Note: Descriptions are shown in the official language in which they were submitted.
CA 02685964 2011-02-10
Industry industrle 41hq/J 'rihwD
Canada Canada
2011102110
IILIIiIIiIIIIIII~IIIIIIIIIIiIIIIIIIIIII~IIIIIIIILIIIIIilllill II 041- 11
CPO oPiC 19880550
Patent Application of
John Archie Gillis
for
TITLE: METHODS OF PRESERVATION, FABRICATION AND PERFUSION OF
TISSUE CONSTRUCTS
APPLICATION NUMBER 2,685,964
CROSS-REFERENCE TO RELATED APPLICATIONS Not Applicable
FEDERALLY SPONSORED RESEARCH Not Applicable
SEQUENCE LISTING OR PROGRAM Not Applicable
BACKGROUND OF THE INVENTION-FIELD OF INVENTION
The present invention relates to methods of fabrication, preservation,
banking,
transporting, vascularization and perfusion of tissue constructs.
BACKGROUND OF THE INVENTION-PRIOR ART
Tissue engineering in its early days was considered a sub-field of
biomaterials. It has
recently grown in both importance and potential and is now considered to be a
field of its own.
It generally uses a combination of cells, engineering, materials methods, and
suitable
CA 02685964 2011-02-10
biochemical and physio-chemical factors to improve or replace biological
functions. Tissue
engineering is usually describes as an interdisciplinary field incorporating
elements of
engineering, material and life sciences.
Most recently tissue engineering has begun to incorporate elements of computer
aided
design and rapid prototyping. The names currently most in use are bioprinting
and organ
printing.
Tissues are often fabricated in the laboratory using stem cells, growth and
differentiation
factors, biomaterials, printing devices and biomimetic environments. It is
with these
combinations of engineered extracellular matrices (or scaffolds), cells, and,
biologically active
molecules that researchers in this field have propelled this area of research
forward.
One of the major challenges facing tissue engineering today is the requirement
for more
complex functionality. For a greater number of tissue engineered structures to
be considered
useful in areas such as transplantation, more biomechanical stability is
required along with an
advanced means of supplying these structures with nutrients, especially when
discussing thick
tissue structures.
A cryoprotectant is a substance that is used to protect biological tissue from
freezing
damage. This damage often occurs due to the formation of ice. Cryoprotectants
in common
use include glycols, such as ethylene glycol, propylene glycol and glycerol
and dimethyl
sulfoxide (DMSO), 2-methyl-2, 4-pentanediol (MDP) Sucrose and Trebalose.
Cryobiologists have been using both glycerol and dimethyl sulfoxide for
decades to reduce ice
formation in sperm and embryos that are cold-preserved in liquid nitrogen.
Mixtures of cryoprotectants have less toxicity and are more effective than
single-agent
cryoprotectants. A mixture of formaxnide with DMSO, propylene glycol and a
colloid, was for
many years the most effective of all artificially created cryoprotectants.
Cryoprotectant
mixtures have been used for vitrification, i.e. solidification without any
crystal ice formation.
Vitrification has important application in preserving embryos, biological
tissues and organs for
transplant. Vitrification is also used in cryonics in an effort to eliminate
freezing damage.
CA 02685964 2011-02-10
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Some cryoprotectants function, by lowering a solution's or a material's glass
transition
temperature. In this way, the cryprotectants prevent actual freezing, and the
solution maintains
some flexibility in a glassy phase.
Vitrification techniques utilize low toxicity solutions and optimized cooling
and warming
curves that, when applied under sterile conditions, allow for better, longer,
safer and more
convenient storage of complex living systems.
An example of a method of cryopreservation of tissues by vitrification is
Khirabadi; Bijan
S., Song; Ying C., Brockbank; Kelvin G. M. "Method of cryopreservation of
tissues by
vitrification", Organ Recovery Systems, Inc. US 7,157,222, (2007) or US
6,740,484
This prior art teaches a method that includes vascularized tissues and
avascular tissues, or
organs. The method comprises immersing the tissue or organ. in increasing
concentrations of
cryoprotectant to a cryoprotectant concentration sufficient for vitrification;
rapidly cooling the
tissue or organ to a temperature between -80° C. and the glass
transition temperature
(Tg); and further cooling the tissue or organ from a temperature above
the glass transition
temperature to a temperature below the glass transition temperature to vitrify
the tissue or
organ.
This prior art also describes a method for removing a tissue or organ, from
vitrification in a
cryoprotectant solution. The method comprises slowly warning a vitrified
tissue or organ in
the cryoprotectant solution to a temperature between -80° C. and the
glass transition
temperature; rapidly warming the tissue or organ in the cryoprotectant
solution to a
temperature above -75° C.; and reducing the concentration of the
cryoprotectant by
immersing the tissue or organ in decreasing concentrations of cryoprotectant.
With this method for treating tissues or organs, viability is retained at a
high level. For
example, for blood vessels, the invention provides that smooth muscle
functions and graft
patency rate are maintained.
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These and similar methods are great for protecting existing and fabricated
tissues from
damage, but are not always successful at penetrating deep into thick tissue
constructs. These
methods have not been used in tissue engineering processes such as those
described by the
present invention. It is an object of the present invention to prepare
cellular compositions with
both intracellular and extracellular cryoprotectant solution mixtures prior to
a bio printing
process, thus allowing precise placement of solutions. In fact cryoprotectants
are rarely if ever
used in tissue engineering. Most cryoprotectants have been used in protecting
existing
structures. It can be very difficult to position the protective solutions deep
within these
already existing structures. This ability of the protective solutions to be
selectively located is
one of the key benefits of the described invention.
