U.S. patent application number 11/723529 was filed with the patent office on 2007-08-23 for antibody recognizing proliferative human liver cells, proliferative human liver cells and functional human liver cells.
Invention is credited to Chise Mukaidani, Chihiro Yamasaki, Katsutoshi Yoshizato.
Application Number | 20070196345 11/723529 |
Document ID | / |
Family ID | 28449202 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070196345 |
Kind Code |
A1 |
Mukaidani; Chise ; et
al. |
August 23, 2007 |
Antibody recognizing proliferative human liver cells, proliferative
human liver cells and functional human liver cells
Abstract
The invention provides a monoclonal antibody which specifically
recognizes proliferative human hepatocytes, a method for isolating
proliferative human hepatocytes by using the antibody, and a method
for inducing to differentiate the proliferative human hepatocytes
to functional human hepatocytes. Further, the invention provides
the proliferative human hepatocytes and functional human
hepatocytes obtained by the methods, as well as a cell kit and a
hybrid artificial liver comprising those hepatocytes.
Inventors: |
Mukaidani; Chise;
(Hiroshima, JP) ; Yoshizato; Katsutoshi;
(Hiroshima, JP) ; Yamasaki; Chihiro; (Hiroshima,
JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
28449202 |
Appl. No.: |
11/723529 |
Filed: |
March 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10509096 |
Feb 9, 2005 |
7220839 |
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PCT/JP03/03624 |
Mar 25, 2003 |
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11723529 |
Mar 20, 2007 |
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Current U.S.
Class: |
424/93.7 ;
435/370; 435/7.23 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12N 5/067 20130101; C12N 5/0697 20130101; C07K 16/18 20130101;
C07K 16/28 20130101; C12N 2501/38 20130101 |
Class at
Publication: |
424/093.7 ;
435/007.23; 435/370 |
International
Class: |
A61K 35/407 20060101
A61K035/407; C12N 5/08 20060101 C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 25, 2002 |
JP |
2002-084280 |
Claims
1-4. (canceled)
5. A method for isolating proliferative human hepatocytes, which
comprises separating cells recognized by an antibody, from a human
hepatocyte population, wherein said antibody specifically
recognizes proliferative human hepatocytes that exist in a
hepatocyte population isolated from an adult human liver and have
clonal proliferative ability and differentiation ability to
functional hepatocytes.
6. Proliferative human hepatocytes isolated by the method of claim
5.
7. A method for inducing the differentiation of the proliferative
human hepatocytes of claim 6, which comprises performing at least
one of the following means: (a) spheroid culture of the
proliferative human hepatocytes; and (b) transfer of hepatic
nuclear factor 4 (HNF4) gene into the proliferative human
hepatocytes.
8. Functional human hepatocytes induced to differentiate by the
method of claim 7.
9. A cell kit comprising the functional human hepatocytes of claim
8.
10. A hybrid artificial liver packed with the functional human
hepatocytes of claim 9.
11. The method of claim 5, wherein said antibody is produced by
hybridoma cell, Mouse-Mouse hybridoma K8223 (FERM BP-8334).
Description
TECHNICAL FIELD
[0001] The present invention relates to an antibody recognizing
proliferative human hepatocytes, and human hepatocytes. More
particularly, the present invention relates to a monoclonal
antibody specifically recognizing human hepatocytes having clonal
proliferative potency and useful for separating proliferative
hepatocytes from a human hepatocyte population, proliferative human
hepatocytes separated by using this antibody, a method of inducing
the differentiation of the proliferative human hepatocytes to
differentiated hepatocytes, functional human hepatocytes induced to
differentiate by such a method, and a hepatocytes kit containing
the functional hepatocytes and an extracorporeal artificial
liver.
BACKGROUND ART
[0002] A liver has 500 or more types of various specific functions.
Major functions of a liver include plasma protein synthesis and
secretion, blood sugar control by gluconeogenesis and glycogen
metabolism, lipogenesis, ureogenesis, bile synthesis and secretion
and detoxication.
[0003] Most substances incorporated into a body are mainly
metabolized in a liver. In the filed of pharmaceutical development,
what type of metabolism pharmaceutical candidate substances will be
received and what type of effect is given to a liver or other
organs and tissues are essential data. Further, many chemical
substances have been synthesized and discharged into environment up
to now. To elucidate what kind of effect these substances have
exerted individually or in combination to a human body is socially
very important. Toxicological tests on liver function are essential
for evaluation of the effects of such chemical substances to a
human body.
[0004] Mice, rats, rabbits, dogs, monkeys, etc are used at present
for safety tests and drug metabolism tests of chemical substances
including pharmaceutical candidate substances. Especially in
pharmaceutical development, toxicological tests and safety tests
using animals are compulsive before entering phase I study for
human, and long period and efforts as well as huge costs are
required in these tests.
[0005] However, there is no guarantee that data obtained by these
animal experiments can be applicable to human. In fact, many cases
are known wherein substances not recognized toxicity in animal
experiments exhibited toxicity in human or vice versa.
