U.S. patent application number 13/909320 was filed with the patent office on 2013-12-26 for immortal unipotent porcine picm-19h and picm-19b stem cell lines.
The applicant listed for this patent is U.S. Department of Agriculture. Invention is credited to Thomas J Caperna, Neil C. Talbot, Ryan R. Willard.
Application Number | 20130344154 13/909320 |
Document ID | / |
Family ID | 41340769 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344154 |
Kind Code |
A1 |
Talbot; Neil C. ; et
al. |
December 26, 2013 |
Immortal Unipotent Porcine PICM-19H and PICM-19B Stem Cell
Lines
Abstract
Two cell lines, PICM-19H and PICM-19B, were derived from the
bipotent ARS-PICM-19 pig liver stem cell line. The unipotent
porcine stem cell line PICM-19H differentiates exclusively into
hepatocytes and can be induced to express CYP450 enzymes. The
growth rate and cell density in culture, morphological features,
and hepatocyte detoxification functions, i.e., inducible CYP450
activity, ammonia clearance, and urea production of the PICM-19H
cells were evaluated for their application in artificial liver
devices. PICM-19H cells contain numerous mitochondria, Golgi
apparatus, smooth and rough endoplasmic reticulum, vesicular bodies
and occasional lipid vacuoles and display inducible CYP450
activity, clear ammonia, and produce urea in a glutamine-free
medium. The data indicate that both cell lines, either together or
alone, may be useful as the cellular substrate for an artificial
liver device. The results demonstrate the potential for the use of
PICM-19H cells in drug biotransformation and toxicity testing.
Inventors: |
Talbot; Neil C.;
(Clarksville, MD) ; Caperna; Thomas J; (Arnold,
MD) ; Willard; Ryan R.; (Halethorpe, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
U.S. Department of Agriculture |
Washington |
DC |
US |
|
|
Family ID: |
41340769 |
Appl. No.: |
13/909320 |
Filed: |
June 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12154631 |
May 23, 2008 |
8486699 |
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13909320 |
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Current U.S.
Class: |
424/490 ;
424/93.7; 435/177; 435/325 |
Current CPC
Class: |
C12N 11/04 20130101;
C12N 11/00 20130101; A61P 43/00 20180101; C12N 5/0672 20130101;
C12N 11/02 20130101 |
Class at
Publication: |
424/490 ;
435/325; 435/177; 424/93.7 |
International
Class: |
C12N 5/071 20060101
C12N005/071; C12N 11/00 20060101 C12N011/00 |
Claims
1-3. (canceled)
4. An isolated, immortal, unipotent porcine stem cell line wherein
cells of said cell line are capable of differentiating exclusively
into functional bile duct cells expressing cholangiocyte
functions.
5. The stem cell line of claim 4 wherein said cells exhibit all the
identifying characteristics of PICM-19B cells and said
characteristics are that the cells are basolaterally polarized
cells exhibiting basal membrane fluid transport, high GGT activity
and a lack of serum protein production.
6. The stem cell line of claim 26 wherein the stem cell line is the
immortal, unipotent PICM-19B stem cell line deposited as ATCC
PTA-9173.
7. The cell line according to claim 4 wherein a culture of said
cells further comprises feeder cells.
8. A composition comprising cells of the cell line according to
claim 4, said cells being attached to microbeads.
9. A composition comprising cells of the cell line according to
claim 4, said cells being encapsulated in alginate
microcapsules.
10. A composition comprising cells of the cell line according to or
claim 4, said cells being attached to and between single hollow
fibers.
11. (canceled)
12. A bioartificial liver device comprising cells of an isolated,
immortal, unipotent porcine stem cell line wherein cells of said
cell line are capable of differentiating exclusively into
functional bile duct cells expressing cholangiocyte functions, and
a support for said cells.
13. A bioartificial liver device comprising cells of an isolated,
immortal, unipotent porcine stem cell line wherein cells of said
cell line are capable of differentiating exclusively into
hepatocytes, cells of an isolated, immortal, unipotent porcine stem
cell line wherein cells of said cell line are capable of
differentiating exclusively into functional bile duct cells
expressing cholangiocyte functions, and a support for said
cells.
14. The bioartificial liver device of any one of claims 12 and 13,
wherein said support comprises a collection of single hollow
fibers, microbeads, or alginate microcapsules.
15. (canceled)
16. The method of using the cell lines of any one of claims 4-6 as
a bioartificial liver device, wherein said cells are capable of
functioning as functional bile duct cells expressing cholangiocyte
functions, for a patient in need of functioning cholangiocytes.
17-18. (canceled)
19. The method of using a composition comprising cells of the cell
lines of any one of claims 4-6, said cells being attached to
microbeads, attached to inorganic or organic porous supports,
attached to single hollow-fibers or encapsulated in hydrogel-based
supports to treat body fluids, such as whole blood, blood plasma,
and peritoneal fluids as a bioartificial liver device, wherein said
cells are capable of functioning as functional be duct cells
expressing cholangiocyte functions, for a patient in need of
functioning cholangiocytes.
20-24. (canceled)
25. A screening assay kit comprising PICM-19H cells, PICM-19B cells
or both.
26. An isolated, immortal, unipotent porcine stem cell line wherein
cells of said cell line are capable of differentiating exclusively
into functional bile duct cells expressing cholangiocyte functions
exhibiting all the identifying characteristics of PICM-19 Bile duct
(PICM-19B) cells and said characteristics are that the cells are
basolaterally polarized cells exhibiting basal membrane fluid
transport, high GGT cholangiocyte-like activity observed as a
recognized functional marker of bile duct (cholangiocyte) cells,
and a lack of serum protein production.
27. The method of using the immortal, unipotent PICM-19 Hepatocyte
(PICM-19H) stem cell line deposited as ATCC PTA-9174 as a
bioartificial liver device, wherein said cells are capable of
functioning as hepatocytes, for a patient in need of functioning
hepatocytes.
28. The method of using the immortal, unipotent PICM-19 Hepatocyte
(PICM-19H) stern cell line deposited as ATCC PTA-9174 together with
the cell lines of any one of claims 4-6 as bioartificial liver
device, wherein said cells are capable of functioning as
hepatocytes and as functional bile duct cells expressing
cholangiocyte functions, for a patient in need of functioning
hepatocytes and cholangiocytes.
29. The method of using a composition comprising cells of the
immortal, unipotent PICM-19 Hepatocyte (PICM-19H) stem cell line
deposited as ATCC PTA-9174, said cells being attached to
microbeads, attached to inorganic or organic porous supports,
attached to single hollow-fibers or encapsulated in hydrogel-based
supports to treat body fluids, such as whole blood, blood plasma,
and peritoneal fluids as a bioartificial liver device, wherein said
cells are capable of functioning as hepatocytes, for a patient in
need of functioning hepatocytes.
30. The method of using the immortal, unipotent PICM-19 Hepatocyte
(PICM-19H) stem cell line deposited as ATCC PTA-9174, together with
the cell lines of any one of claims 4-6, said cells being attached
to microbeads, attached to inorganic or organic porous supports,
attached to single hollow-fibers or encapsulated in hydrogel-based
supports to treat body fluids, such as whole blood, blood plasma,
and peritoneal fluids as a bioartificial liver device, wherein said
cells are capable of functioning as hepatocytes and as functional
bile duct cells expressing cholangiocyte functions, for a patient
in need of functioning hepatocytes and cholangiocytes.
31. The method of using the immortal, unipotent PICM-19 Hepatocyte
(PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell
line of claim 4 to screen for compounds or new chemical entities
which inhibit or promote an enzyme activity involved in the
metabolism of xenobiotics in the liver.
32. The method of using the immortal, unipotent PICM-19 Hepatocyte
(PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell
line of claim 4 to screen for compounds or new chemical entities
which results in cytotoxicity due to the metabolism of xenobiotics
and/or endogenous substrates in the liver.
33. The method of using the immortal, unipotent P1CM-1Hepatocyte
(PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell
line of claim 4 to screen for compounds or new chemical entities
which results in hepatotoxicity due to metabolism of xenobiotics
and/or endogenous substrates in the liver.
34. The method of using the immortal, unipotent PICM-19 Hepatocyte
(PICM-19H) stem cell line deposited as ATCC PTA-9174 and the cell
line of claim 4 to screen for compounds or new chemical entities
which results in hepatic dysfunction due to metabolism of
xenobiotics and/or endogenous substrates in the liver.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to an immortalized derivative porcine
stem cell line, PICM-19H, capable of differentiating exclusively
into hepatocyte cells expressing hepatocyte function, for example,
inducible enzyme activity, such as cytochrome P450 (CYP450)
activity; an immortalized derivative porcine stem cell line,
PICM-19B, capable of differentiating exclusively into bile duct
cells expressing bile duct cell function and forming a complete
(confluent) cell monolayer of basolaterally polarized cells; a
bioartificial liver device comprising either the PICM-19H cells or
the PICM-19B cells or both, a method of using the PICM-19H and/or
PICM-19B stem cell lines in a bioartificial liver device or support
to alleviate liver dysfunction, a method of using the PICM-19H
and/or PICM-19B stem cell lines in a screening assay to detect a
compound or new chemical entity which inhibits or promotes an
enzyme activity involved in the metabolism of xenobiotics in the
liver and/or to detect a compound or new chemical entity which
results in cytotoxicity, hepatotoxicity, or hepatic dysfunction due
to the metabolism of xenobiotics and/or endogenous substrates in
the liver; and a screening assay kit comprising PICM-19H cells
and/or PICM-19B cells.
[0003] 2. Description of the Relevant Art
[0004] Cell lines that possess in vivo-like hepatocyte functions
are needed for the biological component of bioartificial liver
devices that are currently in development (Strain and Neuberger.
2002. Science 295: 1005-1009; Chamuleau et al. 2005. Metab. Brain
Dis. 20: 327-335). Tumor-derived cell lines, of human or animal
origin, are without exception compromised in their liver functions,
presumably because of their lack of normal differentiation and
uncontrolled growth characteristics (Nyberg et al. 1994. Ann. Surg.
220(1): 59-67; Wang et al. 1998. Cell Transplant 7: 459-468;
Kobayashi et al. 2003a. J. Artif. Organs. 6: 236-244; Kobayashi et
al. 2003b. Keio J. Med. 52: 151-157; Rodriguez-Antona et al. 2002.
Xenobiotica 32: 505-520; Filippi et al. 2004. J. Hepatol. 41:
599-605). Although new cell lines transfected with immortalizing
transgenes are being developed and tested, there is no assurance
that these cell lines won't suffer from similar problems for
similar reasons (Hoekstra and Chamuleau. 2002. Int. J. Artif.
Organs. 25: 182-191; Kobayashi et al. 2003b, supra). To date, most
clinically tested bioartificial liver devices have used fresh or
frozen porcine hepatocytes as the cell component in the device
(Hoekstra and Chamuleau, supra; Demetriou et al. 2004. Ann. Surg.
239 (5): 660-670). While some efficacy in patient support has been
achieved using these "liver-harvested" hepatocytes (Demetriou et
al., supra), they are also compromised as cell components of
bioartificial liver devices because the harvested hepatocyte cells
rapidly die within the bioartificial liver device, and in addition,
the cells can be under attack by the patient's preformed antibodies
and complement factors, and further, such cell preparations are
variable, and, therefore, are a potentially unsafe, cell source
(Rodriguez-Antona et al., supra; Filippi et al., supra; Di Nicuolo
et al. 2005. Xenotransplantation 2: 286-292).
[0005] Presently, most testing of new pharmacological and chemical
agents in vitro for the purpose of investigating any adverse
reactions with liver cells and liver cell function is performed
with primary hepatocyte cultures, hepatocyte cell lines, or
microsomal preparations derived from liver tissue or cells (Bertz
and Granneman. 1997. Clin. Pharmaokinet. 32: 210-258; Yan and
Caldwell. 2001. Curr. Top. Med. Chem. 1: 403-425; Vermeir et al.
2005. Expert Opin. Drug Metab. Toxicol. 1: 75-90). Microsomal
preparations, while useful for some assessment, cannot be used to
assess and predict cellular enzyme inductions or transport
processes (Shimada et al. 1994. J. Pharmacol. Exp. Ther. 270:
414-423; Gomez-Lechon et al. 2004. Curr. Drug Metab. 5: 443-462).
Fresh primary hepatocyte cultures can provide in vitro models of
liver cellular function and can be prepared from a variety of
species, including from specific disease state animal models
(Guillouzo, A. 1998. Environ. Health Perspect. 106 (Suppl. 2):
511-532; Ulrichova et al. 2001. Toxicol. Lett. 125: 125-132;
Gomez-Lechon et al., supra). However, even hepatocyte preparations
of excellent quality are limited in their growth and survival in
vitro, and this therefore necessitates the continual acquisition of
new hepatocytes from source liver tissue (Guillouzo, supra;
Hoekstra and Chamuleau, supra; Rodriguez-Antona et al., supra).
Good quality human liver tissue is frequently in short supply and
must always be handled as if potentially infectious (Guillouzo,
supra; Hoekstra and Chamuleau, supra). Animal source liver tissue
can be obtained in steady quantity and is usually not an infectious
disease hazard, but even here, reproducibility problems may exist
as a result of animal-to-animal genetic variation, animal health,
nutritional status, and stress levels, and, perhaps most
importantly, the cell culturist's skill in preparing the hepatocyte
cell suspension (Guillouzo, supra; Di Nicuolo et al., supra).
