Note: Descriptions are shown in the official language in which they were submitted.
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I
IN VITRO MICRO-ORGANS, AND USES RELATED THERETO
Background of the Invention
Eukaryotic cell culture was first achieved in the early 1950s. Since that
time, a
wide range of transformed and primary cells have been cultivated using a wide
variety
of media and defined supplements, such as growth factors and hormones, as well
as
undefined supplements, such as sera and other bodily extracts. For example,
fibroblasts
obtained from the skin of an animal can be routinely cultivated through many
cell
to generations as karyotypically diploid cells or indefinitely as established
cell lines.
Epithelial cells, however, have morphological and proliferative properties
that differ
from fibroblasts and are more difficult to cultivate. Moreover, when
epithelial cells and
fibroblasts are grown in the same culture, the epithelial cells are commonly
overgrown
by the fibroblasts.
While the growth of cells in two dimensions is a convenient method for
preparing, observing and studying cells in culture, allowing a high rate of
cell
proliferation, it lacks the cell-cell and cell-matrix interactions
characteristic of whole
tissue in vivo.
In order to study such functional and morphological interactions, a few
investigators have explored the use of three-dimensional substrates such as
collagen gel
(Douglas et al., (1980) In Vitro 16:306-312; Yang et al., (1979) Proc. Natl.
Acad. Sci.
76:3401; Yang et al. (1980) Proc. Natl. Acad. Sci. 77:2088-2092; Yang et al.,
(1981)
Cancer Res. 41:1021-1027); cellulose sponge, alone (Leighton et al., (1951) J.
Natl
Cancer Inst. 12:545-561) or collagen coated (Leighton et al., (1968) Cancer
Res.
28:286-296); a gelatin sponge, Gelfoam (Sorour et al., (1975) J. Neurosurg.
43:742-749).
For growing epithelial cells in a clonally competent manner, a variety of
culture
conditions have been employed. For example, epithelial cells, and in
particular, skin
epithelial cells (keratinocytes), have been cultivated on feeder layers of
lethally
irradiated fibroblasts (Rheinhardt et al. (1975) Cell 6:331-343) and on semi-
synthetic
collagen matrices (U.S. Patent No. 5,282,859; European Patent Application No.
0361957). In some cases, the media used to grow such cells is manipulated by
adding
biological extracts, including pituitary extracts and sera, and growth
supplements, such
as epidermal growth factor and insulin (Boisseau et al. (1992) J. Dermatol.
Sci
3(2):111-120; U.S. Patent No. 5,292,655).
Numerous attempts at growing skin in vitro have been undertaken. These
attempts typically include the step of separating the keratinocytes in the
epidermis from
fibroblasts and fat cells in the dermis. After separation, the keratinocytes
are generally
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grown in a manner that permits the formation of a stratified epidermis. The
epidermis
prepared in this manner, however, lacks hair follicles and sweat glands.
Moreover, in
such cultures, the natural relationship between the epidermis and the dermis
is not
preserved. Cultivation methods including growing keratinocytes on non-viable
fibroblasts (Rheinwald et al. (1975) Cell 6:331-343 or placing keratinocytes
on a dermal
substrate of collagen and fibroblasts that is synthetic or has been derived
from an
alternative source from that of the epidermis (Sugihara et al. (1991) Cell.
Dev. Biol.
27:142-146; Parenteau et al. (1991) J. Cell Biochem. 45(3):245-251) have also
been
undertaken. In some cases, however, separation of keratinocytes is not
performed and
the whole organ is placed in culture. Attempts to cultivate organs in vitro
have been
limited to incubating organs in a serum-containing medium (Li et al. (1991)
Proc. Natl.
Acad. Sci. 88(5):108-112).
Most existing in vitro models of the epidermis lack hair follicles, sweat
glands
and sebaceous glands (for a view of epidermal cell culture, see Coulomb et al.
(1992)
Pathol. Biol. Paris 40(2):139-146). Exceptions include the gel-supported skin
model of
Li et al. ((1992) Proc. Natl. Acad. Sci 89:8764-8768) in which skin explants
with
dimensions of 2 x Smmz and 2.0 mm thick remained viable for several days in
the
presence of serum-containing media.
In addition to the drawbacks of cell damage, bio-reactors and other methods of
culturing mammalian cells are also very limited in their ability to provide
conditions
which allow cells to assemble into tissues which simulate the spatial three-
dimensional
form of actual tissues in the intact organism. Conventional tissue culture
processes limit,
for similar reasons, the capacity for cultured tissues to express a highly
functionally
specialized or differentiated state considered crucial for mammalian cell
differentiation
and secretion of specialized biologically active molecules of research and
pharmaceutical interest. Unlike microorganisms, the cells of higher organisms
such as
mammals form themselves into high order multicellular tissues. Although the
exact
mechanisms of this self assembly are not known, in the cases that have been
studied
thus far, development of cells into tissues has been found to be dependent on
orientation
of the cells with respect to each other (the same or different type of cell)
or other
anchorage substrate and/or the presence or absence of certain substances
(factors) such
as hormones, autocrines, or paracrines. In summary no conventional culture
process is
capable of simultaneously achieving sufficiently low shear stress, sufficient
3-dimensional spatial freedom, and sufficiently long periods for critical cell
interactions
(with each other or substrates) to allow excellent modeling of in vivo tissue
structure.
There is a need, therefore, for in vitro methods of generating and maintaining
portions of organs in cultures in which the cells of the culture preserve
their natural
intercellular relationships for extended periods of time. The availability of
tissue and
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organ models in which cell differentiation, cell proliferation, and cell
function mimics
that found in the whole organ in vivo would have utility in understanding the
mechanisms by which organs are maintained in a healthy state and consequently
how
abnormal events may be reversed.
Summary of the Invention
The present invention provides an in-vitro micro-organ culture which addresses
the above-cited needs. Salient features of the subject micro-organ cultures
include the
to ability to be maintained in culture for relatively long periods of time,
e.g., at least about
twenty four hours, preferably for at least seven days or longer, as well as
the
preservation of an organ microarchitecture which facilitates, for example,
cell-cell and
cell-matrix interactions analogous to those occurring in the source organ.
Typically, at least one cell of the population of cells of the micro-organ
culture
has the ability to proliferate. The population of cells in the micro-organ
culture can,
overall, be in a state of equilibrium, i.e., the ratio of cell proliferation
to cell loss in the
population of cells is approximately one, or the cells in the micro-organ
culture can be
proliferating at a greater rate than they are lost, resulting in a ratio of
cell proliferation to
cell loss in the population of cells which is greater than one, e.g., as in a
population of
cells obtained from neoplastic tissue, or, e.g., a progenitor cell population
induced to
proliferate in an explant.
Preferred organs from which the cells of the micro-organ culture can be
isolated
include lymphoid organs, e.g., thymus and spleen; digestive tract organs,
e.g., gut, liver,
pancreas, gallbladder and bile duct; lung; reproductive organs, e.g., prostate
and uterus;
zs breast, e.g., mammary gland; skin; urinary tract organs, e.g., bladder and
kidney; cornea;
and blood-associated organs such as bone marrow. The isolated population of
cells of
the micro-organ culture can, in certain embodiments, be encapsulated within
polymeric
devices, e.g., for delivery of the cells or cell products, e.g., gene
products, to a subject.
The present invention also pertains to conditioned medium isolated from the
micro-organ cultures of the present invention.
In one embodiment of the present invention, the micro-organ culture includes a
population of cells which is a section of an organ. Preferably, the micro-
organ explant
includes epithelial and connective tissue cells. In one embodiment of the
invention, the
organ explant is obtained from a pancreas, e.g., the microarchitecture of the
population
of cells is substantially the same as the microarchitecture of the original
pancreas from
which the explant was derived, and includes pancreatic epithelial cells, e.g.,
islet cells,
and pancreatic connective tissue cells.
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In another embodiment of the invention, the micro-organ explant is obtained
from skin, e.g., microarchitecure of the population cells is substantially the
same as the
microarchitecture of skin in vivo, and includes skin epithelial, e.g.,
epidermal cells, and
skin connective tissue cells, e.g., dermal cells. The micro-organ culture
which is
obtained from a skin explant can also include a basal lamina supporting the
epidermal
cells, an extracellular matrix which includes the dermal cells, and at least
one
invagination, e.g., at least one hair follicle or gland.
In another embodiment of the present invention, the micro-organ culture
includes an isolated population of cells infected with a virus, such as a
hepatitis virus,
e.g., hepatitis B or hepatitis C, or a human papilloma virus (HPV), e.g., HPV-
6, HPV-8,
or HPV-33. When infected with a virus, the micro-organ culture can be used in
a
method for identifying an inhibitor of viral infectivity. This method includes
isolating a
micro-organ explant according to the method of the present invention, which
explant is
derived from a virally-infected organ, or is subsequently infected in vitro
with a virus to
t5 produce a population of virus-infected cells in the explant. The explant
can then be
contacted with a candidate agent, e.g., agent which is being tested for anti-
viral activity,
and the level of infectivity (e.g., viral loading, new infectivity, etc) in
the presence of the
candidate agent is measured and compared to the level of infectivity by the
virus in the
absence of the candidate agent. A decrease in the level of infectivity of the
virus in the
presence of the candidate agent is indicative of an inhibitor of viral
infectivity.
The present invention also pertains to a method for producing a micro-organ
culture. This method includes isolating, from a mammalian donor subject, a
micro-organ
explant having dimensions which provide the isolated population of cells as
maintainable in a minimal medium for at least about twenty-four hours. The
micro-organ explant is then placed in culture. Typically, the explant includes
an isolated
population of cells having a microarchitecture of the organ from which the
explant is
isolated. In one embodiment of the present invention, at least one cell of the
explant has
the ability to proliferate. The cells of the subject micro-organ culture can
be in a state of
equilibrium, i.e., the ratio of cell proliferation to cell loss in the
population of cells is
one, or the cells in the micro-organ culture can be proliferating at a greater
rate than they
are lost resulting in a ratio of cell proliferation to cell loss in the
population of cell loss
in the population of cells which is greater than one, e.g., the micro-organ
explant
includes a population of cells obtained from neoplastic tissue.
Preferred organs from which the cells of the micro-organ culture can be
isolated
include lymphoid organs, e.g., thymus and spleen; digestive tract organs,
e.g., gut, liver,
pancreas, gallbladder and bile duct; lung; reproductive organs, e.g., prostate
and uterus;
breast; skin; urinary tract organs, e.g., bladder; kidney; cornea; and blood-
associated
organs such as bone-marrow. In each of these examples, the microarchitecture
of the
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organ is maintained by the cultured explant. The micro-organ culture can be a
tissue
section, e.g., a pancreatic tissue section which includes (3-islet cells,
e.g., a skin tissue
section which includes epidermal and dermal cells and other skin-specific
architectural
features, e.g., hair follicles.
5 Cells in the micro-organ explants can also be modified to express a
recombinant
protein, which protein may or may not be normally expressed by the organ from
which
the explant is derived. For example, gene products normally produced by the
pancreas,
and which can be augmented by the subject transgenic method, e.g., to correct
a
deficiency, include insulin, amylase, protease, lipase, trypsinogen,
chymotrypsinogen,
carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase,
phospholipase AZ, elastase, and amylase; likewise, gene products normally
produced by
the liver, and which can be complemented by replacement gene therapy, include
blood
clotting factors, such as blood clotting Factor VIII and Factor IX, UDP
glucuronyl
transferase, ornithine .transcarbamoylase, and cytochrome p450 enzymes; gene
products
normally produced by thymus include serum thymic factor, thymic humoral
factor,
thymopoietin and thymosin al.
The micro-organ culture of the present invention can be used in a method for
delivering a gene product to a recipient subject. This method includes
providing an
isolated population of cells from a donor subject, the population of cells
having a
microarchitecture of an organ or tissue from which the cells are isolated and
a surface
area to volume which provides the isolated population of cells as maintainable
in a
minimal medium for at least about twenty-four hours. A recombinant nucleic
acid which
encodes and directs expression of a desired gene product can then be
introduced into the
population of cells to produce a population of transgenic cells in the micro-
organ
explant, e.g., a transgenic explant. The transgenic explant can be
administered to a
recipient subject. The donor subject and the recipient subject can be of the
same species
or of different species.
The micro-organ culture of the present invention can also be used in a method
for identifying agents which induce proliferation of cells of a given organ,
including
progenitor cells. This method includes generating a micro-organ explant
culture
according to the present invention, which explant includes at least one cell
which has
the ability to proliferate. After being placed in culture, the explant is
contacted with a
candidate compound, e.g., a compound to be tested for cell proliferative
capacity, and
the level of cell proliferation in the presence of the candidate compound is
measured.
The measured level of cell proliferation in the presence of the candidate
compound is
then compared to the level of cell proliferation in the absence of the
candidate
compound. An increase in the level of cell proliferation in the presence of
the candidate
compound is indicative of a cell proliferative agent. Inhibitors of cell
proliferation can
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be identified using a similar method. Specifically, when the measured level of
cell
proliferation in the presence of the candidate compound is determined using
the
above-described method, it can be compared to the level of cell proliferation
in the
absence of the candidate compound. A decrease in the level of cell
proliferation in the
presence of the candidate compound is indicative of an inhibitor of cell
proliferation.
Another method in which the micro-organ culture of the present invention can
be
used is in a method for identifying an agent which induces, or inhibits,
differentiation of
one or more cell types in a given organ, or an agent which maintains a
particular
differentiated state (prevent dedifferentiation). This method includes
generating a
micro-organ explant from the organ of interest, the population of cells making
up the
explant having a microarchitecture of that organ, as described hereonbelow,
aleph of at
least about 1.5 mm -~, and including at least one cell which has the ability
to
differentiate or is differentiated and has the ability to dedifferentiate.
Once in culture,
the population of cells is contacted with a candidate compound and the level
of cell
t5 differentiation in the presence of this compound is measured. The measured
level of cell
differentiation in the presence of the candidate compound is compared with the
level of
cell differentiation in the absence of the candidate compound. An increase in
the level of
cell differentiation in the presence of candidate compound is indicative of
cell
differentiating agent. Inhibitors of cell differentiation can be identified
using a similar
2o method. In particular, when the measured level of cell differentiation in
the presence of
the candidate compound is determined using the above-described method, it can
be
compared to the level of cell differentiation in the absence of the candidate
compound.
A decrease in the level of cell differentiation in the presence of the
candidate compound
is indicative of an inhibitor of cell differentiation.
25 Yet another aspect of the present invention provides a method for
identifying,
and isolating, stem cell or progenitor cell populations from an organ. This
method
generally provides isolating, in a culture, an explant of a population of
cells from an
organ. As described herein, the explant is characterized by (i) maintenance,
in the
culture, of a microarchitecture of the organ from which the explant is
derived, (ii) a
3o surface area to volume index (aleph) of at least about 1.55 mrri ~, and
(iii) at least one
progenitor or stem cell which has the ability to proliferate. The explant is
contacted
with an agent which induces proliferation of the progenitor or stem cell,
e.g., a growth
factor or other mitogen, in order to amplify discrete populations of cells in
the explant.
Subsequently, the amplified progenitor cells can be isolated from the explant.
Such
35 sub-populations of the explant can be identified by virtue of their
proliferative response.
In other embodiments, the progenitor/stem cells will proliferate spontaneously
in the
culture even without addition of an exogenous agent. In other embodiment,
progenitor
or stem cells from the explant that proliferate in response to the agent can
be isolated,
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such as by direct mechanical separation of newly emerging buds from the rest
of the
explant or by dissolution of all or a portion of the explant and subsequent
isolation of
the amplified cell population.
Still another method in which the micro-organ culture of the present invention
can be used is in a method for promoting wound healing in a recipient subject.
This
method includes isolating, from a donor subject, a population of cells having
an aleph of
at least approximately 1.5 mm-~ and applying the population of cells to a
wound of the
recipient subject. The donor subject and the recipient subject can be of the
same species
or of different species. In one embodiment, the tissue from which the cells
are isolated is
to skin and the wound of the recipient subject is an ulcer, e.g., an ulcer
associated with
diabetes.