Consequently, there might exist many cases wherein development of
many pharmaceutical candidate substances were terminated after
entering in phase I study on human, or also many substances were
terminated development before entering clinical trials due to
exhibiting strong toxicity in animal experiments but recognized
actually no toxicity in human.
[0006] This may be caused by difference in metabolic function in a
human liver and in metabolic function in livers of mice and rats.
Recently, in vitro metabolic tests and toxicity tests using human
hepatocytes have been performed. However, amount of livers from
brain death patients which were not used for transplantation and
amount of human hepatocytes obtained from hepatectomy in tumor
excision are far fewer than demanded. Consequently, development of
technology for human hepatocyte proliferation is essential for
pharmaceutical development.
[0007] Necessity of high amount of human hepatocytes is very much
alike in an extracorporeal artificial liver. The artificial liver
is medical device acting liver function artificially. Development
of a hybrid artificial liver combining with the artificial function
based on physico-chemical principle such as adsorption, dialysis
and filtration, along with biological actions using perfusion of an
excised liver and liver tissue is strongly progressing. In
development of the artificial liver, performance improvement of
membrane and circuit to enhance physico-chemical function is
essential, along with supplying high amount of hepatocytes
applicable to human use.
[0008] However, subcultivation of primary cultured cells of a human
liver isolated from adults has been considered impossible. Namely,
the matured hepatocytes with adhesion dependency are largely
damaged when cells are detached from culture substrate for
subcultivation operation and are difficult to re-adhere to culture
substrate. Contrary to that, the present inventors have invented a
method for proliferative hepatocytes comprising isolating small
hepatocytes, having clonal proliferative ability, from normal
hepatocytes isolated from human liver, performing primary culture
of the small hepatocytes, and subculturing further the cultured
hepatocytes, and the invention was granted patents (JP-A-08-112092;
JP No.3266766; U.S. Pat. No. 6,004,810, JP-A-10-179148; JP No.
3211941, JP-A-07-274951; JP No. 3157984, and JP-A-09-313172; JP No.
3014322). Relating articles published are as follows: (Tateno, C.
and Yoshizato, K.; "Growth and differentiation in culture of
clonogenic hepatocytes that express both phenotypes of hepatocytes
and biliary epithelial cells", Am. J. Phathol. 149: 1593-1605,
1996; Hino, H. Tateno, C. Sato, H. Yamasaki, C. Katayama, S.
Kohashi, T. Aratani, A. Asahara, T. Dohi, K. and Yoshizato, K.; "A
long-term culture of human hepatocytes which show a high growth
potential and express their differentiated phenotypes", Biochem.
and Biophys. Res. Commun. 256: 184-191, 1999; Tateno, C. Kajihara,
K. T. Yamasaki, C. Sato, H. and Yoshizato, K. "Heterogeneity of
growth potential of adult rat hepatocytes in vitro", Hepatology 31:
65-74, 2000; and Katayama, S. Tateno, C. Asahara, T. and Yoshizato,
K.; "Size-dependent in vivo growth potential of adult rat
hepatocytes", Am. J. Pathol. 158: 97-105, 2001).
[0009] The method of the above prior patented inventions provides
novel means for obtaining high amount of human hepatocytes by
proliferative hepatocytes in vitro, however, the human hepatocytes
obtained by this method have a problem that various liver functions
are impaired during long-term subculture. There is further a
problem that the small hepatocytes can not differentiate to
hepatocytes (functional hepatocytes) having function equivalent to
normal hepatocytes, for example, albumin expression level and
metabolic activity of cytochrome P450 for chemical substance and so
on. By those reasons, they are still insufficient as hepatocytes
for alternative human liver function or as a material for a hybrid
artificial liver, although they are useful as a screening system of
drugs for maintaining specific liver function or as a system for
testing toxicity and pharmacological effect of drugs on specific
functions which are conserved after long-term subculture.
[0010] Small hepatocytes having proliferation potency can be
collected by using not only a method for centrifugal separation as
described in the above prior patented invention (a method for
separating cells in supernatant obtained by low speed
centrifugation) but also a cell sorter such as an elutriator and
FACS, however, cells obtained by such means are a mixture of not
only proliferative hepatocytes but also other cells (such as
non-hepatic parenchymal cells such as stellate cells contained in
supernatant by low speed centrifugation). Consequently, means to
obtain substantially proliferative human hepatocytes only has been
required.
[0011] The present invention has been completed considering the
above circumstances, and an aspect of the present invention is to
provide a monoclonal antibody which specifically recognizes
proliferative human hepatocytes.
[0012] Further, other aspect of the present invention is to provide
a method for isolating proliferative human hepatocytes and the
proliferative human hepatocytes obtained by such a method.
[0013] Further, other aspect of the present invention is to provide
a method for inducing to differentiate the proliferative human
hepatocytes to functional human hepatocytes, the functional human
hepatocytes obtained by such a method and utilization of the
functional human hepatocytes.
