DECELLULARIZED TISSUE ENGINEERED CONSTRUCTS AND TISSUES

CONSTRUCTIONS DE TISSU DECELLULARISE ISSUES DE L'INGENIERIE TISSULAIRE ET TISSUS AINSI PRODUITS

English Abstract

New methods for producing tissue engineered constructs and engineered native
tissues are disclosed. The methods include producing a tissue engineered
construct by growing cells in vitro on a substrate and then decellularizing
the
construct to produce a decellularized construct consisting largely of
extracellular
matrix components. The construct can be used immediately or stored until
needed. The decellularized construct can be used for further tissue
engineering,
which may include seeding the construct with cells obtained from the intended
recipient of the construct. During any of the growth phases required for
production of the construct, the developing construct may be subjected to
various
tissue engineering steps such as application of mechanical stimuli including
pulsatile forces. The methods also include producing an engineered native
tissue
by harvesting tissue from an animal or human, performing one or more tissue
engineering steps on the tissue, and subjecting the tissue to
decellularization.
The decellularized, engineered native tissue may then be subjected to further
tissue engineering steps.

Claims

59
WHAT IS CLAIMED IS:
1. A construct for use in the manufacture of a medicament for implanting
into a
subject comprising: a freeze-thawed, proteinaceous extracellular matrix,
wherein the
proteinaceous extracellular matrix has a thickness of greater than 50 pm, and
wherein the proteinaceous extracellular matrix was formed around a plurality
of
individual cells which were cultured in a three dimensional mass of living
tissue on a
flat or tubular artificial substrate.
2. The construct of claim 1, wherein the individual cells are smooth muscle
cells.
3. The construct of claim 1 or 2, wherein the individual cells are
allogeneic to the
subject.
4. The construct of any one of claims 1 to 3, which comprises no living
cells
seeded on the proteinaceous extracellular matrix.
5. Use of the construct as defined in any one of claims 1 to 4 in the
manufacture
of a medicament for the treatment of tissue damage or tissue loss.
6. Use of the construct as defined in any one of claims 1 to 4 for the
treatment of
tissue damage or tissue loss.

Description

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DECELLULARIZED TISSUE ENGINEERED CONSTRUCTS AND TISSUES
GOVERNMENT SUPPORT
The U. S. government has a paid-up license in this invention and the right
under certain circumstances to require the patent owner to license others on
reasonable terms as provided for by the terms of grant number HL-03492 awarded
by the NHLBI.
BACKGROUND OF THE INVENTION
Tissue damage, dysfunction, or loss is a feature of a wide variety of medical
conditions. Atherosclerosis, in which formation of fatty plaques in blood
vessel walls
leads to narrowing of the vessels, is one well-known example. Accidents
frequently
result in damage to tendons, ligaments, and joints. Degenerative diseases such
as
arthritis represent another source of injury to such tissues. Systemic
diseases such
as diabetes, cancer, and cirrhosis are yet another cause of organ destruction
or
dysfunction.
In many of the situations described above, replacement of the damaged tissue
or organ is the best or even the only option. Transplantation from human
donors
(either live or cadaveric) has enjoyed significant success, and procedures
such as
liver, heart, and kidney transplants are becomingly increasingly common.
However,
the severe shortage of donors, the complexity of harvesting organs and
delivering
them to the recipient, and the potential for transmission of infectious agents
are
significant shortcomings of this approach. In some situations, such as
replacement of
blood vessels, vessels are removed from one portion of the body and grafted
elsewhere to bypass sites of obstruction. However, the number of available
vessels
is limited, and those available may not be optimal in terms of strength or
other
parameters.

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Use of synthetic materials or tissues derived from animals offer alternatives
to
the use of human tissues. For example, grafts made of synthetic polymers such
as
Dacron find use in the replacement of vessels. Mechanical prostheses are
widely
used to replace damaged heart valves. However, use of synthetic materials has
a
number of disadvantages. Frequently the material is immunogenic and can serve
as
a nidus for infection or inflammation. Use of animal tissues also poses
problems of
immunogenicity as well as the potential to transmit diseases. In addition,
harvested
animal tissues may be suboptimal in terms of size, shape, or other properties,
thus
limiting the utility and flexibility of this approach. There is a need for
innovative
approaches to the problem of replacing damaged or dysfunctional organs and
tissues.
Tissue engineering is an evolving field that seeks to develop techniques for
culturing replacement tissues and organs in the laboratory (See, for example,
Niklason, L. and Langer, R., Advances in tissue engineering of blood vessels
and
other tissues, Transplant Immunology, 5,303-306,1997). The general strategy
for
producing replacement tissues utilizes mammalian cells that are seeded onto an
appropriate substrate for cell culture. The cells can be obtained from the
intended
recipient (e.g., from a biopsy), in which case they are often expanded in
culture
before being used to seed the substrate. Cells can also be obtained from other
sources (e.g., established cell lines). After seeding, cell growth is
generally continued
in the laboratory and/or in the patient following implantation of the
engineered tissue.
Tissue engineered constructs may be used for a variety of purposes including
as prosthetic devices for the repair or replacement of damaged organs or
tissues.
They may also serve as in vivo delivery systems for proteins or other
molecules
secreted by the cells of the construct or as drug delivery systems in general.
Tissue
engineered constructs also find use as in vitro models of tissue function or
as models
for testing the effects of various treatments or pharmaceuticals.
Tissue engineering technology frequently involves selection of an appropriate
culture substrate to sustain and promote tissue growth. In general, these
substrates

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should be three-dimensional and should be processable to form scaffolds of a
desired shape for the tissue of interest. Several classes of scaffolds are
known.
These scaffolds fall into five general categories: (1) non-degradable
synthetic
polymers; (2) degradable synthetic polymers; (3) non-human collagen gels,
which are
non-porous; (4) non-human collagen meshes, which are processed to a desired
porosity; and (5) human (cadaveric) decellularized collagenous tissue.
These different scaffold types are further discussed below.
Non-degradable synthetic polymers, e.g., Dacron* and Teflon*, may be
processed into a variety of fibers and weaves. However, these materials are
essentially non-biodegradable and thus represent a nidus for infection or
inflammation following implantation into the body. Degradable synthetic
polymers,
including substances such as polyglycolic acid, polylactic acid,
polyanhydrides, etc.,
may also be processed into various fibers and weaves and have been used
extensively as tissue culture scaffolds. These materials may be modified
chemically
to "tune" their degradation rate and surface characteristics. However,
fragments of
degradable polyesters can trigger significant and undesirable inflammatory
reactions.
Non-human collagen gels, e.g., gels made from bovine collagen and rat-tail
collagen are convenient materials to work with in the laboratory, but suffer
from
significant drawbacks including poor tensile strength, no void volume to allow
cell
growth and tissue development, and sensitivity to collagenases that weaken the
gels
over time. Non-human collagen meshes consist of porous meshes made from
processed bovine collagen. While the utility of these meshes for tissue
engineering
applications has been little studied, as with all materials made from bovine
proteins
they carry the risk of immunologic and/or inflammatory reactions when
implanted into
a human patient as well as the risk of contamination with agents of prion-
based
disease.
* trademarks

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In summary, none of the tissue culture scaffolds presently available is fully
satisfactory from all points of view. Thus there exists a need for improved
tissue
culture scaffolds for use in tissue engineering.
In general, tissue culture scaffolds represent an intermediate in the
production
of tissue engineered products. The need for improved tissue culture scaffolds
represents one aspect of the broader need for improved tissue engineered
products
for implantation into a human or animal to replace or supplement diseased,
damaged, or absent tissues and/or organs. As in the case of animal tissues,
tissue
engineered tissues created using cells that are not obtained from the intended
recipient may be antigenic. On the other hand, when using cells obtained from
the
intended recipient, a considerable period of time may be required to produce
the
tissue engineered tissue or organ, given that only a limited number of cells
can be
harvested. There is therefore a need for improved methods of producing tissue
engineered tissues and organs with minimal antigenicity. There is also a need
for
more flexible methods of producing tissue engineered tissues and organs, for
example, methods that would allow use of cells from the intended recipient
while
minimizing the time required to produce the engineered tissue or organ.
SUMMARY OF THE INVENTION
The present invention provides methods for producing scaffolds for use in
tissue engineering and for producing tissue engineered constructs and
engineered
tissues for implantation into the body. The invention also provides scaffolds
for use in
tissue engineering, tissue engineered constructs, and engineered tissues
suitable for
implantation into the body based on the inventive methods. In addition, the
invention
provides methods for treating an individual in need of replacement or
enhancement
of a tissue or organ by implantation of the engineered constructs or tissues
of the
invention.

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In one aspect, the invention provides methods for producing decellularized,
tissue engineered constructs and also provides decellularized, tissue
engineered
constructs produced according to the inventive methods. In a preferred
embodiment
of the method a substrate is seeded (i.e., contacted) with a first population
of cells,
preferably cells known to secrete extracellular matrix molecules such as
collagen and
elastin. The substrate can be flat, tubular, or, in general, can be configured
to
assume any desired three-dimensional shape. In a particularly preferred
embodiment
of the invention the substrate is tubular. Preferably the substrate consists
of a
biocompatible material, e. g., a biocompatible polymer having properties or
incorporating modifications conducive to cell adherence and/or growth.
In one aspect, the invention provides a construct for use in the manufacture
of
a medicament for implanting into a subject comprising: a freeze-thawed,
proteinaceous extracellular matrix, wherein the proteinaceous extracellular
matrix
has a thickness of greater than 50 pm, and wherein the proteinaceous
extracellular
matrix was formed around a plurality of individual cells which were cultured
in a three
dimensional mass of living tissue on a flat or tubular artificial substrate.
In one aspect, the invention provides a use of the construct of the invention
in
the manufacture of a medicament for the treatment of tissue damage or tissue
loss.
In one aspect, the invention provides a use of the construct of the invention
for
the treatment of tissue damage or tissue loss.
Appropriate cell types for seeding the substrate include fibroblasts and
smooth
muscle cells. In certain embodiments of the invention, the cells used to seed
the
substrate are derived from an individual of the same species as the individual
into
which the construct will ultimately be implanted in order to minimize
immunogenicity.
For example, if the construct is to be implanted into a human being, then
human cells
may be used to form the primary cell-seeded construct.
The construct is maintained in culture under conditions appropriate for growth
of the cells for a growth period during which the cells secrete extracellular
matrix
molecules. In certain embodiments of the method multiple seedings and growth

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periods are employed. In certain embodiments of the invention more than one
cell
type is employed. For example, one or more seedings may be performed with a
mixture of cells of different types. Alternatively, each seeding may employ
cells of
only one type but the same type is not necessarily used for all seedings. In
certain
embodiments of the invention growth conditions, e. g., tissue culture media,
are
selected to promote deposition of extracellular matrix. In certain embodiments
of the
invention stimuli, e. g., pulsatile forces, are applied to the construct
during the growth
period (s). Such stimuli may be selected to promote the development of desired
properties such as mechanical strength.
After the cells have formed a tissue of the desired thickness, the construct
is
decellularized. Decellularization may be accomplished using any of a variety
of
detergents, emulsification agents, proteases, and/or high or low ionic
strength
solutions. In certain embodiments of the invention decellularization is
performed
under conditions and for sufficient times so that antigenic cells and cellular
components are substantially removed, leaving a decellularized tissue
engineered
construct (scaffold) consisting primarily of extracellular matrix components
such as
collagen and elastin. In certain embodiments of the invention the substrate
that was
initially seeded is substantially or entirely removed from the scaffold.
Following the decellularization process, the decellularized construct may be
washed to remove components of the decellularization solution. The
decellularized
construct may be subjected to additional tissue engineering steps as described
below. In certain embodiments of the invention the decellularized construct is
stored
for later use. Different methods may be used to preserve the decellularized
construct
during storage including cryopreservation and drying according to a variety of
protocols. Alternatively, the decellularized construct can be used immediately
for
further tissue engineering or implanted into the body of a subject. In certain
embodiments of the invention the construct is treated with a biologically
active agent
prior to implantation.