[0006] To address these problems liver cell models based on
hepatocyte cell lines that grow continuously, i.e., are
functionally immortal, have been used. Unfortunately, immortal
hepatocyte cell lines, human or otherwise, are functionally
compromised as a result of their intrinsic character of unabated
growth and lack of normal differentiation, and they are therefore
poor model systems with which to measure normal hepatocyte
metabolism; particularly the phase I and II enzymatic reactions and
the cellular transport properties that are used as a basis for
estimating in vivo toxicokinetics and pharmacokinetics (Guillouzo,
supra; Hoekstra and Chamuleau, supra; Wilkening et al. 2003. Drug
Metab. Dispos. 31: 1035-1042; Yan and Caldwell, supra; Chandra and
Brouwer. 2004. Pharm. Res. (NY) 21: 719-735). Thus, improved in
vitro models for the prediction of in vivo liver biotransformation
and toxicity are needed to enable faster biological evaluation of
new chemical entities and to reduce controversial and costly animal
testing (Bertz and Granneman, supra; Guillouzo, supra; Yan and
Caldwell, supra; Chandra and Brouwer, supra).
[0007] Given the limitations of the in vitro liver cell models
discussed above, it is generally accepted that a cell line that
exhibits unlimited growth, and yet which differentiates normally,
e.g., a liver stem cell line, would provide the best biological
component for a cell based extracorporal bioartificial liver
assistance device. For similar reasons, a liver stem cell line
having such characteristics would also be the best in vitro model
with which to conduct pharmacological and toxicological assessments
of new chemical entities and would enable assessments that are
standardized and repeatable.
[0008] Here, we describe the porcine liver stem cell lines of the
invention, PICM-19H and PICM-19B, two derivative cell lines of the
ARS-PICM-19 cell line, that fulfill these needs. The ARS-PICM-19
parental cell line and an artificial liver device comprising them
have been patented in U.S. Pat. No. 5,532,156 and U.S. Pat. No.
5,866,420, respectively, and are hereby incorporated by reference
in their entirety. One derivative cell line, the PICM-19H cell
line, is capable of differentiating into hepatocytes and no longer
exhibits the ability to differentiate and self-organize into
multi-cellular bile ductules. The other cell line, PICM-19B,
appears to spontaneously arise from the bile duct differentiating
cells, but results in a unique cell phenotype, i.e., a dome-forming
polarized epithelium, not seen within the parental ARS-PICM-19 cell
line population.
SUMMARY OF THE INVENTION
[0009] We have derived (1) a unipotent porcine liver stem cell
line, the PICM-19H cell line, which differentiates only into
functional hepatocyte cells, from a bipotent stem cell line capable
of differentiating into both hepatocytes and bile duct cells and
established the PICM-19H cells as a cell line and confirmed its
differentiation into hepatocytes as evidenced by its morphology,
inducible CYP450 activity, serum protein production, low
gamma-glutamyl transpeptidase (GGT) activity, ammonia clearance
ability and urea production ability and (2) a unipotent porcine
liver stem cell line, the PICM-19B cell line, which differentiates
only into functional bile duct cells (cholangiocytes), from a
bipotent stem cell line capable of differentiating into both
hepatocytes and bile duct cells and established the PICM-19B cells
as a cell line and shown that the cells form confluent (complete)
cell monolayers in culture, are basolaterally polarized cells
exhibiting basal membrane fluid transport, have high GGT activity
and have greatly reduced serum protein production.
[0010] In accordance with this discovery, it is an object of the
invention to provide an immortalized derivative porcine stem cell
line capable of differentiating exclusively into hepatocyte cells
expressing hepatocyte functions, namely, inducible enzyme activity
involved in the metabolism of xenobiotics.
[0011] It is another object of the invention to provide the
unipotent PICM-19H stem cell line wherein the major enzyme activity
is CYP450 activity, including CYP1A1, CYP1A2 or CYP3A activity.
Other characteristics of PICM-19H cells are low levels of GGT
activity, serum protein production, urea production, and ability to
clear ammonia.
[0012] It is yet another object of the invention to provide the
unipotent PICM-19H stem cell line wherein the cell culture is
deposited as ATCC PTA-9174.
[0013] It is an additional object of the invention to provide the
unipotent porcine stem cell line which differentiates only into
cholangiocytes expressing bile duct function, namely vectorial
fluid transport.
[0014] It is a further object of the invention to provide the
PICM-19B stem cell line which form confluent cell monolayers of
basolaterally polarized cells exhibiting basal membrane fluid
transport and have high GGT activity, low CYP450 activity, and no
serum protein production.
[0015] It is yet another object of the invention to provide the
unipotent PICM-19B stem cell line wherein the cell culture is
deposited as ATCC PTA-9173,
[0016] It is an object of the invention to provide a method of
culturing the PICM-19H and PICM-19B stem cells with or without
feeder cells.
[0017] It is an object of the invention to provide a method of
culturing the PICM-19H and PICM-19B stem cells under serum-free
conditions.
[0018] It is still another object of the invention to provide a
method of using the unipotent PICM-19H and PICM-19B stem cell lines
in a screening assay to detect a compound which inhibits or
promotes an enzyme activity involved in the metabolism of
xenobiotics in the liver, or which inhibits or promotes the
expression of a gene encoding an enzyme involved in the metabolism
of xenobiotics in the liver.
[0019] It is another object of the invention to provide a method of
using the unipotent PICM-19H and PICM-19B stem cell lines in a
screening assay to detect a compound which results in cytotoxicity
due to the metabolism of xenobiotics and/or endogenous
substrates.
[0020] It is yet another object of the invention to provide a
method of using the unipotent PICM-19H and PICM-19B stem cell lines
in a screening assay to detect a compound which results in
carcinogenicity due to the metabolism of xenobiotics and/or
endogenous substrates.
[0021] It is still another object of the invention to provide a
method of using the unipotent PICM-19H and PICM-19B stem cell lines
in a screening assay to detect a compound which results in
mutagenicity due to the metabolism of xenobiotics and/or endogenous
substrates.
[0022] Another object of the invention is to provide a method of
using the unipotent PICM-19H and PICM-19B stem cell lines in a
screening assay to detect a compound which results in
hepatotoxicity due to the metabolism of xenobiotics and/or
endogenous substrates.
[0023] An additional object of the invention is to provide a method
of using the unipotent PICM-19H and PICM-19B stem cell lines in a
screening assay to detect a compound which results in hepatic
dysfunction.
[0024] A further object of the invention is to provide a method of
using the unipotent PICM-19H and PICM-19B stem cell lines in a
bioartificial liver device, such as a hollow fiber bioreactor, to
extracorporally treat body fluids such as whole blood, blood
plasma, and isotonic lavage-peritoneal fluid of individuals in need
of such treatment to alleviate liver dysfunction.
[0025] A still further object of the invention is to provide a
method of using cultures of the unipotent PICM-19H and PICM-19B
stem cell lines attached to microbeads, attached to inorganic or
organic porous supports, attached to hollow-fibers or encapsulated
in hydrogel-based supports to treat body fluids, such as whole
blood, blood plasma, and peritoneal fluids of individuals in need
of such treatment to alleviate liver dysfunction.
[0026] Other objects and advantages of this invention will become
readily apparent from the ensuing description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the U.S.
Patent and Trademark Office upon request and payment of the
necessary fee.
[0028] FIG. 1 shows a phase-contrast micrograph of parental
ARS-PICM-19 cells culture after 3-4 wk post-passage (FIG. 1A) in
comparison to a culture of the PICM-19H cells after 3 wk
post-passage (FIG. 1B) at 200.times. magnification.
[0029] FIG. 2 depicts the PICM-19H growth curve at passage 66 from
a 1:6 split ratio passage.
[0030] FIGS. 3A-F show phase-contrast micrographs (200.times.) of
an independent PICM liver stem cell line (B, D and F) and the
parental ARS-PICM-19 (A, C and E) pig liver stem cell line
undergoing phenotypic conversion to dome-forming epithelial cells.
The primary event is depicted in A to C and B to D. Note the PICM
cells symmetrically arranged in a densely packed columnar
morphology along a line of axis (arrows in B) that is a lumenal
space between the cells. The lumenal space is filled with a dark
material that in A has a yellow color (arrows). Panels C and D
depict early expansions with a curved arrow indicating early
dome-formation and arrowheads mark the edge of a colonial expansion
in C. In Panel E, the PICM-19B cells have flattened and spread, and
a small dome is beginning (double arrows). In Panel F the PICM
variant cells have multiplied extensively, are flattening out, and
a large dome has occurred in the monolayer (arrows). Bar .about.50
.mu.m.
[0031] FIG. 4 depicts the PICM-19B growth curve at passage 64 from
a 1:12 split ratio passage.
[0032] FIG. 5A shows a transmission electron micrograph of PICM-19H
cells arranged in a monolayer situated on top of a large STO feeder
cell at 6000.times. magnification. FIG. 5B shows a biliary
canaliculi (c) occurring between the PICM-19H cells with associated
tight junctions (arrowheads). Also, note the STO feeder cells are
both above and below the PICM-19H cells (magnification
33,800.times.). FIG. 5C shows that some lipid vacuoles (L) were
found in PICM-19H cells (magnification: 60,000.times.).
[0033] FIG. 6 shows a transmission electron micrograph of PICM-19H
cells highlighting the extensive Golgi complexes (G), rough
endoplasmic reticulum (RER), and numerous mitochondria (M) found
within the cells (magnification: 60,000.times.).
[0034] FIG. 7 shows a transmission electron micrograph of PICM-19H
cell showing what appeared to be remnants of poorly fixed areas of
glycogen rosettes (GLY). Also, note the layer of collagen fibrils
(Col), presumably produced by the adjacent STO feeder cell, and the
lamellar cristae characteristically traversing the PICM-19H cell's
mitochondrion (M) (magnification: 94,500.times.).
[0035] FIG. 8A shows a transmission electron micrograph of
PICM-19B-like cells with FIG. 8A showing the apical (facing the
culture medium) surface of two cells joined by a tight junction (t)
and an associated desmosome-like junction (d). Also, note in FIG.
8A the secretory vesicles (arrows) just below the apical membrane
which are typical of mucin containing vesicles with their
eccentrically located condensed spherule (double
arrows)(magnification: 48,000.times.). FIG. 8B shows two PICM-19B
cells growing on top of the STO feeder layer and its matrix of
collagen fibers. Note the microvilli at the cells apical membrane,
the interdigitations of the lateral cell membranes and the typical
indented or singularly crenulated nuclei (magnification:
6,000.times.). FIG. 8C shows that adhesion belt type junctions can
become robust between the cells particularly in areas of the
monolayer where domes form through the transport and accumulation
of fluid under their basal membrane (magnification:
60,000.times.).
[0036] FIG. 9 shows a transmission electron micrograph of
PICM-19B-like cells. Apical surface of cells with microvilli
(arrow) and cilium basal body (double arrows). Note the staining of
the glycocalyx, which also held precipitated stain (arrowheads)
(magnification: 120,000.times.).
[0037] FIG. 10 shows a transmission electron micrograph of the
basal aspect of a PICM-19B-like cell showing a microbody (arrow)
containing numerous parallel prismatic plates. The microbody
appears to have a double membrane like a mitochondrion. The
crystalloid array may be the nucleoid of a peroxisome or perhaps a
condensation within a lysosome. Note the sparse basal lamina below
the bottom of the cells (double arrows)(N=nucleus; magnification:
240,000.times.).
[0038] FIG. 11 shows a transmission electron micrograph of
PICM-19B-like cells. Note the long mitochondria with matrix
granules (arrows) and the well developed Golgi apparatus (G) that
were frequently seen around the nucleus (N)(magnification:
48,000.times.).
[0039] FIG. 12 shows a phase-contrast photomicrograph of PICM-19H
cell monolayer histochemically stained for GGT activity. Arrows
denote biliary canaliculi between PICM-19H cells showing positive
staining (red color) for GGT activity.
[0040] FIG. 13 shows bright field (FIG. 13A) and phase-contrast
(FIG. 13B) photomicrographs of parental ARS-PICM-19 liver stem cell
cultures histochemically stained for GGT activity. FIG. 13A shows a
monolayer expansion of PICM-19B-like variant cells (large arrows)
as they grow out and over the parental phenotypes of biliary
epithelial cells (ductal-forming; small arrows) and hepatocyte-like
cells (monolayer patches of cuboidal cells with canaliculi;
arrowheads). Note that these two parental or usually occurring
differentiated phenotypes never approach confluency since they
terminally differentiate well before that happens, as depicted
here. In contrast, the variant PICM-19B-like cells will continue
growing until confluency is achieved as depicted in FIG. 13B. Bar
in (A) .about.95 .mu.m and Bar in (B) .about.50 .mu.m.
[0041] FIG. 14 depicts 3-methylcholanthrene-induced CYP450 activity
in PICM-19 cell lines as measured by EROD assay. Un-induced CYP450
values were below the limit of detection and are not shown. ND=not
detectable.
[0042] FIG. 15 shows a two-dimensional polyacrylamide gel of
serum-free medium samples conditioned for 48 h by nearly confluent
monolayers of PICM-19H cells (FIG. 15A) or PICM-19B cells (FIG.