DISCLOSURE OF INVENTION
[0014] The present invention provides, as the first invention for
solving the above problem, a monoclonal antibody specifically
recognizing proliferative human hepatocytes. An example of a
monoclonal antibody of the first invention is the monoclonal
antibody produced by a hybridoma cell, Mouse-Mouse hybridoma K8223
(FERM BP-8334).
[0015] The second invention provides a hybridoma cell producing the
monoclonal antibody of the first invention. An example of the
hybridoma cell is Mouse-Mouse hybridoma K8223 (FERM BP-8334).
[0016] The third invention provides a method for isolating
proliferative human hepatocytes, which comprises separating cells
recognized by the monoclonal antibody of the first invention from a
human hepatocyte population.
[0017] The fourth invention provides proliferative human
hepatocytes isolated by the method of the third invention.
[0018] Further the fifth invention provides a method for inducing
the differentiation of the proliferative human hepatocytes of the
fourth invention, which comprises performing at least one of the
following means:
[0019] (a) spheroid culture of the proliferative human hepatocytes;
and
[0020] (b) transfer of hepatic nuclear factor 4 (HNF4) gene into
the proliferative human hepatocytes.
[0021] The sixth invention provides functional human hepatocytes
induced to differentiate by the method of the fifth invention.
[0022] The seventh invention provides a cell kit comprising the
functional human hepatocytes of the sixth invention.
[0023] The eighth invention provides a hybrid artificial liver
filled with the functional human hepatocytes of the sixth
invention.
[0024] In the present invention, a term "proliferative human
hepatocytes" means human hepatocytes forming colonies as a
population and proliferative so as to increase the colonies under
in vitro culture condition. Further, "proliferation" may be
designated as "clonal proliferation" because colony component cells
are single species. Further, such cells can increase cell numbers
by subculture.
[0025] In the present invention, a term "functional human
hepatocytes" means cells having function level equivalent to that
of human normal hepatocytes in human body (in vivo) or in the
primary culture (in vitro) thereof, and more particularly, it means
that at least level of "albumin expression" and "cytochrome P450
activity" thereof are substantially equal to those of human normal
hepatocytes.
[0026] Other terms and concepts in the present invention will be
defined in detail in the description of embodiments and examples of
the present invention. Various techniques used for carrying out the
present invention can easily be available based on known references
by those skilled in the art except for those indicating specific
sources thereof. For example, technologies of genetic engineering
and molecular biology of the present invention are described in
Sambrook and Maniatis, in "Molecular Cloning--A Laboratory Manual",
Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F. M.
et al., "Current Protocols in Molecular Biology", John Wiley &
Sons, New York, N.Y. 1995, etc.
BRIEF DESCRIPTION OF DRAWINGS
[0027] FIG. 1 illustrates a growth curve in culturing hepatocytes
isolated from patients of various ages.
[0028] FIG. 2 is a phase-contrast microscopic images of cultured
human hepatocytes used as an antigen for preparing a monoclonal
antibody.
[0029] FIG. 3 is an image showing reactivity of culture supernatant
of hybridoma K8223 on human liver tissues observed by
immunofluorescence staining.
[0030] FIG. 4 is analysis result on reactivity of culture
supernatant of hybridoma No. 23 on isolated human hepatocyte by
FACS.
[0031] FIG. 5 is a phase-contrast microscopic images of cell
populations which reacted, No. 23 (R2), and non-reacted (R3) with
culture supernatant of hybridoma are sorted and cultured.
[0032] FIG. 6 is analysis result on reactivity of culture
supernatant of hybridoma No. 23 on subcultured human hepatocyte
surface, by FACS.
[0033] FIG. 7 is a phase-contrast microscopic images of monolayer
culture and spheroid culture of hepatocytes after 1 day (upper
column) and 7 days (lower column).
[0034] FIG. 8 is a graph exhibiting expression of human P450 gene
or GAPDH gene on hepatocytes immediately after separation from a
human liver, a monolayer cultured and spheroid cultured hepatocytes
of subcultured hepatocytes, and spheroid cultured hepatocytes.
[0035] FIG. 9 is a graph showing time-dependent changes in albumin
secretion from hepatocytes obtained by monolayer culture and
spheroid culture.
[0036] FIG. 10 is a graph showing expression of human P450 gene on
cultured human cells, to which human HNF4 gene is introduced.
BEST MODE FOR CARRYING OUT THE INVENTION
[0037] A monoclonal antibody of the first invention is
characterized by specifically recognizing human hepatocytes
proliferating while forming colonies. More particularly, the
monoclonal antibody is characterized by specifically recognizing
proliferative human hepatocytes while forming colonies and having
potency of differentiating to functional human hepatocytes. In this
case, a term "specifically recognizing" means to bind only to human
hepatocytes defined hereinbefore and not to bind to other cells
and/or human hepatocytes without having characteristics mentioned
hereinbefore.
