultures grown in the
presence of either combined HGF/SF (40 ng/ml)and EGF (20 ng/ml)
(FIGS. 5A and 5B) or with TGF.alpha. alone (20 ng/ ml) (data not
shown). Total RNA was isolated from cultures at days 0, 6, 10, 15,
and 21. No expression of HGF/SF or TGF.beta.1 mRNA was seen at any
of the time points examined.
[0092] As can be seen, albumin and cytochrome IIB1 mRNA were
present at time zero and subsequently decreased. Albumin mRNA
increased by day 21 at which time mRNA for a feto protein (AFP) was
also detected. mRNA for cytokeratins, 14, 18, and 19 increased
through culture. There was a steady increase in mRNA for aFGF and
TGF.alpha.. The mRNA of receptors for HGF/SF (MET, FIG. 5A) and
aFGF (FIG. 5B aFGFR) remained present throughout the culture time.
The EGF receptor (EGFR) mRNA declined from day zero but remained
expressed. GAPDH mRNA expression was used as a reference
"housekeeping" gene. Dramatic increases were noted in expression of
urokinase as well as cytokeratins 14 and 19. Some differences from
the above pattern were seen in the cultures that, instead of HGF/SF
and EGF, were maintained in the presence of TGF.alpha. (20.0
ng/ml). In these cultures albumin expression and expression of the
HGF/SF receptor were better preserved during proliferation whereas
AFP appeared earlier. (Data not shown.) Despite the observed
differences in gene expression patterns no morphologic differences
were seen between cells growing in the presence of TGF.alpha. or
HGF/SF plus EGF, once confluency was achieved.
Proliferating Hepatocytes Revert to Mature Hepatocytes under the
Influence of Matrigel or in the Presence Nonparenchymal Cells or
With Time in Culture
[0093] When Matrigel was overlaid on cultures at day 8, there was a
rapid (within 2 days) appearance of bile canaliculi and
organization of the cells into cord-like structures. The features
of these cells are shown in FIG. 6A. As shown by electron
microscopy in FIGS. 6B and 6C, these cells had typical markers of
mature hepatocytes, including wrapping of the endoplasmic reticulum
around mitochondria, bile canaliculi, and the presence of glycogen.
Preparations of mRNA were made from cultures exposed to Matrigel
for 10 days (days 8-18 in culture). The expression of albumin was
compared between day zero in culture (immediately after collagenase
perfusion), at day 8 in culture (before the overlay by Matrigel),
and cultures at day 3 and 7 after Matrigel overlay as shown in FIG.
6D. Addition of Matrigel caused dramatic increases in expression of
albumin mRNA, compared to control proliferating cultures in which
it was minimally detectable. The effect of Phenobarbitol (PB) on
the levels of cytochrome P450 IIB1 mRNA in the Matrigel-treated
cultures was also measured as shown in FIG. 6E. Matrigel was added
to the cultures at day 8. PB was added 2 days later (day 10 of
culture). The cells were harvested 5 days after addition of PB (day
15 in culture). Addition of PB induced cytochrome IIB1 mRNA only in
the Matrigel-treated cultures. Induction of this mRNA by
phenobarbitol is typical of hepatocytes and does not occur in any
other cell as reported by Michalopoulos, G., et al., Science (Wash.
D.C.) 193:907 (1976), the disclosure of which is incorporated
herein by reference. Typically, hepatocyte cultures rapidly lose
the capacity to respond to PB. This finding is supporting evidence
that addition of Matrigel to the cultures of proliferating
hepatocytes induces a mature hepatocyte phenotype, as attested to
by the electron microscopic structure shown in FIGS. 6B and 6C. The
expression of cytokeratin 19 (CK19) (FIG. 6F), a bile duct marker
expressed by the proliferating hepatocytes before introducing
differentiating conditions, also ceased expression after addition
of Matrigel as seen in FIG. 5B.
[0094] DNA synthesis was measured in the cultures exposed to
Matrigel and there was a substantial decrease. To assess whether
differentiation to mature hepatocyte morphology required DNA
synthesis, 20 mM hydroxurea was added to the HBM media. This has
been shown (Michalopoulos, G. K., et al., Cancer Res. 38:1866
(1978), the disclosure of which is incorporated herein by
reference) to abolish scheduled semiconservative DNA synthesis in
hepatocytes by inhibiting ribonucleotide reductase. Hydroxyurea was
added to cultures before the Madrigel overlay and maintained
throughout the next 5-day period. DNA synthesis decreased down to
3.93% of the control (without hydroxyurea) in the proliferating
cultures maintained in the absence of Matrigel and down to 6.27% of
control (without hydroxyurea) in the cultures maintained in the
presence of Matrigel. Though DNA synthesis was decreased down to
6.27% of control (+Matrigel, no hydroxyurea) levels, the conversion
of the proliferating hepatocytes to mature hepatocyte morphology
was entirely unaffected and involved the entire population.