15B). Gels were stained with colloidal Coomassie-Blue and some
serum proteins, as identified by MALDI-TOF and LC-MS, are indicated
(see also Table 2).
[0043] FIG. 16 depicts ammonia clearance and urea production
without (control) and with the addition of 12 .mu.moles of
NH.sub.4Cl to the cell culture medium of cultures of the three
PICM-19 cell lines tested. The value above each bar is the
percentage of added NH.sub.4Cl nitrogen that was converted to urea
nitrogen by the cells.
[0044] FIG. 17 depicts the induction of multiple CYP450 isoforms in
PICM-19H cells with phenobarbital, rifampicin and
3-methylcholanthrene (3-MC). Cultures were exposed to equivalent
volumes of PBS, DMSO (0.1% final), 1 mM Phenobarbital (PHB), 50
.mu.M rifampicin (Rif) or 5 .mu.M 3-MC in complete medium. After 48
h, medium was replaced with specific enzyme substrate medium and
the activities of the specific CYPs (EROD, MROD, MFCD, and BFCD)
were determined. Fresh cultures (n=3) were used for each CYP450
analysis and values are means.+-.SEM of three independent
assays.
[0045] FIG. 18 depicts determination of testosterone metabolites in
control and rifampicin-induced PICM-19H cells. Cultures were
exposed to 50 .mu.M Rif or 0.1% DMSO for 48 hr and then incubated
for 1 h with testosterone. The concentrations of
6-.beta.-hydroxy-testosterone (6.beta.OH T), 2-.alpha. hydroxyl
testosterone (2.alpha.OH T) and 2-.beta. hydroxy testosterone
(2.beta.OH T) were determined by quantitative LC-MS. Values are
means.+-.SEM of three independent experiments which were performed
in triplicate cultures.
[0046] FIG. 19 shows a comparison of CYP450 activities in STO
(mouse embryonic fibroblast cell line) cells, adult porcine
hepatocytes (APH), PICM-19H cells and human hepatoma-derived
HepG2-C3A cells. Triplicate cultures were exposed to 0.1% DMSO
(control) and either 50 .mu.M Rif or 5 .mu.M 3-MC for 48 hr and
BFCD and EROD activities were determined, respectively. Values are
means.+-.SEM of three independent experiments. ND; not
detectable.
[0047] FIG. 20 shows an analysis of Phase II metabolism in adult
porcine hepatocytes (APH), PICM-19H cells and HepG2 C3A cells.
Activities of induced EROD and BFCD activities were determined with
and without incubation of media samples in
.beta.-glucuronidase/arylsulfatase cocktail. Fluorescent products
(resorufin or 7-HCF) were determined and the amount of product
released by the cocktail is reported as a percentage of the total.
Values are means.+-.SEM of three independent experiments performed
in triplicate cultures. ND; not detectable.
[0048] FIG. 21 depicts the bioactivation of aflatoxin B1 in
PICM-19H cells. Toxicity of aflatoxin B1 was determined in 0.1%
DMSO-treated (control) and 3-MC-induced PICM-19H cells grown in
96-well microplates. Following addition of aflatoxin B1, viability
was determined by WST-1 activity in each well. Each concentration
of aflatoxin was added to ten wells for each condition. Pooled
response curves were analyzed by non-linear regression; exponential
model R.sup.2 values for DMSO and 3-MC-treated cultures were 0.967
and 0.998, respectively.
[0049] FIG. 22 shows the toxicity determination of acetaminophen in
PICM-19H cells. Toxicity of acetaminophen was determined by WST-1
activity in PICM-19H cells grown in 96-well microplates. Nine
independent dose response curves were prepared and analyzed by
nonlinear regression using a sigmoidal response curve model.
[0050] FIGS. 23A and 23B show PICM-19H cells cultured in a
3D-hollow-fiber bioreactor. FIG. 23A shows a single hollow-fiber
that was fixed with 4% paraformaldehyde after 14 days of perfusion
culture and then stained with Hoechst nuclear-specific fluorescent
stain, magnification:100.times.. FIG. 23B shows a single
hollow-fiber (as in FIG. 23A) with a detached portion of the
PICM-19H monolayer (arrowheads) and the STO feeder cells situated
underneath the PICM-19H cells and left attached to the hollow-fiber
surface (individual STO nuclei are indicated by arrows),
magnification: 100.times.
[0051] FIGS. 24A and 24B show the PICM-19H cells growing on
microbeads in the presence of STO-GFP cells (STO feeder cells
expressing green fluorescent protein, GFP). STO-GFP cells were
allowed to attach to the beads first; PICM-19H cells were then
added to the culture. FIG. 24A depicts the STO cells excited by
blue light (used to excite the GFP fluorescence). FIG. 24B shows
the fluorescent nuclei of all the cells, STO and PICM-19H, because
the ultraviolet light excites the Hoescht nuclear stain (light blue
nuclear fluorescence) that the culture was stained with. Comparing
FIG. 24B with 24A allows identification of the STO and PICM-19H
cells.
[0052] FIG. 25 shows PICM-19H cells grown in culture as a spheroid,
a common method of culturing primary pig hepatocytes. The PICM-19H
spheroid was cultured for 2 weeks in suspension culture, and then
allowed to reattach to a monolayer of STO feeder cells, as
shown.
DISCLOSURE OF THE INVENTION
[0053] The objective of the present invention is to provide the
unipotent porcine liver stem cell lines, PICM-19H, which exhibits
unlimited growth, yet differentiates normally, and PICM-19B, which
forms a complete monolayer with basolateral cell polarization and
basal membrane fluid transport activity; a bioartificial liver
device which contains the PICM-19H cell line, the PICM-19B cell
line, or both, and rapid screening assays, comprising the cell
lines, for estimating in vivo toxicokinetics and pharmacokinetics.
The Phase I and Phase II metabolic functions of the PICM-19H and
the PICM-19B cell lines have been characterized; the PICM-19H and
the PICM-19B cell lines can be used to assess cellular enzyme
induction and transport processes.
[0054] The ARS-PICM-19 cell line, the bipotent parental line of the
unipotent PICM-19H and PICM-19B cell lines of the invention, had
been derived from the in vitro culture of the totipotent embryonic
stem cells of the preimplantation pig blastocyst, i.e., the
epiblast cells, and was just one of many cell lines of specific
cell types that spontaneously differentiated from the porcine
embryonic stem cells (Talbot et al. 1993. In Vitro Cell. Dev. Biol.
29A: 543-554; Talbot et al. 1994a. In Vitro Cell. Dev. Biol. 30A:
843-850). Early in their passage history, the parental ARS-PICM-19
cells were observed to form monolayers of fetal hepatocyte-like
cells as well as areas where the cells self-organized into
multi-cellular ductular structures composed of cholangiocyte-like
cells. ARS-PICM-19 hepatocytes have the characteristic morphology
of fetal pig hepatocytes, i.e., cuboidal cells with centrally
located nuclei joined by tight junctions and desmosomes to form
canalicular structures between the cells (Talbot et al. 1994b. In
Vitro Cell. Dev. Biol. 30A: 851-858). The ARS-PICM-19 cells could
be single-cell cloned without loss of differentiation and division
potential. ARS-PICM-19 cultures were found to have both inducible
CYP450 activity, a marker of hepatocytes, and high GGT activity, a
marker of cholangiocytes (Talbot et al. 1996a. Exp. Cell Res.
225-22-34). They also expressed alpha-fetoprotein along with
albumin and other liver-specific proteins (Talbot et al. 1994a,
1996a, supra). The culture of fetal pig liver tissue (Talbot et al.
1994b, supra) and adult pig liver tissue (Talbot and Caperna. 1998.
In Vitro Cell. Dev. Biol. 34A: 785-798; unpublished data) resulted
in cell cultures that closely resembled the ARS-PICM-19 cells in
their differentiation potential, morphology, and protein/enzyme
expression. In vivo-like responses of the ARS-PICM-19 ductules to
secretin and cAMP inducers were also demonstrated, i.e.,
basolateral to apical transport of culture fluid with in vivo-like
kinetics (Talbot et al. 2002. Cells Tissues Organs 171: 99-116).
ARS-PICM-19 differentiation into bile duct epithelium is marked by
unique in vitro intercellular and intracellular changes, i.e.,
self-organization into functional multi-cellular ductal structures
of columnar epithelium (Talbot et al. 1994a, 1996a, 2002, supra).
These in vitro-produced bile ductules closely resembled similar
bile ductules that were produced in vitro from the culture of both
fetal and adult pig liver tissue (Talbot et al. 1994b, 1998,
supra). Thus, the ARS-PICM-19 cells had been shown to be
functionally immortal (Talbot et al. 1994a, supra) and to possess
characteristics of both parenchymal hepatocytes and bile duct
epithelium cells (Talbot et al. 1994a, 1996a, supra). This was most
directly manifest in the ARS-PICM-19 cells spontaneously stopping
cell division (approximately 10 days after each passage) and
differentiating into at least two strikingly different
morphological phenotypes, one resembling hepatocytes and the other,
self-organizing, multi-cellular, functional ductules that behaved
like in vivo bile ducts (Talbot et al. 1994a, 1996a, 2002,
supra).
[0055] PICM-19H and PICM-19B are two variant unipotent cell lines
derived from the ARS-PICM-19 cell line described above. The
PICM-19H unipotent cell line is capable of differentiating into
hepatocytes and no longer exhibits the ability to differentiate and
self-organize into multi-cellular bile ductules. The other cell
line, PICM-19B, appears to have spontaneously arisen from the bile
duct differentiating cell component of the parental ARS-PICM-19
cells and is a unique differentiated phenotype, i.e., dome-forming
basolaterally polarized epithelium, not usually seen within the
parental ARS-PICM-19 cell population. The isolation of these
morphologically variant cell lines is described (see Example 1).
The derivative cell lines were evaluated to assess to what extent
they retained either hepatocyte or cholangiocyte cellular
functions. Specifically, since the cells of the parental
ARS-PICM-19 cell line can differentiate into either hepatocytes or
bile duct cells (cholangiocytes), the nature of the cells of these
particular derivative cell lines were evaluated to determine if
they are more or less hepatocyte-like in their cellular functions,
and whether either of them possess new unique cellular features
that would enhance their utility in a bioartificial liver device
and/or in vitro rapid liver toxicity assays.
[0056] As stated above, one specific use for a liver cell line is
for it to act as the biological component of an extracorporeal
bioartificial liver device. The bioartificial liver contains living
cells, usually hepatocytes or some combination of hepatocytes and
non-parenchymal accessory cells, within an ex vivo "bioreactor"
through which the patient's blood or blood plasma is pumped to
interact with the cells in an extracorporeal circulatory loop
(Sussman and Kelly. 1995. Scientific American 2: 68-77; Strain and
Neuberger, supra; Sen and Williams. 2003. Seminars in Liver Disease
23(3): 283-294). Such a device is needed for the treatment of acute
liver failure because no effective treatment options are currently
available that reduce the high mortality associated with this
condition except liver transplantation (Sussman and Kelly, supra;
Strain and Neuberger, supra; Sen and Williams, supra).
[0057] Thus, the PICM-19H and PICM-19B derivative liver cell lines
of the invention are candidates for use in a bioartificial liver
device because they have the particular characteristic of being
immortal unipotential stem cells. That is, PICM-19H cells
differentiate only into hepatocytes and do not differentiate into
bile ductules. Thus, PICM-19H can provide better hepatocyte
function for treatment of acute liver failure than can be provided
by the parental ARS-PICM-19 cells in those situations where
hepatocyte functions are the primary need, and not bile transport
and conditioning functions (Sussman and Kelly, supra; Strain and
Neuberger, supra; Sen and Williams, supra). Similarly, the PICM-19H
cell line provides an excellent model for screening assays for the
biological evaluation of new chemical entities. Inducible CYP450
activity is a marker of hepatocytes. The data set forth illustrate
the specificity of the inducible CYP450 activity of the PICM-19H
cell line and include a comparison to that described previously for
the parental ARS-PICM-19 cell line (Talbot et al. 1996a, supra),
thus demonstrating the utility of the PICM-19H cell line for in
vitro toxicity testing. The potential application of PICM-19H for
bioartificial liver devices is also set forth.
[0058] Qualities that make the PICM-19H cells more favorable than
the parental ARS-PICM-19 cell line for application to a
bioartificial liver device are that the PICM-19H cells retain
critical hepatocyte functions, they are non-tumorigenic and display
normal differentiation in vitro, they may be maintained in the
bioartificial liver device's bioreactor for relatively long periods
of time, their phenotypic stability, (i.e., no spontaneous
occurrences of PICM-19B-like cells over extensive culture,
approximately 450 population doublings), then pathogen-free status
can be defined and routinely assessed, and they can be genetically
engineered for enhancement of function.
[0059] The two PICM-19 derivatives have contrasting characteristics
that make each more suitable for the cellular component of a
bioartificial liver device in different ways. PICM-19H cells appear
to be more "hepatocyte-like" by all measures; including cell and
colony morphology, ultrastructure features, serum protein
production, and metabolic enzymatic functions. The data show that
the PICM-19H cells are superior in the critical hepatic functions
of CYP450 activity, urea production, and ammonia clearance.