[0038] The monoclonal antibody of the first invention can be
obtained from the hybridoma cells of the second invention by means
of a known method. The hybridoma cells and the monoclonal antibody
can be prepared according to a known method for preparation of a
monoclonal antibody, described in "Monoclonal antibody", by
Nagamune, K. and Terada, H.; Hirokawa Publ. Co., 1990, and
"Monoclonal Antibody" James W. Goding, 3rd. Ed., Academic Press,
1996, for example, by the following procedures.
1. Preparation of Hybridoma Cells
[0039] Mammalians are immunized by using an immunogen containing
subcultured human normal hepatocytes, and if necessary, animals are
sufficiently sensitized by immunizing properly and additionally.
Antibody producing cells (lymphocytes or spleen cells) are then
isolated from the animals, and antibody producing cells and myeloma
cell lines are fused to obtain hybrid cells. Cells which produce an
objective monoclonal antibody are selected from these hybrid cell
lines and cultured to obtain hybridoma cells. Each process is
explained in detail below.
a) Preparation of an Immunogen
[0040] Human hepatocytes isolated from normal liver tissues using
collagenase are subcultured to prepare an immunogen. The human
hepatocytes isolated from human liver tissues of subjects not older
than 15 years old, which are able to subculture for not less than 4
passages, preferably not less than 6 passages, are preferably
used.
b) Immunization of Animals
[0041] Animals to be immunized can be mammals used for known
hybridoma preparation and include typically, for example, mice,
rats, goats, sheeps, equines and bovines. Mice or rats are
preferable animals for immunization from the standpoint of easy
availability of myeloma cells to be fused with separated antibody
producing cells. There is no specific limitation on strains of mice
and rats actually used, and strains of mice such as A, AKR, BALB/c,
BDP, BA, CE, C3H, 57BL, C57BR, DBA, FL, HTH, HT1, LP, NZB, NZW, RF,
RIII, SJL, SWR, WB and 129, and strains of rats such as Low, Lewis,
Sprague, Daweley, ACI, BN and Fisher can be used. Considering
compatibility with myeloma cells which will be explained
hereinbelow, BALB/c strain for mouse and low strain for rat are
particularly preferable for animals to be immunized. Ages of these
mice and rats are preferably 5-12 weeks old.
[0042] Immunization of animals can be performed by administering
subcultured human hepatocytes of the immunogen, about
10.sup.4-10.sup.8 cells, intracutaneously or intraperitoneally into
the animals. Administering schedule of the immunogen depends on
types of animals to be immunized, individual differences, etc.,
however generally frequency of administering the immunogen is 1-6
times and an administering interval is 1-2 weeks in multiple
administrations.
c) Cell Fusion
[0043] Spleen cells or lymphocytes containing antibody producing
cells are aseptically collected from the animals to be immunized
after 1-5 days from the final immunization date on the above
administering schedule. Separation of the antibody producing cells
from the spleen cells or the lymphocytes can be performed according
to the known method.
[0044] The antibody producing cells and the myeloma cells are then
fused. The myeloma cells used are not specifically limited and can
be selected from known cell lines for use. However, considering
convenience in selection of hybridoma from the fused cells, use of
HGPRT (Hypoxanthine-guanine phosphoribosyltransferase) deficient
strain, wherein selective procedure thereof has been established,
is preferable. Namely, those are X63-Ag8(X63), NS1-Ag4/1(NS-1),
P3X63-Ag8.U1(P3U1), X63-Ag8.653(X63.653), SP2/0-Ag14(SP2/0),
MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO, BU.1, etc. derived from
mice; 210.RSY3.Ag.1.2.3(Y3), etc. derived from rats; and
U266AR(SKO-007), GM1500 GTG-A12(GM1500), UC729-6,
LICR-LOW-HMy2(HMy2), 8226AR/NIP4-1(NP41), etc. derived from
human.
[0045] Fusion of the antibody producing cells and the myeloma cells
can be preferably performed under conditions without extremely
reducing cell viability according to the known method. Such methods
include, for example, a chemical method for admixing the antibody
producing cells and the myeloma cells in a high concentration
solution of polymer such as polyethylene glycol, and a physical
method utilizing electric stimulation.
[0046] Selection of fused cells and non-fused cells are preferably
performed, for example, by a known HAT
(hypoxanthine-aminopterin-thymidine) selection method. This method
is effective for obtaining fused cells by using the myeloma cells
of HGPRT deficient strain which can not viable in the presence of
aminopterin. Namely, fused cells with resistant to aminopterin can
selectively be left and proliferated by culturing non-fused cells
and fused cells in HAT medium.
d) Screening of Hybridoma
[0047] Screening of hybridoma cells producing an objective
monoclonal antibody can be performed by known methods such as
enzyme immunoassay (EIA), radio immunoassay (RIA) and fluorescent
antibody technique.
[0048] The hybridoma cells producing a monoclonal antibody
specifically bound to highly proliferative human hepatocytes can be
obtained by such a screening method.
[0049] The screened hybridoma cells are cloned by known methods
such as a methylcellulose method, a soft agarose method and a
limiting dilution method and are used for antibody production.