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In another aspect, the invention provides methods for producing engineered
constructs suitable for implantation into the body. In certain embodiments of
the
invention, a decellularized tissue engineered construct is prepared from a
tissue
engineered construct as described above. The decellularized tissue engineered
construct is implanted into the body and may recellularize in vivo. In certain
embodiments of the invention, prior to implantation, the decellularized
construct is
treated with any of a variety of agents to enhance the recellularization
process.
In an alternative method of the invention, prior to implantation into the body
the decellularized tissue engineered construct is seeded with a population of
cells to
form a seeded decellularized tissue engineered construct. Before this seeding,
the
construct may be treated in various ways to enhance recellularization. The
seeded
construct may be implanted into the body of a subject (e.g., an animal, or
preferably
a human) in need thereof or may be maintained under conditions suitable for
the
growth and/or differentiation of the cells for a growth period prior to
implantation.
In certain embodiments of the invention the cells employed for the seeding are
derived from an individual of the same species as the individual into which
the
engineered construct is to be implanted. The cells may be derived from the
same
individual into which the engineered construct is to be implanted. A
combination of
different cell types can be used. For example, the decellularized tissue
engineered
construct can be seeded with a mixture of cells. Different cell types can be
used to
seed different portions or surfaces of the construct. In certain embodiments
of the
invention the cell type(s) are selected in accordance with the ultimate use of
the
engineered construct. For example, if the construct is to be used as an
artery, then
appropriate cell types may include vascular cells such as endothelial cells,
smooth
muscle cells, and fibroblasts. If the construct is to be used to repair a
cartilagenous
structure, appropriate cell types may include chondrocytes and fibroblasts. In
certain
embodiments of the invention precursor cells are used to seed the
decellularized
culture scaffold. The precursor cells may differentiate during the second
growth
period and/or after implantation into an individual. In certain embodiments of
the

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,
8
method multiple seedings and growth periods are employed. In certain
embodiments
of the invention more than one cell type is employed. For example, one or more
seedings may be performed with a mixture of cells of different types.
Alternatively,
each seeding may employ cells of only one type but the same type is not
necessarily
used for all seedings. In certain embodiments of the invention stimuli, e.g.,
pulsatile
forces, are applied to the construct during the growth period (s). Such
stimuli may be
selected to promote the development of desired properties such as mechanical
strength.
In another aspect, the invention provides methods for producing
decellularized, engineered native tissues and also provides decellularized,
engineered native tissues produced according to the inventive methods. The
method
includes the steps of harvesting native tissue from an animal or human donor,
subjecting the native tissue to one or more tissue engineering steps, and
decellularizing the engineered native tissue. The tissue engineering step can
comprise seeding the native tissue with cells and maintaining the seeded
tissue for a
growth period under conditions suitable for the growth of the cells. The
tissue
engineering step can comprise applying a mechanical or electrical stimulus to
the
native tissue, e.g., a pulsatile stimulus.
Following decellularization the tissue may be implanted into the body of a
subject or subjected to further tissue engineering steps. Such steps may
include any
of the steps mentioned above, e.g., seeding with a population of cells and
maintaining the seeded tissue for a growth period under conditions conducive
to
growth of the cells. The tissue may also be stored and subsequently retrieved
for
use. In those embodiments of the invention in which the decellularized,
engineered
native tissue is seeded with cells, the cells may be derived from the
individual into
whom the tissue is to be implanted. Seeding with cells from the individual
into whom
the tissue is to be implanted may decrease the likelihood of immune system
rejection.

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In another aspect, the invention provides methods of treating an individual in
need of replacement or enhancement of a tissue or organ. In certain
embodiments,
the methods comprise producing a decellularized tissue engineered construct or
a
decellularized engineered native tissue and implanting the construct or tissue
into the
body of the individual in accordance with standard surgical procedures. In
certain
embodiments, the methods comprise producing a decellularized tissue engineered
construct or a decellularized engineered native tissue, seeding the construct
or tissue
with cells, and implanting the construct or tissue into the body of the
individual in
accordance with standard surgical procedures. In certain embodiments of the
inventive method the construct or tissue is maintained in culture for a growth
period
under conditions conducive to growth of the seeded cells prior to implantation
into the
body of the individual. In certain embodiments of the invention the construct
or tissue
is seeded with cells that are derived from the individual. After implantation,
cells from
the individual may migrate into the tissue in vivo, complementing the seeded
cell
population. The migration of cells into the construct may be enhanced, e. g.,
by
treating the construct with growth factors, chemotactic agents, or other
compounds
prior to or after implantation. The construct may include cells that are
genetically
engineered to produce one or more such growth factors, chemotactic agents,
etc.
DEFINITIONS
In order to more clearly and concisely point out the subject matter of the
claimed invention, the following definitions are provided for specific terms
used in the
description and appended claims.
Allogeneic -- With respect to a recipient, an allogeneic cell or tissue is a
cell or tissue
that originates from or is derived from a donor of the same species as the
recipient.
Animal -- As used herein, the term animal includes humans. Thus when referring
to
processes such as harvesting tissue from an animal, it is intended that the
animal

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,
can be a human. Although at times reference will be made herein to "an animal
or
human", this is not intended to imply that the term "animal" does not include
a
human.
Artificial substrate -- As used herein, the term artificial substrate includes
materials
such as degradable or non-degradable polymers synthesized in vitro (i.e., not
produced by a living animal or plant). Note that the polymer may be identical
to a
polymer produced by a living plant or animal, e.g., the polymer may be a
protein
produced using recombinant DNA technology. The substrate can also be, for
10 example, a length of tubing, which may be coated with any of a variety
of artificial
materials or materials obtained from natural sources. Artificial substrate
also
encompasses certain materials obtained by isolating and processing substances
produced by a living source. In particular, the term encompasses materials
obtained
by harvesting tissue from an organism and isolating and/or processing one or
more
extracellular matrix proteins produced by a living source and therefore
includes
collagen sponges or rafts. However, a tissue that remains substantially intact
and
substantially retains the structure in which it is naturally found within the
body of an
organism is not considered an artificial substrate but is instead considered a
native
tissue. Other than this exception, the term "artificial substrate" is not
intended to
impose any limitation with respect to either material or configuration.
Autologous -- With respect to a recipient, an autologous cell or tissue is a
cell or
tissue that originates with or is derived from the recipient.
Biologically active agent -- A naturally occurring or synthetic chemical
entity that is
capable of inducing a change in the phenotype or genotype of a cell, tissue,
organ, or
organism when contacted with the cell, tissue, organ, or organism.

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Cellular component -- This term refers to substances that constitute a portion
of a
cell, including cell membranes and macromolecules (e.g., nucleic acids or
polypeptides) normally found enclosed within a cell membrane, embedded within
a
cell membrane, or attached to a cell membrane. The term does not include
molecules that have been secreted by cells, e.g, extracellular matrix
components
such as collagen, elastin, and proteoglycans even if such molecules are linked
to the
cell surface.
Conditions suitable for growth -- Conditions suitable for growth of a
particular cell
type means an environment with conditions of temperature, pressure, humidity,
nutrient and waste exchange, and gas exchange, that are permissive for the
survival
and reproduction of the cells. With respect to any particular type of cells,
an
environment suitable for growth may require the presence of particular
nutrients or
growth factors needed or conducive to the survival and/or reproduction of the
cells.
Native tissue -- As used herein a native tissue is a tissue that is harvested
from an
animal or human and that remains substantially intact and substantially
retains the
structure in which it is naturally found within the body of the animal or
human.
Non-cellular structural components -- As used herein a non-cellular structural
component refers to a substance present within a biological tissue (either a
native
tissue or a tissue-engineered construct), the substance being derived from a
cell that
is or was present within the tissue but is not contained within the plasma
membrane
of a cell. Examples include collagen, elastin, proteoglycans, fibronectin, and
laminin.
Precursor cell -- The term "precursor cell" refers to a cell that is not fully
differentiated
but that has the capacity to either become more fully differentiated itself or
to give
rise to a cell (or cells) that is able to further differentiate. The precursor
cell may give
rise to one or more different cell types. The process by which the precursor
cell gives

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rise to a cell (or cells) that is able to further differentiate may involve
one or more
rounds of cell division. A stem cell is one type of progenitor cell. However,
the term
"progenitor cell" also includes cells that may have undertaken one or more
steps
along a differentiation pathway, e.g., that express one or more
differentiation
markers.
Primary cell-seeded construct -- A construct comprising an artificial
substrate that
has been seeded with a population of cells and maintained in culture under
conditions suitable for growth and/or division of the cells for a period of
time.
Secondary cell-seeded construct -- A primary cell-seeded construct that has
been
seeded with a second population of cells. The second population of cells may
be
substantially equivalent to the population of cells that was used to produce
the
primary cell-seeded construct or may differ therefrom.
Tissue engineered construct -- This term is generally used herein to refer to
a two or
three dimensional mass of living mammalian tissue produced primarily by growth
in
vitro. The construct may include one or more types of tissue, and each tissue
may
include one or more types of cells. The term also encompasses a two or three
dimensional mass of living mammalian tissue produced at least in part by
growth in
vivo on an artificial substrate. A tissue-engineered construct is
distinguished from an
explant of a corresponding natural tissue, e.g., a native tissue, in that the
primary
growth of the construct occurs in vitro.
Xenogeneic -- With respect to a recipient, a xenogeneic cell or tissue is a
cell or
tissue that originates from or is derived from a donor of a different species
than the
recipient.

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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a bioreactor including a tubular substrate suitable for growth
of a
tissue engineered construct.
Figure 2A shows a photomicrograph of an untreated tissue engineered small
caliber
artery stained with hematoxylin and eosin at 66X magnification.
Figure 2B shows a photomicrograph of an untreated tissue engineered small
caliber
artery stained with hematoxylin and eosin at 100X magnification.
Figure 3A shows a photomicrograph of a decellularized tissue engineered small
caliber artery stained with hematoxylin and eosin at 66X magnification.
Figure 36 shows a photomicrograph of a decellularized tissue engineered small
caliber artery stained with hematoxylin and eosin at 100X magnification.
Figure 4A shows a photomicrograph of a decellularized tissue engineered small
caliber bovine artery stained with hematoxylin and eosin at 66X magnification.
Figure 4B shows a photomicrograph of a decellularized tissue engineered small
caliber bovine artery stained with hematoxylin and eosin at 100X
magnification.
Figure 5A shows a photomicrograph of a tissue engineered small caliber porcine
artery prior to decellularization, stained with hematoxylin and eosin.
Figure 5B shows a photomicrograph of a tissue engineered small caliber porcine
artery following decellularization, stained with hematoxylin and eosin.

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Figure 6A shows a phase contrast view of a cross section of a seeded
decellularized
porcine artery cross section.
Figure 6B shows a fluorescent cross section of the same sample shown in Figure
6B.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
The invention provides a variety of new methods and products of utility in the
field of tissue engineering and replacement tissues and organs. More
specifically, in
one aspect the invention provides methods for producing tissue engineered
constructs that can be implanted into the body, e.g., as treatments for
conditions
involving tissue damage or dysfunction. As used herein, implanting into the
body
includes implanting on the body surface and/or attaching onto the body in
addition to
implanting within the body so that the implanted construct or tissue is
entirely
enclosed within the body. Thus the constructs and tissues of the present
invention
can include replacements for skin, cornea, and other tissues that are not
strictly
within the body.
In general, the constructs of the present invention are produced by first
growing a tissue engineered construct according to any of a variety of methods
as
described below. Typically these methods involve seeding (i.e., contacting) a
substrate with cells and culturing the seeded substrate under conditions
suitable for
growth of the cells to form a tissue engineered construct. As the cells grow
and divide
on and/or in the substrate they secrete extracellular matrix proteins such as
collagen
and elastin. The construct is cultured for a period of time sufficient to
produce a
construct of desired thickness and/or properties, consisting primarily of
secreted
proteins and cells. Various growth conditions can be selected to enhance this
process and/or to stimulate the development of desirable mechanical, physical,
or
biochemical properties, etc. Such growth conditions may include the use of
particular
growth media, the application of mechanical, electrical, and/or chemical
stimuli, etc.