However, because the PICM-19B cells grow to a greater cell density
and also display these key metabolic functions (albeit at lower
levels), the PICM-19B cells are a good choice for utilization in a
bioartificial liver device as well, particularly where vectorial
(i.e., basolateral) transport is an engineered quality of the
bioartificial liver device's bioreactor. Also, PICM-19B's relative
lack of serum protein secretion is of benefit in that the human
patient's blood would not be exposed to so many foreign antigens
(depending on the molecular weight cut-off of the dialysis membrane
in the bioartificial liver device). PICM-19B's apical to basal cell
membrane polarization affords an advantage in that its demonstrated
directional transport (i.e., dome-formation) could potentially move
toxins out of the human patient's plasma to an external waste flow
circuit if the PICM-19B cells were properly configured on a
bioartificial liver device's dialysis membrane. In any case and in
summation, the data presented here indicate enhanced functions of
the PICM-19H and PICM-19B cell lines for use in a bioartificial
liver device.
[0060] Having the availability of two unipotent cell lines offers
the unique opportunity to design and target the contents of the
bioreactor to suit the particular functional needs of the patient.
Each of the cell lines, PICM-19H and PICM-19B, can be cultured
individually in the bioreactor or they can both be seeded into the
bioreactor together, resulting in the opportunity to manipulate the
cell numbers of each, i.e., seed in differing ratios, depending on
the functions required by the patient.
[0061] It has been suggested that porcine cells would be useful for
in vitro modeling of hepatic metabolic functions and as the
cellular component of bioartificial liver devices due to their
human hepatocyte metabolism similarities (Donato et al., 1999. J.
Hepatol. 31: 542-549). In the present study, we have characterized
the PICM-19H cell line with respect to the presence and induction
of the major CYP450 activities (CYP1A, 2 and 3A). Additionally, the
extent of phase II conjugation is shown to be significant with test
substrates and comparable to adult pig hepatocytes. Known
hepatotoxins, acetaminophen and aflatoxin B1, are shown to be
metabolized in a dose-dependent manner. These data, combined with
the other demonstrated hepatic differentiated functions and the
robust culture characteristics of the PICM-19H cell line indicate
that PICM-19H cells can provide a cellular component in a
bioartificial liver device and also provide an improved model
system for hepatic cells in in vitro toxicological testing.
[0062] CYP450 comprises a family of cellular enzymes having key
enzyme activities involved in the liver-specific metabolism of
xenobiotic substances, i.e., chemical substances that are foreign
to the body of a living organism. Xenobiotic substances include
naturally occurring compounds, drugs, environmental agents,
carcinogens, insecticides, etc. CYP450 represents the class of
enzymes most important from the viewpoint of distribution and
functions involved in the metabolism of xenobiotics. CYP450 is a
generic name for a large number of enzymatic proteins; CYP1A1,
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP3A
(specifically CYP3A4) are known members of the CYP450 enzyme family
involved in the metabolism of xenobiotics in the human liver.
[0063] In addition, a large number of xenobiotic-metabolizing
enzymes are known to be induced under particular conditions.
Well-known examples of inducers include polycyclic aromatic
compounds such as benzo[A]pyrene, benzanthracene,
3-methylcholanthrene and dioxin which induce the expression of
CYP1A1 and CYP1A2; phenobarbital and phenobarbitone which induce
CYP2B (e.g., CYP2B6); and rifampicin, dexamethasone, phenyloin and
phenylbutazone which induce CYP3A (C. G. Gibson et al. 1995. New
Metabolomics of Xenobiotics, Kodansha Ltd., Tokyo, Japan).
[0064] Test compounds (new chemical entities and xenobiotic
substances) include, for example, peptides, proteins, non-peptide
compounds, synthetic compounds, fermentation products, cell
extracts, plant extracts, animal tissue extracts, and plasma. Thus,
they include naturally occurring compounds, drugs, environmental
agents, carcinogens, pesticides, herbicides, etc. These compounds
may be new compounds or commonly known compounds.
[0065] Specifically, the PICM-19H or PICM-19B cells of the present
invention can be treated with the test compound and compared with
an untreated control PICM-19 culture to evaluate the
therapeutic/preventive effects of the test compound with changes
such as those in (1) an enzyme activity involved in the metabolism
of xenobiotics in the liver or (2) the expression (activation) of a
gene encoding an enzyme involved in the metabolism of xenobiotics
in the liver, in the immortal PICM-19H or PICM-19B cells.
[0066] A test therapeutic compound identified as safe by using the
screening method of the present invention can be used as a safe
therapeutic/preventive or other pharmaceutical of low toxicity for
diseases associated with abnormalities of the metabolism of
xenobiotics in the liver (e.g., hepatic insufficiency) because of
its therapeutic/preventive effects on such diseases.
[0067] A compound obtained by said screening method may have formed
a salt. Said salt is exemplified by salts with physiologically
acceptable acids (e.g., inorganic acids, organic acids), bases
(e.g., alkali metals), etc., with preference given to
physiologically acceptable acid adduct salts. Such salts include,
for example, salts with inorganic acids (e.g., hydrochloric acid,
phosphoric acid, hydrobromic acid, sulfuric acid) and salts with
organic acids (e.g., acetic acid, formic acid, propionic acid,
fumaric acid, maleic acid, succinic acid, tartaric acid, citric
acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid,
benzenesulfonic acid).
[0068] Promotion of the activity of enzymes which metabolize
xenobiotics and/or endogenous substrates can be analyzed, for
example, by exposing a test substance to cells and detecting the
increase in the activity of enzymes which metabolize xenobiotics
and/or endogenous substrates, the increase in the amount of the
enzyme and/or the increase in the amount of transcription of the
gene encoding the enzyme. Specifically, this is possible by
detecting the elevation of CYP450 enzyme activity, an increase in
CYP450 protein content, or an increase in CYP450 mRNA in the
PICM-19H or PICM-19B cells. Useful methods of detection include
commonly known techniques such as assays of enzyme activities
corresponding to various types of CYP450, Western blotting
techniques corresponding to various CYP450 proteins, Northern
hybridization techniques corresponding to various types of CYP450
mRNA, and the CYP450-specific RT-PCR methods.
[0069] Hepatotoxicity due to the metabolism of xenobiotics and/or
endogenous substrates can be determined by exposing a test
substance to PICM-19H or PICM-19B cells and observing or measuring
the resulting cytotoxicity, or by exposing the test substance to
the PICM-19 cells and subsequently administering the test substance
altered by the cells to another hepatocyte, or other target cell
type, and observing the changes caused thereby in the target
cells.
[0070] The PICM-19H and PICM-19B unipotent cell lines as obtained
in Examples 2 and 3 have been deposited as cell lines ATCC PTA-9174
and ATCC PTA-9173, respectively, on Apr. 24, 2008, under the
Budapest Treaty, with the American Type Culture Collection (ATCC),
located at 10801 University Boulevard, Manassas, Va. 20110.
[0071] The subject cultures have been deposited under conditions
that assure that access to the cultures will be available during
the pendency of this patent application to one determined by the
Commissioner of Patents and Trademarks to be entitled thereto under
37 CFR 1.14 and 35 USC 122. The deposits are available as required
by foreign patent laws in countries wherein counterparts of the
subject application, or its progeny, are filed. However, it should
be understood that the availability of a deposit does not
constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
[0072] Further, the subject culture deposits will be stored and
made available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e., they will
be stored with all the care necessary to keep them viable and
uncontaminated for a period of at least five years after the most
recent request for the furnishing of a sample of the deposit, and
in any case, for a period of at least 30 (thirty) years after the
date of deposit or for the enforceable life of any patent which may
issue disclosing the cultures. The depositor acknowledges the duty
to replace the deposits should the depository be unable to furnish
a sample when requested, due to the condition of the deposit(s).
All restrictions on the availability to the public of the subject
culture deposits will be irrevocably removed upon the granting of a
patent disclosing them.
EXAMPLES
[0073] Having now generally described this invention, the same will
be better understood by reference to certain specific examples,
which are included herein only to further illustrate the invention
and are not intended to limit the scope of the invention as defined
by the claims.
Example 1
Reagents Utilized for Stem Cell Culture and for Screening
Assays
[0074] All cells were grown on 25 cm.sup.2 tissue culture flasks
(T25; Greiner, Frickenhausen, Germany). Fetal bovine serum (FBS)
and iron-supplemented calf serum were purchased from Hyclone, Logan
Utah. Cell culture reagents including Dulbecco's phosphate buffered
saline (PBS) without Ca.sup.++ and Mg.sup.++, media, trypsin-EDTA
(0.025% trypsin, 0.43 mM EDTA), antibiotics, non-essential amino
acids, and L-glutamine were purchased from InVitrogen,
Gaithersburg, Md. PICM-19H cells were grown on irradiated STO mouse
fibroblast (CRL 1503, American Type Culture Collection, Rockville,
Md.) feeder cell layers. Feeder-layers were prepared by exposing a
suspension of STO cells to 8 krad of gamma radiation and plating
the cells at 6.times.10.sup.4 cells/cm.sup.2. STO feeder-layers
were maintained by refeeding with 10% DMEM every 6-7 d. The growth
and differentiation medium for PICM-19 cultures was a 50:50 mixture
of DMEM low glucose and Medium 199 supplemented with 10% FBS,
2-mercaptoethanol, and nucleosides as described in Talbot and Paape
(1996. Methods in Cell Science 18: 315-327). PICM-19 cultures were
refed with fresh medium every 2-3 days after passage. Cultures were
routinely maintained at 37.degree. C. and in a 3-4% CO.sub.2
atmosphere.
[0075] HepG2 C3A human hepatoblastoma cells were obtained from ATCC
(Manassas, Va.; CRL-10741). The cells were passaged sub-confluently
in Minimal Essential Medium (MEM) supplemented with 10% FBS, 1 mM
sodium pyruvate, non-essential amino acids, and antibiotics and
grown at 37.degree. C. in 5% CO.sub.2. All experiments with HepG2
C3A cells were performed between passages 3 and 12.
[0076] Except where noted, all chemical reagents including
aflatoxin B1, 3-methylcholanthrene (3-MC), rifampicin (rif),
resorufin, phenobarbital (PHB), 7-methoxy resorufin (7-MRF),
7-ethoxyresorufin (7ERF) and dimethylsulfoxide (DMSO) were obtained
from Sigma Chemical Co., St. Louis, Mo.
7-methoxy-4-(trifluoromethyl) coumarin (7MFC) and
7-benzyloxy-4-(trifluoromethyl) coumarin (7BFC) were from BD
Gentest, Woburn, Mass.
[0077] Care and treatment of pigs in this study (n=3) were approved
by the Institutional Animal Care and Use Committee of the U.S.
Department of Agriculture. Crossbred barrows (.about.45 kg) were
stunned by electric shock and exsanguinated. Adult pig hepatocytes
were prepared from a portion of the left lateral hepatic lobe by a
two-step collagenase digestion procedure as previously described
(Caperna et al. 1985. J. Anim. Sci. 61:1576-1586; Fernandez-Figares
et al. 2004. Domest. Anim. Endocrinol. 27: 125-140). Hepatocytes
(4.5.times.10.sup.6 cells) were seeded into T25 flasks pre-coated
with pig tail collagen and cultured as previously described
(Caperna et al. 2005. Domest. Anim. Endocrinol. 29: 582-592).
Briefly, cells were initially maintained in William's E medium
containing insulin-transferrin-selenium (ITS; Sigma) and 10% FBS.
Following a 3 h attachment period, flasks were washed to remove
non-attached and non-viable cells, and William's E medium
containing 5% FBS and ITS was added to each flask. On the following
day, flasks were washed twice and medium was replaced with 10%
DMEM/199. All experiments were terminated approximately 72 h after
cell isolation and initiation of cultures.
Example 2
Establishment of PICM-19H Stem Cell Line
[0078] The PICM-19H cell line was established from a T25 mass
culture of parental ARS-PICM-19 cells, i.e., approximately one
million cells, that were subjected to hypothermic selection
(33-34.degree. C.) for approximately 3 wk at passage 37. The
PICM-19H cell line was derived from approximately 50-100 cells that
survived the temperature selection.
[0079] After expansion to mass cultures, by culturing at
37-38.degree. C., it was observed that the PICM-19H cell line did
not self-organize into multi-cellular bile ductules under standard
or elevated pH culture conditions as is characteristic of the
parental ARS-PICM-19 population (FIGS. 1A and B; Talbot et al.
2002, supra). Phase-contrast microscopy showed the PICM-19H cells
to be generally cuboidal cells with distinct, centrally located
nuclei (FIG. 1B). Like the parental ARS-PICM-19 cells, the fetal
hepatocyte-like PICM-19H cells grew as a patch work of small
monolayer colonies nestled in amongst the STO (mouse embryonic
fibroblast cell line) feeder cells, but, unlike the parental cells
that could only achieve approximately 50% confluency, the PICM-19H
cells reached approximately 85% confluency before terminal
differentiation and contact inhibition markedly slowed their growth
(FIG. 1B and FIG. 2).
[0080] It was necessary to passage the PICM-19H cultures at least
every two weeks in order to keep the majority of the culture's cell
cycling, as is the case with parental ARS-PICM-19 cells. The
PICM-19H subpopulation was passaged over several years, at 1:3 to
1:10 split ratios, to the current passage level of approximately
P175. The PICM-19H cells were cryopreserved at various times over
their passage history.