[0050] The hybridoma cells obtained by methods explained
hereinabove can be stored under freezing state in liquid nitrogen
or a freezer at not higher than -80.degree. C. The present
application provides Mouse-Mouse hybridoma K8223 (FERM BP-8334), as
a typical example of the hybridoma cell.
2. Obtaining a Monoclonal Antibody and Purification Thereof
[0051] A monoclonal antibody specifically bound only to
proliferative human hepatocytes can be obtained by culturing the
hybridoma cells prepared in the above 1 step with a known
method.
[0052] Cultivation can preferably be performed in medium having the
same composition used in the above cloning method, or in a mouse
intraperitoneally cultured by injecting the hybridoma cells and
collecting the monoclonal antibody from ascites.
[0053] Thus obtained monoclonal antibody can be purified, for
example, by ammonium sulfate fractionation, gel filtration, ion
exchange chromatography and affinity chromatography methods.
[0054] The present application provides the above-described
hybridoma cell, Mouse-Mouse hybridoma K8223 (FERM BP-8334) as a
typical example of the hybridoma cells and a monoclonal antibody
produced by the hybridoma cell, Mouse-Mouse hybridoma K8223 (FERM
BP-8334), as a typical example of a monoclonal antibody.
[0055] The third invention relates to a separation method for
proliferative human hepatocytes, and is characterized by isolating
cells, which are recognized by the monoclonal antibody of the first
invention, from a human hepatocyte population. The human hepatocyte
population can be prepared, for example, as primary cultured cells,
which are isolated from a human liver by a known method such as
collagenase perfusion method and is cultured in vitro, or can be
prepared as subcultured cells for 1-8 passages. Further, the human
hepatocyte population can be "a cell population containing small
human hepatocytes" disclosed in the patented inventions invented by
the present inventors (JP-A-08-112092; JP No. 3266766; U.S. Pat.
No. 6,004,810, JP-A-10-179148; JP No. 3211941, JP-A-07-274951; JP
No. 3157984 and JP-A-09-313172; JP No. 3014322). That is, the small
human hepatocytes in these patented inventions include
non-parenchymal hepatocytes such as stellate cells or Swiss 3T3
cells which are co-cultured for culturing and subculturing small
human hepatocytes. The proliferative human cells can be separated
more efficiently from such cell populations by using the monoclonal
antibody of the first invention.
[0056] Separation of the proliferative human hepatocytes can be
performed by contacting the monoclonal antibody labeled with
enzyme, radioisotope, magnetic beads or fluorescence with the above
human hepatocyte population and separating cells indicating labeled
signal. Since this method exhibits almost no cell loading caused by
centrifugation, cell fractionation, etc., the proliferative human
hepatocytes can be obtained without any damage. Enzyme used for
labeling is not specifically limited, as long as it can satisfy
conditions such as having large turnover number, exhibiting
stability in binding to an antibody and specifically coloring
substrate. Enzymes used for conventional EIA, such as peroxidase,
.beta.-galactosidase, alkaline phosphatase, glucose oxidase,
acetylcholinesterase, glucose-6-phosphate dehydrogenase and malate
dehydrogenase can be used. Enzyme inhibitors and coenzymes can also
be used. Binding between an enzyme and an antibody can be achieved
by a conventional method using a crosslinking agent such as a
maleimide compound. With regard to substrate, known substance can
be used depending on types of enzyme. For example, when peroxidase
is used as an enzyme, 3,3',5,5'-tetramethylbenzidine can be used,
and when alkaline phosphatase is used as an enzyme, such as
p-nitrophenol can be used. With regard to a radioisotope,
conventionally used radioisotope in RIA such as .sup.125I and
.sup.3H can be used. With regard to a fluorescence pigment, those
conventionally used for fluorescence antibody technique such as
fluorescence isothiocyan ate (FITC) and tetramethylrhodamine
isothiocyanate(TRITC) can be used. When enzyme is used, detection
of labeled signal can be performed by adding substrate, which
develops color by decomposition due to enzyme action, followed by
assaying enzyme activity by measuring the decomposed amount of the
substrate by photometry, converting enzyme activity to an amount of
bound antibody, and calculating the amount of an antibody by
comparing with the standard value. When a radioisotope is used,
radiation quantity emitted by the radioisotope is measured by using
a scintillation counter, etc. When a fluorescence pigment is used,
fluorescence yield is measured by apparatus combined with a
fluorescence microscope.
[0057] According to the method described hereinabove, "the
proliferative human hepatocytes" of the fourth invention can be
obtained. The proliferative human hepatocytes grow actively while
forming colonies under culture condition, and have differentiation
potency to functional human hepatocytes. Such functional human
hepatocytes can be induced to differentiate from the proliferative
human hepatocytes by the method of the fifth invention. The
functional human hepatocytes can also be obtained by the method for
obtaining the human hepatocytes used as the immunogen in the
preparation of the monoclonal antibody in Example 1, however, there
is possibility to contain non-proliferative cells in the method of
Example 1. The method using a monoclonal antibody in the present
invention is an excellent method on the point that substantially
the proliferative human hepatocytes only can be isolated.