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of cell biology, cell culture, molecular biology,
transgenic
biology, microbiology, recombinant DNA, and immunology, which are within the
skill
~s of the art. Such techniques are explained fully in the literature. See, for
example,
Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and
Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I
and II
(D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);
Mullis et al.
U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.
Higgins
20 eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984);
Culture of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., N.Y.); Gene
Transfer
Vectors for Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold
Spring
Harbor Laboratory); Methods in Enzymology, Vols. 154 and 155 (Wu et al. eds.),
lmmunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds.,
25 Academic Press, London, 1987); and Handbook of Experimental Immunology,
Volumes
I-IV (D.M. Weir and C.C. Blackwell, eds., 1986).
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
3o Brief Description of the Drawings
Figure 1 is a diagrammatic representation of a micro-organ depicting the
dimensions that determine Aleph where x = thickness and a = width of tissue.
Figure 2 is a histogram showing cell proliferation in a guinea pig micro-organ
35 culture as determined by BrdU labeling after incubation for different time
periods.
Figure 3 is a histogram showing cell proliferation in a human back skin
micro-organ culture as determined by BrdU labeling after incubation of
cultures for 1-8
days.
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Figures 4A-4D are micrographs showing immunofluorescence corresponding to
replicating cells of mouse skin (mag. 50x) (Figure 4A), guinea pig skin (mag.
75x)
(Figure 4B) human foreskin (mag. 50x) (Figure 4C) and human foreskin (mag.
75x)
(Figure 4D).
Figures 5A-5C are transverse sections of human epidermal micro-organ
explants. (mag x75) showing tissue architecture at zero (Figure 5A), three
(Figure 5B)
and six (Figure 6D) days in culture.
Figure 6 is a histogram demonstrating the effect on epidermal proliferation of
varying thickness (x) of guinea pig skin micro-organ cultures using BrdU
incorporation
to where (a) has been kept constant at 4mm.
Figure 7A-7B are micrographs showing immunofluorescence corresponding to
proliferating cells in pancreas-derived micro-organ cultures (mag 75x).
Figure 8 is a histogram showing amounts of insulin released by adult pig
pancreas micro-organ cultures.
Figure 9 is a histogram showing 3H-Thymidine incorporation in proliferating
cells in micro-organ cultures of the colon, liver, kidney, duodenum and
esophagus, at
three days, four days and six days of culture.
Figures l0A-IOC are micrographs showing active proliferation of hair follicles
in micro-organ cultures as determined by immunofluorescence. Magnification 40x
(Figure I OA), 40x (Figure I OB), and 75x (Figure lOC).
Figure II is a histogram showing the size distribution of hair shafts at the
beginning and end of the microculture.
Figure 12 is a histogram showing the inhibition of mitogenesis in micro-organ
cultures in the presence of 2.5 ng/ml TGF-~i in guinea-pig skin cultures.
Figure 13 is a diagrammatic representation of a micro-organ explant for
treatment of chronic skin ulcers showing incomplete sectioning of tissue
slices so as to
maintain a structure that can be readily manipulated in vivo.
Figure 14 is a photograph of the surface of a mouse after replacement of a
piece
of normal skin with a micro-organ culture; healing, generation of new hair
shafts in the
3o implant, and incorporation of the implant into the normal mouse skin can be
observed
(mag 10x).
Figure 15 is a graphic representation of the expression of a luciferase
reporter
gene in a guinea pig skin micro-organ culture after transfection ( of the
culture with a
plasmid encoding the luciferase reporter gene.
Figure 16 is a graphic representation of the expression of a luciferase gene
in rat
lung and thymus micro-organ cultures after cationic lipid mediated
transfeetion of the
culture with plasmid encoding the luciferase reporter gene.
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Figure 17 is a graphic representation of the activation of telogen follicles
upon
treatment with FGF in micro-organ cultures of the present invention.
Figure 18 is a graphic representation of the expression of a transgenic
luciferase
gene in micro-organ explants of the present invention.
Detailed Description of the Invention
The present invention is directed to a three-dimensional organ explant culture
system. This culture system can be used for the long term proliferation of
micro-organ
l0 explants in vitro in an environment that closely approximates that found in
the whole
organ in vivo. The culture system described herein provides for proliferation
and
appropriate cell maturation to maintain structures analogous to organ
counterparts in
VIVO.
The micro-organ cultures of the present invention provide in vitro culture
systems in which tissue or organ sections can be maintained and their function
preserved
for extended periods of time. These culture systems provide in vitro models in
which
cell differentiation, cell proliferation, cell function, and methods of
altering such cell
characteristics and functions can be conveniently and accurately tested. The
resulting
cultures have a variety of applications ranging from transplantation or
implantation in
2o vivo, to screening cytotoxic compounds and pharmaceutical compounds in
vitro, to the
production of biologically active molecules in "bioreactors", and to isolating
progenitor
cells from a tissue.
For example, and not by way of limitation, specific embodiments of the
invention include (i) micro-organ bone marrow culture implants used to replace
bone
marrow destroyed during chemotherapeutic treatment; (ii) micro-organ liver
implants
used to augment liver function in cirrhosis patients; (iii) genetically
altered cells grown
in the subject micro-organ culture (such as pancreatic micro-organs which
express a
recombinant gene encoding insulin); and (iv) dental prostheses joined to a
micro-organ
culture of oral mucosa.
In yet other illustrative non-limiting embodiments, the subject micro-organ
cultures may be used in vitro to screen a wide variety of compounds, such as
cytotoxic
compounds, growth/regulatory factors, pharmaceutical agents, etc. To this end,
the
micro-organ cultures are maintained in vitro and exposed to the compound to be
tested.
The activity of cytotoxic compound can be measured, for example, by its
ability to
damage or kill cells in the explant.
This may readily be assessed by vital staining techniques. The effect of
growth/regulatory factors may be assessed by analyzing the cellular content of
the
explant, e.g., by total cell counts, and differential cell counts. This may be
accomplished
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using standard cytological and/or histological techniques including the use of
immunocytochemical techniques employing antibodies that define type-specific
cellular
antigens. The effect of various drugs on normal cells cultured in the three-
dimensional
system may be assessed. For example, drugs that increase red blood cell
formation can
5 be tested on the bone marrow micro-organ cultures. Drugs that affect
cholesterol
metabolism, e.g., by lowering cholesterol production, could be tested on the
liver
micro-organs. Micro-organ cultures of abnormal tissue can also be employed,
such as to
facilitate study of hyperproliferative or neoproliferative disorders. For
instance,
micro-organ explants of organs invaded by tumor cell growth may be used as
model
to systems to test, for example, the efficacy of anti-tumor agents.
For convenience, certain terms employed in the specification, examples, and
appended claims are collected here.
The term "explant" refers to a collection of cells from an organ, taken from
the
body and grown in an artificial medium. When referring to explants from an
organ
having both stromal and epithelial components, the term generally refers to
explants
which contain both components in a single explant from that organ.
The term "tissue" refers to a group or layer of similarly specialized cells
which
together perform certain special functions.
The term "organ" refers to two or more adjacent layers of tissue, which layers
of
tissue maintain some form of cell-cell and/or cell-matrix interaction to
generate a
microarchitecture. In the present invention, micro-organ cultures were
prepared from
such organs as, for example, mammalian skin, mammalian pancreas, liver,
kidney,
duodenum, esophagus, bladder, cornea, prostrate, bone marrow, thymus and
spleen.
The term "stroma" refers to the supporting tissue or matrix of an organ.
The term "micro-organ culture" as used herein refers to an isolated population
of
cells, e.g., an explant, having a microarchitecture of an organ or tissue from
which the
cells are isolated. That is, the isolated cells together form a three
dimensional structure
which simulates/retains the spatial interactions, e.g. cell-cell, cell-matrix
and
cell-stromal interactions, and the orientation of actual tissues and the
intact organism
from which the explant was derived. Accordingly, such interactions as between
stromal
and epithelial layers is preserved in the explanted tissue such that critical
cell
interactions provide, for example, autocrine and paracrine factors and other
extracellular
stimuli which maintain the biological function of the explant, and provide
long term
viability under conditions wherein adequate nutrient and waste transport
occurs
throughout the sample.
The subject micro-organ cultures have a microarchitecture of an organ or
tissue
from which the cells or tissue explant are isolated. As used herein, the term
"microarchitecture" refers to an isolated population of cells or a tissue
explant in which
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at least about 50%, preferably at least about 60%, more preferably at least
about 70%,
still more preferably at least about 80 %, and most preferably at least about
90% or more
of the cells of the population maintain, in vitro, their physical and/or
functional contact
with at least one cell or non cellular substance with which they are in
physical and/or
functional contact in vivo and form a cell culture of at least about one, more
preferably
at least about five, and most preferably at least about ten layers or more.
Preferably, the
cells of the explant maintain at least one biological activity of the organ or
tissue from
which they are isolated.
The term "isolated" as used herein refers to an explant which has been
separated
l0 from its natural environment in an organism. This term includes gross
physical
separation from its natural environment, e.g., removal from the donor animals,
e.g., a
mammal such as a human or a miniature swine. For example, the term "isolated"
refers
to a population of cells which is an explant, is cultured as part of an
explant, or is
transplanted in the form of an explant. When used to refer to a population of
cells, the
term " isolated" includes population of cells which result from proliferation
of cells in
the micro-organ culture of the invention.
The term "ectoderm" refers to the outermost of the three primitive germ layers
of
the embryo; from it are derived the epidermis and epidermal tissues such as
the nails,
hair and glands of the skin, the nervous system, external sense organs and
mucous
2o membrane of the mouth and anus.
The terms "epithelia" and "epithelium" refer to the cellular covering of
internal
and external body surfaces (cutaneous, mucous and serous), including the
glands and
other structures derived therefrom, e.g., corneal, esophageal, epidermal and
hair follicle
epithelial cells. Other exemplary epithelial tissues include: olfactory
epithelium, which
is the pseudostratified epithelium lining the olfactory region of the nasal
cavity, and
containing the receptors for the sense of smell; glandular epithelium, which
refers to
epithelium composed of secreting cells; squamous epithelium, which refers to
epithelium composed of flattened plate-like cells. The term epithelium can
also refer to
transitional epithelium, which is that characteristically found lining hollow
organs that
3o are subject to great mechanical change due to contraction and distention,
e.g. tissue
which represents a transition between stratified squamous and columnar
epithelium. The
term "epithelialization" refers to healing by the growth of epithelial tissue
over a
denuded surface.
The term "skin" refers to the outer protective covering of the body,
consisting of
the corium and the epidermis, and is understood to include sweat and sebaceous
glands,
as well as hair follicle structures. Throughout the present application, the
adjective
"cutaneous" may be used, and should be understood to refer generally to
attributes of the
skin, as appropriate to the context in which they are used.
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The term "epidermis" refers to the outermost and nonvascular layer of the
skin,
derived from the embryonic ectoderm, varying in thickness from 0.07-l.4mm. On
the
palmar and plantar surfaces it comprises, from within outward, five layers:
basal layer
composed of columnar cells arranged perpendicularly; prickle-cell or spinous
layer
composed of flattened polyhedral cells with short processes or spines;
granular layer
composed of flattened granular cells; clear layer composed of several layers
of clear,
transparent cells in which the nuclei are indistinct or absent; and horny
layer composed
of flattened, cornified non-nucleated cells. In the epidermis of the general
body surface,
the clear layer is usually absent. An "epidermoid" is a cell or tissue
resembling the
1o epidermis, but may also be used to refer to any tumor occurring in a
noncutaneous site
and formed by inclusion of epidermal elements.
The "corium" or "dermis" refers to the layer of the skin beneath deep to the
epidermis, consisting of a dense bed of vascular connective tissue, and
containing the
nerves and terminal organs of sensation. The hair roots, and sebaceous and
sweat glands
are structures of the epidermis which are deeply embedded in the dermis.
The term "gland" refers to an aggregation of cells specialized to secrete or
excrete materials not related to their ordinary metabolic needs. For example,
"sebaceous
glands" are holocrine glands in the corium that secrete an oily substance and
sebum.
The term "sweat glands" refers to glands that secrete sweat, situated in the
corium or
2o subcutaneous tissue, opening by a duct on the body surface. The ordinary or
eccrinesweat glands are distributed over most of the body surface, and promote
cooling
by evaporation of the secretion; the apocrine sweat glands empty into the
upper portion
of a hair follicle instead of directly onto the skin, and are found only in
certain body
areas, as around the anus and in the axilla.
The term "hair" (or "pilus") refers to a threadlike structure, especially the
specialized epidermal structure composed of keratin and developing from a
papilla sunk
in the corium, produced only by mammals and characteristic of that group of
animals.
The term also refers to the aggregate of such hairs. A "hair follicle" refers
to one of the
tubular-invaginations of the epidermis enclosing the hairs, and from which the
hairs
3o grow; and "hair follicle epithelial cells" refers to epithelial cells which
are surrounded by
the dermis in the hair follicle, e.g., stem cells, outer root sheath cells,
matrix cells, and
inner root sheath cells. Such cells may be normal non-malignant cells, or
transformed/immortalized cells.
The term "alopecia" refers generally to baldness, e.g., the absence of hair
from
skin areas where it is normally present. Various forms of alopecia are noted
in the art.
For instance, alopecia areata refers to hair loss, usually reversible, in
sharply defined
areas, usually involving the beard or scalp; alopecia mediacamentosa refers to
hair loss
due to ingestion of a drug; and male pattern alopecia, or male pattern
baldness, refers to
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13
loss of scalp hair genetically determined and androgen-dependent, generally
beginning
with frontal recession and progressing symmetrically to leave ultimately only
a sparse
peripheral rim of hair.
Throughout this application, the term "proliferative skin disorder" refers to
any
disease/disorder of the skin marked by unwanted or aberrant proliferation of
cutaneous
tissue. These conditions are typically characterized by epidermal cell
proliferation or
incomplete cell differentiation, and include, for example, X-linked
ichthyosis, psoriasis,
atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis
and
seborrheic dermatitis. For example, epidermodysplasia is a form of faulty
development
of the epidermis, such as "epidermodysplasia verruciformis", which is a
condition due to
a virus identical with or closely related to the virus of common warts.
Another example
is "epidermolysis", which refers to a loosened state of the epidermis with
formation of
blebs and bullae either spontaneously or at the site of trauma.
As used herein, the term "psoriasis" refers to a hyperproliferative skin
disorder
which alters the skin's regulatory mechanisms. In particular, lesions are
formed which
involve primary and secondary alterations in epidermal proliferation,
inflammatory
responses of the skin, and an expression of regulatory molecules such as
lymphokines
and inflammatory factors. Psoriatic skin is morphologically characterized by
an
increased turnover of epidermal cells, thickened epidermis, abnormal
keratinization,
zo inflammatory cells infiltrates into the dermis layer and polymorphonuclear
leukocyte
infiltration into the epidermis layer resulting in an increase in the basal
cell cycle.
Additionally, hyperkeratotic and parakeratotic cells are present.
As used herein, "proliferating" and "proliferation" refer to cells undergoing
mitosis.
The term "progenitor cell" refers to an undifferentiated cell which is capable
of
proliferation and giving rise to more progenitor cells having the ability to
generate a
large number of mother cells that can in turn give rise to differentiated, or
differentiable
daughter cells. As used herein, the term "progenitor cell" is also intended to
encompass
a cell which is sometimes referred to in the art as a "stem cell". In a
preferred
3o embodiment, the term "progenitor cell" refers to a generalized mother cell
whose
descendants (progeny) specialize, often in different directions, by
differentiation, e.g., by
acquiring completely individual characters, as occurs in progressive
diversification of
embryonic cells and tissues. For instance, a "hematopoietic progenitor cell"
(or stem
cell) refers to progenitor cells arising in bone marrow and other blood-
associated organs
and giving rise to such differentiated progeny as, for example, erythrocytes,
lymphocytes
and other blood cells.
As used herein, "transformed cells" refers to cells which have spontaneously
converted to a state of unrestrained growth, i.e., they have acquired the
ability to grow
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14
through an indefinite number of divisions in culture. Transformed cells may be
characterized by such terms as neoplastic, anaplastic and/or hyperplastic,
with respect to
their loss of growth control.