Example 3
Establishment of PICM-19B Stem Cell Line
[0081] The PICM-19B cell line was developed from a spontaneously
arising morphological variant of the parental PICM-19 cells which
occurred in the culture at low frequency at approximately passage
35 (FIG. 3; Talbot et al. 1994a, supra). The PICM-19B cell line was
established by micropipette-mediated colony-cloning of a single
colony of this variant cell type. Independent, spontaneously
forming, "PICM-19B-like" outgrowths occurred at various passage
levels of the parental PICM-19 cells when initiated from frozen
stocks of the cells. Observation of the occurrence of the PICM-19B
cells, and of many similar independent occurrences, indicated that
this morphological variant appeared to arise from PICM-19 cells
that were differentiating and forming into bile ductules and not
from PICM-19 cells forming monolayer patches of hepatocyte-like
cells (Talbot et al. 1994a, supra). To illustrate this point more
clearly, FIG. 3B, C, and D illustrates the genesis of this variant
from another independent, epiblast-derived pig liver cell line.
[0082] The occurrence of the "19B" phenotype could first be
recognized within the parental PICM-19 culture as colonies of cells
that appeared as closely packed and mounded up columnar cells
symmetrically arranged around a central point or line (FIGS. 3A and
B). The central point or line was a 3-dimensional space that was
usually filled with a material that often had a yellowish color
under phase-contrast microscopic observation (FIG. 3A). As the
cells continued their outgrowth over several weeks, they formed a
monolayer of cells that grew over the top of the STO feeder cells
or "bull-dozed" the feeder cells at the colony's periphery as the
colony expanded (FIGS. 3C and D). Unlike the parental ARS-PICM-19
hepatocyte-like monolayers, the 19B variant cell monolayer did not
have canalicular connections between its cells. Instead, PICM-19B
cells were joined by tight-junctions and displayed
apical/basolateral morphological polarity (FIG. 8). The monolayer
frequently developed domes (FIGS. 3E and F) indicating apical to
basal fluid transport. Also, in some areas of the 19B variant cell
monolayers, particularly where domes were present, the cell-to-cell
unions had a typical "prickle cell" morphology (FIG. 3F) similar to
skin epithelium where robust desmosomal connections join adjacent
cells (Alberts et al. 1994. Molecular Biology of the Cell, Second
Edition, Garland Publishing, New York, Pages 789 and 954-955).
[0083] In contrast to the terminally differentiating and
self-organizing bile ductule cells of the parental ARS-PICM-19 cell
line, the 19B variant cells grew to 100% confluency which resulted
in a final cell density of approximately 2.61.times.10.sup.5
cells/cm.sup.2 (FIG. 4). The parental ARS-PICM-19 cells have never
achieved more than 60-75% confluency before terminally
differentiating (Talbot et al. 1994a, 2002, supra). Also, despite
their basolaterally polarized monolayer characteristic, the 19B
variant cells were found to be more easily dissociated from one
another after trypsin-EDTA treatment than the parental ARS-PICM-19
cells. The PICM-19B cell line was passaged at 1:5 to 1:10 split
ratios until the 89.sup.th passage, a time period of greater than
one year. The PICM-19B cells were cryopreserved at various times
over their passage history.
Example 4
Cell Growth Assays
[0084] PICM-19H and PICM-19B cell growth was assayed at passage 66
and 64, respectively, by counting the increase in the total cells
per T25 flask over a 3 wk period at 2-4-day intervals post-passage.
Duplicate T25 flasks were counted at each time interval. Single
cell suspensions of the contents of each flask were produced by
trypsin-EDTA dissociation. The cells were suspended to a total
volume of 2 ml in 10% DMEM for cell counts. The total number of
cells per T25 flask was determined by averaging the counts of 16
hemocytometer squares (1 mm.sup.2). Input of the number of PICM-19H
and PICM-19B cells at the start of the growth assay was undefined,
but was a 1:6 split ratio for PICM-19H and a 1:12 split ratio for
PICM-19B, each from nearly confluent stock cultures. STO
feeder-cells surviving the trypsin/EDTA dissociation were similarly
enumerated from a parallel group of feeder-cell T25 flasks that had
not received any PICM cell input.
[0085] The growth curve of the PICM-19H cells indicated a lag
period of 3-5 days post-passage, a logarithmic growth phase with a
doubling time of approximately 48 h and a differentiation driven
plateau phase where the final cell number reached per T25 flask was
nearly 4 million cells or approximately 1.49.times.10.sup.5
cells/cm.sup.2 (FIG. 2).
[0086] The PICM-19B variant cells grew to 100% confluency which
resulted in a final cell density of approximately
2.61.times.10.sup.5 cells/cm.sup.2. The 19B's doubling time during
logarithmic growth was approximately 48-72 h (FIG. 4).
Example 5
Ultrastructure Analysis; Transmission Electron Microscopy
[0087] Transmission electron microscopy (TEM) sample preparation
and photomicroscopy were done with the assistance of JFE
Enterprises, Brookeville, Md. as previously described (Talbot et
al. 1998, supra; Talbot et al. 2000. Tissue and Cell 32: 9-27).
Ultrastructural analysis was performed on samples processed from
T25 flask cultures that were 6-wk post-passage for PICM-19H and
8-wk post-passage for PICM-19B-like cells that had spontaneously
formed within the parental ARS-PICM-19 culture.
[0088] The ultrastructure features of the PICM-19H cells are
similar to those observed in the hepatocyte-like cells of the
parental ARS-PICM-19 cultures (Talbot et al. 1996a, supra). The
ultrastructural feature perhaps most defining of hepatocytes is the
specialized cell-to-cell union that occurs between hepatocytes to
form a biliary canaliculus (Wanson et al. 1977. J. Cell Biol. 74:
858-877). PICM-19H cells are closely associated with one another by
extensive plasma membrane foldings that are interdigitated along
their lateral surfaces (FIG. 5A) and are often are found sandwiched
between STO feeder cells (FIG. 5B). Junctional apparati typical of
polarized epithelial cells are found between adjacent PICM-19H
cells at their lateral apical surfaces, i.e., facing the lumen of
the biliary canalicular spaces (FIG. 5B). These tight-junction-like
unions establish the impermeable boundaries of the biliary
canaliculi that exist between adjacent PICM-19H cells as has been
previously shown by ruthenium red staining (Talbot et al. 1996a,
supra). The biliary canalicular surface has numerous microvilli
that protrude into the canalicular space (FIG. 5B).
[0089] The biliary canaliculi formed by PICM-19H are similar to
those found in vivo in thin sections of human embryonic, piglet,
and rodent liver (Enzan et al. 1974. Acta Pathol. Jap. 24: 427-447;
Singh and Shahidi. 1987. Ultrastructure of Piglet Liver In: Swine
in Biomedical Research, ME. Tumbleson (Ed), Volume 1, Plenum Press,
New York, page 84; Wanson et al., supra), and, as with the parental
cells, are responsive to added secretin or glucagon, i.e., they
display transcellular movement of fluid into the canaliculus (not
shown; Talbot et al. 2002, supra).
[0090] The nuclei of the cells are oval and often display a single
deep invagination (FIG. 5A). Rough endoplasmic reticulum (RER) is
particularly well represented in the cells and is often found in
extensive stacks that surround some of the mitochondria of the
cells (FIG. 6). However, the RER cisternae are relatively collapsed
indicating that they contain relatively little secretory material.
While other PICM-19H and PICM-19B ultrastructural features are
typical of hepatocytes or bile duct epithelium, the extensive RER
arranged in long laminar cisternae found in both cell lines is not.
This feature is most like that reported in hepatoblasts of human
fetal liver where extensive multi-zonal collections of RER with
long cisternae are present. In contrast, fetal and adult bile duct
epithelium cells are found to have very little and short tubular
RER (Enzan et al. 1974. Acta Pathol. Jap. 24: 427-447.; Ishii et
al. 1989. Physiol. Rev. 69: 708-764; Phillips et al. 1987. In: The
Liver: An Atlas and Text of Ultrastructural Pathology, Phillips et
al., Eds., Raven Press, New York, pp. 1-35. This distinction is not
apparent in comparing PICM-19H and PICM-19B cells, and, therefore,
like the parental ARS-PICM-19 cells, it may indicate that the
derivative cell lines are either fundamentally different from in
vivo fetal liver cells because of the in vitro environment, or that
they still display a transitional morphology similar to
hepatoblasts that can "mature" given the proper environment.
[0091] Smooth ER also appears to be present in the cells, but it is
difficult to discriminate from Golgi complexes with certainty.
Golgi complexes which are often found in a supranuclear position
are well developed and numerous (FIG. 6). Mitochondria are elongate
(2-3 .mu.m in length) in longitudinal section and oval (0.2-0.3
.mu.m in diameter) in cross-section (FIG. 6). Their lamellar
cristae characteristically traverse the mitochondrion and electron
dense granules are sometimes present within their matrixes.
Numerous peroxisome-like organelles are also present throughout the
cell's cytoplasm although their identification as peroxisomes is
not proven. Relatively "empty" areas of cytoplasm with residual
glycogen-like granules in them are frequently observed in the
PICM-19H cells (FIG. 7). Presumably these are areas of glycogen
storage that are mostly lost as a result of inadequate glycogen
rosette fixation. Finally, no monocilia are observed in the
PICM-19H cells examined.
[0092] The ultrastructure of PICM-19B cells show features more
typical of hepatic biliary epithelium (cholangiocytes) or
gallbladder epithelium. In contrast to PICM-19H cells, PICM-19B
cells form a monolayer of interdigitating cells with a clear
basal/apical polarization, but without canalicular formations,
i.e., the cells are arranged in a continuous epithelial sheet with
tight junctions, as evidenced by dome-formation (FIGS. 1 and 8B).
Microvilli of a moderate, uniform height (0.4-0.6 .mu.m) are found
on the apical surfaces of the cells (FIGS. 8A and 8C). The
microvilli have particularly distinct internal actin filaments that
run deep into the underlying cytoplasm before joining the
adhesion-belt actin filaments that run parallel to the apical
cytoplasmic surface. Tight junctional elements and desmosomes join
the cells together at their apical and lateral unions, and
interdigiting cytoplasmic foldings are also common at lateral cell
surfaces (FIGS. 8A and 8C). The cells also have numerous and well
developed perinuclear Golgi apparatus, and secretory vesicles are
frequently observed in the cytoplasm between the nucleus and apical
cell membrane (FIGS. 9 and 8A). The contents of the secretory
vesicles are usually similar to or darker in electron density than
that of the surrounding cytoplasm and, as is typical of the muscin
containing vesicles of gallbladder epithelium (Gilloteaux et al.
1997. Microsc. Res. Tech. 38: 643-659), they often contain an
eccentrically located dense spherule (FIG. 8A). The cells display
cilia, although infrequently, and therefore, the cells are probably
monociliated (FIG. 9).
[0093] Cilia are not observed by electron microscopy in the
PICM-19H ultrathin sections, but are observed, mostly likely as
monocilia on the apical surface of the PICM-19B cells. Cilia have
not been found in human hepatoblasts or piglet hepatocytes (Enzan
et al., supra; Singh and Shahidi, supra). In contrast, the presence
of monocilia was shown to be a characteristic feature of fetal
human, neonatal pig, and adult rat bile duct epithelium cells
(Enzan et al., supra; Ishii et al., supra; Singh and Shahidi,
supra) or gallbladder epithelium cells (Nakanuma et al. 1997.
Microsc. Res. Tech. 39: 71-84). Therefore, the absence and presence
of monocilia on PICM-19H cells and PICM-19B cells, respectively, is
consistent with the PICM-19H cells being more hepatocyte-like in
phenotype and the PICM-19B cells being more bile duct- or
gallbladder-epithelium-like.
[0094] Membrane-bound bodies resembling peroxisomes are numerous in
PICM-19B cells and some have prismatic parallel plate-like
structures in their interior (FIG. 10). Also, mitochondria with
lamellar cristae are numerous and are sometimes found to contain
one or more granules, apparently distributed randomly in their
matrix (FIG. 11). Both smooth and rough endoplasmic reticulum (RER)
are found in the cells, although the former is difficult to
distinguish from the many Golgi complexes present (FIG. 11). The
RER sometimes occurs in extensive stacks, although their cisternae
are relatively collapsed, indicating that they contained relatively
little secretory material. As found in PICM-19H cultures, a matrix
of collagen fibrils is evident between the STO feeder cells and the
PICM-19B cells, presumably having been produced by the STO
fibroblasts. However, in contrast with the PICM-19H culture, the
STO feeder cells are mostly situated underneath the PICM-19B cell
monolayer (FIG. 8B). Finally, a thin basal lamina can be discerned
below the basal membrane of the PICM-19B cells (FIG. 10).
Example 6
Assay of .gamma.-Glutamyltranspeptidase (GGT) Activity and CYP450
Content
[0095] T25 cultures of PICM-19H and PICM-19B cells were grown for
approximately 3 wk post-passage and the cultures were scraped and
harvested for whole cell homogenates and microsomes. Two days prior
to harvest the cultures were exposed to metyrapone to stimulate
CYP450 expression. CYP450 content and GGT activity were determined
as previously described (Talbot et al. 1996, supra) from a pool of
three flasks.