[0058] The method according to the fifth invention is characterized
by performing the following means (a) or (b), or (a) and (b).
(a) Spheroid Culture of Proliferative Human Hepatocytes
[0059] "Spheroid" means a cell population forming tissue structure
by aggregation of several hundreds of cells. Typically, cells are
collected to form a three-dimensional cell population by a method
shown below and are cultured in an animal cell culture medium.
Known methods for obtaining spheroid include culturing using
Matrigel (MATRIGEL.TM. Matrix, Becton, Dickinson and Co.), a
positive charged incubator (Primaria, Becton, Dickinson and Co.)
and a U-shaped incubator (Spheroid MS-0096S, Sumiron) and rotary
culture using a roller bottle. Culture is performed for about 6-15
days.
(b) Transfer of Hepatocyte Nuclear Factor 4 (HNF4) Gene into
Proliferative Human Hepatocytes
[0060] Hepatocyte nuclear factor 4 (HNF4) is a protein relating to
transcription of gene expressing specific function in hepatocytes
(e.g. Bell, G. I. et al., Proc. Natl. Acad. Sci. USA 88(4),
1484-1488, 1991) and cDNA sequence of human HNF4.alpha. is known
(e.g. GeneBank/NM.sub.--000457). cDNA sequences of HNF4b
(GenBank/X87871) and HNF4c (GenBank/X87872) is also known. Mouse
HNF4 cDNA (GenBank/NM.sub.--008261) and rat HNF4 cDNA
(GenBank/X57133) are also known. Consequently, these known HNF4
cDNAs are recombined with a eukaryotic cell expression vector and
the resultant expression vector is introduced into proliferative
human hepatocytes by known method such as an electroporation
method, a calcium phosphate transfection method, a liposome method
and a DEAE dextran method, to introduce HNF4 gene. HNF4 gene can
also be introduced by a method according to a gene therapy method
(an ex vivo method) by using hollow nano-particles having a
biorecognizing molecule, retro virus, lentivirus, adenovirus and
adeno-associated virus. HNF4 gene cDNA can be obtained by screening
human and other cDNA libraries using a probe DNA prepared based on
known sequence thereof. Thus obtained cDNA can be used by
amplifying with a conventionally used gene amplification method
such as PCR (Polymerase Chain Reaction) method, NASBN (Nucleic acid
sequence based amplification) method, TMA (Transcription-mediated
amplification) method and SDA (Strand Displacement Amplification)
method. Sufficient amount of objective cDNA can also be obtained by
RT-PCR (Reverse Transcriptase-PCR) method with a template of mRNA
isolated from mammalian cells using a primer set based on known
sequence.
[0061] According to the above-described differentiation inducing
method, as will be described in Examples hereinafter, the
functional hepatocytes of the sixth invention having function level
equivalent to that of human normal hepatocytes in vivo or in
primary culture can be obtained, typically expressing sufficient
amount of albumin and having sufficient cytochrome P450 activity.
The functional human hepatocytes obtained by the above
differentiation induction can be replaced, for example, by
subcultured cells obtained by a similar method for preparing the
immunogenic human hepatocytes in Example 1 (hepatocytes which can
be subcultured while forming colonies). In such subcultured cells,
non-proliferative cells may be contained.
[0062] The human hepatocytes kit (the seventh invention), which can
be used for drug metabolizing tests and safety tests, and the
hybrid artificial liver (the eighth invention) are provided by
using the functional human hepatocytes of the sixth invention
obtained by the above methods.
[0063] Various hepatocytes kits are known depending on types of
cells and applications. Those skilled in the art can easily prepare
the cell kit of the seventh invention by employing a constitution
of the human hepatocytes of the sixth invention and a known cell
kit. Further, a constitution of a module type or a hybrid type
artificial organ is known, and those skilled in the art can easily
prepare the artificial liver of the eighth invention.
EXAMPLES
[0064] The present invention in this application is explained by
describing Examples in detail and concretely, however, the present
invention is by no means limited thereto.
Example 1
Preparation of a Monoclonal Antibody Recognizing Human
Proliferative Hepatocytes
1. Culture of Human Hepatocytes
[0065] A cell suspension was obtained from human liver tissue by a
collagenase perfusion method. The cell suspension was centrifuged
in low speed (50 g, 2 min.). The precipitated fraction was
co-cultured with mitomycin C treated Swiss 3T3 cells using
Dulbecco's modified Eagle's medium (DMEM) added with fetal calf
serum, human serum, EGF, nicotinamide and long-acting vitamin C
derivative. Swiss 3T3 cells were added every ten days. Human
hepatocyte colonies were observed after about 7 days of culture.
Confluent growth hepatocytes were subcultured by using
EDTA/Trypsin. Hepatocytes of children could be subcultured for 6-9
passages, however, hepatocytes of patients, not younger than 60
years old, could only be subcultured for 3-4 passages (FIG. 1).
Hepatocytes of the child (12 years old) exhibiting the highest
proliferation ability were used as an antigen (FIG. 2).