As used herein, "immortalized cells" refers to cells which have been altered
via
chemical and/or recombinant means such that the cells have the ability to grow
through
an indefinite number of divisions in culture.
The term "carcinoma" refers to a malignant new growth made up of epithelial
cells tending to infiltrate surrounding tissues and to give rise to
metastases. Exemplary
carcinomas include: "basal cell carcinoma", which is an epithelial tumor of
the skin that,
to while seldom metastasizing, has potentialities for local invasion and
destruction;
"squamous cell carcinoma", which refers to carcinomas arising from squamous
epithelium and having cuboid cells; "carcinosarcoma", which include malignant
tumors
composed of carcinomatous and sarcomatous tissues; "adenocystic carcinoma",
carcinoma marked by cylinders or bands of hyaline or mucinous stroma separated
or
surrounded by nests or cords of small epithelial cells, occurring in the
mammary and
salivary glands, and mucous gland of the respiratory tract; "epidermoid
carcinoma",
which refers to cancerous cells which tend to differentiate in the same way as
those of
the epidermis; i.e., they tend to form prickle cells and undergo
cornification;
"nasopharyngeal carcinoma", which refers to a malignant tumor arising in the
epithelial
lining of the space behind the nose; and "renal cell carcinoma", which
pertains to
carcinoma of the renal parenchyma composed of tubular cells in varying
arrangements.
Another carcinomatous epithelial growth is "papillomas", which refers to
benign tumors
derived from epithelium and having a papillomavirus as a causative agent; and
"epidermoidomas", which refers to a cerebral or meningeal tumor formed by
inclusion
of ectodermal elements at the time of closure of the neutral groove.
As used herein, a "transgenic animal" is any animal, preferably a non-human
mammal, bird or an amphibian, in which one or more of the cells of the animal
contain
heterologous nucleic acid introduced by way of human intervention, such as by
transgenic techniques well known in the art. The nucleic acid is introduced
into the cell,
3o directly or indirectly by introduction into a precursor of the cell, by way
of deliberate
genetic manipulation, such as by micro injection or by infection with a
recombinant
virus. The term genetic manipulation does not include classical cross-
breeding, or in
vitro fertilization, but rather is directed to the introduction of a
recombinant DNA
molecule. This molecule may be integrated within a chromosome, or it may be
extrachromosomally replicating DNA. This term also includes transgenic animals
in
which the recombinant gene is silent, as for example, the FLP or CRE
recombinase
dependent constructs described in the art. Transgenic animals also include
both
constitutive and conditional "knock out" animals. The "non-human animals" of
the
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invention include vertebrates such as rodents, non-human primates, swine,
sheep, dog,
cow, chickens, amphibians, reptiles, etc. Preferred non-human animals are
miniature
swine, or are selected from the rodent family including rat and mouse, most
preferably
mouse. The term "chimeric animal" is used herein to refer to animals in which
the
5 recombinant gene is found, or in which the recombinant is expressed in some
but not all
cells of the animal.
I. Establishment ofthe micro-organ culture
to A salient feature of the present micro-organ cultures and methods,
according to
the invention, is the ability to preserve the cellular microenvironment found
in vivo for
a particular tissue. The invention is based, in part, upon the discovery that
under defined
circumstances growth of cells in different tissue layers of an organ explant,
e.g.,
mesenchymal and epithelial layers, can be activated to proliferate and mature
in culture.
~ 5 Moreover, the cell-cell and cell-matrix interactions provided in the
explant itself are
sufficient to support, cellular homeostasis, e.g., maturation, differentiation
and
segregation of cells in explant culture, thereby sustaining the
microarchitecture and
function of the tissue for prolonged period of time.
An example of physical contact between a cell and a noncellular substrate
(matrix) is the physical contact between an epithelial cell and its basal
lamina. An
example of physical contact between a cell and another cell includes actual
physical
contact maintained by, for example, intercellular cell junctions such as gap
junctions
and tight junctions. Examples of functional contact between one cell and
another cell
includes electrical or chemical communication between cells. For example,
cardiomyocytes communicate with other cardiomyocytes via electrical impulses.
In
addition, many cells communicate with other cells via chemical messages, e.g.,
hormones which either diffuse locally (paracrine signaling and autocrine
signaling) or
are transported by the vascular system to more remote locations (endocrine
signaling).
Examples of paracrine signaling between cells are the messages produced by
various
cells (known as enteroendocrince cells) of the digestive tract, e.g., pyloric
D cells which
secrete somatostatin which in turn inhibits the release of gastrin by nearby
pyloric
gastric (G) cells.
Not wishing to be bound by any particular theory, this microarchitecture can
be
extremely important for the maintenance of the explant in minimal media, e.g.,
without
exogenous sources of serum or growth factors, because the tissue can be
sustained in
such minimal media by paracrine and autocrine factors resulting from specific
cellular
interactions within the explant.
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Moreover, the phrase "maintain, in vitro, their physical and/or functional
contact" is not intended to exclude an isolated population of cells in which
at least one
cell develops physical and/or functional contact with at least one cell or
noncellular
substance with which it is not in physical and/functional contact in vivo. An
example of
such a development is proliferation of at least one cell of the isolated
population of cells.
In preferred embodiments, the populations of cells which make up the explant
are isolated from an organ in a manner that preserves the natural affinity of
one cell to
another, e.g., to preserve layers of different cells if present in the
explant. For example,
in skin micro-organ cultures, keratinocytes of the epidermis remain associated
with the
to stroma and the normal tissue architecture is preserved including the hair
follicles and
glands. This basic structure is common to all organs, for instance, which
contain an
epithelial component. Moreover, such an association facilitates intercellular
communication. Many types of communication take place among animal cells. This
is
particularly important in differentiating cells where induction is defined as
the
t 5 interaction between one (inducing) and another (responding) tissue or
cell, as a result of
which the responding cells undergo a change in the direction of
differentiation.
Moreover, inductive interactions occur in embryonic and adult cells and can
act to
establish and maintain morphogenetic patterns as well as induce
differentiation (Gurdon
(1992) Cell 68: 185-199).
2o Furthermore, the micro-organ cultures prepared according to the invention
preserve normal tissue architecture even when cultured for prolonged periods
of time.
This includes the maintenance of hair follicles, sweat glands and sebaceous
glands in
skin micro-organs in vitro according to their normal occurrence in vivo (see
Examples
VIII and Figures IOA -lOC), or islets of Langerhans in the pancreas according
to the
25 normal occurrence in vivo (see Examples IV, V and VI). Because these
cultures can be
maintained in controlled and uniform conditions and yet closely resemble
tissue in vivo,
they provide a unique opportunity to observe, measure and control natural
phenomena
and the perturbation of natural phenomena arising from disease, aging or
trauma.
Furthermore, the ready availability of techniques to study individual cells at
identified
3o sites on the culture provide insights into the functioning of individual
components of the
tissue as they interact with each other as well as the whole tissue.
Examples of micro-organ cultures prepared according to the invention are
described in the appended Examples, and can include a population of cells
grouped in a
manner that may include a plurality of layers so as to preserve the natural
affinity of one
35 cell to another. The proliferation of individual cells or groups of cells
can be observed
and followed by autoradiography or immunofluorescence.
As merely further exemplification, the appended examples demonstrate that the
subject culture system provides for the replication of epithelial and stromal
elements in
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17
vitro, in a system comparable to physiologic conditions. Importantly, the
cells which
replicate in this system segregate properly to form morphologically and
histologically
normal epidermal and dermal components.
In addition to isolating an explant which retains the cell-cell, cell-matrix
and
cell-stroma architecture of the originating tissue, the dimensions of the
explant are
important to the viability of the cells therein, e.g., where the micro-organ
culture is
intended to be sustained for prolonged periods of time, e.g., 7-21 days or
longer.
Accordingly, the dimensions of the tissue explant are selected to provide
diffusion of
adequate nutrients and gases, e.g., OZ , to every cell in the three
dimensional
to micro-organ, as well as diffusion of cellular waste out of the explant so
as to minimize
cellular toxicity and concomitant death due to localization of the waste in
the
micro-organ. Accordingly, the size of the explant is determined by the
requirement for a
minimum level of accessibility to each cell in the absence specialized
delivery structures
or synthetic substrates. It has been discovered, as described herein, that
this
accessibility can be maintained if Aleph, an index calculated from the
thickness and the
width of the explant, is at least greater than approximately 1.5 mm-~
As used herein, "Aleph" refers to a surface area to volume ratio given by a
formula 1/x + 1/a > 1.5 mm~~; wherein x= tissue thickness and a= width of
tissue in
millimeters. In preferred embodiments, the aleph of an explant is in the range
of 1.5 to
25mm~~, more preferably in the range of 1.5 to 15 mm-~, and even more
preferably in
the range of 1.5 to 10 mm ~, though alephs in the range of 1.5 to 6.67 mm-~,
1.5 to 3.33
mm~~ are contemplated.
Accordingly, the present invention provides that the surface area to volume
index of the tissue explant is maintained within a selected range. This
selected range of
surface area to volume index provides the cells access to nutrients and to
avenues of
waste disposal by diffusion in a manner similar to cells in a monolayer. This
level of
accessibility can be attained and maintained if the surface area to volume
index, defined
herein as "Aleph or Aleph index" is at least about 1.5 mm ~. The third
dimension has
been ignored in determining the surface area to volume index because variation
in the
3o third dimension causes radiometric variation in both volume and surface
area. However,
when determining Aleph, a and x should be defined as the two smallest
dimensions of
the tissue slice.
Examples of Aleph are provided in Table I wherein, for example, a tissue
having
a thickness (x) of 0.1 mm and a width (a) of 1 mm would have an Aleph index of
11. In
Example I, the tissue had x=0.3 mm and a=4 mm such that Aleph = 3.48. In
Example
III, x is varied and a is constant at 4 mm. As illustrated in Figure 6,
proliferative activity
is substantially reduced as the thickness of the explant increases.
Accordingly, at 900
pm thickness, the number of proliferating cells in a micro-organ culture is
about 10 fold
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18
less then in tissue from a similar source having a thickness of 300 pm. The
Aleph index
for a tissue having a thickness of 900 pm is 1.36 mm ~, below the minimum
described
herein whereas the Aleph index for tissue having a thickness of 300 Ean is
3.58 mm ~,
which is well within the range of defined herein.
s
TABLE 1: Different values for the surface area to volume ratio index "Aleph",
as a
function of a (width) and x (thickness) in mm ~
WIDTH
x(mm) a = 1 mm a = 2mm a = 3mm a=4mm a=5mm
0.1 11 10.5 10.33 10.25 10.2
0.2 6 5.5 5.33 5.25 5.2
0.3 4.3 3.83 3.67 3.58 3.53
0.4 3.5 3 2.83 2.75 2.7
0.5 3 2.5 2.33 2.25 2.2
0.6 2.66 2.16 2 1.91 1.87
0.7 2.4 1.92 1.76 1.68 1.63
~
0.8 2.25 1.75 1.58 1.5 1.45
0.9 2.11 1.61 1.44 1.36 1.31
1 2 1.5 1.33 1.25 1.2
1.2 1.83 1.3 1.16 1.08 1.03
1.3 1.77 1.26 1.1 1.02 0.96
1.6 1.625 1.13 0.96 0.88 0.83
2 1.5 1 0.83 0.75 0.7
to Again, not wishing to be bound by any particular theory, a number of
factors
provided by the three-dimensional culture system may contribute to its
success:
(a) The appropriate choice of the explant size, e.g., by use of the above
Aleph calculations, three-dimensional matrix provides appropriate surface area
to
volume ratio for adequate diffusion of nutrients to all cells of the explant,
and adequate
IS diffusion of cellular waste away from all cells in the explant.
(b) Because of the three-dimensionality of the matrix, various cells continue
to actively grow, in contrast to cells in monolayer cultures, which grow to
confluence,
exhibit contact inhibition, and cease to grow and divide. The elaboration of
growth and
regulatory factors by replicating cells of the explant may be partially
responsible for
2o stimulating proliferation and regulating differentiation of cells in
culture, e.g., even for
the micro-organ culture which is static in terms of overall volume.
(c) The three-dimensional matrix retains a spatial distribution of cellular
elements which closely approximate that found in the counterpart tissue in
vivo.
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19
(d) The cell-cell and cell-matrix interactions may allow the establishment of
localized microenvironments conducive to cellular maturation. It has been
recognized
that maintenance of a differentiated cellular phenotypes requires not only
growth/differentiation factors but also the appropriate cellular interactions.
The present
invention effectively mimics the tissue microenvironment.
As described in the illustrative examples below, micro-organ cultures from
animals (including humans), such as derived from skin, pancreas, liver,
kidney,
duodenum, esophagus, bladder, bone marrow, thymus or spleen, have been
isolated and
grown for up to 21 days in culture. However, it is within the scope of the
invention to
to maintain cultures for extended periods of time beyond 21 days.
II. Source of explants for the micro-organ culture
The subject micro-organ culture can be derived using explants isolated from,
for
example: skin and mucosa (including oral mucosa, gastrointestinal mucosa,
nasal tract,
respiratory tract, cervix and cornea); pancreas; liver; gallbladder; bile
duct; lung;
prostate; uterus; mammary gland; bladder tissue; and blood-associated organs
such as
thymus, spleen and bone marrow. Accordingly, in vitro culture equivalents of
such
organs can be generated. The tissue forming the explants can be diseased or
normal
(e.g., healthy tissue). For example, the organs from which the micro-organ
explants of
the invention are isolated can be affected by hyperproliferative disorders,
e.g., psoriasis
or keratosis; proliferation of virally-infected cells, e.g., hepatitis
infected or
papillomavirus infected; neoproliferative disorders, e.g., basal cell
carcinoma, squamous
cell carcinoma, sarcomas, or Wilm's tumors; or fibrotic tissue, e.g., from a
cirrhotic liver
or a pancreas undergoing panereatitis.
Examples of animals from which the cells of the invention can be isolated
include humans and other primates, swine, such as wholly or partially inbred
swine
(e.g., miniature swine and transgenic swine), rodents, etc.
III. The growth media
There are a large number of tissue culture media that exist for culturing
cells
from animals. Some of these are complex and some are simple. While it is
expected that
micro-organ cultures may grow in complex media, it has been shown here that
the
cultures can be maintained in a simple medium such as Dulbecco's Minimal
Essential
Media. Furthermore, although the cultures may be grown in a media containing
sera or
other biological extracts such as pituitary extract, it has been shown here
that neither
serum nor any other biological extract is required. Moreover, the organ
cultures can be
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maintained in the absence of serum for extended periods of time. In preferred
embodiments of the invention, growth factors are not included in the medium
during
maintenance of the cultures in vitro.
The point regarding growth in minimal media is important. At present, most
5 media or systems for prolonged growth of mammalian cells incorporate
undefined
proteins or use feeder cells to provide proteins necessary to sustain such
growth.
Because the presence of such undefined proteins can interfere with the
intended end use
of the subject micro-organ cultures, it will generally be desirable to culture
the explants
under conditions to minimize the presence of undefined proteins.
t0 As used herein the language "minimal medium" refers to a chemically defined
medium which includes only the nutrients that are required by the cells to
survive and
proliferate in culture. Typically, minimal medium is free of biological
extracts, e.g.,
growth factors, serum, pituitary extract, or other substances which are not
necessary to
support the survival and proliferation of a cell population in culture. For
example,
I5 minimal medium generally includes at least one amino acid, at least one
vitamin, at least
one salt, at least one antibiotic, at least one indicator, e.g., phenol red,
used to determine
hydrogen ion concentration, glucose, and other miscellaneous components
necessary for
the survival and proliferation of the cells. Minimal medium is serum-free. A
variety of
minimal media are commercially available from Gibco BRL, Gathersburg, MD, as
20 minimal essential media.
However, while growth factors and regulatory factors need not be added to the
media, the addition of such factors, or the inoculation of other specialized
cells may be
used to enhance, alter or modulate proliferation and cell maturation in the
cultures. The
growth and activity of cells in culture can be affected by a variety of growth
factors such
as insulin, growth hormone, somatomedins, colony stimulating factors,
erythropoietin,
epidermal growth factor, hepatic erythropoietic factor (hepatopoietin), and
liver-cell
growth factor. Other factors which regulate proliferation and/or
differentiation include
prostaglandins, interleukins, and naturally-occurring negative growth factors,
fibroblast
growth factors, and members of the transforming growth factor -~i family.