[0096] CYP450 is present in the microsomal fraction of PICM-19H and
PICM-19B cells that had been exposed to metyrapone for 48 h prior
to assay (Table 1). In contrast, CYP450 is undetectable in PICM-19
cell culture homogenates that had not been exposed to metyrapone
(data not shown). The crude homogenate and microsomal fraction of
STO feeder cells grown under identical conditions and treated with
metyrapone have no detectable CYP450 content. Because the
microsomal protein associated with STO cells represent more than
one-third of the total harvested microsomal protein, the actual
specific content of CYP450 (nmoles/mg/protein) in the PICM-19H and
PICM-19B cells is greater than reported. PICM-19B CYP450 content
was also compared to freshly harvested pig hepatocytes (Table 1)
and the results show the cell line to contain approximately
one-sixth the CYP450 of freshly harvested adult pig
hepatocytes.
[0097] GGT activity is found in both PICM-19H and PICM-19B cells by
histochemical staining. PICM-19H GGT activity is specifically
expressed at the biliary canaliculi visible in PICM-19H monolayers
(FIG. 12), whereas in PICM-19B cells the GGT staining is diffuse
and is associated with all of the cells (FIG. 13). GGT activity was
also measured in the homogenates of PICM-19H and -19B cells
cultured for three to four weeks (Table 1). Total GGT activity was
markedly higher in PICM-19B cells compared to that found in the
homogenates of freshly harvested pig hepatocytes or PICM-19H cells
(Table 1). The STO feeder cell homogenates show very low or no GGT
activity when grown alone under the same conditions as the PICM-19
cells (not reported). The reported specific activity of GGT in
PICM-19, total GGT, is therefore underestimated because
approximately two-fifths of the cell culture homogenate protein is
derived from the STO feeder cells. PICM-19B cells had 84 times as
much GGT activity as the freshly harvested pig hepatocyte
preparation. PICM-19B cells had approximately 6 times more GGT
activity than PICM-19H cells (Table 1).
TABLE-US-00001 TABLE 1 Levels of CYP450 and activity of GGT in
PICM-19H, PICM-19B, and Adult Porcine Hepatocytes. CYP450
(pmoles/mg GGT Cell Type microsomal protein) (munits/mg protei n)
PICM-19H *136 .+-. 35 (n = 3) 51.2 .+-. 1.7 (n = 9) PICM-19B *#BD
(n = 2) 311.9 .+-. 9.4 (n = 9) Adult Porcine Hepatocytes 617 .+-.
47 (n = 4) 3.7 .+-. 0.4 (n = 4) (freshly prepared) *48 hr post
metyrapone addition #BD: Below Detection
[0098] GGT is highly expressed in bile duct epithelium and thought
to be a good marker for this cell type (Tanaka, M. 1974. Acta.
Path. Jpn. 24: 651-665; Ishii et al., supra). Previously the
parental ARS-PICM-19 cells were demonstrated by histochemical
staining to have GGT activity localized to the plasma membranes of
their biliary canaliculi as has been found in primary hepatocyte
cultures (Meister et al. 1976. In: The Enzymes of Biological
Membranes, Martinosi, A. (Ed.), Plenum, New York, pages 315-347;
Talbot et al. 1996a, supra). As shown here, PICM-19H cells have a
similar expression pattern (FIG. 12).
[0099] In comparison, the PICM-19B cells have a more robust GGT
expression (Table 1) and, when viewed microscopically, a seemingly
ubiquitous GGT expression. This overall cell-surface histochemical
GGT staining probably results from the GGT expression being
associated with the apical membranes of PICM-19B cells which, in
aspect, are parallel with the focal plane of the microscopic image.
The majority of PICM-19H cells appear devoid of GGT staining
because of the localization to biliary canaliculi, and perhaps it
is not localized to all biliary canaliculi, and because the
canaliculus constitutes a discrete side-to-side polarization within
the PICM-19H monolayer. This polar distribution of GGT is
indicative of the transport function of the enzyme in the
specialized apical areas of hepatocytes and cholangiocytes (Meister
et al., supra). The enhanced GGT expression of PICM-19B, and
conversely the relatively low expression found in PICM-19H, are
again indicative of a more bile duct-like or gallbladder
epithelium-like phenotype for the PICM-19B cells and a more
hepatocyte-like phenotype for the PICM-19H cells.
Example 7
CYP450 EROD Activity Assay
[0100] Three nearly confluent T25 flask cultures of PICM-19H and
PICM-19B cells were pre-incubated with 5 .mu.M 3-methylcholanthrene
(3-MC) in culture medium for 48 h to induce CYPA1 activity.
3-MC-induced CYP450 activity was measured by EROD assay, i.e., by
conversion of 7-ethoxyresorufin (7-ERF) to the highly fluorescent
product resorufin, in PICM-19H cells, PICM-19B cells, parental
ARS-PICM-19 cells, and in STO feeder cells alone (FIG. 14). Cells
were exposed to Medium 199 medium with Hank's salts without
L-glutamine or sodium bicarbonate and containing 7-ERF (8 .mu.M),
dicumerol (10 .mu.M), and bovine serum albumin for 30 min as
described by Donato and coworkers (1993. Anal. Biochem. 213:
29-33). The medium was harvested and the concentration of the
fluorescent product, resorufin, was assayed in the presence and
absence of .beta.-glucuronidase/arylsulfatase (Roche Applied
Sciences, Mannhein, Germany) to determine the extent of possible
conjugation reactions. All reagents were from Sigma-Aldrich (St.
Louis, Mo.) and activity is presented as pmole product formed per
30 min/mg cell protein in cultures prepared with and without 3-MC.
T25 flasks of STO feeder cells only were also assayed as to control
for their presence in the PICM-19 cultures.
[0101] PICM-19H cells converted 7-ERF to resorufin at rates of
approximately 5.times.10.sup.3 pmole per 30 min/mg cell protein.
Induced EROD activity is also found in PICM-19B cells, but its
levels are comparatively reduced at approximately 50% of that
measured in PICM-19H cells. ARS-PICM-19 parental cells have only
marginally higher rates of resorufin production than the PICM-19B
cells. STO feeder cells show no detectable CYP450 activity. EROD
activity is below the level of detection in all of the PICM-19 cell
cultures when not induced by exposure to 3-MC (not shown).
[0102] PICM-19H cells display higher inducible CYP450 content when
exposed to 3-MC for 48 h then do PICM-19B cells and parental
ARS-PICM-19 cells. Similarly, CYP450 activity, measured by
conversion of 7-ER to resorufin, is also higher in the PICM-19H
cells (FIG. 14). The ability to induce relatively high amounts of
CYP450 in the PICM-19H cells is consistent with a hepatic phenotype
(Murray et al., 1987. Gastroenterology 93: 141-147; Brill et al.
1993. Proc. Soc. Exp. Bio. Med. 204: 261-269), whereas the lower
amounts seen in the PICM-19B cells indicates the cells trending
towards a phenotype typical of cholangiocytes (Sirica, A. E. 1992.
Progress in Liver Diseases 10: 63-87; Alpini et al. 1994. The
Biology of Biliary Epithelia. In: The Liver: Biology and
Pathobiology, Arias et al. (Eds.), Raven Press, New York, pages
623-653; Talbot et al. 1998, supra). In the parental ARS-PICM-19
cell line it was previously suggested that CYP450 levels probably
depended upon the relative proportions and extent of the
alternative differentiated phenotypes, i.e., the hepatocyte
monolayer differentiated cells having more CYP450 activity than the
PICM-19 cells that differentiated into bile ductules (Talbot et al.
1996a, supra). Thus, the loss of the bile ductule differentiation
phenotype in the PICM-19H cell line can enhance total CYP450
activity of these cultures compared to those of the parental
ARS-PICM-19 cell line, cell numbers being equal, since no PICM-19H
cells differentiate to form bile ductules which largely lack CYP450
activity relative to hepatocytes (Sirica, supra; Alpini et al.,
supra; Talbot et al. 1998, supra).
Example 8
Two-Dimensional Electrophoretic Analysis of Conditioned Medium;
Mass Spectrophotometric Analysis of Proteins
[0103] PICM-19H or PICM-19B cells were seeded into T25 flasks and
cultured as previously described (Talbot et al. 1996, supra). At
approximately 2 wk post-passage, medium was removed and the flasks
were rinsed four times with serum-free DMEM medium, to remove
FBS-related proteins, and the flasks were culture 48 h in 4 ml of
serum-free DMEM. The conditioned medium (CM) was collected and cell
debris was pelleted by centrifugation at .about.500.times.g for 15
min. The proteins of the CM were concentrated and separated by
isoelectric focusing as previously described (Talbot et al. 2007.
In Vitro Cell dev. Biol. Anim. 43: 72-86). Second dimension
separations were also done as previously described (Talbot et al.
2007, supra) on 10% polyacrylamide gels (8.times.10 cm). The
proteins in the gel were visualized by staining with Colloidal
Coomassie Blue G-250 (Gradipure.RTM.; Life Therapeutics, Frenchs
Forest, Australia) and the gel was scanned using laser densitometry
(PDSI, GE Healthcare). The CM from STO feeder cells alone was
similarly analyzed as a control for their presence in the PICM-19
cultures and for FBS-related proteins.
[0104] Protein spots were excised from 2D gels using standard
pipette tips and the gel "plugs" were processed as previously
described (Talbot et al. 2007, supra). A Voyager DE-STR MALDI-TOF
mass spectrometer (Applied Biosystems, Framingham, Mass.) operated
in positive ion reflector mode was used to analyze tryptic
peptides, and spectra were acquired with 75 shots of a 337 nm
Nitrogen Laser operating at 20 Hz. Spectra were calibrated using
the trypsin autolysis peaks at m/z 842.51 and 2,211.10 as internal
standards.
[0105] Analysis of serum-free medium conditioned by PICM-19H cells
for 48 h showed that the cell line was secreting a spectrum of
proteins similar to that found in fetal pig serum (FIG. 15A). No
secretion was seen in STO feeder-cells alone (not shown; Talbot et
al. 1994, 2000a, 2005, supra). Several of the protein spots were
identified by MALDI-TOF and LC-MS/MS mass spectroscopy. The
serum-proteins identified included alpha-2-HS-glycoprotein
precursor (fetuin-A), transthyretin, albumin, alpha-fetoprotein
(AFP), transferrin, apolipoprotein-A1, and retinol-binding protein
(FIG. 15A and Table 2). The Coomassie Blue total protein staining
indicated that transferrin, AFP, alpha-1-anti-trypsin, and fetuin-A
were the most abundantly secreted proteins.
TABLE-US-00002 TABLE 2 Serum Proteins Identified in the Conditioned
Medium of PICM-19H cells by MALDI-TOF and LC-MS/MS. Spot expected
ID No. MW PI Protein ID Peptides SC MO value NCBI method 1 76918
6.93 Chain A, porcine serum transferrin 17 23% 119 6.10E-07
gi|18655907 Maldi Tof 2 47164 5.54 alpha-1-antitrypsin [Sus scrofa]
7 20% 84 0.0018 gi|975230 Maldi Tof 3 68580 5.47 alpha-fetoprotein
[Sus scrofa] 10 14% 100 4.90E-05 gi|47523700 Maldi Tof 4 38424 5.5
Alpha-2-HS-glycoprotein (Fetuin-A) 4 12% 268 gi|231467 MS/MS 5
15792 6.34 Transthyretin (prealbumin) [Sus scrofa] 6 58% 380
gi|975233 MS/MS 6 69366 5.92 Albumin [Sus scrofa] 15 26% 152
3.50E-10 gi|833798 Maldi Tof 7 30312 5.38 apolipoprotein A-I 16 54%
220 4.90E-17 gi|164359 Maldi Tof 8 21142 5.6 Retinol-Binding
Protein (Rbp) 2 11% 88 gi|2914422 MS/MS 9 20885 5.83 Alpha-1-acidic
glycoprotein 5 27% 69 0.068 gi|164302 Maldi Tof 10 27746 6.59
Properdin Factor D (Adipsin) 8 37% 105 1.80E-05 gi|1705760 Maldi
Tof MW: predicted molecular weight; PI: predicted isoelectric
point; Peptides: the number of peptides matched; SC: the percentage
of sequence coverage; MO: MOWSE score; Expected value: the number
of matches with equal or better scores that are expected to occur
by chance alone (http://www.matrixscience.com); NCBI: Accession
number; ID method: mass spectroscopy identification method. The
assigned protein of the best matched was given with the species in
which it has been identified and its accession number.
[0106] Analysis of PICM-19B CM showed the cells are secreting much
less protein and that only traces of transferrin, AFP,
apolipoprotein-A1 and retinol-binding protein are observable after
Coomassie Blue staining (FIG. 15B).
[0107] Thus, perhaps the most striking difference found between the
two derivative PICM-19 cell lines was in their production of serum
proteins. Serum protein production by hepatocytes is a defining
characteristic shared by only one other cell type, the
extraembryonic visceral endoderm cells of the early mammalian
embryo (Junqueira et al. 1992. Basic Histology, Appleton and Lange,
Norwalk, Conn., page 406; Talbot et al. 2007, supra). In that the
PICM-19H cell line retains this hepatocyte function (as found in
the parental ARS-PICM-19 cell line; Talbot et al. 1994a, supra) and
it is greatly reduced in the PICM-19B cell line, the PICM-19H cell
line is hepatocyte-like, while the PICM-19B cell line is not.