2. Immunization of Animals
[0066] The hepatocytes of the child (12 years old) subcultured for
3-5 passages by the above-described method were proliferated on
culture dishes. After confluent growth cells (about
1.times.10.sup.7 cells) were washed with PBS (a phosphate buffer
salt solution), PBS was removed and the cells were recovered by
scraping with a cell scraper to suspend in about 1 ml PBS. The
suspension was administered intraperitoneally in Balb/c mice, 6
weeks old. Immunization was further performed by a similar method
after 20 days or 30 days.
3. Cell Fusion
[0067] After twice immunization, increased antibody titer was
observed. After 72 hours of the third immunization (boost), a
spleen was extracted from an immunized animal to collect spleen
cells. These spleen cells and mouse myeloma cells (Name of cell:
NS-1) were fused, plating into 372 wells of a 96-well plate and
cultured.
4. Screening of Hybridoma
Primary Screening (ELISA, Tissue Staining)
[0068] Reactivity of culture supernatant of the fused cells thus
obtained to an antigen was assayed by ELISA. Assay was performed by
the following method. The culture supernatant was treated with the
subcultured hepatocytes, which were obtained by seeding the
subcultured hepatocytes used, as an antigen, on the 96-well plate,
followed by washing with PBS after cultivation, drying and storing
at -80.degree. C. An enzyme-labeled anti-mouse IgG antibody or an
anti-mouse IgM antibody was then reacted therewith, followed by
developing color by adding substrate to measure optical absorption.
As the results, mean absorbance of 372 samples of the fused cell
was 0.149 (SD: 0.099), and samples with absorbance of not lower
than 0.20 (81 samples, about 20%) were defined as positive samples.
Since color development was confirmed in naked eye observation in
samples with absorbance of not lower than 0.15, samples with the
absorbance of from 0.15 to 0.20 (46 samples) was treated with
tissue staining to confirm reactivity. Among them, 13 samples
showing interesting staining pattern were judged as positive
samples. Selected 94 positive samples were further cultured in
large scale, and culture supernatant was collected and cells were
cryopreserved.
5. Secondary Screening (ELISA, Tissue Staining)
[0069] From 94 samples selected by the primary screening, 88
positive samples were selected by measuring reactivity against an
antigen in culture supernatant after the large scale cultivation,
by ELISA similarly as in the primary screening. Reactivity in
tissue of these samples was studied by tissue staining. Samples
containing hybridoma, which specifically reacts with cell membrane
of hepatocytes and hepatocytes in portal region, or supernatant of
the clone obtained by cloning therefrom, were studied on reactivity
against freshly isolated human hepatocytes.
[0070] Supernatant No. 23 of cultured hybridoma, in which the
hepatocyte membrane of the portal region was stained in a
histological test, was analyzed on reactivity at cell surface of
freshly isolated hepatocyte, by using FACS (fluorescence activated
cell sorting). Hepatocytes of adult males, 46 years old and 49
years old, obtained by a collagenase perfusion method under low
speed centrifugation, were treated with culture supernatant of this
sample at 4.degree. C. for 30 minutes, and an FITC labeled
anti-mouse IgG antibody was then treated at 4.degree. C. for 30
minutes to make detection by FACS possible. As the result, a part
of cells (1-2%) in a hepatocyte population reacted with the sample
(FIG. 4). The reacted cell population, designated as R2 fraction,
and the non-reacted cell population, designated as R3 fraction,
were sorted and cultured. Hepatocytes before fractionation were
also cultured. As the result, colony formation was observed on
culture of about 7 days in the hepatocytes before fractionation as
described hereinbefore. On the other hand, colony-forming cells
were not observed in the R3 fraction, but large numbers of colony
were observed in the R2 fraction reacted with No. 23 (FIG. 5).
Reactivity with the subcultured human hepatocytes was examined by
FACS, and about 80% of the cells were found to be positive (FIG.
6). Namely, it was considered that among the subcultured human
hepatocytes, the differentiated cells during culturing process were
not recognized and only the proliferative hepatocytes were
recognized. From these results, No. 23 was suggested to contain
hybridoma which specifically recognized colony-forming cells.
Clones obtained by cloning from No. 23 sample were analyzed by
using FACS on reactivity at cell surface of freshly isolated
hepatocytes. As the result, three clones showing similar reactivity
were obtained. Among these clones, a clone (Mouse-Mouse hybridoma
K8233) was deposited in The International Patent Organism
Depositary, National Institute of Advanced Industrial Science and
Technology (Deposition No. FERM P-18752) on Mar. 6, 2002, and was
subjected to international deposition on Mar. 20, 2002 (Deposition
No. FERM BP-8334).
6. Preparation of a Monoclonal Antibody
[0071] The above hybridoma cell (K8223 strain) was cultured,
followed by intraperitoneal injection in mice and collection of a
monoclonal antibody from ascites to obtain a monoclonal antibody
which specifically binds to proliferative human hepatocytes.