The micro-organ cultures may be maintained in any suitable culture vessel such
0
as 24 or 96 well microplates and may be maintained at 37 C in 5% COZ. The
cultures
may be shaken for improved aeration, the speed of shaking being for example 12
rpm.
With respect to the culture vessel in/on which (optionally) the subject
micro-organ cultures are provided, it is noted that in the preferred
embodiment such
vessel may generally be of any material and/or shape. A number of different
materials
may be used to form the vessel, including but not limited to: nylon
(polyamides), dacron
(polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds
(e.g.,
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21
polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE;
teflon),
thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut
sutures,
cellulose, gelatin, dextran, etc. Any of these materials may be woven into a
mesh. Where
the micro-organ culture is itself to be implanted in vivo, it may be
preferable to use
biodegradable matrices such as poly glycolic acid, catgut suture material, or
gelatin, for
example. Where the cultures are to be maintained for long periods of time or
cryopreserved, non-degradable materials such as nylon, dacron, polystyrene,
polyacrylates, polyvinyls, teflons, cotton, etc. may be preferred. A
convenient nylon
mesh which could be used in accordance with the invention is Nitex, a nylon
filtration
to mesh having an average pore size of 210pm and an average nylon fiber
diameter of
90pm (#3-210/36, Tetko, Inc., N.Y.). Yet other embodiments are discussed
below.
In an exemplary embodiment, pancreatic micro-organs containing islets of
Langerhans are prepared as cultures of the present invention. The cultures are
then
provided in encapsulated form so as to avoid immune rejection. Three general
(exemplary) approaches for encapsulation might be used. In the first, a
tubular
membrane is coiled in a housing that contains the micro-organ explants. The
membrane
is connected to a polymer graft that in turn connects the device to blood
vessels. By
manipulation of the membrane permeability, so as to allow free diffusion of
glucose and
insulin back and forth through the membrane, yet block passage of antibodies
and
lymphocytes, normoglycemia can be maintained in pancreatectomized animals
treated
with this device (Sullivan et al (1991) Science 252:718).
In a second approach, hollow fibers containing the pancreatic explants are
(optionally) immobilized in the polysaccharide alginate. When the device is
placed
intraperitoneally in diabetic animals, blood glucose levels can be lowered and
good
tissue compatibility observed (Lacey et al. (1991) Science 254:1782; see also
Example
VI). Accordingly, fibers can be pre-spun and subsequently loaded with the
micro-organexplants (Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et
al. U.S.
Patent No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger
et al.
(1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J. Biomech Eng.
113:178-183).
Third, the micro-organ islet explants can be placed in microcapsules composed
of alginate or polyacrylates (see, for example, Lim et al. (1980) Science
210:908; O'Shea
et al. (1984) Biochim. Biochys. Acta 840:133; Sugamori et al (1989) Trans Am.
Soc.
Artif. Intern. Organs 35:791; Levesque et al. (1992) Endocrinology 130:644;
and Lim et
al. (1992) Transplantation 53:1180).
Finally, it is noted that the culture medium in which the micro-organ cultures
of
the present invention are maintained can be collected as a source of
conditioned
medium. The term "conditioned media" refers to the supernatant, e.g. free of
the
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cultured cells/tissue, resulting after a period of time in contact with the
cultured cells
such that the media has been altered to include certain paracrine and/or
autocrine factors
produced by the cells and secreted into the culture. Examples of such products
are
insulin, various growth factors, and hormones. This conditioned medium can be
used as
culture medium for other types of cell and tissue culture. Alternatively, the
conditioned
medium can be employed as a source of novel cell products such as growth
factors.
Such products can be fractionated and purified or substantially purified from
the
conditioned medium.
IV. Measuring the biological properties of micro-organ culture
The micro-organ cultures of the present invention derived from normal tissue
have been shown to maintain a state of homeostasis with proliferation of
constituent
cells without overall growth of the tissue.
~ 5 Methods of measuring cell proliferation are well known in the art and most
commonly include determining DNA synthesis characteristic of cell replication.
There
are numerous methods in the art for measuring DNA synthesis, any of which may
be
used according to the invention. In an embodiment of the invention, DNA
synthesis has
been determined using a radioactive label (3H-thymidine) or labeled nucleotide
2o analogues (BrdU) for detection by immunofluorescence.
Micro-organ cultures can be formed and maintained not only by the
proliferation
of mature cells but also by the active participation of precursor cells
including in some
instances, embryonic cells. The micro-organ cultures have been shown to
present-a
suitable environment for preserving, identifying, isolating and facilitating
the natural
25 evolution of these precursor cells. For example, the immature cells of the
basal layer
have been observed to become mature keratinocytes in skin micro-organ
cultures.
Similarly, embryonic pancreatic cells can provide a mature pancreatic
epithelium in
micro-organ cultures. The maturation of precursor cells and their subsequent
functioning
as adult cells can be monitored by measuring secretion of specialized products
such as
30 specific keratins in epidermal cells and insulin, Glut 2 and glucagon in
pancreatic
epithelia, and albumin and Factor VIII in liver micro-organ cultures.
The micro-organ cultures prepared according to the invention preserve the
normal tissue architecture that is present in vivo. As set out above, this
includes
maintenance of hair follicles, sweat glands and sebaceous glands in skin micro-
organs
35 in vitro, according to the normal occurrence in vivo and insulin and
glucagon secreting
cells in pancreatic micro-organs. Because these cultures can be maintained in
controlled
and uniform conditions and yet they closely resemble the microarchitecture of
the organ
in vivo, they provide a unique opportunity to observe, measure and control
natural
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23
phenomena and the perturbation of natural phenomena arising from disease,
aging or
trauma. Furthermore, the ready availability of techniques to study individual
cells at
identified sites on the culture, provides insights into the functioning of
individual
components of the organs and their interact with each other as well as the
whole organ.
Furthermore, the subject micro-organ cultures are maintainable in culture for
extended periods of time. Preferably, the micro-organ cultures are
maintainable in
culture for at least about twenty-four hours, more preferably for at least
about two days,
yet more preferably for at least about five days, still more preferably at
least about seven
days, still further preferably for at least about two weeks or more. The micro-
organ
cultures of the invention are typically maintained in culture for at least
seven days. To
illustrate, skin micro-organ cultures from human, mouse, guinea pig, and rat
skin have
been maintained in culture for at about least twenty-one days.
As used herein, the language "maintainable in culture" refers to the
population of
cells of a tissue explant of which at least about 60%, preferably at least
about 70%, more
~ 5 preferably at least about 80%, yet more preferably at least about 90%,
most preferably
95% or more of the cells remain viable in culture after a certain period of
time.
In a preferred embodiment, the ratio of cell proliferation to cell loss, e.g.,
by
death or sloughing, of the cells in the micro-organ cultures is equal to one,
i.e., the
number of cells proliferating is equal to the number of cells lost. In another
embodiment
20 of the present invention, the ratio of cell proliferation to cell loss of
the cells in the
micro-organ cultures is greater than one, i.e., the cells are proliferating at
a greater rate
than the cells are being lost. In the instance of the latter, the micro-organ
culture is
understood to include a population of cells which is being amplified.
25 V. Application of micro-organ cultures
Exemplary applications for the micro-organ cultures of the present invention
include the following:
(a) identification of factors involved in normal homeostasis of tissues and
30 organs;
(b) studying the effect on the normal homeostasis of tissues and cells of an
organ with respect to changes in the environment including changes in
nutrients and the
presence of potentially toxic agents;
(c) understanding the pathway of changes in the tissues and cells of an organ
35 that are triggered at the beginning and during pathogenesis or trauma;
(d) identification of repair mechanisms that reverse the adverse effects in an
altered environment associated with pathogenesis or trauma;
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24
(e) developmental regulation of cells that differentiate during the normal
homeostasis of the tissue.
(f) developmental regulation of specialized structures within an organ, such
as hair follicles;
(g) organ supplementation/transplantation where parts of an individual's
organ remain but are insufficient for replacing or regenerating damaged tissue
such as
occurs in patients with chronic skin ulcers, various forms of diabetes, or
chronic liver
failure;
(h) as a tissue/organ equivalent for drug screening and cytotoxicity studies;
l0 (i) as a diagnostic assay for proliferative disorders;
(j) as a source of novel growth factors;
(k) as a source of stem/progenitor cells;
(1) as a source of inducing molecules;
(m) as a screen for inducing molecules;
t 5 To further illustrate, the present method can be used to generate skin
equivalents
in the form of micro-organ cultures. By way of background, it is noted that
numerous
attempts have been described for growing epithelial cells in such a way as to
mimic
human skin for purposes of wound treatment, in particular treatment of burns.
The skin
consists of two types of tissue. These are: (1) the stroma or dermis which
includes
2o fibroblasts that are loosely dispersed within a high density collagen
matrix as well as
nerves, blood vessels and fat cells; (2) the epidermis which includes an
epidermal basal
layer of tightly packed, actively proliferating immature epithelial cells. As
the cells of
the basal layer replicate, some of the young cells remain in the basal layer
while others
migrate outward, increase in size and eventually develope an envelop resistant
to
25 detergents and reducing agents. In humans, a cell born in the basal layer
takes about 2
weeks to reach the edge or outer layer after which time the cells die and are
shed. The
skin contains various structures including hair follicles, sebaceous glands
and sweat
glands. Hair follicles are formed from differentiating keratinocytes that
densely line
invaginations of the epidermis. The open ended vesicles that formed from such
30 invaginations collect and concentrate the secreted keratin and a hair
filament results.
Alternatively, epidermal cells lining an invagination may secrete fluids
(sweat gland) or
sebum (sebaceous gland). The regulation of formation and proliferation of
these
structures is unknown. The constant renewal of healthy skin is accomplished by
a
balanced process in which new cells are being produced and aged cells die.
There is a
35 need to understand how this precise regulation comes about in order to
counteract
abnormal events occurring in aging, and also through disease and trauma that
disrupt the
balance.
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In one embodiment of the invention, the microarchitecture of the micro-organ
culture mimics or is substantially the same as that of skin in vivo, e.g., it
has an
epithelial tissue/connective tissue structure. For example, in skin micro-
organ cultures,
keratinocytes of the epidermis remain associated with the connective tissue
and the
5 normal tissue architecture is preserved including the hair follicles. The
micro-organ
culture which is obtained from a skin tissue section can also include a basal
lamina
supporting the epidermal cells, an extracellular matrix which includes the
dermal cells,
and at least one invagination, e.g., at least one hair follicle. The
association between skin
epithelial tissue and the skin connective tissue facilitates intercellular
communication.
t 0 Moreover, full thickness skin can be grown in a variety of ways allowing
an air
interface. Exposure of the keratinocytes of the explant to air promotes a more
rapid
differentiation of keratinocytes and more extensive secretion of keratin
layers, which
may be very important in skin penetration studies.
Finally, it is noted that recent studies have indicated that the skin is an
integral
15 . and active element of the immune system (Cooper et al., (1987) The
mechanobullous
disease. In: Dermatology in General Medicine, 3d. Ed., McGraw Hill, NY (pp.610-
626).
One of the major cell types in the skin which is responsible for various
immune
activities is the Langerhans cell. These cells may be prepared from fresh skin
samples
and added to the three-dimensional skin culture to produce an immunologically
2o complete tissue system. Growth of these cells in the culture for long
periods of time by
conventional tissue culture techniques is difficult. The ability to grow these
cells in a
three-dimensional system would be of great importance in all aspects of study
including
engraftment, cytoxicity, and disease mechanisms. This type of skin culture
system
would have the greatest impact on research involving auto-immune disorders
which
25 have direct or indirect cutaneous involvement (lupus erythematosis, bullous
pemphigoid, etc.). Accordingly, the micro-organ cultures of the present
invention can be
used to study proliferative/differentiative disorders under conditions in
which
immunological aspects of the disease are minimized. An exemplary drug
screening
assay can be derived using psoriatic skin explants in order to identify agents
which can
inhibit proliferation of the hyperplastic epithelial cells.
The skin is merely an example of a tissue which can be grown as a micro-organ
culture having epithelial tissue which is supported by stromal tissue. Other
tissues
including epithelial tissue can be grown as micro-organ cultures of the
present
invention. Epithelial tissues are found in every part of the body where an
interface
between an organ and the environment arise. Epithelial cells cycle
continuously in an
uninjured body and form the covering tissue for all the free surfaces in the
body
including the skin. In some cases, such as in the pancreas, the epithelial
cells line
numerous invaginations and secrete enzymes into open spaces that enable the
organ to
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26
function. The lung is another example of a highly invaginated organ, each
invagination
in the lung being lined with epithelial cells through which air diffuses from
the
environment in to the body. Once again, these epithelial cells have
characteristic
properties. The lining of the gut is also composed of specialized epithelial
cells that not
only form a barrier but also contain specialized structures for selectively
absorbing food.
All the epithelia are supported by connective tissue. Still another organ
comprising
important cell-stromal interactions is the bone marrow.
Thus, in another embodiment of the present invention, microarchitecture of a
micro-organ pancreas culture mimics or is substantially the same as that of
the source
pancreas in vivo, e.g., it has an epithelial tissue/connective tissue
structure. For
example, pancreas micro-organ cultures include pancreatic epithelial cells,
e.g., islet
cells, remain associated with the pancreatic connective tissue. In the
pancreas
micro-organ culture, therefore, the normal tissue architecture is preserved
and the
normal pancreatic epithelial cell products, e.g., insulin and glucagon are
produced.
t 5 In another embodiment, the present invention provides for the generation
of
micro-organ cultures derived from the bone marrow, which cultures preserve the
microarchitecture of the in vivo organ. As described in Example XV, bone
marrow
micro-organs have been isolated in culture to derive a system comparable to
physiologic
conditions.
2o The bone marrow cultures of the present invention may be used for treating
diseases or conditions which destroy healthy bone marrow cells or depress
their
functional ability. Implantation of the subject micro-organs can be effective
in the
treatment of hematological malignancies and other neoplasias which involve the
bone
marrow. This aspect of the invention is also effective in treating patients
whose bone
25 marrow has been adversely affected by the environmental factors (e.g.,
radiation, toxins,
etc). While reimplantation of explants derived from the patients own marrow
are
generally preferable, it is noted that such explants can be allogenic, e.g.,
from another
member of the same species, or xenogenic, e.g., from another organism. An
exemplary
xenogenic implant could be a micro-organ culture derived from a miniature
swine for
3o implantation in a human.
Moreover, long-term growth of human hematopoietic progenitors is possible if
they are provided with the necessary stromal-derived growth/regulatory
factors. Such
interactions are provided by the subject micro-organs, rendering these
explants as
sources of stem and progenitor cells. In general, hematopoietic progenitor
cells of the
35 marrow colonize ("seed") the natural packets formed in the stromal matrix
of the bone
marrow micro-organ. The primary rate limiting factor in the growth of marrow
stromal
cells is the relatively low mitotic index of the fibroblasts included among
the marrow
stromal cells. Accordingly, where the growth of these cells and their
disposition of
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27
extracellular matrix components is desired to be enhanced, the explant can be
contacted
with such agents as hydrocortisones or other fibroblast growth factors.
If the bone marrow is to be cultured in order to treat certain patients with
metastatic disease or hematological malignancies, the marrow obtained from the
patients
should be "purged" of abnormally proliferating cells by physical or
chemotherapeutic
means prior to culturing.
The conditioned medium from a bone marrow micro-organ culture of the present
invention can be used as a source of novel or known lymphokines, e.g., as a
source of
interleukins.
to The invention contemplates, in one aspect, the use of the subject micro-
organ
cultures for transplantation in an organism. As used herein the terms
"administering","
introducing", and "transplanting" are used interchangeably and refer to the
placement of
the cell populations of the invention into a subject, e.g., an allogeneic or a
xenogeneic
subject, by a method or route which results in localization of the cells to a
desired site.