HGF/SF (But Not TGF.alpha. or EGF) Induces Proliferating
Hepatocytes to Differentiate into Ductular/Acinar Structures in
Type I Collagen Gels
[0095] Hepatocytes maintained between two collagen gel layers
retain their morphology and differentiation for prolonged time
periods as described by Michalopoulos, G. K., et al., J. Cell.
Physiol. 156:443 (1993), the disclosure of which is incorporated
herein by reference. Hepatocytes maintained in collagen gel
sandwiches in cultures with convential media containing HGF/SF
undergo intense proliferation, form prominent projections, and
eventually become organized in structures reminiscent of the
hepatic plates. The behavior of hepatocytes maintained between two
layers of collagen gel as previously described was examined, but in
the presence of HBM supplemented with either HGF/SF or EGF as
described above. It was noted that in the EGF supplemented media
hepatocytes underwent the typical phenotypic transitions as
described above. On the other hand, in hepatocytes in HEM
supplemented with HGF/SF alone, following expansion of cells
identical in appearance to the proliferating hepatocytes described
above, there appeared multiple duct-shaped structures between days
10 and 15. These became prominent and encompassed most of the cells
present in the cultures. Starting from approximately day 10 and by
day 15 most of the cells in the culture were arranged in such
ductular structures. The appearance of these structures is shown in
FIG. 7A. Histologic sections are shown in FIG. 7B (light
microscopy) and FIGS. 7C and 7D (electron microscopy). The
structures had a ductular or acinar configuration. Some of the
cells surrounding these structures were very attenuated and had
light and electron microscopic appearance identical to bile duct
epithelium. Others however are larger and resemble more closely the
ductular hepatocytes described in previous studies of in vivo
models. The proliferation of cells (data not shown) under either
EGF or HGF/SF in the collagen gel sandwiches was much less (<25%
at the highest peak) than that seen in the cultures on plastic
coated with dry collagen. Most proliferation ceased by day 10 and
the duct-like structures appeared after cell proliferation had
ceased (days 10-15). Ductular acinar structures were also noted in
these cultures when HGF/SF and EGF were combined but were fewer
than with HGF/SF alone. As with the Matrigel overlay, additional of
hydroxyurea to inhibit DNA synthesis (inhibition down to 5.1% of
control) did not affect the formation of the ductular structures
(data not shown). FIG. 7E shows that these cells express
cytokeratin 19 which is characteristic of duct cells. (See, Sirica,
A. E., Prog. Liver Dis. 10:63 (1992) and Sirica, A. E., Histol.
Histopathol. 10:433 (1995), the disclosures of which are
incorporated herein by reference.) A small amount of albumin
expression was also retained, consistent with the presence of
hepatocyte-like cells within the ductular structures as seen in
FIG. 7D. It was noted that the ductular cells maintain CK19
expression in the nonproliferating state (FIG. 7F) in contrast to
the cells differentiating toward the mature hepatocyte lineage
which cease expressing this bile duct marker.
Sustained Growth and Population Expansion of Human Hepatocytes in
HGM in the Presence of HGF/SF and EGF
[0096] Though human hepatocytes cultures have not been
characterized as extensively as those of the rat, the literature
available has shown that these cells also undergo a limited round
of DNA synthesis after stimulation by growth factors and rapidly
degenerate in culture. See, Ismail, T., et al., Hepatology 14:1076
(1991), the disclosure of which is incorporated herein by
reference. The response of human hepatocytes to HGF/SF (40 ng/ml)
and EGF (20 ng/ml) in HBM medium was studied. Results similar to
those described for rat hepatocytes were found in primary cultures
of human hepatocytes, as seen in FIG. 8. The human cells begin to
rapidly proliferate at day 3-4 in culture and reach confluency by
day 19.
[0097] As stated above, there is a present need for a media that
allows hepatocytes that are cultured in vitro to expand as a cell
population. The HBM of the present invention allows such expansion
and therefore enables much needed research of proliferating
hepatocytes. The present invention also will be useful in numerous
other applications outlined below.