Example 2
Isolation of Proliferative Human Hepatocytes
[0072] Using the monoclonal antibody obtained in Example 1,
proliferative human hepatocytes were separated from the human
hepatocyte population which was isolated from human liver tissue by
a collagenase perfusion method. Concretely, said cells obtained
from the human liver tissue by collagenase perfusion following low
speed centrifugation were treated with the monoclonal antibody
obtained in Example 1 at 4.degree. C. for 30 minutes and an FITC
labeled anti-mouse IgG antibody was treated at 4.degree. C. for 30
minutes to detect reaction by FACS. The cell population reacted
with this antibody, designated as R2 fraction, and the non-reacted
cell population, designated as R3 fraction, were sorted and
cultured similarly as in Example 1. The hepatocytes before sorting
were also cultured. As the result, colony formation was observed on
culture of about 7 days in the hepatocytes before fractionation. On
the other hand, colony-forming cells were not observed in the R3
fraction, but large numbers of colony were observed in the R2
fraction reacted with this monoclonal antibody. From the above
results, it was confirmed that proliferative hepatocytes could
efficiently be isolated from a isolated hepatocyte population,
using the method of the present invention.
Example 3
Differentiation Induction of Proliferative Human Hepatocytes by
Spheroid Formation
1. Spheroid Formation of Hepatocytes
[0073] The proliferative human hepatocytes obtained in Example 2
were seeded, 1.times.10.sup.5 cells/cm.sup.2, on matrigel
(MATRIGEL.TM. Matrix, Becton, Dickinson and Co.) and cultured in
DMEM medium added with fetal calf serum, EGF and long-acting
vitamin C. The hepatocytes formed spheroid on the matrigel at 1 day
of culture (FIG. 7, upper column).
2. Analysis of Human P450 Gene Expression
[0074] After seeding the cultured human hepatocytes on matrigel,
the spheroid was recovered after 7 days (FIG. 7, lower column).
Total RNA was extracted from the spheroidal hepatocytes, and cDNA
was synthesized by reverse transcription reaction. Primers
corresponding to each gene cDNA of six types of human P450
molecular species (CYP1A1, 1A2, 1C9, 2C19, 2D6 and 3A4) were
synthesized, and expression amount of each mRNA was measured
quantitatively by using PRISM 7700 Sequence Detector (ABI
PRISM.TM., Applied Biosystems Co.). P450 gene expression was also
quantitatively measured in the hepatocytes freshly isolated from a
human liver and monolayer cultured proliferative human
hepatocytes.
[0075] As the result, it was confirmed that spheroid cultured
hepatocytes exhibited higher gene expression of each human P450
than monolayer cultured hepatocytes. Some human P450 genes
exhibited P450 gene expression level equivalent to that of human
normal hepatocytes freshly isolated (FIG. 8).
3. Analysis of Albumin Secretion
[0076] Each supernatant of the spheroid cultured hepatocytes and
the monolayer cultured hepatocytes was collected on every 3 days
and human albumin concentration in the culture medium was assayed
by using Quantitative ELISA immunoassay (Bethyl Laboratories
Inc.).
[0077] As the result, the hepatocytes by spheroidal culture
exhibited higher albumin secretion at culture period not shorter
than 6 days as compared hepatocytes by monolayer culture (FIG.
9).
4. Analysis of P450 Enzyme Activity
[0078] On 22 days of the spheroid culture, lidocaine hydrochloride
(500 .mu.g/ml) was added to the culture medium and incubated for 24
hours. The medium was collected and amount of MEGX, a metabolite of
lidocaine, in the medium was assayed by HPLC. Similar assay was
performed on monolayer cultured hepatocytes.
[0079] As the result, MEGX was not detected in the medium of the
monolayer cultured hepatocytes, but MEGX was detected in the medium
of the spheroid cultured hepatocytes (8.3 .mu.g/ml/24 hours, 1.7
.mu.g/10.sup.5 cells/24 hours). From this result, it was confirmed
that the spheroid cultured hepatocytes have P450 enzyme activity
(chemical substance metabolizing enzyme activity).
Example 4
Differentiation Induction of Proliferative Human Hepatocytes by
HNF4 Gene Transfer
1. Gene Transfer
[0080] The proliferative human hepatocytes obtained in Example 2
was infected with Human HNF4.alpha. cDNA (GenBank/X87870) through
an adenovirus vector (Qbiogene, Inc.) with a ratio of multiplicity
of infection (MOI) 0, 1, 5, 10, 20, 50 and 100 on 1 day of the
culture.
2. Analysis of Human P450 Gene Expression
[0081] Cells on 7 days after the adenovirus vector infection were
collected, and expression of each human P450 gene was assayed
quantitatively similarly as in Example 3-2.
[0082] As the result, increase in the expression of human P450 gene
was recognized with infective dose dependent manner (FIG. 10).
INDUSTRIAL APPLICABILITY
[0083] As explained in detail hereinabove, the present invention
provides a monoclonal antibody which specifically recognizes
proliferative human hepatocytes, proliferative human hepatocytes
isolated by use of the antibody, and functional human hepatocytes
obtained by differentiation induction of the cells.
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