The cell populations can be administered to a subject by any appropriate route
which
results in delivery of the cells to a desired location in the subject where at
least a portion
of the cells remain viable. It is preferred that at least about 5%, preferably
at least about
10%, more preferably at least about 20%, yet more preferably at least about
30%, still
more preferably at least about 40%, and most preferably at least about SO% or
more of
2o the cells remain viable after administration to a subject. The period of
viability of the
cells after administration to a subject can be as short as a few hours, e.g.,
twenty-four
hours, to a few days, to as long as a few weeks to months. Methods of
administering
populations of cells of the invention include implantation of cells into the
visceral or the
parietal peritoneum, for example into a pouch of the omentum, implantation of
cells into
or onto an organ of the recipient subject, e.g., pancreas, liver, spleen,
skin. The
micro-organs of the invention can also be administered to a subject by
implantation
under, e.g., a kidney capsule.
As used herein, the term "subject" refers to mammals, e.g., primates, e.g.,
humans. A "xenogeneic subject" as used herein is a subject into which cells of
another
3o species are introduced or are to be introduced. An "allogeneic subject" is
a subject into
which cells of the same species are introduced or are to be introduced. Donor
subjects
are subjects which provide the cells, tissues, or organs, which are to be
placed in culture
and/or transplanted to a recipient subject. Recipient subjects can be either
xenogeneic or
allogeneic subject. Donor subjects can also provide cells, tissues, or organs
for
reintroduction into themselves, i.e. for autologous transplantation.
To facilitate transplantation of the cell populations which may be subject to
immunological attack by the host, e.g., where xenogenic grafting is used, such
as
swine-human transplantations, the micro-organ can be inserted into or
encapsulated by
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28
rechargeable or biodegradable devices and then transplanted into the recipient
subject.
Gene products produced by such cells can then be delivered via, for example,
polymeric
devices designed for the controlled delivery compounds, e.g., drugs, including
proteinaceous biopharmaceuticals. A variety of biocompatible polymers
(including
hydrogels), including both biodegradable and non-degradable polymers, can be
used to
form an implant for the sustained release of a gene product of the cell
populations of the
invention at a particular target site. The generation of such implants is
generally known
in the art. See, for example, Concise Encyclopedia of Medical & Dental
Materials, ed.
By David Williams (MIT Press: Cambridge, MA, 1990); the Sabel et al. US Patent
No.
4,883,666; Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S.
Patent No.
5,106,627; Lim U.S. Patent No. 4,391,909; and Sefton U.S. Patent No.
4,353,888. Cell
populations of the invention can be administered in a pharmaceutically
acceptable
carrier or diluent, such as sterile saline and aqueous buffer solutions. The
use of such
carriers and diluents is well known in the art.
In one embodiment, the micro-organ cultures of the present invention can be
employed for wound healing. Repair of skin lesions is known to be a highly
complex
process that includes primary epithelial cell migration as well as replication
of epidermal
cells in response to molecular signals from underlying connective tissue. Skin
micro-organ cultures are described herein as a model for wound healing. Under
2o controlled culture conditions, factors controlling healing can be carefully
monitored.
Furthermore, because the micro-organ culture is isolated from the natural
blood supply,
analysis of the healing process can be done without the additional complexity
of blood
borne factors or cells. Normal epidermis has a low mitotic activity with cells
cycling
every 200-300 hours. When the epidermis is wounded, a burst of mitotic
activity takes
place so that the cells divide up to 10 times faster depending on the
conditions and
severity of the wound (Pinkus H.(1951) J. Invest. Dermatol. 16:383-386).
As demonstrated in Example II, skin micro-organ cultures show increased
proliferation of up to 10 fold for several days. In this example, the edge of
a wound is
comparable to the micro-organ culture. This increased proliferation mimics the
events
3o that are associated with wounding and provides a unique opportunity to
study the
process of wound healing. Moreover, the appended examples demonstrate in vivo
that
the epidermal explants of the present invention can be applied to chronic
wounds
(example IX) and can form a viable implant capable of growing hair (example
XI).
Moreover, the subject epidermal micro-organs can be used in the treatment of
burn patients. The need for a skin replacement for burn patients is evident.
Several
centers in the United States and Europe have utilized cultured human
keratinocyte
allografts and autografts to permanently cover the wounds of burns and chronic
ulcers
(Eisinger et al., (1980) Surgery 88:287-293; Green et al., (1979) Proc. Natl.
Acad. Sci.
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29
USA 76:5665-5668; Cuono et al., (1987) Plast. Reconstr. Surg. 80:626-635).
These
methods are often unsuccessful and recent studies have indicated that
blistering and/or
skin fragility in the healed grafts may exist because of an abnormality in one
or more
connective tissue components formed under the transplanted epidermal layer
(Woodley
et al., (1988) JAMA 6:2566-2571). The skin culture system of the present
invention
provides a skin equivalent of both epidermis and dermis and should overcome
problems
characteristic of currently used cultured keratinocyte grafts.
In yet another embodiment, the micro-organ culture system of the invention may
afford a vehicle for introducing genes and gene products in vivo for use in
gene
1o therapies. For example, using recombinant DNA techniques, a gene for which
a patient
is deficient could be placed under the control of a viral or tissue-specific
promoter. The
recombinant DNA construct can be used to transform or transfect all or certain
of the
cells in the subject micro-organ culture system. The micro-organ culture which
expresses the active gene product could be implanted into an individual who is
deficient
for that product.
The use of the subject micro-organ culture in gene therapy has a number of
advantages. Firstly, since the culture comprises eukaryotic cells, the gene
product will
be properly expressed and processed in culture to form an active product.
Secondly,
gene therapy techniques are useful only if the number of transfected cells can
be
2o substantially enhanced to be of clinical value, relevance, and utility; the
subject cultures
allow for expansion of the number of transfected cells and amplification.
In a further embodiment of the invention, the transgenic micro-organ cultures
may be used to facilitate gene transduction. For example, and not by way of
limitation, a
micro-organ culture comprising a recombinant virus expression vector may be
used to
transfer the recombinant virus into cells brought into contact with the
culture, e.g., by
implantation, thereby simulating viral transmission in vivo. Accordingly, this
system
can be a more efficient way of accomplishing gene transduction than are
current
techniques for DNA transfection.
Accordingly, the cells of the micro-organ cultures of the present invention
can
3o be modified to express a gene product. As used herein, the phrase "gene
product" refers
to proteins, peptides and functional RNA molecules. Generally, the gene
product
encoded by the nucleic acid molecule is the desired gene product to be
supplied to a
subject. Examples of such gene products include proteins, peptides,
glycoproteins and
lipoproteins normally produced by an organ of the recipient subject. For
example, gene
products which may be supplied by way of gene replacement to defective organs
in the
pancreas include insulin, amylase, protease, lipase, trypsinogen,
chymotrypsinogen,
carboxypeptidase, ribonuclease, deoxyribonuclease, triacylglycerol lipase,
phospholipase A2, elastase, and amylase; gene products normally produced by
the liver
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include blood clotting factors such as blood clotting Factor VIII and Factor
IX, UDP
glucuronyl transferase , ornithine transcarbanoylase, and cytochrome p450
enzymes, and
adenosine deaminase, for the processing of serum adenosine or the endocytosis
of low
density lipoproteins; gene products produced by the thymus include serum
thymic
5 factor, thymic humoral factor, thymopoietin, and thymosin a~; gene products
produced
by the digestive tract cells include gastrin, secretin, cholecystokinin,
somatostatin, and
substance P. Alternatively, the encoded gene product is one which induces the
expression of the desired gene product by the cell (e.g., the introduced
genetic material
encodes a transcription factor which induces the transcription of the gene
product to be
to supplied to the subject). In still another embodiment, the recombinant gene
can provide
a heterologous protein, e.g., not native to the cell in which it is expressed.
For instance,
various human MHC components can be provided to non-human micro-organs to
support engraftment in a human recipient. Alternatively, the transgene is one
which
inhibits the expression or action of a donor MHC gene product normally
expressed in
~ 5 the micro-organ explant.
A nucleic acid molecule introduced into a cell is in a form suitable for
expression in the cell of the gene product encoded by the nucleic acid.
Accordingly, the
nucleic acid molecule includes coding and regulatory sequences required for
transcription of a gene (or portion thereof) and, when the gene product is a
protein or
zo peptide, translation of the nucleic acid molecule include promoters,
enhancers and
polyadenylation signals, as well as sequences necessary for transport of an
encoded
protein or peptide, for example N-terminal signal sequences for transport of
proteins or
peptides to the surface of the cell or secretion.
Nucleotide sequences which regulate expression of a gene product (e.g.,
zs promoter and enhancer sequences) are selected based upon the type of cell
in which the
gene product is to be expressed and the desired level of expression of the
gene product.
For example, a promoter known to confer cell-type specific expression of a
gene linked
to the promoter can be used. A promoter specific for myoblast gene expression
can be
linked to a gene of interest to confer muscle-specific expression of that gene
product.
30 Muscle-specific regulatory elements which are known in the art include
upstream
regions from the dystrophin gene (Klamut et al., (1989) Mol. Cell
Biol.9:2396), the
creatine kinase gene (Buskin and Hauschka, (1989) Mol. Cell Biol. 9:2627) and
the
troponin gene (Mar and Ordahl, (1988) Proc. Natl. Acad. Sci. USA. 85:6404),
Negative
response elements in keratin genes mediate transcriptional repression (Jho Sh
et al,
(2001).1 Bioi Chem). Regulatory elements specific for other cell types are
known in the art
(e.g., the albumin enhancer for liver-specific expression; insulin regulatory
elements for
pancreatic islet cell-specific expression; various neural cell-specific
regulatory elements,
including neural dystrophin, neural enolase and A4 amyloid promoters).
Alternatively, a
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31
regulatory element which can direct constitutive expression of a gene in a
variety of
different cell types, such as a viral regulatory element, can be used.
Examples of viral
promoters commonly used to drive gene expression include those derived from
polyoma
virus, Adenovirus 2, cytomegalovirus and Simian Virus 40, and retroviral LTRs.
Alternatively, a regulatory element which provides inducible expression of a
gene linked
thereto can be used. The use of an inducible regulatory element (e.g., an
inducible
promoter) allows for modulation of the production of the gene product in the
cell.
Examples of potentia:ly useful inducible regulatory systems for use in
eukaryotic cells
include hormone-regulated elements ( e.g., see Mader, S. and White, J.H.
(1993) Proc.
l0 Natl. Aca~l Sci. USA 90:5603-5607), synthetic ligand-regulated elements
(see, e.g.,
Spencer, D.M. et al 1993) Science 262:1019-1024) and ionizing radiation-
regulated
elements (e.g., see Manome, Y. Et al. (1993) Biochemistry 32:10607-10613;
Datta, R. et
al. (1992) Proc. Natl. Acad. Sci. USA89:1014-10153). Additional tissue-
specific or
inducible regulatory systems which may be developed can also be used in
accordance
with the invention.
There are a number of techniques known in the art for introducing genetic
material into a cell that can be applied to modify a cell of the invention. In
one
embodiment, the nucleic acid is in the form of a naked nucleic acid molecule.
In this
situation, the nucleic acid molecule introduced into a cell to be modified
consists only of
2o the nucleic acid encoding the gene product and the necessary regulatory
elements.
Alternatively, the nucleic acid encoding the gene product (including the
necessary
regulatory elements) is contained within a plasmid vector. Examples of plasmid
expression vectors include CDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman, et al. (1987) EMBO J. 6:187-195). In another embodiment, the nucleic
acid
molecule to be introduced into a cell is contained within a viral vector. In
this situation,
the nucleic acid encoding the gene product is inserted into the viral genome
(or partial
viral genome). The regulatory elements directing the expression of the gene
product can
be included with the nucleic acid inserted into the viral genome (i.e., linked
to the gene
inserted into the viral genome) or can be provided by the viral genome itself.
Naked nucleic acids can be introduced into cells using calcium-phosphate
mediated transfection, DEAE-dextran mediated transfection, electroporation,
liposome-mediated transfection, direct injection, and receptor-mediated
uptake.
Naked nucleic acid, e.g., DNA, can be introduced into cells by forming a
precipitate containing the nucleic acid and calcium phosphate. For example, a
HEPES-buffered saline solution can be mixed with a solution containing calcium
chloride and nucleic acid to form a precipitate and the precipitate is then
incubated with
cells. A glycerol or dimethyl sulfoxide shock step can be added to increase
the amount
of nucleic acid taken up by certain cells. CaP04-mediated transfection can be
used to
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32
stably (or transiently) transfect cells and is only applicable to in vitro
modification of
cells. Protocols for CaP04-mediated transfection can be found in Current
Protocols in
Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates,
(1989),
Section 9.1 and in Molecular Cloning: A Laboratory Manual, 2nd Edition,
Sambrook et
al. Cold Spring Harbor Laboratory Press, (1989), Sections 16.32-16.40 or other
standard
laboratory manuals.
Naked nucleic acid can be introduced into cells by forming a mixture of the
nucleic acid and DEAE-dextran and incubating the mixture with the cells. A
dimethylsulfoxide or chloroquine shock step can be added to increase the
amount of
to nucleic acid uptake. DEAE-dextran transfection is only applicable to in
vitro
modification of cells and can be used to introduce DNA transiently into cells
but is not
preferred for creating stably transfected cells. Thus, this method can be used
for short
term production of a gene product but is not a method of choice for long-term
production of a gene product. Protocols for DEAF-dextran-mediated transfection
can be
found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.)
Greene
Publishing Associates, (1989), Section 9.2 and in Molecular Cloning: A
Laboratory
Manual, 2nd Edition, Sambrook et al. Cold Spring Harbor Laboratory Press,
(1989),
Sections 16.41-16.46 or other standard laboratory manuals.
Naked nucleic acid can also be introduced into cells by incubating the cells
and
the nucleic acid together in an appropriate buffer and subjecting the cells to
a
high-voltage electric pulse. The efficiency with which nucleic acid is
introduced into
cells by electroporation is influenced by the strength of the applied field,
the length of
the electric pulse, the temperature, the conformation and concentration of the
DNA and
the ionic composition of the media. Electroporation can be used to stably (or
transiently)
transfect a wide variety of cell types. Protocols for electroporating cells
can be found in
Current Protocols in Molecular Biology, Ausubel F.M. et al. (eds.) Greene
Publishing
Associates, (1989), Section 9.3 and in Molecular Cloning: A Laboratory Manual,
2nd
Edition, Sambrook et al. Cold Spring Harbor Laboratory Press, (1989), Sections
16.54-16.55 or other standard laboratory manuals.
3o Another method by which naked nucleic acid can be introduced into cells
includes liposome-mediated transfection (lipofection). The nucleic acid is
mixed with a
liposome suspension containing cationic lipids. The DNA/liposome complex is
then
incubated with cells. Liposome mediated transfection can be used to stably (or
transiently) transfect cells in culture in vitro. Protocols can be found in
Current
Protocols in Molecular Biology, Ausubel F.M. et al. (eds.) Greene Publishing
Associates, (1989), Section 9.4 and other standard laboratory manuals.
Additionally,
gene delivery in vivo has been accomplished using liposomes. See for example
Nicolau
et al. (1987) Meth. Enz. 149:157-176; Wang and Huang (1987) Proc. Natl. Acad.
Sci.
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33
USA 84:7851-7855; Brigham et al. (1989) Am. JMed. Sci. 298:278; and Gould-
Fogerite
et al. (1989) Gene 84:429-438.
Naked nucleic acid can also be introduced into cells by directly injecting the
nucleic acid into the cells. For an in vitro culture of cells, DNA can be
introduced by
microinjection. Since each cell is microinjected individually, this approach
is very labor
intensive when modifying large numbers of cells. However, a situation wherein
microinjection is a method of choice is in the production of transgenic
animals
(discussed in greater detail below). In this situation, the DNA is stably
introduced into a
fertilized oocyte which is then allowed to develop into an animal. The
resultant animal
contains cells carrying the DNA introduced into the oocyte. Direct injection
has also
been used to introduce naked DNA into cells in vivo (see e.g., Acsadi et al.
(1991)
Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468). A delivery
apparatus
(e.g., a "gene gun") for injecting DNA into cells in vivo can be used. Such an
apparatus
is commercially available (e.g., from BioRad).