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The cells may be derived from an animal or cell line of the same species as
the intended recipient, so that the resulting construct contains proteins that
will be
minimally antigenic and maximally compatible in the body. For example, if the
construct is to be implanted into a human, the cells may be human cells.
Although in
general production of the tissue engineered construct involves culturing the
developing tissue primarily in vitro, tissue engineered constructs produced at
least in
part by culturing the tissue in vivo are also within the scope of the
invention.
In certain embodiments of the invention, production of the tissue engineered
construct involves multiple rounds of cell seeding and intervening growth
periods.
10 The cells used in different seedings may be of the same or different
types and/or may
consist of multiple cell types. The growth periods and culture conditions may
be the
same or may vary between different growth periods. For example, in certain
embodiments of the invention, to produce a tissue engineered blood vessel, a
substrate is seeded with smooth muscle cells and cultured for a period of
time, e.g., 6
weeks. After this first growth period the construct is seeded with endothelial
cells and
cultured for a further growth period, e.g., 1-2 weeks.
Following production of the tissue engineered construct, regardless of the
particular steps employed in its production, the construct is decellularized.
Appropriate decellularization techniques remove cellular components while
leaving
the secreted proteins, e.g., collagen and elastin, substantially intact. Thus,
one
method of the present invention includes the steps of (i) producing a tissue
engineered construct by seeding a substrate with cells, allowing the cells to
grow in
culture, and optionally subjecting the construct to one or more additional
rounds of
cell seeding and growth; and (ii) decellularizing the tissue engineered
construct,
thereby producing a decellularized construct. The first step can include
performing
various tissue engineering manipulations such as applying mechanical or
electrical
stimuli to the developing construct, applying selected biologically active
agents to the
construct (e.g., growth factors). In certain embodiments of the invention the
decellularized construct retains substantially the same shape and physical
properties

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16
as prior to decellularization. In particular, connective tissues such as blood
vessels,
muscle, bone, tendon, and ligament, all of which have substantial components
of
extracellular matrix proteins, derive most of their mechanical strength from
their
extracellular matrix components. The contribution of cells to the physical
characteristics of connective tissues is rather small. Thus treatments that
remove
cells while causing little damage to the extracellular matrix are preferable.
In preferred embodiments of the invention the decellularized construct is
thoroughly washed to remove residual decellularization solution that may
reduce
biocompatibility or inhibit subsequent growth of cells on or in the construct.
In certain
embodiments of the invention the decellularization process and/or subsequent
washing steps result in removal of most or substantially all of the substrate
(i.e., the
material on which the cells were initially seeded) that remains after the
growth period.
Following decellularization, the construct can be implanted into the body of a
subject
or stored before further use. In the latter case, when a patient is in need of
an
implanted tissue, the construct can readily be reconstituted. Such
reconstitution may
include the removal of residual storage solution, etc. Thus the inventive
method
includes one or more of the optional steps of (i) washing the decellularized
construct;
(ii) removing some or all of the remaining substrate; (iii) storing the
decellularized
construct; (iv) reconstituting the decellularized construct; and (v)
implanting the
decellularized construct into a subject.
In contrast to tissues harvested from animal or human donors, the substrate
(and therefore the decellularized construct itself) can be configured to
assume a
particular desired shape and size without the constraints that are imposed by
the
shape or size of harvested tissue. For example, engineered blood vessels can
be
grown to a certain desired length or diameter without undesired structures
such as
side branches or valves. Also, in contrast to decellularized animal tissues,
the cell-
derived proteins in the decellularized construct can come from cells of the
same
species as the intended recipient. While not wishing to be bound by any
theory,
human extracellular matrix proteins are expected to be essentially non-
immunogenic

CA 02861285 2014-08-25
17
when implanted into a human, which may make them preferable to proteins
derived
from animal sources and also preferable to synthetic degradable or non-
degradable
polymers. This is so because the extracellular matrix components that make up
the
non-cellular structural components of a decellularized construct are highly
conserved
within a species. For example, genetic variants in collagens and elastins are
quite
rare. Furthermore, cells (e.g., fibroblasts or smooth muscle cells) can be
obtained
from a single donor and used to produce large numbers (e.g., hundreds) of
constructs. These cells may be rigorously screened for transmissible diseases
(e.g.,
HIV or hepatitis), thus decreasing the infectious risk associated with the
products.
Engineered tissues can be produced using cells that have desirable properties
such
as an ability to grow well in culture, that have been genetically modified to
alter, for
example, their secretion of extracellular matrix components, etc.
In certain embodiments of the invention the decellularized construct is used
as
a scaffold for further tissue engineering. In the case that the scaffold was
stored after
the decellularization process, the scaffold may be reconstituted as
appropriate
depending upon the storage technique employed. The decellularized construct,
also
referred to herein as a scaffold, is seeded with a population of cells, which
may be
substantially equivalent to the population of cells that was used to seed the
substrate
or may be different in one or more respects. For example, the cells used to
seed the
scaffold may be of a different cell type or species from the cells that were
used to
produce the decellularized construct. In general, the cells are of the same
species as
the intended recipient and are of a cell type characteristic of the tissue or
organ that
the construct is intended to replace or augment. For example, if the construct
is a
blood vessel, the cells preferably include endothelial cells and smooth muscle
cells.
As in the case of initial production of the tissue engineered construct,
multiple rounds
of cell seeding and intervening growth periods can be employed. The cells used
in
different seedings may be of the same or different types and/or may consist of
multiple cell types. The growth periods and culture conditions may be the same
or
may vary between different growth periods. Various growth conditions can be

CA 02861285 2014-08-25
,
18
selected to enhance this process and/or to stimulate the development of
desirable
mechanical, physical, or biochemical properties, to stimulate migration of
cells into
the wall of the construct, etc. Such growth conditions may include the use of
particular growth media, the application of mechanical, electrical, and/or
chemical
stimuli, etc. Thus, in general, the decellularized construct (scaffold) may be
subjected
to any of the tissue engineering steps involved in production of a tissue
engineered
construct.
In certain embodiments of the invention the decellularized construct is seeded
with cells obtained from the individual who is the intended recipient of the
construct.
This approach minimizes the likelihood that the construct will cause an
immunological or inflammatory reaction when implanted into the recipient. This
embodiment of the invention represents an especially advantageous strategy for
the
production of a cell-based implantable tissue. For example, using current
techniques
it takes approximately 6-10 weeks of culture time to produce an implantable
tissue
engineered artery from cells that are seeded and grown on degradable polymer
scaffolds (Niklason, et al., Functional arteries grown in vitro, Science, 284:
48993,1999). The availability of decellularized and mechanically robust
collagenous
scaffolds as provided by the present invention dramatically shortens this
production
time. According to one embodiment of the inventive methods, when a patient who
would benefit from an implantable vessel is identified, a small biopsy is
taken from
the patient and the cells isolated. The cells are then seeded onto a
decellularized
scaffold and grown in culture for a period of several days to one or two
weeks. Then
the complete, essentially autologous vessel is implanted. This approach
reduces the
total culture time to produce an autologous vessel from 6-10 weeks to 1-2
weeks, a
reduction with profound implications from the point of view of clinical
applicability. Of
course the methods have similar benefit with respect to other implantable
tissues,
e.g., heart valves, bladders, etc.
The scaffold may be treated in any of a variety of ways either before or after
seeding. For example, agents selected to enhance the adherence or growth of
the

CA 02861285 2014-08-25
19
cells may be applied to the scaffold. After seeding, the seeded scaffold may
be
implanted into a subject or may be cultured for one or more additional growth
periods
(i.e., in addition to the period(s) of growth prior to decellularization). In
the latter case,
various growth conditions can be selected to enhance cell growth and division
and/or
to stimulate the development of desirable mechanical, physical, or biochemical
properties, to stimulate migration of cells into the wall of the scaffold,
etc. Such
growth conditions may include the use of particular growth media, the
application of
mechanical, electrical, and/or chemical stimuli, etc.
Thus in summary the inventive methods optionally include the additional steps
of (i) seeding the scaffold (i.e., the decellularized construct) with a
population of cells,
thereby obtaining a cell-seeded decellularized construct; and (ii) implanting
the
cellseeded decellularized construct into a subject. Prior to the second of
these steps
the construct may be maintained in culture for a period of time under
conditions
suitable for growth and/or division of the cells, thereby producing a tissue
engineered
decellularized construct. As in the case of production of the initial tissue
engineered
construct, the tissue engineered, recellularized, decellularized construct may
be
subjected to multiple rounds of cell seeding and growth, each of which may
involve
different cell type(s) and/or different growth conditions. During one or more
growth
periods the tissue engineered decellularized construct may be subjected to
various
tissue engineering manipulations such as the application of mechanical or
electrical
stimuli.
In general, the constructs of the present invention can be treated with any of
a
variety of biologically active agents prior to implantation into a subject. In
certain
embodiments of the invention these agent(s) are selected to enhance the
properties
of the construct following implantation, e.g., to facilitate the ability of
endogenous
cells (i. e., cells present within the subject) to populate the construct, to
enhance the
growth of seeded cells, to facilitate vascularization of the construct, to
reduce the
likelihood of thrombus formation, etc. Appropriate biologically agents
include, but are
not limited to, thrombomodulators, agents that increase hemocompatibility, and

CA 02861285 2014-08-25
antibiotics. In certain embodiments of the invention the biologically active
agent
comprises a pharmaceutical composition. In this case the construct may serve
as a
drug delivery vehicle. The pharmaceutical composition may be intended for
treatment
of the same condition as that being treated by implanting the construct or for
treatment of a different condition.
In addition to the decellularization of constructs obtained through tissue
engineering techniques (e.g., constructs obtained by seeding an artificial
substrate),
the present invention also encompasses the decellularization of native tissue
that has
been subjected to certain tissue engineering step(s) prior to
decellularization. For
10 example, the native tissue may be cultured for a period of time under
conditions
suitable for growth and division of the cells contained therein after
harvesting. The
native tissue may be seeded with additional cells. The growth conditions
(e.g., the
growth medium) may be selected to enhance cell growth and division and/or to
stimulate the development of desirable mechanical, physical, or biochemical
properties. For example, mechanical or electrical forces may be applied to the
native
tissue. Following the tissue engineering steps the native tissue is
decellularized and
may then be stored, implanted into the body of a subject, or used as a
scaffold for
further tissue engineering. In the latter case, the decellularized native
tissue is
seeded with a population of cells and may then be used or further processed in
20 essentially the same manner as the decellularized constructs described
above.
In the following sections, techniques and conditions for production of a
primary
cell-seeded construct, techniques for decellularization of the primary
cellseeded
construct to produce a decellularized construct (scaffold), methods for
storage of the
scaffold, and methods for reconstitution of the scaffold after storage are
described in
further detail. Methods for using the scaffold to produce a tissue engineered
construct for implanting into the body are also described in more detail
below. In
addition, tissue engineering steps that may be applied to a harvested native
tissue
prior to decellularization are described.

CA 02861285 2014-08-25
21
In certain embodiments of the invention the construct to be decellularized
comprises a tissue engineered construct produced as described in the pending
patent application entitled, "Tissue-Engineered Constructs", Ser. No.
09/109,427,
filed 07/02/98.
Production of a Tissue Enqineered Construct
Numerous methods and techniques for producing tissue engineered
constructs are known in the art and are appropriate for use in conjunction
with the
present inventive methods. Examples of suitable seeding and culturing methods
for
the growth of three-dimensional cell cultures are disclosed in pending
application
"Tissue-Engineered Constructs" Ser. No. 09/109,427; U. S. Pat. No. 5,266,480,
and
U. S. Patent No. 5,770,417. These references disclose techniques for
establishing a
three-dimensional matrix, inoculating the matrix with the desired cells, and
maintaining the culture. In general, a tissue engineered construct is produced
by
seeding cells onto an appropriate substrate and culturing the cells under
conditions
suitable for growth. The substrate can be flat, tubular, or, in general, can
be
configured to assume any desired three-dimensional shape. For example, the
substrate may be formed into shapes including but not limited to spheres,
ellipsoids,
disks, sheets, or films as well as hollow spheres, hollow ellipsoids, and open-
ended,
hollow tubes. In certain embodiments of the invention the substrate is
tubular.
In certain embodiments of the invention the substrate comprises a
biocompatible material, e.g., a biocompatible polymer having properties or
incorporating modifications conducive to cell adherence and/or growth.
Suitable
materials include materials that are biodegradable or bioerodable, such as
materials
that hydrolyze slowly under physiological conditions. Porous materials are
preferred
in certain embodiments of the invention. Among the various suitable materials
are
synthetic polymeric materials such as polyesters, polyorthoesters, or
polyanhydrides,
including polymers or copolymers of glycolic acid, lactic acid, or sebacic
acid.
Substrates comprising proteinaceous polymers are also suitable for production
of
tissue engineered constructs. Collagen gels, collagen sponges and meshes, and

CA 02861285 2014-08-25
,
22
substrates based on elastin, fibronectin, laminin, or other extracellular
matrix or
fibrillar proteins may be employed. Either synthetic polymers or proteinaceous
polymers may be modified or derivatized in any of a variety of ways, e.g., to
increase
their hydrophilicity and/or provide improved cell adhesion characteristics. In
certain
embodiments of the invention the substrate is coated with a material, e.g.,
denatured
collagen, prior to seeding in order to increase adherence of the cells
thereto.
Materials useful as substrates for growing cells to produce tissue engineered
substrates, and methods of producing such substrates are known in the art and
are
described in pending application "Tissue-Engineered Constructs", Ser. No.
09/109,427, and in U. S. Patent No. 5,770,417.
In certain embodiments of the invention some or all of the substrate degrades
during the growth period and/or is removed prior to implantation of the
construct into
a subject. Removal may be accomplished by application of a fluid flow and may
be
enhanced by decellularization. In certain embodiments of the invention a
tissue
engineered construct is grown on a structure from which it is completely
removed
after a growth period. For example, a vascular construct may be grown on a
length of
silicone tubing that has been coated with a thin layer of dilute, denatured
human
collagen to which cells can adhere. After the growth period the silicone
tubing is
removed from the vascular construct, resulting in a tissue engineered
construct
entirely free of substrate.
A number of different cell types or combinations thereof may be employed in
the present invention, depending upon the intended function of the tissue
engineered
construct being produced. These cell types include, but are not limited to:
smooth
muscle cells, cardiac muscle cells, epithelial cells, endothelial cells,
urothelial cells,
fibroblasts, myoblasts, chondrocytes, chondroblasts, osteoblasts, osteoclasts,
keratinocytes, hepatocytes, bile duct cells, pancreatic islet cells, thyroid,
parathyroid,
adrenal, hypothalamic, pituitary, ovarian, testicular, salivary gland cells,
adipocytes,
and precursor cells. For example, smooth muscle cells and endothelial cells
may be
employed for muscular, tubular constructs, e.g., constructs intended as
vascular,