[0098] For example, all current methods in liver-targeted gene
therapy that achieve stable, long-term expression of transferred
genes require actively dividing cells during the initial
transfection. Normal liver has only 1 in 20,000 hepatocytes in S
phase growth on any particular day. In order to increase cell
proliferation, a large portion (2/3) of the patient's liver must be
removed. Subsequently the liver-targeted gene carrier source is
intravascularly injected. The alternative to this drastic measure
is to remove a small piece of liver (10%), culture the hepatocytes,
transfect them in culture, and then reinfuse the cells back into
the liver. This latter method, while safer, less expensive and
better controlled than the former, is currently hampered by the
lack of culture media such as HBM which allows prolonged
proliferation, clonal expansion of essentially all cells cultured,
and long term viability of the cells.
[0099] The design of current extracorporea 5(b), and a linear-valve
pressure difference .DELTA.P1 (additional linear-valve pressure
difference .DELTA.Pdadd) necessary for generating a hydraulic
braking force corresponding to the above-described "decrease in the
linear-valve-pressure-difference braking force Fval" for the front
wheel cylinders Wfr and Wfl (.DELTA.Pd+.DELTA.Pdadd, i.e., a value
larger than a value when both of the linear solenoid valves PC1 and
PC2 are normal and smaller than a value twice as large as that)
(refer to .DELTA.P1').
[0100] In this case, the linear-valve-pressure-difference braking
force Fval is equal to that when both of the linear solenoid valves
PC1 and PC2 are normal. As a result, as indicated by the solid line
of FIG. 11(a), also the total braking force (=Fvb+Fcomp) becomes
equal to that when both of the linear solenoid valves PC1 and PC2
are normal (becomes constant at a value Ft).
[0101] For this reason, when the brake pedal BP is operated in the
case where only the linear solenoid valve PC2 fails (e.g., a break
in wire), the device sets the linear-valve pressure difference
.DELTA.P1 of the rear-wheel-side linear valve PC1 (specifically,
the command pressure difference .DELTA.Pd to the front-wheel-side
linear valve PC1) to a value larger than a value when both of the
linear solenoid valves PC1 and PC2 are normal and smaller than a
value twice as large as that) (.DELTA.Pd+.DELTA.Pdadd).
[0102] In this way, even when either of the linear solenoid valves
PC1 and PC2 fails (e.g., a break in wire), the characteristic of
the total braking force relative to the brake-pedal pressure Fp can
be agreed with the target characteristic indicated by the solid
line A of FIG. 4. The means for increasing the linear-valve
pressure difference (pressurization) of normal one when one of the
linear solenoid valves PC1 and PC2 fails corresponds to the
pressurization-intensifying section.
Actual Operation of Second Embodiment
[0103] The actual operation of the vehicle braking device according
to the second embodiment will be described. The HV ECU 60 of this
device executes the routine shown in FIG. 8 for the HV ECU 60 of
the first embodiment. The brake ECU 50 of this device executes the
routine shown in the flowchart of FIG. 12, in place of the routine
of FIG. 7 executed by the brake ECU 50 of the first embodiment. The
routine shown in FIG. 12 specific to the second embodiment will be
described hereinbelow.
[0104] The brake ECU 50 of the device repeats the routine of
controlling the hydraulic braking force, shown in FIG. 12, at a
fixed interval (a time interval .DELTA.t, e.g., 6 msec). In the
routine of FIG. 12, the same steps as those of FIG. 7 are given the
same step numbers as those of FIG. 7.
[0105] Accordingly, the brake ECU 50 starts the operation from step
700 at a predetermined time. Assuming that the brake pedal BP is in
operation, the brake ECU 50 executes the operation from steps 705
to 735, as in FIG. 7, wherein it determines in step 735 whether or
not one of the linear solenoid valves PC1 and PC2 fails.
[0106] When only the front-wheel-side linear-valve PC1 fails, the
brake ECU 50 makes a positive determination in step 735, and then
moves to step 1205, wherein it determines whether or not the
front-wheel-side linear valve PC1 fails. In this case, the brake
ECU 50 makes a positive determination, and moves to step 1210,
wherein it determines an additional linear-valve pressure
difference .DELTA.Pdadd from the command pressure difference
.DELTA.Pd obtained in step 730, and a table Map.DELTA.P2 havtion
except as it may be limited by the claims.
* * * * *