~5 Naked nucleic acid can be complexed to a canon, such as polylysine, which
is
coupled to a ligand for a cell-surface receptor to be taken up by receptor-
mediated
endocytosis (see for example Wu, G. and Wu, C.H. (1988) J. Biol. Chem. 263:
14621;
Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Patent No.
5,166,320).
Binding of the nucleic acid-ligand complex to the receptor facilitates uptake
of the DNA
by receptor-mediated endocytosis. Receptors to which a DNA-ligand complex have
targeted include the transferrin receptor and the asialoglycoprotein receptor.
A
DNA-ligand complex linked to adenovirus capsids which naturally disrupt
endosomes,
thereby releasing material into the cytoplasm can be used to avoid degradation
of the
complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc.
Natl.
Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA
90:2122-2126). Receptor-mediated DNA uptake can be used to introduce DNA into
cells either in vitro or in vivo and, additionally, has the added feature that
DNA can be
selectively targeted to a particular cell type by use of a ligand which binds
to a receptor
selectively expressed on a target cell of interest.
Generally, when naked DNA is introduced into cells in culture (e.g., by one of
the transfection techniques described above) only a small fraction of cells
(about 1 out
of 105) typically integrate the transfected DNA into their genomes (i.e., the
DNA is
maintained in the cell episomally). Thus, in order to identify cells which
have taken up
exogenous DNA, it is advantageous to transfect nucleic acid encoding a
selectable
marker into the cell along with the nucleic acids) of interest. Preferred
selectable
markers include those which confer resistance to drugs such as 6418,
hygromycin and
methotrexate. Selectable markers may be introduced on the same plasmid as the
genes)
of interest or may be introduced on a separate plasmid.
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34
A preferred approach for introducing nucleic acid encoding a gene product into
a
cell is by use of a viral vector containing nucleic acid, e.g. a cDNA,
encoding the gene
product. Infection of cells with a viral vector has the advantage that a large
proportion of
cells receive the nucleic acid which can obviate the need for selection of
cells which
have received the nucleic acid. Additionally, molecules encoded within the
viral vector,
e.g. a cDNA contained in the viral vector, are expressed efficiently in cells
which have
taken up viral vector nucleic acid and viral vector systems can be used either
in vitro or
tn vtvo.
Defective retroviruses are well characterized for use in gene transfer for
gene
t0 therapy purposes (for review see Miller, A.D. (1990) Blood 76:271). A
recombinant
retrovirus can be constructed having a nucleic acid encoding a gene product of
interest
inserted into the retroviral genome. Additionally, portions of the retroviral
genome can
be removed to render the retrovirus replication defective. The replication
defective
retrovirus is then packaged into virions which can be used to infect a target
cell through
the use of a helper virus by standard techniques. Protocols for producing
recombinant
retroviruses and for infecting cells in vitro or in vivo with such viruses can
be found in
Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene
Publishing
Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
Examples
of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known
to
those skilled in the art. Examples of suitable packaging virus lines include
yCrip, yr2
and ytAm. Retroviruses have been used to introduce a variety of genes into
many
different cell types, including epithelial cells endothelial cells,
lymphocytes, myoblasts,
hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example
Eglitis, et al.
(1985) Science 230:1395-1398; Danosand Mulligan (1988) Proc. Natl. Acad Sci.
USA
85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci USA 85:3014-3018;
Armentano et al., (1990) Proc. Natl. Acad. Sci. USA 87: 6141-6145; Huber et
al. (1991)
Proc. Natl. Acad. Sci. USA 88:8039-8043; Feri et al. (1991) Proc. Natl. Acad.
Sci. USA
88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et
al.
(1992) Proc. Natl. Acad. Sci USA 89:7640-7644; Kay et al. (1992) Human Gene
3o Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-
10895; Hwu
et al (1993) J. Immunol. 150:4104-4115; US Patent No. 4,868,116; US Patent No.
4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT
Application WO 89/05345; and PCT Application WO 92/07573). Retroviral vectors
require target cell division in order for the retroviral genome (and foreign
nucleic acid
inserted into it) to be integrated into the host genome to stably introduce
nucleic acid
into the cell. Thus, it may be necessary to stimulate replication of the
target cell.
The genome of an adenovirus can be manipulated such that it encodes and
expresses a gene product of interest but is inactivated in terms of its
ability to replicate
CA 02464460 2004-04-22
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in a normal lytic viral life cycle. See for example Berkner et al. ( 1988)
BioTechniques
6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al.
(1992) Cell
68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad
type 5
d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known
to those
5 skilled in the art. Recombinant adenoviruses are advantageous in that they
do not require
dividing cells to be effective gene delivery vehicles and can be used to
infect a wide
variety of cell types, including airway epithelium (Rosenfeld et al. (1992)
cited supra),
endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad Sci. USA 89:6482-
6486),
hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad Sci. USA 90:2812-2816)
and
1o muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-
2584).
Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is
not
integrated into the genome of a host cell but remains episomal, thereby
avoiding
potential problems that can occur as a result of insertional mutagenesis in
situations
where introduced DNA becomes integrated into the host genome (e.g., retroviral
DNA).
15 Moreover, the carrying capacity of the adenoviral genome for foreign DNA is
large (up
to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited
supra;
Haj-Ahmand and Graham ( 1986) J. Virol 57:267). Most replication-defective
adenoviral
vectors currently in use are deleted for all or parts of the viral E1 and E3
genes but retain
as much as 80% of the adenoviral genetic material.
20 Adeno-associated virus (AAV) is a naturally occurring defective virus that
requires another virus, such as an adenovirus or a herpes virus, as a helper
virus for
efficient replication and a productive life cycle. (For a review see Muzyczka
et al. Curr.
Topics In Micro. And Immunol. (1992) 158:97-129). It is also one of the few
viruses that
may integrate its DNA into non-dividing cells, and exhibits a high frequency
of stable
25 integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol.
Biol.
7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al
(1989)
J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV
can be
packaged and can integrate. Space for exogenous DNA is limited to about 4.5
kb. An
AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol.
30 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic
acids have
been introduced into different cell types using AAV vectors (see for example
Hermonat
et al. (1984)Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985)
Mol. Cell
Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39;
Tratschin et al.
(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-
3790).
35 The efficacy of a particular expression vector system and method of
introducing
nucleic acid into a cell can be assessed by standard approaches routinely used
in the art.
For example, DNA introduced into a cell can be detected by a filter
hybridization
technique (e.g., Southern blotting) and RNA produced by transcription of
introduced
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36
DNA can be detected, for example, by Northern blotting, RNase protection or
reverse
transcriptase-polymerase chain reaction (RT-PCR). The gene product can be
detected by
an appropriate assay, for example by immunological detection of a produced
protein,
such as with a specific antibody, or by a functional assay to detect a
functional activity
of the gene product, such as an enzymatic assay. If the gene product of
interest to be
interest to be expressed by a cell is not readily assayable, an expression
system can first
be optimized using a reporter gene linked to the regulatory elements and
vector to be
used. The reporter gene encodes a gene product which is easily detectable and,
thus, can
be used to evaluate efficacy of the system. Standard reporter genes used in
the art
to include genes encoding (3-galactosidase, chloramphenicol acetyl
transferase, luciferase,
GFP/EGFP and human growth hormone.
When the method used to introduce nucleic acid into a population of cells
results
in modification of a large proportion of the cells and efficient expression of
the gene
product by the cells (e.g., as is often the case when using a viral expression
vector), the
modified population of cells may be used without further isolation or
subcloning of
individual cells within .the population. That is, there may be sufficient
production of the
gene product by the population of cells such that no further cell isolation is
needed.
Alternatively, it may be desirable to grow a homogenous population of
identically
modified cells from a single modified cell to isolate cells which efficiently
express the
2o gene product. Such a population of uniform cells can be prepared by
isolating a single
modified cell by limiting dilution cloning followed by expanding the single
cell in
culture into a clonal population of cells by standard techniques.
As used herein, the phrase "transgenic cell" referred to a cell into which a
nucleic
acid sequence which is partially or entirely heterologous, i.e., foreign, to
the cell in
which it has been inserted or introduced. A transgenic cell can also be a cell
into which
an nucleic acid which is homologous to an endogenous gene of the cell has been
inserted. In this case, however, the homologous nucleic acid is designed to be
inserted ,
or is inserted, into the cell's genome in such a way as to alter the genome of
the cell into
which it is inserted. For example, the homologous nucleic acid is inserted at
a location
which differs from that of the natural gene or the insertion of the homologous
nucleic
acid results in a knockout of a particular phenotype. The nucleic acid
inserted into the
cells can include one or more transcriptional regulatory sequences and any
other nucleic
acid, such as an intron, that may be necessary for optimal expression of a
selected
nucleic acid.
In yet another aspect of the present invention, the subject micro-organ
cultures
may be used to aid in the diagnosis and treatment of malignancies and
diseases. For
example, a biopsy of an organ (e.g. skin, kidney, liver, etc.) may be taken
from a patient
suspected of having a hyperproliferative or neoproliferative disorder. If the
biopsy
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37
explant is cultured according to the present method, proliferative cells of
the explant
will be clonally expanded during culturing. This will increase the chances of
detecting
such disorders, and, therefore, increase the accuracy of the diagnosis.
Moreover, the
patient's micro-organ culture could be used in vitro to screen cytotoxic
and/or
pharmaceutical compounds in order to identify those that are most efficacious;
i.e. those
that kill malignant or diseased cells, yet spare the normal cells. These
agents could then
be used to therapeutically treat the patient.
A further aspect of the invention pertains to a method of using the subject
micro-organ cultures to screen a wide variety of compounds, such as cytotoxic
compounds growth/regulatory factors, pharmaceutical agents, etc. For example,
the need
for thorough testing of chemicals of potentially toxic nature is generally
recognized and
the need to develop sensitive and reproducible short-term in vitro assays for
the
evaluation of drugs, cosmetics, food additives and pesticides is apparent. The
micro-organ cultures described herein permits the use of a tissue-equivalent
as an assay
substrate and offers the advantages of normal cell interactions in a system
that closely
resembles the in vivo state.
To this end, the cultures are maintained in vitro and exposed to the compound
to
be tested. The activity of a cytotoxic compound can be measured by its ability
to
modulate the phenotype (including killing) of cells in the explant. This may
readily be
2o assessed by vital staining techniques, expression of markers, ete. For
instance, the effect
of growth/regulatory factors may be assessed by analyzing the cellular content
of the
culture, e.g., by total cell counts, and differential cell counts. This may be
accomplished
using standard cytological and/or histological techniques including the use of
immunocytochemical techniques employing antibodies that define type-specific
cellular
antigens. The effect of various drugs on normal cells cultured in the present
system may
be assessed. For example, drugs that decrease proliferation of psoriatic
tissue can be
identified.
In an exemplary embodiment of this method, derived for detecting agents which
stimulate proliferation of a cell in the explant, the method includes
isolating a tissue
3o explant from a subject, wherein the population of cells of the explant
retains a
microarchitecture of the organ or tissue from which the explant was isolated,
e.g., the
explant is characterized by Aleph of at least about 1.5 mm~~, and includes at
least one
cell which has the ability to proliferate. The explant is cultured and
contacted with a
candidate compound. The level of cell proliferation in the presence of the
candidate
compound is then measured and compared with the level of cell proliferation in
the
absence of the candidate compound. A statistically significant increase in the
level of
cell proliferation in the presence of the candidate compound is indicative of
a cell
proliferative agent.
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38
The phrase "candidate compound" or "candidate agent'.' as used herein refers
to
an agent which is tested or to be tested for proliferative, anti-
proliferative,
differentiating, anti-differentiating, or anti-viral activity. Such agents can
be, for
example, small organic molecules, biological extracts, and recombinant
products or
compositions.
Methods of measuring cell proliferation are well known in the art and most
commonly include determining DNA synthesis characteristic of cell replication.
There
are numerous methods in the art for measuring DNA synthesis, any of which may
be
used according to the invention. In one embodiment of the invention, DNA
synthesis
to has been determined using a radioactive label (3H-thymidine) or labeled
nucleotide
analogues (BrdU) for detection by immunofluorescence.
Yet another embodiment provides a method for identifying an inhibitor of cell
proliferation. This method includes providing a tissue explant as above,
contacting that
explant with a candidate compound, and measuring the level of cell
proliferation in the
presence of the candidate compound. A statistically significant decrease in
the level of
cell proliferation in the presence of the candidate compound is indicative of
an inhibitor
of cell proliferation.
In an illustrative embodiment, both potentiators and inhibitors of cell
proliferation (also referred to herein as anti-proliferative agents) can be
used, for
example to control hair growth depending on the desired effect.
The growth of hard keratin fibers such as wool and hair is dependent on the
proliferation of dermal sheath. cells. Hair follicle stem cells of the sheath
are highly
active, and give rise to hair fibers through rapid proliferation and complex
differentiation. The hair cycle involves three distinct phases: anagen
(growing), catagen
(regressing), and telogen (resting). The epidermal stem cells of the hair
follicle are
activated by dermal papilla during late telogen. This is termed "bulge
activation".
Moreover, such stem cells are thought to be pluripotent stem cells, giving
rise not only
to hair and hair follicle structures, but also the sebaceous gland and
epidermis. Cell
proliferative agents and inhibitors of cell proliferation provide means for
altering the
dynamics of the hair growth cycle to, for example, induce quiescence of
proliferation of
hair follicle cells, particularly stem cells of the hair follicle.
Inhibitors of hair follicle cell proliferation can be employed as a way of
reducing
the growth of human hair as opposed to its convention removal by cutting,
shaving, or
depilation. For example, inhibitors of hair follicle cells identified using
the method of
the present invention can be used in the treatment of trichosis characterized
by
abnormally rapid or dense growth of hair, e.g., hypertrichosis. In an
illustrative
embodiment, such inhibitors can be used to manage hirsutism, a disorder marked
by
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39
abnormal hairiness. Use of such inhibitors can also provide a process for
extending the
duration of depilation.
Inhibitors of hair follicle cell proliferation can also be used to protect
hair follicle
cells from cytotoxic agents which require progression into S-phase of the cell-
cycle for
efficacy, e.g. radiation-induced death. Treatment with such inhibitors
provides
protection by causing the hair follicle cells to become quiescent, e.g., by
inhibiting the
cells from entering S phase, and thereby preventing the follicle cells from
undergoing
mitotic catastrophe or programmed cell death. For example, inhibitors of hair
follicle
cell proliferation can be used for patients undergoing chemo- or radiation-
therapies
which ordinarily result in hair loss. By inhibiting cell-cycle progression
during such
therapies, the inhibitor treatment can protect hair follicle cells from death
which might
otherwise result from activation of cell death programs. After the therapy has
concluded,
inhibitor treatment can also be removed with concomitant relief of the
inhibition of
follicle cell proliferation.
However, in order to start characterizing the molecular mechanisms underlying
hair growth control, as well as to test potential hair affecting drugs,
appropriate in vitro
models for hair growth are required. In one aspect of the present invention,
the subject
method is used to generate hair follicle micro-organ explants which retain the
microarchitecture of the follicle, e.g., the interaction between the hair
follicle epithelial
2o layer and stromal components (the dermal papilla) of the hair follicle,
e.g., one or more
of the stem cells, outer root sheath cells, matrix cells, and inner root
sheath cells. As
demonstrated in the appended examples, hair growth can be observed in these
micro-organ cultures even in the absence of serum, e.g., in a minimal media.
Importantly, the present invention also provides a hair follicle culture which
provide the
hair follicles in a substantially telogenic phase, e.g., resting. As
demonstrated below, the
telogenic hair follicle explants can be activated in the in vitro culture to
growing anagen
follicles, and in a certain embodiment, in a synchronized manner. The early
transient
proliferation of the epidermal stem cells of the follicle provide a unique
opportunity to
understand the activation of anagenic phase as mediated, for example, by
paracrine
3o and/or autocrine factors produced by the various tissues of the hair
follicle organ.