CA 02861285 2014-08-25
23
esophageal, intestinal, rectal, or ureteral constructs; chondrocytes may be
employed
in cartilaginous constructs; cardiac muscle cells may be employed in heart
constructs; hepatocytes and bile duct cells may be employed in liver
constructs;
epithelial, endothelial, fibroblast, and nerve cells may be employed in
constructs
intended to function as replacements or enhancements for any of the wide
variety of
tissue types that contain these cells. In general, any cells may be employed
that are
found in the natural tissue to which the construct is intended to correspond.
However,
in some cases it may be advantageous to employ cells of a type that is not
naturally
found in the tissue to which the construct is intended to correspond. In
addition,
progenitor cells, such as myoblasts or stem cells, may be employed to produce
their
corresponding differentiated cell types. In some instances it may be preferred
to use
neonatal cells or tumor cells.
In certain embodiments of the invention the cells are allogeneic to the
intended recipient rather than xenogeneic. Cells may be obtained from a donor
(either living or cadaveric) or derived from an established cell line. To
obtain cells
from a donor (e.g., a potential recipient of a tissue engineered construct),
standard
biopsy techniques known in the art may be employed. Representative techniques
are
described, for example, in pending application "Tissue-Engineered Constructs",
Ser.
No. 09/109,427 and in Oberpenning, F., et al., De novo reconstitution of a
functional
mammalian urinary bladder by tissue engineering, Nature Biotechnology, 17, 149-
155,1999. The contents of this article, which also describes appropriate
materials
and techniques for creation of three-dimensional substrates, cell culture and
cell
seeding techniques, and methods for evaluation of tissue engineered organs.
Cells
so obtained may be expanded in culture, although preferably cells of a low
passage
number (e.g., less than 5 or, more preferably, less than 3) are used to
produce the
construct in order to minimize loss of the differentiated phenotype.
Preferably cells
isolated from a donor are screened to eliminate the potential for transmission
of
infectious diseases. Cells derived from established cell lines (e.g., those
available
from the ATCC, Rockville, MD) may also be used. In certain embodiments of the

CA 02861285 2014-08-25
24
invention cells (either obtained from a donor or from an established cell
line) that
have been genetically manipulated by the introduction of exogenous genetic
sequences or the inactivation or modification of endogenous sequences are
employed. For example, genes may be introduced to cause the cells to make
proteins that are otherwise lacking in the host. Production of scarce but
desirable
proteins (in the context of certain tissues) such as elastin may be enhanced.
As mentioned above, in order to minimize antigenicity in certain embodiments
of the invention cells from the same species as the intended recipient of the
final
construct are employed to create the initial tissue engineered construct
(i.e., the
construct that is to be decellularized). Thus if the construct is to be
implanted into a
human, preferably cells derived from a human are used to create the initial
construct.
In those embodiments of the invention in which the decellularized construct is
employed as a scaffold for further tissue engineering (i.e., those embodiments
in
which the decellularized construct is seeded with cells), cells from the same
species
as the intended recipient of the final construct are preferably used to seed
the
decellularized construct. In certain embodiments of the invention the
decellularized
construct is seeded with cells harvested from the intended recipient of the
construct.
General mammalian cell culture techniques, cell lines, and cell culture
systems that
may be used in conjunction with the present invention are described in Doyle,
A.,
Griffiths, J. B., Newell, D. G., (eds.) Cell and Tissue Culture: Laboratory
Procedures,
Wiley, 1998.
In certain embodiments of the invention mammalian cells are seeded onto
and/or within a substrate from a suspension so that, preferably, they are
evenly
distributed at a relatively high surface and/or volume density. The substrate
may be,
but need not be, a porous substrate. The cell suspensions may comprise
approximately 1 X 104 to 5 X 107 cells/nil of culture medium, more preferably
approximately 2 X 106 cells/ml to 2 X 107 cells/ml, and yet more preferably
approximately 5 X 106 cells/ml. The optimal concentration and absolute number
of

CA 02861285 2014-08-25
cells may vary with cell type, growth rate of the cells, substrate material,
and a variety
of other parameters. The suspension may be formed in any physiologically
acceptable fluid, preferably one that does not damage the cells or impair
their ability
to adhere to the substrate. Appropriate fluids include standard cell growth
media
(e.g., DMEM with 10% FBS).
The cells may be seeded onto and/or within a substrate by any standard
method. For example, the substrate may be seeded by immersion in a cell
suspension for a period of time during which cells adhere to the substrate,
followed
by washing away the nonadherent cells. The substrate may be seeded with cells
10 using a syringe, pipet, or other sterile delivery apparatus. According
to a preferred
method the cell suspension is dripped onto the substrate, and the substrate is
subsequently rotated, e.g., in a rotating vessel to promote even distribution
of the
cells.
Following seeding of the cells, in certain embodiments of the invention the
cells are allowed to adhere to the substrate for a period of time (seeding
time) prior to
placing the seeded substrate in tissue culture medium. The optimum seeding
time
varies with cell type and substrate. For example, when using the synthetic
hydrophilic
polymeric substrates disclosed in pending application "Tissue-Engineered
Constructs", Ser. No. 09/109,427, seeding times of approximately 20 minutes
may be
20 used. For other substrates, seeding times of an hour or more may be
appropriate and
have been employed in the prior art.
Various treatments may be applied to enhance adherence of cells to the
substrate and/or to each other. Appropriate treatments are described, for
example, in
the above-mentioned pending application and in U.S. Patent No. 5,613,982. Such
treatments include the application of various proteins, e.g., growth factors
or
extracellular matrix proteins to the substrate or to the growing construct.
For
example, collagen, elastin, fibronectin, laminin, or proteoglycans may be
applied to
the substrate. The substrate can be impregnated with growth factors such as
aFGF,

CA 02861285 2014-08-25
26
bFGF, PDGF, TGFb, VEGF, etc., or these agents may be provided in the culture
medium.
Appropriate growth conditions for mammalian cells in culture are well known in
the art. Cell culture media generally include essential nutrients and,
optionally,
additional elements such as growth factors, salts, minerals, vitamins, etc.,
that may
be selected according to the cell type(s) being cultured. Particular
ingredients may be
selected to enhance cell growth, differentiation, secretion of specific
proteins, etc. In
general, standard growth media include Dulbecco's Modified Eagle Medium, low
glucose (DMEM), with 110 mg/L pyruvate and glutamine, supplemented with 10-20%
fetal bovine serum (FBS) or calf serum and 100 Wm! penicillin are appropriate
as are
various other standard media well known to those in the art. A particularly
preferred
culture medium for producing a muscular, tubular tissue engineered construct
such
as a small caliber blood vessel is described in Example 2 below. Preferably
cells are
cultured under sterile conditions in an atmosphere of 5-15% CO2, preferably
10%
CO2, at a temperature at or near the body temperature of the animal of origin
of the
cell. For example, human cells are preferably cultured at approximately 37 C.
In general, the length of the growth period will depend on the particular
tissue
engineered construct being produced. The growth period can be continued until
the
construct has attained desired properties, e.g., until the construct has
reached a
particular thickness, size, strength, composition of proteinaceous components,
and/or
a particular cell density. Methods for assessing these parameters are
described in
pending application "Tissue-Engineered Constructs", Ser. No. 09/109,427, and
in
U.S. Patent No. 5,613,982.
Following a first growth period the construct can be seeded with a second
population of cells, which may comprise cells of the same type as used in the
first
seeding or cells of a different type. The construct can then be maintained for
a
second growth period which may be different in length from the first growth
period
and may employ different growth conditions. Multiple rounds of cell seeding
with
intervening growth periods may be employed.

CA 02861285 2014-08-25
27
In certain embodiments of the invention a muscular, tubular tissue engineered
construct is grown in a biomimetic system such as that described in pending
application "Tissue-Engineered Constructs", Ser. No. 09/109,427 and in
Niklason, et
al., Functional arteries grown in vitro, Science, 284: 489-93,1999. As
described
therein, a semi-disposable glass bioreactor similar to that shown in Figure 1
and
discussed in Example 1 of the present application is attached to a pump
system. As
shown in Figure 1, the bioreactor chamber 22 includes side arms 12 through
which a
length of tubing 14 is inserted. The tubing serves as a support for a
substrate 16
which is seeded to produce the construct. Alternately, the tubing itself may
serve as a
substrate either with or without a layer or layers of coating material. Fluid
can be
pumped through the tubing to impart a pulsatile force to the lumen of the
developing
construct as discussed below. The bioreactor includes a stopper 18 that can be
removed to place the substrate within the reactor and to seed the substrate
with
cells. Culture medium and other fluids are added to and removed from the
chamber
via a medium fill port 20. The bioreactor system may be made of glass or of
another
appropriate material such as various plastics. In those embodiments of the
invention
in which the decellularized construct is cryopreserved, the bioreactor is
preferably
made of a material such as plastic capable of withstanding extremely low
temperatures (e.g., that of liquid nitrogen).
Application of stimuli during growth period
Tissues within the body are subjected to a variety of physical stimuli. For
example, arteries, heart valves, and heart chambers are exposed to pulsatile
stretch
and flow forces as blood is pumped through the cardiovascular system.
Components
of the musculoskeletal system are subjected to mechanical forces during
walking and
other physical activities. It is well established that physical stimuli can
exert profound
effects on the properties and development of tissues and of the cells that
produce
these tissues. Without wishing to be bound by any theory, we propose that
exposing
developing tissue engineered constructs to certain stimuli (e.g., mechanical
forces

CA 02861285 2014-08-25
28
that resemble those to which corresponding tissues would normally be exposed
in
vivo) will cause the resulting construct to develop properties and structure
that more
closely resemble those of the corresponding naturally occurring tissue. In
some
instances the application of appropriate stimuli may result in desirable
properties,
e.g., increased strength, that exceed those found in the naturally occurring
tissue.
Therefore, in certain embodiments of the invention a physical stimulus (e.g.,
a
mechanical or electrical stimulus) is applied to the tissue engineered
construct during
the growth periods. The strength and nature of the stimulus may be varied
during the
growth period, and the stimulus need not be applied continuously throughout
the
growth period but may be applied during one or more portions of the growth
period.
In the case of a construct that is produced by performing multiple rounds of
cell
seeding with intervening growth periods, different stimuli may be employed
during
different growth periods.
In certain embodiments of the present invention, as described in detail in
pending application "Tissue-Engineered Constructs" Ser. No. 09/109,427, a
muscular, tissue engineered construct is produced in which a distensible body
is
inserted within the lumen of a substrate to provide pulsatile stretch to
seeded muscle
cells. While the muscle tissue is growing on and/or within the substrate, a
pump in
communication with the interior of the distensible body provides cyclic
increases in
pressure to cause the distensible body to distend within the lumen of the
substrate
and impart a pulsatile stretching force to the substrate and the developing
tissue. The
application of pulsatile stretching forces may be used in the production of
both
vascular tissue engineered constructs and muscular, nonvascular constructs
such as
esophageal, intestinal, rectal, ureteral, or bladder constructs. The forces
applied to
the construct may be selected to mimic corresponding natural forces in terms
of
pulsation and the degree of stretch imparted to the construct.
In certain embodiments of the invention forces are applied to a muscular,
tubular tissue engineered construct without the use of a distensible tube. For
example, fluid such as tissue culture medium can be pumped directly through
the

CA 02861285 2014-08-25
s
29
lumen of the construct, thus mimicking intraluminal flow as found in arteries
in the
body. The flow may be varied as the construct develops, and the intraluminal
pressure and shear forces may even be increased beyond those found in the
body.
Of course the application of physical forces is not limited to muscular and/or
tubular tissue engineered constructs but may be advantageously employed in the
production of a variety of other types of tissue engineered constructs. For
example,
pulsatile flow can be employed in the production of heart valves as described
in U.S.
Patent No. 5,899,937. The application of stimuli is not limited to the
application of
pulsatile stimuli, stretching forces, or stimuli related to fluid flow. For
example,
compressive stimuli, either constant or cyclical may be employed. In addition,
non-
mechanical stimuli such as electrical stimuli may be employed.
Decellularization
The methods discussed in this section may be applied to a tissue engineered
construct produced according to any of the methods described above or to a
native
tissue that has been harvested from a subject. In the former case, the result
of
decellularizing is to produce a decellularized, tissue engineered construct.
The
decellularized, tissue engineered construct can be implanted into a subject,
subjected to further tissue engineering steps that may include seeding with
cells, or
used for other purposes. A native tissue can be subjected to tissue
engineering steps
before decellularization to produce an engineered, decellularized native
tissue. Of
course the engineered, decellularized native tissue can be subjected to
additional
tissue engineering steps after decellularization.
Decellularization has a number of effects. In particular, in the case of a
tissue
engineered construct that is produced using cells that are allogeneic to an
intended
recipient (i.e., cells that are derived from the same species as the
recipient), the
extracellular matrix proteins such as collagen and elastin that make up a
large
portion of the construct are substantially non-immunogenic when implanted into
the
recipient. However, the cells themselves are generally immunogenic when
implanted