Example 9
Ammonia Clearance and Urea Production Assay
[0108] Three nearly confluent T25 flask cultures of PICM-19H or
PICM-19B were exposed to glutamine-free Williams-E medium
supplemented with 10% FBS, 1 mM ornithine, glucagon (100 ng/ml),
2-mercaptothanol (0.1 mM), HEPES (25 mM), and antibiotics for 72 h.
The cells were then exposed to the same base medium with the
addition of 12.mu. moles ammonium chloride (final concentration=2
mM) for 48 h. Medium was collected, centrifuged at 2000.times.g to
remove cellular debris, and frozen at -80.degree. C. prior to
analysis. Ammonia content of experimental (48 h) and initial
(T.sub.0) media samples was determined spectrophotometrically using
a commercial kit (Pointe Scientific, Inc., Canton, Mich., USA)
which was modified for use in a microtiter plate reader. A standard
curve was prepared in base medium without ammonia. T25 flasks of
STO feeder cells only were also assayed to control for their
presence in the PICM-19 cultures.
[0109] PICM-19H cells are able to completely clear the ammonia
added to the cell culture medium of T25 cultures in approximately
24 h (FIG. 16). Both ARS-PICM-19 parental and PICM-19B cells are
nearly able to do so, too (FIG. 16). All of the PICM-19 cell lines
produce urea from added ammonia with PICM-19H converting 36% of
added ammonia nitrogen to urea nitrogen, and PICM-19 parental and
PICM-19B achieving 35% and 30% conversion, respectively.
[0110] A distinct difference between the two PICM-19 derivative
cell lines is in their overall production of urea. In terms of
absolute amounts of urea produced on a specific activity basis,
PICM-19H produces more than twice as much as PICM-19B (FIG. 16).
STO feeder cells alone neither clear added ammonia nor produce urea
(FIG. 16). Urea production in response to the addition of 2 mM
ammonia on a percent nitrogen conversion basis does not appear to
be significantly different between the cell lines. This suggests
that the metabolic machinery for urea production from ammonia is
intact in the PICM-19B cells, but that it is operating at a lower
overall rate. Since it is generally accepted that bile duct
epithelium does not produce urea and that it is the function of the
hepatocyte (Triebwasser and Freedland. 1977. Biochem. Biophys. Res.
Commun. 76: 1159-1165; Jungermann and Katz, 1989. Physiol. Rev. 69:
708-764; Sirica, supra; Van Eyken and Desmet. 1993. Bile Duct
Cells. In: Molecular and Cell Biology of the Liver, LeBouton, A. V.
(Ed.), CRC Press, Baton Raton, Forida, Pages 475-524; Alpini et
al., supra), this finding does not support the classification of
the PICM-19B cells as bile duct or gallbladder epithelium. Perhaps
like biliary "oval cells" or putative facultative liver stem cells
(Newsome et al. 2004. Curr. Top. Dev. Biol. 61: 1-28; Sigal et al.
1992. Am. J. Physiol. 263: G139-148), the PICM-19B cells display a
plasticity of function that crosses over between hepatocytes and
cholangiocytes.
Example 10
Induction of CYP450 PICM-19H Cells; Assay of Induced Phase I and II
Activities
[0111] Several CYP450 enzymatic activities were investigated in
PICM-19H cells to determine the presence and inducible nature of
major CYP450 isoforms. Non-fluorescent substrates were added to
cultures of PICM-19H cells (FIG. 17) which were either non-induced
controls (containing PBS or 0.1% DMSO) or induced cultures from
incubation for 48 h with either 5 .mu.M 3-MC, 50 .mu.M rifampicin
(rif) or 1 mM phenobarbital (PHB) to induce CYP450 1A, 3A, and 2,
respectively, as described above in Example 7 and is described
below. Rifampicin and 3-MC stocks were dissolved in DMSO (0.1%
final concentration) and PHB was dissolved in PBS and diluted in
growth medium to the final concentration noted above; solvent
induction-controls were performed as indicated. Cells were washed
with PBS and appropriate inducer medium was added for 48 h prior to
experimentation.
[0112] CYP450 activity was assessed in T25 cultures of PICM-19H
cells that were grown for 3 wk post-passage. T25 cultures of APH or
HepG2 C3A cells were tested after 3 d of primary culture and at 1
wk post-passage or near confluency, respectively. Cell cultures
were treated for 48 h with an inducing agent, either 3-MC,
rifampicin, PHB or DMSO (as a vehicle control). After the initial
induction, cells were washed and given a non-fluorescent substrate
for CYP1A1, CYP1A2, CYP2, or CYP3A (7-ERF, 7-MRF, MFC, and BFC,
respectively). Final incubation conditions were essentially as
described by Donato et al. (1993, supra; 2004. Drug Metab. Dispos.
32: 699-706), and whole cells were allowed to metabolize substrates
30 min for 7-MRF and 7-ERF, and 60 min for MFC and BFC. Medium
samples were collected, centrifuged to remove cell debris
(14,000.times.g, 2 min) and frozen at -80.degree. C. until
analyzed. Aliquots of medium samples were added to 96-well plates
and the concentrations of fluorescent products were determined
(Donato et al. 1993, 2004, supra). Briefly, samples were incubated
at 37.degree. C. with or without
.beta.-glucuronidase/arylsulphatase (15 Fishman/120 Roy units/ml,
Roche Applied Sciences) in pH 4.5 acetate buffer for 2 h to release
any fluorescent reaction product that had been conjugated via
sulfation or glucurondation phase II conjugation reactions.
Standard curves contained resorufin or 7-HFC were treated
identically to the experimental medium samples. Fluorescence was
determined in an HTS 7000 plate reader; excitation/emission filter
pairs were 530/590 and 410/510 for resorufin and 7-HFC,
respectively. To determine relative conjugation activity,
fluorescence was also determined directly without incubating first
in .beta.-glucuronidase/arylsulphatase. Since conjugated forms of
resorufin and 7-HFC are non-fluorescent, the difference between the
direct and indirect fluorescence measurements represents the amount
of product which had been modified by conjugating enzymes.
[0113] Protein in cell homogenates, prepared by sonication, was
determined by a modified Lowry procedure following NaOH
solubilization of TCA-precipitated material (Nerurkar et al. 1981.
Quantification of selected Intracellular and Secreted Hydrolases of
Macrophages. In: Manual of Macrophage Methodology. Herscowitz et
al. (eds. Marcel Dekker, Inc., New York, N.Y., pages 229-247).
Bovine serum albumin (A6003, Sigma) was used as a standard.
[0114] CYP450 CYP 1A Activity. CYP 1A2 activity, defined here as
methoxy resorufin-O-demethylase (MROD) activity, is mainly
responsible for metabolizing environmental carcinogens, e.g.,
polycyclic aromatic hydrocarbons, into their cancer-causing
DNA-binding forms (Shimizu et al. 2000. Proc. Nat. Acad. Sci. USA
97:779-782). MROD activity was low in non-induced PICM-19H cultures
and in those incubated with PHB and Rif, whereas in those exposed
to 3-MC a 5.5-fold induction of activity was measured (FIG. 17).
CYP1 A1 activity, defined here as ethoxy resorufin-O-deethylase
(EROD) activity, is responsible for metabolizing environmental
carcinogens, is highly expressed in the liver, and represents
.about.13% of human liver CYP450 content (Shimada et al., supra).
EROD activity was low in non-induced cultures and those induced
with Rif, while incubation with PHB and 3-MC resulted in 7- and
198-fold increases in activity, respectively (FIG. 17).
[0115] CYP450 CYP2 Activity. The specific activities induced by the
addition of PHB and quantified by the conversion of the
non-fluorescent MFC to the fluorescent compound 7-HFC are
attributable to several members of the CYP2 super family. The
activity of CYP2 isoforms referred to here as methoxy
trifluoromethyl coumarin demethylase (MFCD) are induced by some
xenobiotics and members of the barbiturate family of drugs. In
PICM19H cells, MFCD activity was equally induced by PHB, Rif and
3-MC (FIG. 17).
[0116] CYP450 CYP3A Activity. In humans, CYP3A4 is responsible for
approximately 50% of all drugs metabolized by CYPs (Bertz and
Granneman, 1997). In addition to its role in xenobiotic metabolism,
CYP3A4 is also important in the biosynthesis of several endogenous
steroid hormones and is reported to represent approximately 30-40%
of total human hepatic CYPs (Shimada et al., supra). The conversion
of the non-fluorescent substrate 7-benzyloxy-4-trifluoromethyl
coumarin (7-BFC) to the fluorescent compound 7-hydroxy
4-trifluoromethyl-coumarin (7-HFC) was used to quantitatively
measure debenzylation (BFCD) activity. Induction of BFCD with Rif
and PHB resulted in an approximate 50% increase in activity
compared to non-induced controls, while 3-MC incubation was
associated with a 5.8-fold induction (FIG. 17).
Example 11
Liquid Chromatography-Mass Spectrometry of Testosterone
Metabolites
[0117] Testosterone is hydroxylated by several CYP450 isozymes to
yield different metabolite derivatives (Watanabe et al. 1997. J.
Mass Spectrom Soc. Jpn. 45: 367-375; Donato et al. 1999, supra).
PICM-19H cells were treated with 0.1% DMSO or 50 uM Rif for 48 h,
followed by a 1 h exposure to 250 .mu.M testosterone (Sigma).
Protein was removed from samples by acetonitrile precipitation (1:1
v/v on ice, 15 min), followed by centrifugation at 10,000.times.g.
Supernatants were taken to dryness under N.sub.2 at 37.degree. C.,
followed by resuspension in 1:1 ethanol:MAF solution (25% methanol,
75% 2 mM ammonium acetate, 0.05% formic acid) at 1:2 of original
medium supernatant. Samples were separated with an Alliance 2695
Separation Model (Waters, Beverly, Mass.) with a Symmetry C18
column (3.5 .mu.m, 2.1.times.100 mm with a 2.1.times.10 mm guard
column containing the same packing material (Waters). The initial
mobile phase was 40% Solvent A (2 mM ammonium acetate, 0.05% formic
acid) and 60% Solvent B (methanol). A linear gradient was run from
0 to 10 min, reaching 100% Solvent B, and held for 3 min, flow rate
0.2 mL min.sup.-1. An Ultima API-US, Quadrupole-Time of Flight
(Q-TOF) mass spectrometer (Waters) equipped with an electro-spray
ionization source, was used in the positive mode to characterize
isolated metabolites. The capillary voltage was 2.60 kV (T),
collision energy 10 eV, cone voltage 35 V, source and desolvation
temperatures were 120 and 350.degree. C., respectively. Cone and
desolvation gas flows were 40 and 500 L h.sup.-1, respectively.
Testosterone metabolite standards were obtained from Steraloids,
Inc. (Newport, R.I.). The concentrations of compounds were
calculated based on concurrently run standard curves, summing peak
areas for the ionized parent and two fragments.
[0118] LC/MS analysis was used to identify and quantify three
testosterone metabolites (6.beta.OHT, 2.alpha.OHT and 2.beta.OHT)
in control and Rif-treated PICM-19Hcultures (FIG. 18). Non-induced
activity levels for the production of each metabolite were
relatively low (<0.08 umol/mg protein/hr) while induction with
Rif was associated with 14.7-, 8.3- and 11-fold increases in the
rates of production of 6.beta.OHT, 2.alpha.OHT and 2.beta.OHT,
respectively (FIG. 18).
[0119] While testosterone metabolism in non-induced PICM-19H
cultures was negligible, Rif treatment was associated with a
greater than 10-fold induction of hydroxylated metabolite
formation. As was evident in the PICM-19H cells, pig liver
microsomes and fresh pig hepatocytes have also been shown to
produce 6.beta.-hydroxylation as the predominant hydroxylated
species (Donato et al, 1999). Relatively high induction of
2.alpha.- and 2.beta.-hydroxylation activities in the PICM-19H
cells was also evident. Interestingly, PICM-19H cells incubated
with Rif, metabolized the natural substrate (testosterone)
two-to-three orders of magnitude greater than the artificial
substrates MFC and BFC, indicating that Rif is indeed capable of
inducing high levels of CYP3A activity relative to baseline
levels.
Example 12
Comparison of EROD and BCFD Activities with Other Hepatic Cell
Types
[0120] Monolayer cultures of adult pig hepatocytes (APH) and HepG2
C3A cells were prepared and EROD and BCFD activities were compared
to PICM-19H cultures following a 48 h induction with 3-MC and Rif,
respectively. In addition, we also compared the relative amounts of
Phase II activity among the different cell cultures by evaluating
the extent to which fluorescent activity was observed before and
after treatment with .beta.-glucuronidase/arylsulfatase. As
expected, primary cultures of APH demonstrated high levels of
inducible EROD and BFCD activities (FIG. 19). Maximal induced EROD
and BFCD activities in PICM-19H cells were 30% and 43%,
respectively, of that in APH while in HepG2 C3A cells, EROD
activity was 7% of APH and BFCD activity was undetectable. STO
cells were also evaluated for EROD and BFCD (FIG. 19) and other
CYP450 activities and in all cases have demonstrated an absence of
detectable activity (not shown). Overall conjugation, or phase II
activity, was similar for resorufin and coumarin-based substrates
(>50% and >95% conjugation, respectively) for both PICM-19H
and APH (FIG. 20). CYP450 was limited to EROD activity in HepG2 C3A
cells and only 20% of the resorufin was determined to be in a
conjugated form.