Moreover, the subject micro-organ cultures supply a system for, identifying
agents which modulate the activation or inactivation of the hair follicles,
e.g., to identify
agents which can either promote or inhibit hair growth. In one embodiment,
telogenic
(resting) hair follicle explants, such as described in Example XVIII below,
are contacted
with various test agents, and the level of stimulation of the hair follicles
is detected. For
example, transition of the hair follicle stem cells from telogen to anagen can
be
monitored by observing the mitotic index of the cells of the follicle, or some
other
similar method of detecting proliferation. To illustrate, Figure 17 shows that
thymidine
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incorporation can be used to measure the relative levels of stem cell
activation in the
explant in the absence or presence of the test compound (FGF in the figure)
with
increased proliferation indicative of a test agent having hair growth
promoting activity.
In the reverse assay, anagenic micro-organ explants are provided in culture,
e.g.,
5 such as the activated Sencar explants described in the appended examples, or
growth
factor stimulated explants (e.g., FGF stimulated). Test agents which inhibit
proliferation
of the hair follicle stem cells, e.g., relative to the untreated anagen
explants, could be
considered further for use as telogenic agents that prevent hair growth.
In still other embodiments, inhibitors of cell proliferation identified by the
t0 subject assay can be employed to inhibit growth of neoplastic or
hyperplastic cells, e.g.,
tumor formation and growth. A preferred embodiment of the invention is
directed to
inhibition of epithelial tumor formation and growth. For a detailed
description of skin
epithelial tumor formation, see United States Patent Application Serial Number
08/385,185, filed February 7, 1995. Tumor formation arises as a consequences
of
~5 alterations in the control of cell proliferation and disorders in the
interactions between
cells and their surroundings that result in invasion and metastasis. A
breakdown in the
relationship between increase in cell number resulting from cell division and
withdrawal
from the cell cycle due to differentiation or cell death lead to disturbances
in the control
of cell proliferation. In normal tissues, homeostasis is maintained by
ensuring that as
20 each stem cell divides only one of the two daughters remains in the stem
cell
compartment, while the other is committed to a pathway of differentiation
(Cairns,
J.(1975)Nature 255: 197-200). The control of cell multiplication will
therefore be the
consequence of signals affecting these processes. These signals may be either
positive or
negative, and the acquisition of tumorigencity results from genetic changes
that affect
25 these control points.
As described in Example IX and illustrated in Figure 12, skin micro-organ
cultures of the present invention have been used for identifying cell
proliferative agents
and inhibitors of cell proliferation. As described in Example IX, TGF- (3 was
tested
and found to act as an inhibitor of cell proliferation. Activin, a protein
which is a
3o member of the TGF-(3 superfamily, has also been shown to inhibit
proliferation of
epidermal cells. These results indicate there may be other members of the TGF-
(3 family
that play a role in inhibition of proliferation of epithelial cells. The data
suggests a role
for proteins in the TGF-/3 family as significant regulators of epidermal
homeostasis and
in inhibiting epithelial tumor formation and growth in vivo.
35 Another aspect of the present invention pertains to a method for
identifying a
cell differentiating agent, i.e., a compound which causes cell
differentiation. This
method includes isolating a population of cells from a subject wherein the
population of
cells having a microarchitecture of an organ or tissue from which the cells
are isolated, a
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41
surface area to volume index of at least about l.5mrri ~, and includes at
least one cell
which has the ability to differentiate. The cells are then placed in culture
for at least
about twenty-four hours and contacted with a candidate compound. The level of
cell
differentiation in the presence of the candidate compound is then measured and
compared with the level of cell differentiation in the absence of the
candidate
compound. A statistically significant increase in the level of cell
differentiation in the
presence of the candidate compound is indicative of a cell differentiating
agent.
Differentiation, as used herein, refers to cells which have acquired
morphologies and/or
functions different from and/or in addition to those that the cells originally
possessed.
Typically, these morphologies and functions are characteristic of mature
cells. The
differentiation of populations of cells of the present invention can be
monitored by
measuring production and/or secretion of specialized cell products.
In similar fashion, the present invention also pertains to a method for
identifying
an inhibitor of cell differentiation. Following the same protocol as above,
the level of
cell differentiation in the presence of the candidate compound is measured and
compared with the level of cell differentiation in the absence of the
candidate
compound. A statistically significant decrease in the level of cell
differentiation in the
presence of the candidate compound is indicative of an inhibitor of cell
differentiation.
In yet another embodiment, the subject cultures permit the generation of in
vitro
2o models for viral infection. For example, epidermal or squamous tissue can
be isolated,
and infected with such viruses as herpes viruses, e.g., herpes simplex virus
1, herpes
simplex virus 2; varicella-zoster virus; or human papilloma viruses, e.g., any
of human
papilloma viruses 1-58, e.g., HPV-6 or HPV-8. Similarly, hepatic models can be
provided for hepatitis infection, e.g., an explant infected with hepatitis
viruses, e.g.,
hepatitis A virus, hepatitis B virus, or hepatitis C virus. The virally-
infected tissue
explants can be used to identify inhibitors of viral infectivity by method of
the present
invention. As above, the particular micro-organ culture is provided, and
contacted
(optionally) with a virus which infects the cells to produce a population of
virus-infected
cells. The virus-infected cells can then be contacted with a candidate
compound and the
3o level of infectivity of the virus in the presence of the candidate compound
measured.
The measured level of viral infectivity in the presence of the candidate
compound is
then compared to the level of viral infectivity in the absence of the
candidate compound.
A statistically significant decrease in the level of infectivity of the virus
in the presence
of the candidate compound is indicative of an inhibitor of viral infectivity.
Methods of measuring viral infectivity are known in the art and vary depending
on the type of virus used. For example, one method which can be used to
measure the
level of viral infectivity is by measuring the level of production in the
infected cells of
the micro-organ culture or in the micro-organ culture medium of gene products
specific
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for the particular virus being tested. For example, to measure the level of
infectivity of
hepatitis virus, e.g., hepatitis B virus, of cells in a micro-organ culture,
hepatitis protein
production and hepatitis DNA can be quantitated. In general, micro-organ
culture
medium can be incubated with antibodies against a selected viral protein and
the
immunoreactive proteins analyzed by a variety of methods known in the art,
e.g., on
SDS-polyacrylamide gels, ELISA. For example, to measure production of
hepatitis B
surface antigen, micro-organ culture medium from micro-organs previously
incubated
with hepatitis B virus can be sampled at daily intervals and assayed for the
surface
antigen by an ELISA (Abbott) method as described by the manufacturer. This
method
can be modified for quantitation using serially diluted standard surface
antigen
(CalBiochem). A statistically significant decrease in the accumulation of
hepatitis B
surface antigen in the culture medium indicates that the candidate compound
tested is an
inhibitor of hepatitis virus infectivity.
In addition to measuring levels of HBsAg in the micro-organ culture medium,
newly synthesized hepatitis B virus DNA from cell extracts from the micro-
organ
culture can be detected and quantitated by PCR amplification of the DNA,
followed by
Southern blot analysis using labeled primer pairs in the HBV pre-S (HBsAg
encoding)
region as probes (see e.g., Sambrook, J. Et al. (1989) Molecular Cloning - A
Laboratory
Manual, Cold Spring Harbor Laboratory, 2nd ed., vol. 2, pp. 10.14-10.15).
Relative
2o quantitation can be achieved by densitometry and confirmed by scintillation
counting of
corresponding bands. Reduction in levels of newly synthesized viral DNA
indicate that
the candidate compound tested is an inhibitor of hepatitis virus infectivity.
In another example, the gag, pol, and env protein products of retroviruses,
e.g.,
human immunodeficiency virus (HIV), can also be measured using the above-
described
and other standard techniques known in the art. For example, pol protein
expression in
cells of micro-organ cultures infected with HIV can be measured by incubating
cell
extracts with anti pol antibodies or pooled AIDS patients sera and
immunoreactive
proteins analyzed on SDS/polyacrylamide gels. To measure infectivity of herpes
virus,
e.g., epstein/barr virus (EBV), in the micro-organ cultures of the present
invention, EBV
3o DNA and EBV -induced nuclear antigen production can be analyzed using the
methods
described herein.
The micro-organ cultures of the present invention can also be used to
promote wound healing in a subject. Thus, the present invention further
pertains to a
method for promoting wound healing in a recipient subject. This method
includes
isolating, from a donor subject, a population of cells having a surface area
to volume
index is at least approximately 1.5 mm -~. Typically, the population of cells
is placed in
culture for at least about twenty-four hours. The population of cells can then
be applied
to a wound of the recipient subject. In one embodiment, the wound or lesion,
is
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slow-healing or chronic, e.g., a wound associated with diabetes , e.g., a
burn, e.g., an
ulcer. As demonstrated in Examples X and XI, skin micro-organ cultures of the
present invention can be used as micro-explants to be applied to chronic
wounds
(Example X) and can form a viable implant capable of growing hair (Example
XI).
In still another embodiment, the subject micro-organ explants are provided in
an
assay to test for cytotoxicity or for irritation. In an exemplary embodiment,
the subject
method provides a technique for in vitro testing of ocular and dermal in-
itants. The
process, much like above, involves the topical application of liquid, solid
granular or
gel-like materials (e.g., cosmetics) to the micro-organ cultures of the
present invention,
to followed by detection of the effects produced in the culture.
Currently, potential eye and skin irritation of many chemicals, household
cleaning products, cosmetics, paints and other materials are evaluated through
direct
application to animals or human subjects. However, as is appreciated by most
in the
industry, such approaches are not met with overwhelming public support. The
present
method provides an alternative assay which does not require sacrifice or
permanent
maiming of an animal and also provides data in an objective format. In an
illustrative
embodiment, skin micro-organ cultures are derived according to the present
invention.
The cultured explants are contacted with a test agent, such as a cosmetic
preparation,
and the cell viability is assessed at some time after the exposure. In a
preferred
embodiment, an MTT assay (based on the reduction of a tetrazolium dye by
functional
mitochondria) is used to score for viability.
The micro-organ cultures of the present application can additionally be used
to
identify factors involved in normal homeostasis of tissues and cells, study
the effect on
the normal homeostatic of tissues and cells of changes in the environment of
the cells
including changes in nutrients and the presence of potentially toxic agents,
study the
pathway of changes in the tissues and cells that are triggered at the
beginning and during
pathogenesis or trauma; identify repair mechanisms that reverse the adverse
effects in an
altered environment associated with pathogenesis or trauma; study
developmental
regulation of cells that differentiate during the normal homeostasis of the
tissue and
3o developmental regulation of specialized structures (e.g., hair follicles)
within the tissue;
and for organ supplementation where pieces of an individual's organ remain but
are
insufficient for replacing or regenerating damaged tissue such as occurs in
patients
which chronic skin ulcers, which have healing deficiencies caused by
inappropriate
blood supply, or where the local skin is unable to heal such as in the
conditions known
as type I or type II diabetes.
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Exemplification
The invention now being generally described, it will be more readily
understood
by reference to the following examples which are included merely for purposes
of
illustration of certain aspects and embodiments of the present invention, and
are not
intended to limit the invention.
Micro-organ cultures from animals including adult human skin, mouse, guinea
pig and rat skin have been isolated and grown for up to 21 days in culture.
However, it is
within the scope of the invention to maintain cultures periods of time beyond
21 days.
l0 Furthermore, it is within the scope of the invention to form micro-organ
cultures
from a wide range of animals. The range of animals is merely exemplified but
is not
limited to the samples provided below.
As described in the appended examples, micro-organ cultures were prepared
from skin and also from organs including the mammalian pancreas, liver,
kidney,
IS duodenum, esophagus and bladder. Similarly, micro-organ cultures of
epithelia from
mammalian cornea, kidney, breast tissue and various gut derived tissues in
addition to
the esophagus such as intestine and colon may also be prepared using the
methods of the
invention. Indeed, it is within the scope of the invention to isolate and
maintain
micro-organ cultures from any site which contains an epithelial/stromal
architecture
20 within the body.
The above notwithstanding, the subject micro-organ culture technique has been
used to preserve tissue explants in long-term culture from tissue not having
epithelial/stromal architecture, such as certain lymphoid tissue, e.g., thymus
and spleen
explants.
Example I
Preparation of Micro-Organ Cultures of Epidermis
Fresh skin was obtained after surgery, cleaned from underlying fat tissue and
cut
into 0.4 x 5 cm flaps, which are then transversely sectioned, using a tissue
chopper or
other suitable cutting means into 3001an sections under sterile conditions so
that the
final tissue segments had dimensions of 4 mm in width and 0.3 mm in thickness
(see
Figure 1). These micrograns were placed in a 24-well microplate in 400p1 of
DMEM in
the absence of serum under 5% COZ at 37°C, under constant shaking at 12
rpm for
periods of one to eight days. Twenty micro-explants were grown per well.
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Example II
Measurement Of Tl:e Proliferation of mouse, Guinea Pig and Human Epidermal
Micro-Organ Cultures
5 Micro-organ cultures were prepared according to Example I and proliferation
of
the cells was measured by analyzing the amount of DNA synthesis as follows.
Mouse
skin and guinea pig skin were grown for two days and human skin grown for four
days
after which BrdU was added to the medium at a final concentration of 100NM for
three
hours, followed by fixation of the cells in 4% formaldehyde. After fixation,
the cultures
t 0 were stained with goat anti-BrdU antibodies followed by anti-goat-FICT
labeled IgG.
Histological preparations were embedded in following fixation in 4%
formaldehyde and
cut into 3 lan slices and stained with methylene blue.
It was found that the fraction of cells synthesizing DNA in vitro after two to
four
days in culture increased up to 10 fold compared with the values observed in
vivo, after
t 5 which the rate of DNA synthesis gradually decreased but remained high for
up to 10
days in culture(see Figures 2, 3 and 4A-4D). Even at six days in culture, the
cells
maintained a steady state of proliferation and differentiation so that the
tissue
architecture was preserved (Figures SA-5C).
20 Example III
Proliferation of Cells in Micro-Organ Cultures of Various Sizes
Guinea pig micro-organs were prepared as in Example I. Whole thickness skin
strips 4 mm in width were sectioned into explants of varying thickness
including slices
25 of 300, 450, 600, 700, 900, 1200 and 3000~an thickness. These slices were
placed
individually into wells containing serum free medium for two days. BrdU was
added for
four hours before termination at a final concentration of 100~M. The explants
were then
fixed in 4% formaldehyde and stained with goat antibodies to BrdU followed by
an
anti-goat IgG FITC labeled secondary antibody preparation. The results of this
30 experiment are illustrated in Figure 6. The amount of BrdU incorporation as
a function
of the number of cells/unit tissue is significantly reduced as the thickness
of the explants
increases.
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Example IV .
Preparation of Pancreatic Micro-Organ Cultures and Measurement of Cell
Proliferation
Guinea-pig pancreas was removed and then cut into sections of 300~m in
thickness, 4 mm in width and 2mm in depth using an appropriate tissue chopper
and in
such a way that the pancreas microarchitecture was maintained. The micro-
explants
were grown in cultere for several time periods from two to eighteen days.
Seven
micro-organs were placed in each of 96 wells of a plate in 150N1 of serum-free
DMEM
1o under 5% COz at 37°C under constant shaking at 12 rpm. BrdU was
added three hours
before termination at a final concentration of 1001.iM and the explants were
then fixed in
4% formaldehyde and stained with goat antibodies to BrdU followed by anti-goat-
F1TC
labeled IgG. Figures 7A-7B illustrate that cells in the pancreas-derived micro-
organs
were actively proliferating.
Example Y
Preparation of Pancreatic Micro-Organ Cultures and
Measurement of Insulin Secretion into the Culture Medium
Adult pig pancreas micro-organ cultures were prepared as in the previous
examples for skin. Pancreases were removed, cut with scissors to an
approximate depth
of 2 mm and sliced into sections 300Ntn thick having a width of 4mm. The micro-
organ
cultures were grown for 14 days in serum free medium. Every two days, the
medium
was removed and fresh medium added. Collected media was assayed for insulin
content
using standard radioimmunoassay methods.