CA 02861285 2014-08-25
into a subject other than the individual from whom the cells were derived (or
a
genetically identical individual). For example, pure human collagen (either
obtained
from human tissue or produced using recombinant DNA technology) is generally
nonimmunogenic when implanted into a human subject. However, human cells that
produce collagen are generally immunogenic when implanted into a human being
other than the individual from which they were derived. In other words, in a
typical
tissue engineered construct the cells constitute the majority of the antigenic
material
in the construct. Therefore, by removing the cells, it is possible to
substantially
reduce or eliminate the likelihood that an immunologic or inflammatory
reaction will
10 be induced upon implanting the construct into a subject.
Any of a number of decellularization methods can be employed. In general the
methods employ a variety of chemical, biochemical, and/or physical means to
disrupt, degrade, and/or destroy cellular components and/or modify the matrix
in
which the cells are embedded so as to facilitate removal of the cells and
cellular
components. Such methods are disclosed, for example, in U.S. Pat. No. 4,
776,853,
U.S. Pat. No. 5,192,312, U.S. Pat. No. 5,336,616, U.S. Pat. No. 5,595,571,
U.S. Pat.
No. 5,613,982, U.S. Pat. No. 5,855,620, U.S. Pat. No. 5,899,936, and U.S. Pat.
No.
5,916,265. Additional decellularization methods are disclosed in Bader, A., et
al.,
Tissue engineering of heart valves-human endothelial cell seeding of detergent
20 acellularized porcine valves, Eur. J. Cardio-thoracic Surg, 14,279-
284,1998 and in
Courtman, D. W., et al., Biomechanical and ultrastructural comparison of
cryopreservation and a novel cellular extraction of porcine aortic valve
leaflets, J.
Biomed. Mat. Res., 29,1507-1516,1996. Of course the invention is not limited
to
these decellularization techniques but also includes modifications of these
techniques, as well as other techniques currently available or developed in
the future.
The decellularization method preferably does not cause gross alteration in the
structure of the tissue engineered construct or native tissue or cause
substantial
alteration in its biomechanical properties. The effects of decellularization
on structure
may be evaluated by light microscopy, ultrastructural examination, etc.

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31
Biomechanical tests, which are well known in the art, may be used to evaluate
the
effects of various decellularization protocols on tissue properties. Selection
and
interpretation of such tests will depend, in general, upon the nature of the
construct
and the purpose for which it is intended. In addition, the treatment
preferably does
not result in a cytotoxic environment that significantly inhibits subsequent
steps such
as seeding in vitro or population of the construct or tissue by cells of a
recipient in
vivo.
In certain embodiments of the invention the construct or tissue to be
decellularized is incubated in one or more decellularization solutions for a
period of
time sufficient to remove a substantial fraction of the cells and/or cellular
components. In general, the decellularization solutions enhance cell lysis and
destruction of cellular components, e.g., they contain agents that disrupt
and/or
degrade cellular constituents such as cell membranes, proteins, nucleic acids,
etc.
Aqueous hypotonic or low ionic strength solutions facilitate cell lysis
through osmotic
effects. Such solutions may comprise deionized water or an aqueous hypotonic
buffer (e.g., at a pH of approximately 5.5 to 8, preferably approximately 7 to
7.5).
Decellularization may be accomplished using a single decellularization
solution, or
the construct may be incubated sequentially in two or more solutions. Another
approach involves immersing the construct in alternating hypertonic and
hypotonic
solutions.
Suitable decellularization agents include salts, detergent/emulsification
agents
and enzymes such as proteases, and/or nucleases. Combinations of different
classes of detergents, e.g., a nonionic detergent such as Triton X-100
(tertoctylphenylpolyoxyethylene) and an ionic detergent such as SDS (sodium
dodecyl sulfate) may be employed. In a particularly preferred embodiment of
the
inventive method, one or more decellularization solutions that include Triton
X-100 ,
CHAPS (3-[(3-cholamidopropyI)-dimethyl-ammonio]-1-propanesulfonate), or SDS in
phosphate buffered saline (PBS) is employed as described in the Examples
below.

CA 02861285 2014-08-25
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32
Other suitable detergents include polyoxyethylene (20) sorbitan mono-oleate
and
polyoxyethylene (80) sorbitan mono-oleate (Tween* 20 and 80), sodium
deoxycholate, and octyl-glucoside.
In general, it is preferable to employ a decellularization technique that
minimizes damage to or alteration of the proteinaceous matrix. Such damage may
result from proteases (e.g., collagenase) that may be released upon lysis of
cells or
that may be present in the matrix extracellularly. Therefore, in certain
embodiments
of the invention various additives such as metal ion chelators, e.g., EDTA
(ethylenediaminetetraacetic acid) and/or protease inhibitors are included in
the
decellularization solution. Suitable protease inhibitors for use in
decellularization
solutions include, for example, one or more of the following:
phenylmethylsulfonyl-
fluoride (PMSF), aprotinin, leupeptin, and N-ethylmaleimide (NEM).
Various enzymes that degrade cellular components may be employed in the
decellularization solution. Such enzymes include nucleases (e.g., DNAses such
as
DNAse I, RNAses such as RNAse A), and phospholipases (e.g., phospholipase A or
C). Certain proteases such as dispase* II, trypsin, and thermolysin may be of
use in
decellularization, particularly in decellularization of native tissues such as
skin. When
employing proteolytic enzymes it may be desirable to take care that removal of
cells
occurs without significant damage to the extracellular matrix. The activity of
proteases is a function of time, temperature, and concentration, and these
variables
may be appropriately adjusted to achieve acceptable decellularization without
unacceptable destruction of the extracellular matrix. Nucleases are typically
employed at a concentration of between 0.1 pg/ml and 50 pg/ml, preferably
approximately 10 pg/ml for DNAse I and approximately 1.0 pg/ml for RNAse A.
The
nucleases are preferably employed in a physiologically buffered solution at a
temperature of between approximately 20 C to 38 C, preferably 37 C, for a time
between approximately 30 minutes to 6 hours.
* trademarks

CA 02861285 2014-08-25
33
As mentioned above, the decellularization solution typically includes a
buffer.
Suitable buffers include organic buffers such as Tris (hydroxymethyl)
aminomethane
(IRIS), (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), etc.
Buffers including sodium phosphate, citrate, bicarbonate, acetate, or
glutamate may
also be used. In general, a pH between about 5.5 and 8.0, between about 6.0
and
7.8, or between about 7.0 and 7.5 is employed.
Physical forces such as the formation of intracellular ice may be employed as
a primary means of accomplishing decellularization or to augment the activity
of
decellularization solutions. One such approach referred to as vapor phase
freezing
involves placing the construct or tissue in an appropriate solution, e.g., a
standard
cryopreservation solution such as Dulbecco's Modified Eagle Medium (DMEM), 10%
dimethylsulfoxide (DMSO), 10% fetal bovine serum (FBS) and cooling at a slow
rate,
e.g., 1-2 C/min. Multiple freeze-thaw cycles may be employed. Colloidforming
materials may be added to the solution to reduce extracellular ice formation
while
allowing formation of intracellular ice. Appropriate materials include
polyvinylpyrrolidone (10% w/v) and dialyzed hydroxyethyl starch (10% w/v).
The examples of decellularization techniques provided above are not intended
to be limiting, and the invention encompasses the use of essentially any
decellularization technique that removes a substantial fraction of the cells
while
leaving the matrix substantially intact. Of course it is to be understood that
certain
techniques will be preferred for particular tissue engineered constructs or
native
tissues, depending upon the properties of these constructs or tissues. One of
ordinary skill in the art will be able to select an appropriate
decellularization technique
and to vary parameters such as temperature and time in order to achieve a
desired
degree of decellularization. In certain embodiments of the invention the
decellularization process removes at least 50% of the cells. In certain
embodiments
of the invention the decellularization process removes at least 60%, at least
70%, or
at least 80% of the cells. In certain embodiments of the invention at least
90%, at
least 95%, or substantially all of the cells are removed. As described above,
there

CA 02861285 2014-08-25
34
may be a tradeoff between the two goals of achieving a high degree of
decellularization and preserving the structure and properties of the
extracellular
matrix. Thus it is not necessarily preferred to achieve maximal possible
decellularization if doing so results in unacceptable damage to the
extracellular
matrix. The optimum degree of decellularization may depend upon the properties
of
the construct and the use for which it is intended.
Regardless of the decellularization method employed, in certain embodiments
of the inventive methods the decellularized construct or tissue is washed in a
physiologically appropriate solution such as PBS, tissue culture medium, etc.,
following removal from the solution in which decellularization was performed.
Washing removes residual decellularization solution that might otherwise cause
deterioration of the decellularized construct or tissue, inhibit the growth of
subsequently seeded cells, and/or reduce biocompatibility.
In certain embodiments of the invention decellularization is performed by
soaking the construct in decellularization solution(s) for a period of time.
The solution
may be stirred or agitated during this period. In addition, it may be
desirable to alter
the pattern of flow of the decellularization solution, e.g., by establishing
convective
currents within the container in which decellularization is performed, by
employing
rotating arms with paddles in appropriate locations in the container, etc.
Modifying
the flow pattern may improve transport of important decellularization agents
in the
solution and increase their transfer, improving percent decellurization. In
addition,
modifying the flow may enhance removal of cells and cellular components from
the
tissue.
In certain embodiments of the invention in which the construct is an
engineered vessel grown in a bioreactor, the decellularization solution can be
pumped through the inner lumen of the vessel to decellularize the inner
portion of the
vessel. In addition, the tissue culture medium can be removed from the
bioreactor
and replaced with decellularization solution to expose the outer portion of
the vessel

CA 02861285 2014-08-25
,
to elecellularizing conditions. Application of pulsatile forces (described
above) during
the decellularization period may be employed to enhance decellularization.
Following decellularization and washing, the decellularized tissue engineered
construct or decellularized engineered native tissue may be implanted into a
subject
in need thereof, e.g., as a replacement blood vessel, heart valve, organ,
etc., or may
be subjected to additional tissue engineering steps including seeding with
cells.
Alternatively, the decellularized construct can be stored for future use as
described
below.
10 Evaluating effects of decellularization
Various methods may be used to assess the effects of a particular
decellularization protocol in regards to the extent of decellularization
achieved and/or
in regards to the alterations in the non-cellular structural components (e.g.,
the
extracellular matrix). Samples of the tissue may be stained, e.g., with
hematoxylin
and eosin, and examined by light microscopy. When hematoxylin and eosin
staining
is employed extracellular matrix components appear pink, and nuclei appear as
purple spots as shown in Figures 2,3, and 4. Staining procedures and stains
that
differentiate between cells and extracellular matrix, and stains that
differentiate
between various extracellular matrix components (e.g. collagen and elastin)
are well
20 known in the art. The number of cells present in the tissue can be
determined by
visual inspection at about 20X to 100X magnification. To assess the percent
decellularization achieved, the number of cells present in a given area of
decellularized tissue is compared with the number of cells present in an
equivalent
area of control tissue that has not been subjected to decellularization. The
integrity of
the non-cellular structural components can also be assessed by visual
inspection.
For example, deterioration in these components may be evidenced by
fragmentation
or separation between fibrils of extracellular matrix material.
Other techniques for assessing the extent of decellularization include
immunohistochemistry and electron microscopy. lmmunohistochemistry may be used

CA 02861285 2014-08-25
36
to detect specific cellular components including components that may be
particularly
immunogenic such as histocompatibility antigens. Details regarding processing
of
tissues for light and electron microscopy and for immunohistochemistry may be
found
in the references cited at the beginning of this section, in particular Bader,
et al.
Other appropriate techniques are known to those of ordinary skill in the art.
An
estimate of the density of cells remaining after decellularization may also be
obtained
by determining the DNA content of the tissue, e.g., by measuring the
fluorescence
intensity of a dye such as Hoechst 33258 upon binding to DNA as described in
pending application "Tissue-Engineered Constructs", Ser. No. 09/109,427.
In certain embodiments of the invention the removal of cells and cellular
components results in reduced immunogenicity of the decellularized construct
as
compared with the construct before decellularization. A variety of approaches
may be
used to demonstrate the reduced immunogenicity of the decellularized
construct. For
example, the humoral immune response to extracts made from decellularized
constructs may be compared with the humoral immune response to extracts made
from control constructs that have not been decellularized (See Example 4 in
U.S.
Patent No. 5,613,982). Briefly, rabbits are immunized with NaCI extracts of
either
decellularized or control constructs, and immune sera are obtained. The immune
sera are screened for the presence of IgG and IgM antibodies against antigens
present in extracts made from non-decellularized (control) constructs. Another
approach to assessing the reduction in immune and inflammatory responses to
decellularized constructs compared with control constructs involves implanting
samples of the constructs into rabbits, removing the implants and surrounding
tissue
after a period of time such as two weeks, and subjecting the removed implants
and
tissue to histopathologic analysis (See Example 5 in U.S. Patent No.
5,613,982). The
presence of inflammatory and immune system cells in the samples serves as an
indicator of the degree of the inflammatory and immunologic response triggered
by
the implants. A variety of other methods known to those skilled in the art may
be