[0121] Induction of specific substrate metabolism in PICM-19H cells
included EROD, MROD, MFCD and BFCD; which correlates with CYP1A1,
CYP1A2, CYP2, and CYP3A activities, respectively, at a minimum.
3-MC, at the concentration utilized, induced all of these specific
activities. In contrast, PHB induced EROD, and to a lesser extent,
BFCD and MFCD activity. Rifampicin was shown to specifically induce
MFCD and BFCD, as well as testosterone hydroxylation
activities.
[0122] In the present study, EROD and BFCD were induced with 3-MC
and Rif, respectively, and the activities were compared between
freshly prepared APH, a well-characterized human hepatoma cell
line, HepG2 C3A, and PICM-19H cells. In comparison to the induced
CYP450 activities found in APH cells, PICM-19H had approximately
30% and 43% of the EROD and BFCD activities. In contrast, these
activities were low to non-existent in the HepG2 C3A cells. These
results were not unexpected. First, in comparison to APH, the
PICM-19H cultures are a "naive" population of liver cells in that
they are not constantly exposed to the numerous chemical entities
that the APH cells are in the in vivo environment of the intact
pig's liver. Second, the HepG2 C3A cells are generally recognized
to be deficient in certain differentiated hepatocyte functions
(Nyberg et al, 1994; Rodriguez-Antona et al 2002), and the C3A
derivative cell line appears to share these deficiencies, at least
in terms of inducible CYP450s. This comparative lack of
differentiated function most likely arises from the fact that the
HepG2 cells, unlike PICM-19H cells, are derived from tumor tissue
and are abnormal in several growth and differentiation
characteristics. For example, 10-14 d after passage, PICM-19H cells
stop dividing and by three weeks in culture have terminally
differentiated before confluency can be reached. In contrast, HepG2
C3A cells appear to continue to divide even after reaching
confluency (data not shown).
[0123] Phase II reactions are mediated by cytoplasmic enzymes which
are responsible for the detoxification of xenobiotics via
conjugation with water-soluble chemical moieties, thus providing a
key function for the body's elimination of various chemical
entities. With both coumarin- and resorufin-based substrates,
PICM-19H cells demonstrated phase II conjugation reactions that
were comparable to those of the primary APH cells. Our standard
methodology incorporates a combined hydrolytic enzyme preparation,
viz., .beta.-glucouronidase/arylsulfatase, so it cannot be readily
determined which specific activity was responsible for degradation
of the conjugated products as it has been confirmed that sulfation
and glucuronidation activities are functional in pig liver tissue
(Diaz and Squires. 2003. Xenobiotica 33: 485-498) This does not
preclude the likely formation of other forms of phase II
conjugation such as glutathione addition or acylation reactions,
which were not measured in the present study.
Example 13
Cell Viability Assay
[0124] The toxic effect of the exposure of cells to chemicals was
assayed using the WST-1 cell proliferation reagent (Roche Applied
Sciences, Mannhein, Germany). The method measures the conversion of
tetrazolium salts to formazan by mitochondrial enzymes in intact
cells. A decrease in the production of the formazan dye can be
correlated with a decrease in viable cells compared to controls
(Ishiyama et al., supra).
[0125] Williams E (Phenol Red-free) supplemented with 2% FCS was
used to prepare a 1:10 dilution of WST-1 stock reagent. The diluted
reagent was added to the cells and incubated at 37.degree. C. for
30 min. Samples of the cell processed reagent were transferred to a
clear 96-well plate, diluted with dH.sub.2O and read in a plate
reader (HTS7000, Perkin Elmer, Norwalk, Conn.) at 450 nm. The
background at 620 nm was measured and then subtracted from the 450
nm result. Results were compared to untreated controls.
Example 14
Metabolism of Known Hepatotoxins by PICM-19H Cells
[0126] Aflatoxin B1 is a well documented mycotoxin and suspected
hepatic carcinogen which is metabolized by several CYP450s to
generate significantly toxic metabolites (Kamdem et al. 2006. Chem.
Res. Toxicol. 19: 577-586). In PICM-19H cells grown in 96-well
plates, the 50% lethal concentration (LC.sub.50) of aflatoxin B1,
as measured by WST-1 viability, was approximately 40 .mu.M (FIG.
21). The induction of CYP450s with 3-MC was associated with a
marked decrease in the LC.sub.50 (1.1 .mu.M). Thus, 3-MC treatment
increased the toxicity of aflatoxin B1 confirming induction of CYPs
which are involved in the bioactivation of this toxin.
[0127] To determine the relative toxicity of acetaminophen in
PICM-19H cells, cultures prepared and maintained in 96-well plates
were exposed to various concentrations of the drug between 0.78 and
100 mM for 24 h. Viability of PICM-19H cells was determined by
WST-1 assay. Regression analysis of ten independent determinations
indicated that the LC.sub.50 for acetaminophen was 12.65.+-.0.75 mM
(FIG. 22).
[0128] The hepatic CYP450 system is associated with detoxification
of absorbed xenobiotics, as well as the metabolism of endogenous
molecules including cholesterol and associated steroids. In
addition, the CYP450 enzymes can metabolize drugs to more active
forms and sometimes into toxic forms. For example, dose-dependent
cytotoxicity following treatment of hepatocytes with acetaminophen
resulted from the cells generating reactive oxygen species in
response, and thereby causing a cytotoxic effect in the hepatocytes
(Michael et al. 1999. Hepatology 30: 186-195). The LC.sub.50 of
acetaminophen for PICM-19H cells was determined to be approximately
13 mM. This LC.sub.50 and the dose-response data are similar to
previously published experimental values obtained from cultures of
human or rodent primary hepatocytes (Wang et al. 2002. J. Toxicol.
Sci. 27: 229-237; Allen et al. 2005. Toxicol. Sci. 84: 110-119).
Also, in preliminary experiments, acetaminophen toxicity was
enhanced in glutathione-depleted PICM-19H cells (buthionine
sulfoxime-treatment) further demonstrating the presence of the
metabolic machinery associated with primary acetaminophen
metabolism (data not shown). In addition, the toxic bioactivation
of aflatoxin B1 in PICM-19H cells demonstrated their hepatic CYP450
metabolism. The cytotoxicity of aflatoxin B1 is enhanced by the
activities associated with CYP1A- and 3A (Gallagher et al. 1996.
Toxicol. Applied Pharmacol. 141: 595-606), and pretreatment of the
PICM-19H cells with 3-MC led to a potentiation in aflatoxin B1
toxicity through an induction of these, and perhaps other
CYP450s.
Example 15
Culture of PICM-19H Cells without Feeder Cells
[0129] A feeder-cell-independent culture of PICM-19H cells was
developed. The STO feeder cells have been replaced by precoating
the tissue culture flasks with polymerizing bovine collagen type I.
The flasks are dried and then stored at 4-8.degree. C. before use.
The concentration of the collagen currently used is 0.15 mg/ml in
complete PICM-19 medium (10% DMEM/199+; Talbot and Paape); however,
various concentrations of collagen can be used. Other species of
collagen are also available, e.g., human. Other types of collagen
are also routinely used for coating, e.g., collagen IV. The base
medium is 10% DMEM high glucose that has been conditioned by STO
feeder cells for 6 days. At the end of the conditioning period an
equal portion of Medium 199 medium (Talbot et al. 1996, supra) is
added to the STO conditioned medium (CM), and 2-mercaptoethanol,
100.times. nucleoside mix, and fetal bovine serum are also added to
bring the CM to equivalency with 10% DMEM/199+ (Talbot and Paape,
supra). In addition the following growth factors are added to the
basic CM medium: (a) ITS liquid medium supplement--insulin,
transferrin, selenium 100.times. (No. 13146, Sigma Chem. Co., St.
Louis, Mo.), (b) Hepatocyte Growth Factor (HGF)--50 ng/ml
(eBioscience, Sigma, Becton-Dickinson, and R&D Systems), (c)
Insulin-like Growth Factor-1 (IGF-1)--25 ng/ml (R&D Systems,
Minneapolis, Minn.), and (d) IGF binding protein-4 (IGFBP-4)--50
ng/ml (R&D Systems, Minneapolis, Minn.). The feeder-independent
PICM-19H cultures are currently passaged by exposure to
trypsin-EDTA (Talbot et al. 1994a, supra); however, cells can also
be passaged by exposure to collagenases. The cultures are refed
with fresh complete medium every 2-3 days between passages.
Example 16
Culture of PICM-19H and PICM-19B in a Hollow Bioreactor
[0130] PICM-19H and PICM-19B cells were cultured individually or as
combined cultures using hollow-fiber bioreactor cartridges. The
cells were inoculated with and without STO feeder cells and the
PICM cell inoculum consisted of either differentiated cells or
undifferentiated cells. The hollow-fiber bioreactors used are of
various designs including but not limited to those containing
integrated oxygenation fibers (Stem Cell Systems, Berlin Germany).
Cells were inoculated in a differentiated or undifferentiated state
and cultured using protocols provided by but not limited to various
suppliers such as FiberCell, Inc. Frederick Md., USA. The protocols
for growth and maintenance of the cells in hollow-fiber culture
included but were not limited to continuous recirculation,
perfusion and fed-batch modes. The hollow-fiber devices were used
without coating or were primed prior to the addition of the
PICM-19H and/or the PICM-19B cells by the addition of substances
that enhance the attachment of the STO feeder cells (if used)
and/or the PICM-19H and/or the PICM-19B cells such as but not
limited to collagen in various forms and concentrations. Samples
were removed at daily intervals and examined for residual glucose
and ammonia levels as well as albumin production. The results of a
bioreactor run are shown in FIGS. 23A and 23B.
Example 17
Culture of PICM-19H and PICM-19B on Microcarrier Beads or
Encapsulated
[0131] PICM-19H and PICM-19B cells were cultured individually or as
combined cultures using microcarrier beads or encapsulated into
hydrogels. The cells were inoculated with and without STO feeder
cells and the PICM cell inoculum consisted of either differentiated
cells or undifferentiated cells. The Microcarrier Beads used were
of various designs including but not limited to those containing
coatings of bioactive materials and/or surface invaginations
(SoloHill Engineering Ann Arbor, Mich. USA). Cells were inoculated
in a differentiated or undifferentiated state and cultured using
protocols provided by but not limited to various suppliers such as
those provided by GE Healthcare Piscataway, N.J. USA. The protocols
for growth and maintenance of the cells on Microcarrier Beads
included, but were not limited to batch, perfusion and fed-batch
modes. The Microcarrier Beads were used without coating or were
primed prior to the addition of the PICM-19H and/or the PICM-19B
cells by the addition of substances that enhance the attachment of
the STO feeder cells (if used) and/or the PICM-19H and/or the
PICM-19B cells such as but not limited to collagen in various forms
and concentrations. Samples were removed at daily intervals and
examined for residual glucose and ammonia levels as well as albumin
production. The results of a Microcarrier and run are shown in
FIGS. 24A and B.
[0132] The Hydrogels used for cell encapsulation were of various
designs including but not limited to those containing alginate with
or without additions of bioactive materials such as collagen. Cells
were encapsulated in a differentiated or undifferentiated state and
cultured using standard suspension cell techniques or injected into
the extracapillary space of a hollow-fiber bioreactor.
Example 18
Culture of PICM-19H as a Spheroid
[0133] A method with which to create spheroid cultures of PICM-19H
cells. PICM-19H cells are released from the tissue culture
plasticware by treatment of a PICM-19H culture with collagenase
(such as but not limited to collagenase II) prepared in cell
culture medium or calcium containing physiological salt solutions
(such as but not limited to DMEM or Hank's Buffer Saline Solution)
at 1 mg/ml for 15-30 min at 37.degree. C. The collagenase treatment
breaks down the collagen fibrils within the culture that is produce
by the STO feeder cells and the STO cells and PICM-19H cells
retract from each other, the PICM-19H cells forming small balls of
cells. The balled up PICM-19H colonies are finally fully released
from the plastic substrate by vigorous agitation of the culture
flask or by scrapping the culture with a sterile cell scrapper of
some type. The balls of PICM-19H cells, nascent spheroids, are
suspended in cell culture medium and subjected to low speed
centrifugation (e.g. 100-200.times.g for 5-10 min) and the
supernatant above the resulting cell pellet is removed and
discarded so as to remove the collagenase. The pellet of nascent
PICM-19H spheroids is resuspended in fresh complete culture medium
(such as but not limited to 10% DMEM/199; Talbot and Paape, 1996)
and cultured with gentle agitation through the use of, but not
limited to, a rocking or orbital shaker so that the nascent
PICM-19H spheroids do not reattach to the plasticware or
plasticware/feeder cell substrate beneath them in the culture
vessel. Fresh medium is supplied to the spheroid culture every 2-3
days. The spheroids may be grown and maintained for at least 2 wk
by this method as demonstrated in FIG. 25.
[0134] All publications and patents mentioned in this specification
are herein incorporated by reference to the same extent as if each
individual publication or patent was specifically and individually
indicated to be incorporated by reference.
[0135] The foregoing description and certain representative
embodiments and details of the invention have been presented for
purposes of illustration and description of the invention. It is
not intended to be exhaustive or to limit the invention to the
precise forms disclosed. It will be apparent to practitioners
skilled in this art that modifications and variations may be made
therein without departing from the scope of the invention.
* * * * *
References