Example VI
Transplantation of Pig Pancreatic Micro-Organs into a Xebogeneic Subject
Adult pig pancreas micro-organ cultures were prepared as in the previous
examples for skin. Pancreases were removed, cut with scissors to an
approximate depth
of 2 mm and sliced into sections 3001 an thick having a width of 4 mm. The
micro-organ
cultures were then grown for different time periods of 0 to 5 days in serum-
free medium,
and after culturing, the pancreatic micro-organs were removed from the culture
and
transplanted into both the visceral and parietal mesoderm of rat hosts. The
micro-organs
survived for at least one month in vivo and became well vascularized. After
three, five,
seven and fourteen days in vivo, extensive cell proliferation could be
detected.
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Moreover, positive insulin staining was observed in vivo after four, seven,
and thirty
days post transplantation.
Example VII
Preparation of Liver, Kidney, Duodenum, Esophagus and Bladder Micro-Organ
Cultures and Measurement of Cell Proliferation in the Micro-Organ Cultures
Guinea-pig micro-organ cultures from several epithelial tissue containing
organs
were prepared as in previous examples for skin. Organs were removed and with
scissors,
t o were cut to an appropriate width of 2 mm, length of 3 mm, and sliced into
sections of
300pm thick. The microcultures were incubated for three, four and six days in
serum-free medium. Twelve hours before termination of the experiment, 3H-
thymidine
was added to the cultures of explants. At termination, the tissue was fixed,
rinsed
several times and counted in a scintillation counter. The results of this
experiment are
~5 illustrated in Figure 9. As shown in Figure 9, all tissues exhibited active
proliferation
which continued for six days as determined by uptake of 3H-thymidine.
Example VIII
Proliferation of Hair Follicles in Micro-Organ Cultures
Skin micro-organ cultures were prepared according to Example I and incubated
for two
days. BrdU was added three hours before termination of incubation. Cells were
fixed in
4% formaldehyde and stained with goat anti-BrdU antibodies followed by
anti-goat-FITC labeled IgG. Intact hair follicles that were present in vivo in
their normal
surroundings could be maintained under precisely controlled culture
conditions, without
the need of adding serum or any other exogenous factor. Hair follicle cells in
these
micro-organs were found to proliferate vigorously for several days under the
conditions
of the present method as indicated by the large number of hair follicles cells
that
incorporated BrdU (Figures l0A-l OC). The size distribution of hair shafts at
time zero
of a micro-organ guinea pig culture and after two weeks is shown in Figure 1
I. The
medium was exchanged every two days. Hair shaft sizehas been arbitrarily
classified as
small, medium and large. After nine days in culture, there was a clear shift
in size
distribution so that the percentage of small hairs decreased from 64% to 28%,
while
large shafts which were not present at the beginning of the culture
represented 30% of
the shaft population.
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Example IX
Preparation ofAssay for Measuring the Effect of a Candidate Compound On Cell
Proliferation
The cultures were prepared and maintained in defined medium in similar growth
conditions as described in Example I. Control samples were analyzed by
immunocytochemistry to determine that the micro-organ culture was maintained
in a
manner that was similar to that occurring in vivo.
1o Duplicated samples of skin micro-cultures were treated with TGF-~3 at 2.5
ng/ml. A
quantitative analysis of the number of BrdU labeled cells/explant was
performed
according to Example II. Greater than 90% inhibition of DNA synthesis was
observed in
the presence of TGF-(3 compared with controls (Figure 12).
Example X
A Method for PromotingHealing Chronic Non-Healing Skin Ulcers
According to this method, a small-area of normal, uninvolved skin graft is
removed
from the patient and full thickness micro explants of 4 mm in width and 0.3 mm
thick
2o are prepared as described in Example I. The preparation however differs
from Example
I in that the sectioning into 0.3 mm slices is deliberately incomplete so that
a series of
sections are held together as indicated in Figure 13, the upper epidermal
layers including
the stratum corneum. The design of this implant is directed to permitting the
nutrients to
reach all the cells but maintaining the tissue slices in a manipulatable
format. The
patient's wound is cleaned and surrounding skin edges are removed. The area
devoid of
skin is then carefully covered by the micro-explants, which are placed on the
wound
such that the non-sectioned edge is facing outward and the opposing sectioned
pieces
are suspended in the fluid within the wound. Sufficient micro-explants are
prepared to
substantially cover the wounded area. The treated region is then covered with
a suitable
3o dressing and allowed to heal.
Example XI
Proliferation of Hair Follicles In Vivo
An in vivo animal experiment was performed where a 1 cmz area of skin was
removed from a mouse and incompletely microsectioned so that the stratum
corneum of
the whole skin area was left intact as described above. The micro-organ was
reimplanted
into its original position in the mouse stitched and allowed to heal. The
implant
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remained viable, became incorporated into the animal tissue and new hair
shafts grew
from the implant after one to two weeks in culture. (See Figure 14).
Example XII
Human Psoriatic Skin Micro-Organ Cultures
Split-thickness psoriatic skin from an 82 year old patient was obtained after
autopsy using a dermatome. The skin was then sectioned into 0.5 x 5 cm flaps
which
were then transversely sectioned using a tissue chopper or other suitable
cutting device
into 300 lan sections. These micro-organ sections were placed in microplates
in
serum-free DMEM under 5% COZ at 37°C under constant shaking for periods
of one to
fourteen days. In some instances, growth factors were added to the culture
medium. The
medium was changed every two days. The human psoriatic skin proliferated
extensively
as micro-organ culture.
Example Xlll
Liver Micro-Organ Cultures Infected with Hepatitis Virus
Human, rat, mouse, and guinea pig liver was sectioned and cultured as
zo micro-organ cultures as described in Example VII. Active proliferation in
these
micro-organ cultures was detected using BrdU incorporation as described
herein. The
hepatocytes in these micro-organ cultures were determined to be functional as
measured
by assay of urea (Sigma Chemical, urea detection kit) and albumin production
(ELISA)
after at least 14 days in culture.
Human liver micro-organ cultures prepared above were incubated with sera from
patients positive for hepatitis B and hepatitis C virus. After 24 hours, the
medium was
removed and fresh DMEM with and without 10% normal fetal calf serum (FCS) was
added. Every two days, the culture medium was exchanged with fresh medium and
the
conditioned medium tested for viral particles using antibodies against the
viral protein
HBs. A significant increase in number of viral particles was detected after 4
days in
those micro-organ culture that were cultured in the presence of FCS.
Example XIV
Thymus and Spleen Micro-Organ Cultures
Mouse and rat micro-organ cultures from thymus and spleen were prepared
essentially
as in the previous examples for skin. Organs were removed and cut with
scissors to an
approximate width of 2mm and length of 3mm. These samples were then spliced
into
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explants of approximately 3001nn thick using an appropriate tissue chopper in
such a
way as to preserve the essential microarchitecture of the organ. The micro-
organs were
then incubated for l, 3, 5 and 10 days in serum free medium. Active
proliferation in
these micro-organ cultures was detected using BrdU incorporation as described
herein.
5
Example XV
Bone marrow Micro-Organ Cultures
Micro-organ cultures from bone marrow were prepared by carefully removing
the bone marrow intact from femurs of rats and mice. Since the diameter of the
marrow
in such explants is only about 1-2 mm, the marrow was directly sliced into
micro-organ
explants using 300Nrr~ thick using a tissue chopper. This method ensured the
microarchitecture of the marrow was preserved while at the same time retaining
a
surface/volume index amenable to long-term culture. The micro-organs were
incubated
15 for 3 days in serum free medium. Active proliferation of marrow cells in
these
micro-organ cultures was detected using BrdU incorporation as described
herein.
Example XVI
Delivery of Geue Products to Skin Micro-Organ Cultures
The high surface area to volume inherent to the micro-organ cultures of the
present invention allows easy access to tissues for a variety of gene transfer
techniques.
In this example, micro-organ cultures are transfected with foreign genes using
electroporation and lipofection. The micro-organ cultures can be transplanted
into
animals and survive for at least about thirty days in vivo and become
vascularized. This
demonstrates the feasibility of using MC cultures of tissues in ex vivo gene
therapy
protocols. A further advantage of the MC culture is that it can be
transplanted to a
defined position in the body, so that if necessary it could be readily removed
in the
future. This contrasts with cell suspension transplantation into the body in
which the
cells can migrate or become "lost" in normal tissue.
Guinea pig skin was dissected and sliced into sections with a width of 2 mm
and
a thickness of 300~m. The skin was cultured as a micro-organ in serum-free
Dulbecco's
minimum essential media with penicillin and streptomycin at the concentrations
recommended by the manufacturer. After one day in culture at 37°C and
5% Co2, the
skin micro-organ cultures were rinsed with DMEM without antibiotics and added
to a
0.4 cm gap disposable electroporation cuvette with 5001 of media on ice.
Ten micrograms of the plasmid DNA containing the indicated reporter genes
were added as shown (each plasmid had a cytomegalovirus promoter driving the
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expression of either a Vii- galactosidase (control) or luciferase reporter
gene. The
luciferase plasmid backbone was pRC-CMV (Invitrogen) fused in frame with the
firefly
luciferase gene.
The samples were electroporated at 220 mV and the capacitance varied as shown
in
Figure 15 (I-Ii=900pF, medium=500pF, low=250~F). NIH3T3 cells were treated at
250pF) with a Bio-Rad electroporation device. The samples were then further
incubated
with DMEM containing 10% bovine calf serum, penicillin, streptomycin, and
glutamic
acid for 2 days in a 24 well culture plate. The media was removed, and the
samples were
suspended in about 7001 of cell culture lysis reagent (Promega). The tissue
pieces were
l0 homogenized, and then 20p1 was added to 100p1 of luciferase assay reagent
(Promega),
and luminescence was detected in triplicate with the Packard TopCount. As a
positive
control, NIH3T3 cells from a 75cm culture flask were trypsinized, and treated
identically to the micro-organ cultures. As illustrated in Figure 15, at the
medium
(500~F) and low (250~F) capacitance settings, significant luciferase activity
was
detected. For comparison, similar amount of NIH3T3
immortal cultured cells were electroporated with the same plasmids at 250pF.
In another experiment, the transfection of the micro-organ explants was
accomplished by lipofection, which was observed to be more efficient than
electroporation. In particular, micro-organ cultures from guinea pig skin,
newborn
mouse skin, and rat lung were transfected with a plasmid containing a
luciferase
reporter. Briefly, the micro-organ cultures were grown at 37°C in 5.5%
COz in DMEN
with 1% penicillin/streptomycin and 1% L-glutamine for one day before
transfection.
The explants were plated on 24 well plates with 20 explants and 400p1 of media
per
well. For transfection, the micro-organ cultures were rinsed twice with
Optimem, and
l Opl of Lipofectin (Gibco BRL) +2pg of DNA + Optimem was added to each well
with
the final volume being 500 pl. The Optimem/Lipofectin/DNA solution was made
according to the Lipofectin manufacturer's directions. The cultures were then
incubated
for 5-6 hours at 37°C in 5.5% CO2. The Optimem/Lipofectin/DNA media was
then
replaced with 400 pl of DMEN with 1% penicillin/streptomycin, 1% L-glutamine
and
10% FCS, and the cultures incubated overnight at 37°C in 5.5% CO2. The
following
morning, the micro-organ cultures were removed, washed twice with 1X PBS, and
ground in a hand-operated glass tissue grinder in 750 pl of 1X cell culture
lysis buffer
(Promega). Luciferase activity from the transgene was detected using
Luciferase Assay
System (Promega), with the results reported in Figure 18.
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Example XVII
Delivery of Gene Products to Micro-Organ Cultures
Lung and thymus from an eight week old female Lewis rat were dissected and
processed
for micro-organ culturing as described in Example XV. The micro-organ cultures
were
placed in culture wells and transfected with cationic lipid/luciferase
encoding plasmid
DNA complexes for five to six hours while incubating at 37°C. The
cationic
lipid/plasmid DNA solution was aspirated, and the cultures were then incubated
in
medium plus 10% serum for two days, and then assayed for luciferase reporter
gene
1o expression (expressed in arbitrary light units). The results of this
experiment are
illustrated in Figure 16. As demonstrated in Figure 16, the lung, but not the
thymus
expresses the transfected luciferase gene under these conditions. As expected,
the
negative control (3-galactosidase transfected lung micro-organ culture (10 pl
cationic
lipid concentrate) was near machine background for light production (23 light
units).
Example XVIII
Hair Shaft Growth in vitro
New born mouse skin was obtained after surgery, cleaned of underlying fat
tissue and cut into 0.4 x 5 cm flaps, which were then transversely sectioned,
using a
tissue chopper or other suitable cutting devise into 3001an sections. The
micro-organs
were placed in microplates in DMEM in the absence of serum under 5% COZ at
37°C
under constant shaking for periods of I to 14 days. Certain of the micro-organ
explants
were contacted with a growth factor, e.g., FGF, which was added to the culture
media.
The medium was changed every 2 days.
New born "hairless" skin can be induced to produce hair shafts when grown in
MC cultures. Micrographs of skin from 30 hr-old mouse, grown in micro-organ
cultures
for 3 days in the presence of 1 ng/ml EGF indicated the development of hair
shafts in
the explants, which growths were not present at the beginning of the culture
period.
3o In another set of experiments, activation of telogen follicles was
observed. The
Sencar mouse provides a useful model to study hair follicle activation because
the
follicles are well synchronized and the cycle stages have been well
characterized. Sencar
mice provide an in vivo model for anagen activation. The removal of the club
from a
telogen follicle can induce new hair formation, the first signs of which, are
well
characterized. Skin from adult Sencar mice was obtained after surgery, cleaned
from
underlying fat tissue and cut into 0.4 x 5 cm flaps, which were then
transversely
sectioned, using a tissue chopper or other suitable cutting device into 300
fan sections.
The micro-organs were placed in microplates in DMEM in the absence of serum 5%
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COZ at 37°C under constant shaking for periods of 1 to 14 days.
Activation of telogenic
follicles, whether induced by club removal or growth factor treatment, was
manifested
by the proliferation of follicle stem cells. Figure 17 illustrates the
activation of a
telogenic explant, as detected by thymidine incorporation.
Example IXX
Preparation of Pancreatic Islets For Transplantation
Several techniques have been developed to prepare islet cells from various
to mammalian sources, in large quantities since they constitute a potentially
transplantable
beta cell mass with which to treat established type 1 diabetes. Two main
drawbacks
have been encountered so far. It has proven difficult to obtain a reproducible
reliable
way of preparing beta cells. Second, the viability of these cells both in
vitro and in vivo
is largely variable . In part due to the first reason and in part due to the
fact that the
IS (3-cells most likely require support from the stroma that underlies the
islets in the normal
pancreas. Attempts of course at maintaining pancreatic organs ex vivo have so
far been
unsuccessful. Using the MC culture technology described herein, success has
been
achieved for establishing micro-organ cultures of mouse, rat, guinea pig and
pig
pancreas in vitro in defined culture medium
20 Pancreas micro-organ cultures have now been grown in vitro for periods of
up to
one month. Within the cultures, explants maintain their tissue
microarchitecture and
certain cell subpopulations proliferate actively as determined by BrdU
incorporation and
' labeling. Furthermore the islet cells secrete insulin into the medium even
after one
month of in vitro culture.
25 Transplantation experiments have been performed in which pig micro-organ
pancreas cultures have been implanted into both the visceral and parietal
mesoderm of
rat hosts. Explants have been kept for periods varying from a few days up to
one month
in vivo. The explants become well vascularized and incorporate into the tissue
host.
30 Example XX
Preparation of Human Psoriatic Skin Micro-organ Cultures
Split-thickness psoriatic skin from a patient was obtained after autopsy,
using a
dermatome. The skin was cut into 0.4 X 5 cm flaps, which were then
transversely
35 sectioned with a tissue chopper into 300~m thick sections. These micro-
organ explants
were cultured in DMEM (no serum) in microplates at 37°C and 5% COZ for
periods of 1
to 14 days. Inspection of the micro-organ explants at various time points
indicated that
the cells of the explant had remained viable, and proliferation was occurring.
CA 02464460 2004-04-22
WO 03/035851 PCT/ILO1/00976
54
All of the above-cited references and publications are hereby incorporated by
reference.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, numerous equivalents to the specific assay and
reagents
described herein. Such equivalents are considered to be within the scope of
this
invention and are covered by the following claims.