CA 02861285 2014-08-25
37
employed to assess the reduction in immunogenicity and inflammatory response
due
to decellularization of the tissue engineered constructs.
Storage of a decellularized construct
A decellularized construct or decellularized native tissue may be stored after
decellularization using any of a number of storage techniques. Storage of
decellularized constructs or tissues would provide ready access to these
materials
when needed. In a particularly advantageous embodiment of the invention, many
tissue engineered constructs are prepared using human cells obtained from a
single
preferred source (e.g., a single human donor whose cells have been screened to
reduce the likelihood of transmission of infectious diseases or a cell line
that exhibits
particularly preferred properties or has been genetically modified to enhance
its
ability to secrete extracellular matrix components). The tissue engineered
constructs
are decellularized and stored. When a subject who would benefit from
implantation of
a tissue engineered construct is identified, a stored construct is
reconstituted and
either implanted directly into the patient or subjected to further tissue
engineering,
e.g., seeding with cells obtained from the patient.
Cryopreservation (i.e., preserving by maintaining at an extremely low
temperature) is a method for storing the decellularized construct or
decellularized
native tissue. Freezing and vitrification are two different approaches
currently being
pursued. In both cases, prevention of destructive ice crystal formation is a
major
goal. For freezing, the tissue or organ to be cryopreserved is perfused with a
solution
containing a sufficient concentration (generally approximately 10% by volume)
of a
cryoprotective agent (CPA) so that ice formation is limited during subsequent
cooling.
Typical cryoprotectants include glycerol, dimethylsulfoxide (DMSO), glycols,
propanediol, polyvinylpyrrolidone, dextran, and hydroxyethyl starch.
Vitrification is a
cryopreservation technique involving solidification in an amorphous glassy
state that
minimizes or eliminates ice crystal formation and growth. In both cases,
tissues must

CA 02861285 2014-08-25
38
be typically cooled to temperatures below-100 C (e.g., in liquid nitrogen) for
long-
term stability. For vitrification, the tissue is perfuse with even higher
concentrations of
CPA than for freezing. Following incubation in the cryopreservation solution,
the
tissue may be packaged in a sterile container. In a preferred embodiment of
the
invention in which a tissue engineered construct is grown and decellularized
in a
bioreactor, the cryopreservation solution is introduced into the bioreactor,
which is
used as the storage container.
The choice and concentration of cryoprotectant, time-course for the addition
of
cryoprotectant, temperature at which the cryoprotectant is introduced, and
rate of
cooling and subsequent rewarming all play an important role in the success of
preservation procedures. A variety of specific procedures and methods for
preservation and reconstitution after storage have been developed and applied
to
various tissues and cells. Techniques for preserving tissues and organs,
including
blood vessels, heart valves, muskuloskeletal tissues, and collagenous tissues,
by
cryopreservation are described, for example, in U.S. Patent Nos. 4,890,4575;
5,131,850; 5,145,769; 5,158,867 and in U.S. Patent No. 5,336,616, which
discloses a
method for preserving an acellular, collagen-based tissue matrix. The method
includes incubating a decellularized tissue comprising a proteinaceous matrix
with a
cryoprotectant solution, followed by freezing at cooling rates such that
minimal
functional damage occurs to the proteinaceous matrix, drying the cryoprepared
tissue
under temperature and pressure conditions that permit removal of water without
substantial ice recrystallization or ultrastructural damage, storage of the
tissue, and
subsequent rehydration.
Techniques and reagents for vitrification are described in U.S. Pat. No.
4,559,298; U.S. Pat. No. 5,217,860, U.S. Patent No. 5,952,168, and U.S. Patent
No.
5,962,214. The methods disclosed in these references will be readily adaptable
to
the decellularized, tissue engineered constructs and decellularized,
engineered
native tissues disclosed herein.

CA 02861285 2014-08-25
39
Although cryopreservation represents a reliable approach to storing a
decellularized tissue engineered construct of the present invention,
alternative
methods are also within the scope of the invention. For example, drying
methods can
also be used, with the addition of stabilizing compounds such as sucrose. A
dextran
and sucrose combination provides desirable physical properties and protein
protection against freeze drying and air drying stresses. Freeze drying may
take
place using a lyophilizer. Air drying may take place under a stream of dry
nitrogen,
and the construct may then be lyophilized under a vacuum at room temperature.
Reconstitution of a decellularized construct
Depending upon the particular storage technique selected, the construct is
appropriately reconstituted before being implanted into a subject or used for
further
tissue engineering. Reconstitution preferably removes cryopreservation agents
that
are potentially toxic to cells and irritating if introduced into the body. In
addition,
preferred reconstitution techniques cause minimal alteration in the structural
components of the construct. In the case of cryopreservation, reconstitution
includes
warming (preferably rapidly) and removal of the cryopreservation solution as
described in the patents listed above. Removal of the cryopreservation
solution may
be accomplished by thorough washing, e.g, in normal saline or standard cell
culture
medium. If drying is employed, reconstitution includes rehydration (e.g., in
normal
saline or standard cell culture medium), as described in U. S. Patent No.
5,336,616.
Antibiotics and/or antifungal agents may be included in the solutions used for
rinsing
and/or rehydration to minimize the chance of contamination. Dried tissue can
be
exposed directly to a cell suspension, thereby reconstituting and seeding in
one step.
The dried tissue can be rinsed, e.g., with media to remove any drying agents
and
then soaked in media if needed before exposing the tissue to a cell
suspension.
Uses and further engineering of a decellularized tissue engineered construct

CA 02861285 2014-08-25
As mentioned above, a decellularized tissue engineered construct can be
implanted into the body of a subject in order to repair, replace, or augment a
tissue or
organ in need thereof. Implantation can be performed using any of a variety of
techniques, e.g., surgical techniques, known to those of skill in the art and
dependent
upon the particular function that the construct is intended to fulfill. For
example, a
decellularized vascular construct may be used in a bypass operation to replace
a
diseased blood vessel. A decellularized heart valve construct may be used in a
valve
replacement operation, e.g., to replace a stenotic or incompetent valve. In
the case
that the decellularized construct is implanted directly into a recipient, the
construct
10 may be repopulated iii vivo with the recipient's own cells. Various
agents such as
growth factors, etc., may be applied to the construct to enhance this process.
Such
agents may be applied, for example, prior to implantation, or after
implantation, e.g.,
by injection into or near the construct, by systemic delivery to the recipient
of the
construct, etc.
In certain embodiments of the invention the decellularized construct is
subjected to further tissue engineering steps prior to implantation into a
recipient.
Such steps can comprise seeding the construct with one or more populations of
cells,
preferably cells obtained from the intended recipient. For example, a
decellularized
vascular construct can be seeded with smooth muscle and/or endothelial cells
20 obtained from a biopsy specimen taken from the intended recipient. After
a period of
time during which the cells are allowed to adhere to the construct the seeded
construct can be implanted into the recipient. In certain embodiments of the
invention
the cells are genetically transformed so that they exhibit desirable
characteristics,
e.g., production of a protein or other molecule that is lacking in the
recipient or
production of a growth factor that stimulates cellularization or angiogenesis
in the
construct. In other embodiments of the invention the construct is impregnated
with a
bioactive agent such as a pharmaceutical composition prior to implantation
into the
recipient and thereby serves as a drug delivery vehicle.

CA 02861285 2014-08-25
41
In certain embodiments of the invention the decellularized construct is seeded
and cultured for a period of time prior to implantation into the recipient.
This growth
period may be relatively short (e.g., 1-2 weeks) compared with the time
required to
grow the construct prior to decellularization. This period allows the seeded
cells to
become established and commence division. However, since the construct already
possesses substantial mass and strength, it is not necessary to culture the
cells for
long enough to generate an extensive extracellular matrix. Of course the
decellularized construct can be subjected to multiple seedings and growth
periods. In
general, any or all of the techniques employed in the growth of the construct
prior to
decellularization may be employed during any growth phases that follow
decellularization. For example, substances described above may be applied to
the
decellularized construct to promote adherence of cells. Growth factors may be
applied to the construct and/or included in the medium to promote the growth
of cells
and/or the development or maintenance of a differentiated phenotype. In
certain
embodiments of the invention stimuli such as those described above (e.g.,
pulsatile
forces and/or fluid flow) are applied to the seeded, decellularized construct
during the
growth period(s) that follow decellularization.
Thus certain embodiments of the present invention involve producing a tissue
engineered blood vessel using a bioreactor system in which pulsatile and fluid
flow
stimuli are applied to a substrate that is seeded with smooth muscle cells and
endothelial cells that are allogeneic to an intended recipient. The substrate
is cultured
with the application of pulsatile stretch for approximately 6-8 weeks, during
which a
substantial proteinaceous extracellular matrix is secreted, and the construct
attains
desired physical properties and thickness. The construct is then
decellularized in the
bioreactor chamber, with the application of pulsatile forces and fluid flow
during the
decellularization period to enhance the decellularization process and
contribute to
removal of the substrate. The decellularized construct is stored until needed.
Following identification of an individual in need of a vascular graft, the
construct is

CA 02861285 2014-08-25
42
retrieved from storage and is seeded with smooth muscle and endothelial cells
obtained from the intended recipient. After a relatively short culture period
(e.g., 1-2
weeks), during which pulsatile stimuli and/or fluid flow may be applied to the
construct, the recellularized construct is implanted into the recipient using
an
appropriate surgical procedure.
Assessing biomechanical properties and cell viability of constructs or tissues
It may be desirable to employ a decellularized construct that displays
biomechanical properties similar to or superior to those of the tissue or
organ to
which they correspond, particularly when the decellularized construct is to be
implanted into the body without being subjected to additional tissue
engineering
steps. A variety of methods may be employed to test the biomechanical
properties of
decellularized tissue engineered constructs or of decellularized constructs
that have
been subsequently cell seeded and cultured. The particular technique selected
will,
in general, depend upon the construct, and the desired biomechanical
properties will
depend upon the intended function of the construct following implantation into
a
recipient. Suitable methods for testing the biomechanical properties of a
vascular
construct are described in pending application "Tissue-Engineered Constructs",
Ser.
No. 09/109,427 (see Examples therein) and include measurement of burst
strengths
and compliances and measurement of suture retention strength. Stress-strain
analyses such as the single load versus elongation test, the stress relaxation
test,
and the tensile failure test are described in U.S. Patent No. 5,613,982 (see
Example
7) are also appropriate and may be applied, in general, to any type of tissue
engineered construct. Additional tests known to those of skill in the art may
also be
used.
In those embodiments of the invention in which the decellularized construct is
seeded and cultured prior to implantation into a recipient, it my be desirable
that the
construct is functional and viable prior to implantation. Various methods may
be used

CA 02861285 2014-08-25
43
to assess the functioning and viability of the construct. For example, cell
viability may
be assessed by trypan blue exclusion assay, by measuring total protein
synthesis
(e.g., by measuring incorporation of [3H] proline) or DNA synthesis (e.g., by
measuring incorporation of [3H] thymidine). More specific assays of cellular
activity
such as measurement of collagen production are also well known in the art as
described in U.S. Patent No. 5,613,982.
Production of a decellularized, engineered native tissue
The inventive methods described above involve the decellularization of a
tissue engineered construct. However, the methods may be extended to include
the
decellularization of a native tissue that has been harvested from a donor and
subjected to tissue engineering steps prior to decellularization. Methods for
harvesting tissues from donors (e.g., living or cadaveric animal or human
donors) are
well known in the art. Various methods have been employed to harvest vascular
tissues, heart valves, skin, organs such as kidneys, livers, lungs, hearts,
etc. In some
cases tissues or organs are harvested for purposes of transplanting them
directly into
a recipient, in which case the goal is generally to preserve the tissue or
organ in a
state as closely approximating the state in which it was removed from the
donor as
possible, and the harvested tissue or organ is subjected to minimal
processing. In
other instances, e.g., the harvesting of porcine heart valves to be used as
replacements for human heart valves, the harvested tissue may be subjected to
extensive chemical processing such as fixation, decellularization, cross-
linking, etc.,
to reduce immunogenicity and/or to improve physical characteristics. Processes
for
decellularizing harvested native tissue and repopulating it with new cells
have been
described (e.g., in U.S. Patent No. 5,192,312 and U.S. Patent No. 5,613,982).
However, treatment of harvested tissues prior to decellularization has
generally been
limited to storage and/or preservation of the tissue.

CA 02861285 2014-08-25
44
According to the present invention, harvested native tissue from an animal or
human donor is grown in culture. In certain embodiments of the invention the
native
tissue is seeded with cells before or after a culture period, although this is
not a
requirement. The cells may be of any of the types described above, but
preferably
the cells are derived from the same species as the intended recipient of the
engineered tissue. In general, any of the culture methods and techniques
described
in the context of producing a tissue engineered construct may be employed in
culturing harvested native tissue. For example, growth factors may be employed
to
promote cell growth or maintenance of a differentiated phenotype. Agents
selected to
promote adherence of cells may be applied to the native tissue. The harvested
native
tissue may be subjected to physical stimuli such as pulsatile stretch or fluid
flow
during the culture period as described above for tissue engineered constructs.
Following one or more culture periods, during which one or more cell seedings
may
be performed, the native tissue is decellularized. The decellularized,
engineered
native tissue may be used for any of the purposes described above for
decellularized
tissue engineered constructs.
Thus in summary this inventive method includes the steps of (i) harvesting a
native tissue; (ii) subjecting the native tissue to one or more tissue
engineering steps
to produce an engineered native tissue; and (iii) decellularizing the
engineered native
tissue to produce a decellularized, engineered native tissue. The tissue
engineering
step (s) comprise culturing the native tissue under conditions suitable for
growth and
can optionally include subjecting the native tissue to one or more cell
seedings with
optional intervening growth periods, subjecting the tissue to mechanical,
electrical,
and/or chemical stimuli. Such stimuli can include the application of pulsatile
stretch or
fluid flow forces, treating the native tissue with growth factors, etc. The
decellularized,
engineered native tissue can be used in any of the ways described above for a
decellularized tissue engineered construct. Thus the decellularized,
engineered
native tissue can be implanted into the body of a subject or can be used for
further
tissue engineering. The decellularized, engineered native tissue can be stored
and

CA 02861285 2014-08-25
reconstituted as described above. The decellularized, engineered native tissue
can
be seeded with cells, e.g., cells derived from the intended recipient, and can
be
maintained in culture prior to implantation into the recipient. Mechanical,
electrical,
and/or chemical stimuli can be applied during the culture period(s) following
decellularization.
EXAMPLES
Example 1
10 Preparation of a Primary Cell-Seeded Construct
This example describes the preparation of a tissue engineered construct
suitable for decellularization, in this case a small caliber artery, using a
bioreactor
system. A more detailed description of many aspects of this process is found
in the
pending application referenced above. As an initial step, a non-woven mesh
made of
fine polyglycolic acid (PGA) fibers (Albany International Research Co.,
Mansfield,
MA) was produced and further processed to yield a porous substrate with a
hydrophilic surface. The processing enhances wettability and increases the
number
of cells which are deposited on the surface during seeding. Briefly, the
treatment
began with three successive 30 minute washes in hexane, dichloromethane, and
20 diethyl ether followed by lyophilization overnight. The PGA mesh was
then placed
briefly in ethanol, removed to distilled water, and placed in a 1.0 normal
solution of
NaOH for 1 minute, during which the solution was agitated. The mesh was then
washed successively in distilled water, changing the solution until the pH of
the wash
solution remained at approximately 7Ø The mesh was then lyophilized
overnight.
The mesh was rolled into tubes with inner diameters of approximately 3-6 mm
and
lengths of approximately 1-10 cm which were then sewn together with uncoated
PGA
suture (Davis & Geck, Inc., Manati, P. R.) to form a tubular substrate.
Figure 1 depicts the bioreactor system (10), assembled appropriately for cell
seeding.

CA 02861285 2014-08-25
46
The bioreactor includes a glass chamber (22) with a volume of approximately
200 ml and hollow side-arms (12) with a 4 mm internal diameter. The side-arms
are
attached to tubular plastic connectors (24) within the vessel. A short tubular
sleeve
(26) of non-degradable Dacron* vascular graft material (Sherwood-Davis & Geck,
St.
Louis, MO) having an approximately 5 mm internal diameter is sutured to the
plastic
connector on either side of the bioreactor. The Dacron* sleeve, which is
highly
porous, functions as an anchor to attach the developing tissue to the plastic
and
glass of the bioreactor system. Smooth muscle cells and fibroblasts grow
easily into
the pores, thus allowing formation of a continuous connection between the
cellular
tissue and the non-degradable elements of the system.
In preparation for production of a vascular scaffold the ends of the tubular
PGA substrate (16) were sutured to the Dacro* sleeves using uncoated Dacron*
suture (Sherwood-Davis & Geck, St. Louis, MO). A length of highly distensible
medicalgrade silicone tubing (14) with a known compliance (Patter Products,
Beaverton, MI) was inserted through the side-arms, plastic connectors, and
Dacron*
sleeves. The bioreactor system, including the tubular substrate, was
sterilized by
exposure to ethylene oxide and allowed to outgas for at least 3 days.
Bovine aortic smooth muscle cells were obtained as follows. Explants of
bovine thoracic aorta were obtained from a local abattoir on ice. Aortas were
placed
in PBS supplemented with penicillin at standard concentrations (100 U/ml). The
intimal layer of the aortas was stripped away with forceps, and the outer
adventitia
was removed along with the outer media. The remaining middle portion of the
media
was laid down in a petri dish with the previously endothelial side down, and
the tissue
was scored at 1 cm intervals. Sufficient DMEM with Pen/Strep and 15% FBS was
added to cover the bottom of the dish, without causing the tissues to float
above the
surface. The tissues were cultured for 7 to 10 days, during which smooth
muscle
cells migrated off the tissues to form a monolayer in the dish at the end of
the culture
* trademarks

. CA 02861285 2014-08-25
47
period. The tissues were then removed, and the cells were cultured for a total
of 2-3
passages. Smooth muscle identity and purity were confirmed by visual
appearance
and by immunostaining for smooth muscle a-actin. Cells were removed from
culture
by trypsinization (0.05% trypsin, 0.02% EDTA), centrifuged to a pellet, and
gently
resuspended to form a single cell suspension in fresh DMEM.
The substrate was seeded by evenly pipetting 1-2 ml of a suspension of
bovine aortic smooth muscle cells at a concentration of approximately 5 X
106/m1
onto the substrate. Cells were allowed to attach for approximately 30 minutes,
and
then fresh medium was added to the bioreactor vessel. The engineered vessel
was
cultured in the bioreactor for 8 weeks in an atmosphere of 10%CO2 at a
temperature
of 37 C in DMEM supplemented with 20% FBS, penicillin G (100 U/ml), 5 mM
HEPES, ascorbic acid (0.05 mg/ml), CuSO4 (3 ng/ml), proline (0.05 mg/ml),
alanine
(0.03 mg/ml), and glycine (0.05 mg/ml) with continuous stirring. Ascorbic acid
was
replenished daily. Approximately half the medium was replaced with fresh
medium
twice per week. Thus a volume of fresh medium equivalent to the volume of the
bioreactor was supplied each week.
Example 2
Decellularization of a Tissue Engineered Bovine Artery Construct Using Ionic
Detergent Solutions
Materials and Methods
Small caliber arteries were engineered using bovine aortic smooth muscle
cells as described in Example 1. After an 8 week culture period a vessel was
removed from the bioreactor, washed with PBS, and sliced into segments 2 mm
thick. The slices were immersed in 50 ml of a decellularization solution
containing 1
M NaCI, 25 mM EDTA, 8 mM CHAPS in sterile PBS at pH 7.2. The samples were

CA 02861285 2014-08-25
48
incubated with continuous stirring for 1 hour at room temperature and were
then
washed three times in PBS. The samples were then placed in 50 ml of a second
decellularization solution containing 1 M NaCl, 25 mM EDTA, 1.8 mM SDS in
sterile
PBS at pH 7.2 and incubated for 1 hour at room temperature with continuous
stirring.
After removal from the decellularization solution the segments were washed
twice
with PBS for 5 minutes to remove residual solution.
The decellularized artery and a control artery that had been produced under
identical conditions but not subjected to decellularization were fixed in
formalin,
embedded in paraffin, sectioned, and stained with hematoxylin and eosin
according
to standard techniques.
Results
Figure 2 shows low-power (66X, panel A) and high-power (100X, panel B)
photomicrographs of untreated control vessels. In panel A, the outer surface
of the
vessel wall is at the right, and the inner (lumina!) surface is to the left.
As shown in
the figure, the outermost portion of the vessel is composed almost entirely of
cells
(visible as small dark purple spots) and extracellular matrix. The innermost
third of
the vessel also contains circular polymer fragments, which are incorporated
into the
engineered vessel in a disorganized fashion. In the higher powered view of
panel B,
the outer surface of the vessel is oriented downward.
Figure 3 shows low-power (66X, panel C) and high-power (100X, panel D)
photomicrographs of the decellularized vessel. The inner surface of the vessel
is
oriented towards the right in panel C. As shown in the figure, polymer
fragments are
more loosely incorporated into the vessel architecture than in the control
artery.
Panel D shows the tissue portion of the vessel with the outer surface on the
right.
Some cellular fragments are visible, especially in the outermost portions of
the vessel

CA 02861285 2014-08-25
49
wall, but the overall number of cells and cellular remnants is significantly
reduced
compared with the control artery.
Example 3
Decellularization of a Tissue Engineered Bovine Artery Construct Using a
Nonionic
Detergent Solution
Materials and Methods
Small caliber arteries were engineered using bovine aortic smooth muscle
cells as described in Example 1. After an 8 week culture period a vessel was
removed from the bioreactor, rinsed with PBS, and sliced into segments 2 mm
thick.
The slices were immersed in 50 ml of a decellularization solution containing
1%
Triton X1000 (Sigma), 0.02% EDTA (Sigma), 20, pg/ml RNAse A (Sigma), and 0.2
mg/ml DNAse (Sigma) in sterile PBS without Ca2 or Mg2+ and incubated for 24
hours in a 10% CO2 atmosphere at 37 C with continuous stirring. After removal
from
the decellularization solution, the segments were washed several times with
PBS to
remove residual solution.
The decellularized artery and a control artery that had been produced under
identical conditions but not subjected to decellularization were fixed in
formalin,
embedded in paraffin, sectioned, and stained with hematoxylin and eosin
according
to standard techniques.
Results
Figure 4 shows low-power (66X, panel E) and high-power (100X, panel F)
photomicrographs of the decellularized vessel. The outer surface of the vessel
is
oriented towards the right in panel E. As shown in the figure, polymer
fragments (34)
are very loosely adherent to the remaining collagen matrix. Panel F shows the
tissue
portion of the vessel, with very few remnants of nuclear material remaining
between

CA 02861285 2014-08-25
collagen strands. In comparison with the control artery shown in Figure 2, the
number
of cells and cellular remnants is significantly reduced.
Example 4
Decellularization of a Tissue Engineered Porcine Artery Construct Using Ionic
Detergent Solutions
Materials and Methods
Porcine carotid artery smooth muscle cells were obtained as follows. Explants
of porcine carotid artery were obtained from a local abattoir on ice. Aortas
were
10 placed in PBS supplemented with penicillin at standard
concentrations (100 U/ml).
The intimal layer of the aortas was stripped away with forceps, and the outer
adventitia was removed along with the outer media. The remaining middle
portion of
the media was laid down in a petri dish with the previously endothelial side
down,
and the tissue was scored at 1 cm intervals. Sufficient DMEM with Pen/Strep
and
101% FBS was added to cover the bottom of the dish, without causing the
tissues to
float above the surface. The tissues were cultured for 7 to 10 days, during
which
smooth muscle cells migrated off the tissues to form a monolayer in the dish
at the
end of the culture period. The tissues were then removed, and the cells were
cultured
for a total of 2-3 passages. Smooth muscle identity and purity were confirmed
by
20 visual appearance and by immunostaining for smooth muscle a-actin. Cells
were
removed from culture by trypsinization (0.05% trypsin, 0.02% EDTA),
centrifuged to a
pellet, and gently resuspended to form a single cell suspension in fresh DMEM.
A small caliber artery was engineered using porcine carotid smooth muscle
cells essentially as described in Example 1 except that the medium used was
supplemented with 10% FBS rather than 20% FBS. After an 8 week culture period
the vessel was removed from the bioreactor, washed with PBS, and sliced into
segments 2 mm thick. The slices were immersed in 50 ml of a decellularization
solution containing 1 M NaCl, 25 mM EDTA, 8 mM CHAPS in sterile PBS at pH 7.2.
The samples were incubated with continuous stirring for 4 hours at room
temperature

CA 02861285 2014-08-25
51
and were then washed three times in PBS. The samples were then placed in 50 ml
of
a second decellularization solution containing 1 M NaCI, 25 mM EDTA, 1.8 mM
SDS
in sterile PBS at pH 7.2 and incubated for 4 hours at room temperature with
continuous stirring. After removal from the decellularization solution the
segments
were washed twice with PBS for 5 minutes to remove residual solution.
The decellularized artery was fixed in formalin, embedded in paraffin,
sectioned, and stained with hematoxylin and eosin according to standard
techniques.
Results
Figure 5A shows a photomicrograph of a portion of the engineered vessel
prior to decellularization. The image shows a donut-shaped cross-section of
the
vessel. The purple cell nuclei are clearly visible, and the extracellular
matrix is
stained pink. Figure 5B shows a photomicrograph of a portion of the same
vessel
after decellularization. The almost complete absence of nuclear material
suggests
that the decellularization protocol effectively removed the great majority of
cells while
leaving the extracellular matrix substantially intact. It is likely that
shorter periods of
immersion in the decellularization solutions (e.g., 30 min to 4 hours) would
also yield
acceptable results.
Using this protocol there is no evidence of remaining polymeric substrate
after
decellularization. This finding suggests that the decellularization periods
employed
with the porcine vessel may more effectively remove the substrate than the
shorter
time periods employed to decellularize the bovine vessel described above.
Example 5
Decellularization and Reseeding of a Cultured Native Tissue Construct
Materials and Methods
Porcine carotid artery smooth muscle cells were obtained as describe in
Example 4. To facilitate visualization after seeding, cells were fluorescently
labeled

CA 02861285 2014-08-25
52
with red fluorescent dye PKH26-GL (Sigma) according to the manufacturer's
instructions.
A segment of native adult porcine carotid artery was decellularized in a
twophase treatment of solutions. The artery was harvested as follows,
according to a
protocol similar to that described in Swindle, M. M., Moody, D. C., Phillips,
L. D.,
Swine as Models in Biomedical Research, Ames, Iowa, Iowa State University
Press,
1992. Anesthesia was induced with Tiletamine 2.0 mg/kg + Zolazepam 8.8 mg/kg
IM,
supplemented with intermittent boluses of Xylazine 2.2 mg/kg IM every 1-2
hours as
needed. Anesthesia was maintained with Forane, 2%, during the length of the
procedures. The left lateral neck was prepped and draped. The left common
carotid
artery was exposed, ligated, and excised for a length of 2-3 cm. The incision
was
closed in layers.
The harvested artery was placed in DMEM for transport (-10 min) and then
immediately placed into decellularization solution. For the first twenty-four
hours, the
artery was submerged in PBS-based 8 mM CHAPS, 1M NaCI, and 25 mM EDTA.
The solution was then changed to 1. 8mM SDS, 1M NaCI, and 25mM EDTA
for an additional 24 hours. Both treatments were conducted under sterile
conditions
at 37 C and 10% CO2 with stirring. After decellularization, the vessel was
rinsed
thoroughly in PBS.
A segment of vessel was suspended in a sterile bioreactor and seeded with
fluorescently labeled porcine carotid artery smooth muscle cells by evenly
pipetting
1-2 ml of cell suspension at a concentration of approximately 7 X 106/m1 onto
the
outer surface of the decellularized vessel. The cells were allowed to attach
for twenty
minutes, and the seeded vessel was placed in a bioreactor as described above.
The
bioreactor was filled with 250 ml of DMEM culture medium supplemented with 10%
FBS, penicillin G (100 U/ml), 5 mM HEPES, ascorbic acid (0.05 mg/ml), CuSO4 (3
ng/ml), proline (0.05 mg/ml), alanine (0.03 mg/ml), and glycine (0.05 mg/ml).
Ascorbic acid was replenished daily.

CA 02861285 2014-08-25
53
The vessel was cultured for three days in a 10% CO2 atmosphere at a
temperature of 37 C with stirring. The vessel was then removed from the
bioreactor,
and segments were frozen for histology by first submerging in OCT- (optimum
cutting
temperature) compound a widely available formulation of water soluble glycols
and
resins and then placing in liquid nitrogen. The segments were sliced in a
frozen state
and mounted on slides, which were kept frozen until examined under the
microscope.
Results
Figure 6 shows (A) a phase contrast view of a vessel cross section and (B) the
corresponding fluorescent cross section. Since they were fluorescently labeled
prior
to seeding, seeded cells can be distinguished from residual cells remaining
after the
decellularization procedure. The presence of fluorescent cells on the vessel
surface
as seen in Figure 6B indicated that seeded cells attached to the
decellularized native
vessel, but no inward migration occurred.
Example 6
Preparation of a Decellularized Tissue Engineered Bovine Construct Using a
Nonionic Detergent Solution
A small caliber artery is engineered using bovine aortic smooth muscle cells
as described in Niklason, et al., Functional arteries grown in vitro, Science,
284: 489-
93,1999. Briefly, 1-2 ml of a suspension of smooth muscle cells (5 X 106
cells/ml)
isolated from the medial layer of bovine aorta (as described in Ross, R., et
al., J Cell.
Biol. 50,172,1999) are pipetted onto a tubular polyglycolic acid substrate
that is
secured in a bioreactor chamber over a length of distensible silicone tubing.
The
surface of the substrate is modified with sodium hydroxide as described in
Gao, J., et
al, J. Biomed Mater. Res. 42,417,1998, to increase surface hydrophilicity.
After an
initial seeding period of 30 min, the bioreactor is filled with medium (DMEM
modified
as described in Example 1). The construct is cultured for 8 weeks during which

CA 02861285 2014-08-25
54
pulsatile radial stress is applied to the developing construct at 165 beats
per minute
and 5% radial distention (strain) by pumping medium through the distensible
silicone
tubing in a pulsatile fashion. Following the 8 week culture period the
silicone tubing is
removed, and the flow of medium is applied directly through the cultured
vessel. To
produce an endothelial layer, a suspension of bovine aortic endothelial cells
(3 X 106
cells/m1) is injected into the lumen, and the cells are allowed to adhere for
90 min.
Luminal flow rate is gradually increased from 0.033 to 0.1 ml/sec over 3 days
of
culture, with corresponding shear stresses at the vessel wall of 1 X 10-2 N/m2
to 3 X
10-2 N/m2. The construct is cultured for an additional two weeks during which
it is
subjected to intraluminal flow.
Following the culture periods, the medium is drained from the bioreactor, and
the construct is rinsed with sterile PBS. The bioreactor vessel is filled with
a
decellularization solution containing 1 /0 Triton X-100 (Sigma), 0.02% EDTA
(Sigma), 20, ug/ml RNAse A (Sigma), and 0.2 mg/ml DNAse (Sigma) in sterile PBS
without Ca2+ or Mg2+. Decellularization solution is also placed in a flow
system
attached to the bioreactor and is pumped through the inner lumen of the vessel
at a
flow rate of approximately 0.1 ml/sec. After 24 hours of exposure to the
decellularization solution at 37 C in a 10% CO2 atmosphere at 37 C with
continuous
stirring, the decellularization solution is removed from the bioreactor and
flow system,
and the system is rinsed with PBS. The application of intraluminal flow
through the
interior of the engineered vessel results in removal of substantially all of
the
remaining fragments of the polymeric substrate.
For cryopreservation, the decellularized construct is first immersed for 20
min
in HEPES-buffered DMEM containing 1 M DMSO, 2.5% chondroitin sulfate, and 10%
fetal bovine serum at 4 C and then cooled at a controlled rate of
approximately
1.0 /min to -80 C and transferred to liquid nitrogen for storage. Thawing is
accomplished by immersing the storage container in a waterbath at 37 C until
all ice
has disappeared, after which the container is transferred sequentially for 5
minute

CA 02861285 2014-08-25
periods to DMEM containing 0.5 M, 0.25 M, and finally 0 M mannitol as an
osmotic
buffer. The decellularized vessel is implanted into the right saphenous artery
of a
Yucatan miniature pig as described in Niklason, et al., Functional arteries
grown in
vitro, Science, 284: 489-93, 1999.
Example 7
Preparation and Subsequent Engineering of a Decellularized Tissue Engineered
Construct
A vascular tissue engineered construct is produced in a plastic bioreactor
10
system similar to that described in Example 1, but without the use of a
polymer
substrate, as follows: Human smooth muscle cells and human endothelial cells
are
obtained using standard biopsy and culture techniques and are maintained in
vitro.
The length of silicone tubing extending between the Dacron sleeves in the
bioreactor
is coated in a sterile fashion with a thin layer of a gelatin material, e.g.,
made from
dilute human collagen. Human collagen (commercially available) is denatured
and
pipetted or applied with a syringe onto the outside of the silicone tubing to
create a
layer approximately 50 pm thick. The tubing may be rotated during application
of the
collagen solution so that a layer of uniform thickness is produced. Thus
according to
this embodiment of the invention the collagen-coated tubing serves as the
substrate.
20
After the collagen layer is allowed to dry, a suspension of 1 to 2 ml of
culture
medium containing human smooth muscle cells at a concentration of
approximately 5
million cells/ml medium, is applied to the coated tubing and the Dacron
sleeves on
either end using a pipet. The tubing is preferably rotated during application
of the
cells to create an even distribution of cells. The thin coating allows an
initial layer of
cells to adhere to the outside of the tubing. The bioreactor stopper is
replaced, and
the cells are allowed to adhere for 30-60 minutes, after which the bioreactor
and fluid
reservoir are filled with culture medium (DMEM supplemented as described in
Example 1). The bioreactor is placed in a tissue culture incubator and
maintained at

CA 02861285 2014-08-25
56
37 C in a 10% CO2 atmosphere for a period of about 1 to 7 days during which
pulsatile radial stress is applied to the developing construct at 165 beats
per minute
and 5% radial distention (strain) by pumping medium through the distensible
silicone
tubing in a pulsatile fashion.
Following this initial growth period, the stopper is removed, and the medium
is
drained from the bioreactor. The surface of the developing tissue on the
tubing is
then reseeded with a suspension of human smooth muscle cells substantially
equivalent to those used in the initial seeding, and these cells are allowed
to adhere
to the developing tissue. The bioreactor is then filled with medium, and the
culture
process is repeated with physical stimuli applied as in the first growth
period. This
sequence (i.e., reseeding followed by a growth period) is continued until a
tissue of
desired thickness (e.g., approximately 0.038 cm) is produced on the tubing
(approximately 8 weeks).
After the vessel has reached the desired thickness the silicone tubing is
removed, and the flow of medium is applied directly through the cultured
vessel. To
produce an endothelial layer, a human endothelial cell suspension of 3 X 106
cells/ml
in DMEM is injected into the lumen of the construct, and the cells are allowed
to
adhere for 90 min. Luminal flow rate is gradually increased from 0.033 to 0.1
ml/sec
over 3 days of culture, with corresponding shear stresses at the vessel wall
of 1 X
10-2 N/m2 to 3 X 10-2 N/m2. The construct is cultured for an additional two
weeks
during which it is subjected to intraluminal flow.
Following this second growth period, the medium is removed from the
bioreactor chamber, and the construct is rinsed with PBS. The chamber is then
filled
with a decellularization solution containing 1 M NaCI, 25 mM EDTA, 1.8 mM SDS
in
sterile PBS at pH 7.2. Decellularization solution is also placed in a flow
system
attached to the bioreactor and is pumped through the inner lumen of the vessel
at a
flow rate of approximately 0.1 ml/sec in a pulsatile fashion. After 30 minutes
of
exposure to the decellularization solution at room temperature, the
decellularization

CA 02861285 2014-08-25
57
solution is removed from the bioreactor and flow system, and the system is
rinsed
with PBS.
For cryopreservation, the bioreactor chamber is first filled with
HEPESbuffered
DMEM containing 1 M DMSO, 2.5% chondroitin sulfate, and 10% fetal bovine serum
at 4 C. Following a 20 minute period at 4 C, the chamber is cooled at a
controlled
rate of approximately 1.0 C/min to a temperature of -80 C and transferred to
liquid
nitrogen for storage. After identification of a subject in need of a vascular
graft, the
decellularized vessel is thawed by immersing the bioreactor chamber in a
waterbath
at 37 C until all ice has disappeared, after which the cryoprotection solution
is
drained from the chamber. For elution of the cryoprotection solution, the
chamber is
then filled sequentially for periods of 5 minutes with DMEM containing 0.5 M,
0.25 M,
and finally 0 M mannitol as an osmotic buffer. The chamber is then filled with
DMEM
supplemented as described in Example 1.
Smooth muscle cells and endothelial cells are obtained from a biopsy
specimen removed from the intended recipient of the construct. These cells are
maintained in tissue culture and expanded. Following thawing of the
decellularized
construct, the outer surface of the construct is seeded with smooth muscle
cells (1-2
ml of a suspension containing 5 X 106 cells/ml), which are allowed to adhere
for 30
minutes. The bioreactor chamber is filled with culture medium, and the
construct is
maintained in culture for a period of 1 week. To produce an endothelial layer,
a
suspension of bovine aortic endothelial cells (3 X 106 cells/m1) is injected
into the
lumen, and the cells are allowed to adhere for 90 min. The construct is
cultured for
an additional three days with the application of pulsatile stimuli as during
the growth
periods prior to decellularization and is then implanted into the recipient.
While the invention has been described and illustrated in connection with
certain embodiments, many variations and modifications as will be evident to
those
skilled in this art may be made therein without departing from the spirit of
the

CA 02861285 2014-08-25
58
invention, and the invention as set forth in the claims is thus not to be
limited to the
precise details set forth above.