Preservation of organs and tissues are commonplace in medicine, but because
organs are
most often donated rather that fabricated it can be difficult to place these
solutions in areas that
can deeply penetrate the structure, especially if the tissue or organ is a
thick structure.
Organ printing is usually assisted by computers, dispenser-based, and has an
emphasis on
three-dimensional fabrication. These methods are aimed at constructing
functional organ
modules however at present there has been limited success and the printing of
entire organs
layer-by-layer has not yet been. realized.
Bio-printing or organ printing is a new area of research and engineering that
involves
printing devices that deposit biological material. Examples of bioprinter
technologies would
be those in development by Organovo and fabricated at Inventech, which use
combinations of
"bio-ink" and "bio-paper" to print complex 3D structures.
A number of developments have been occurring in the field of organ printing.
One such
development is that of Self-Assembling Cell Aggregates. Forgacs; Gabor;
(Columbia, MO) ;
Jakab; Karoly; (Columbia, MO) ; Neagu; Adrian; (Columbia, MO) ; Mironov;
Vladimir; (Mount
Pleasant, SC)"Self-Assembling Cell Aggregates and Methods of Making Engineered
Tissue
Using the Same", The Curators of the Univeristy of Missouri, Columbia MO,
US20080070304,2008
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This prior art describes a composition comprising a plurality of cell
aggregates for use in
the production of engineered organotypic tissue by organ printing. In a method
of organ
printing, a plurality of cell aggregates are embedded in a polymeric or gel
matrix and allowed
to fuse to form a desired three-dimensional tissue structure. An intermediate
product
comprises at least one layer of matrix and a plurality of cell aggregates
embedded therein in a
predetermined pattern. Modeling methods predict the structural evolution of
fusing cell
aggregates for combinations of cell type, matrix, and embedding patterns to
enable selection
of organ printing processes parameters for use in producing an. engineered
tissue having a
desired three-dimensional structure.
Another development is the method of forming an array of viable cells
developed by
James Yoo, Tao Xu and Anthony Atala which decribes a method wherein at least
two different
types of viable mammalian cells are printed on to a substrate. Inventors:
James Yoo, Tao Xu,
Anthony Atala. Application number: 12/293,490 Publication number: US
2009/0208466
A I Filing date: Apr 20, 2007
These methods of tissue engineering still suffers from some of the limitations
of traditional
scaffolding methods. There have been some great successes with this method,
but the issue of
nutrient delivery is still a major, concern.
A common problem with thick tissue structures is that cells deep inside the
structure are
damaged due to a lack of nutrient delivery. One can delay this problem for a
short by
preserving the tissue with. a cryoprotectant solution, but unless the tissue
is prepared as
described in the present invention the problems of getting cryoprotectant
solutions into all the
desired locations, including cells deep within the structure remains a large
and limiting
problem.
If tissue engineering is ever to surpass the tissue thickness limit of 100-200
m, it must
overcome the challenge of creating functional blood vessels to supply cells
with oxygen and
nutrients and to remove waste products.
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A major dilemma with most current tissue engineering technologies is that most
tissues
and organs require vascularization and perfusion to survive. Creating this
vascular supply and
more viable methods of perfusion to a thick-engineered tissue construct
remains one of the
great challenges in the field today.
SUMMARY
By immersing cells and cellular aggregates, in gradually increasing
cryoprotectatnt
solutions prior to their dispensing from a three dimensional printing
technology we can create
a structure that is very well prepared for preservation.
Once cooled the tissues can be transported or banked for drug testing, cell
therapies,
graphs and implantation, reconstructive surgery, wound healing, cardiovascular
treatment and
many others beneficial applications.
When the constructs are taken out of their preserved state they will be moved
from their
vat and into a new holding vessel. The holding vessel will have holes for
transporting
substances; will contain bioreactor and perfusion bioreactor components, a
temperature
specific environment and electronic pin molding capabilities.
The tissue selection located in, the holding vessel will then be attached to a
circulatory
system of a human by connecting the vasculature of the human to an umbilical
cord. The
other and of the umbilical will be attached to the vasculature of the tissue
selection. A tube
casing containing a protective solution will protect the cord.
BREIF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow chart describing how one or more cells may be prepared for
bio-printing
and dispensed as a cellular composition that is ready for cryopreservation.
Fig. 2 is a flow chart describing how a number of tissue constructs prepared
for
preservation can be placed in a vat, preserved, stored and/or transported and
warmed.
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Fig. 3 shows what could be separate sections of a printed heart valve.
Fig. 4 shows what the separate sections of a printed heart valve could look
like if stacked
together in a holding vessel.
Fig. 5 shows what the separate sections could look like once fully self
assembled into a
finished structure.
Fig. 6 shows what a pin molding system capable of adjusting to different
shapes for
compensating tissue compaction, maturation and movement could look like. The
mold would
also be helpful with supporting materials involved in extracellwar matrix
maturation.
Fig. 7 shows a human being perfusing a. tissue structure or organ by means of
attachment
to his/her circulatory system. The holding vessel is a Transm.edic device with
the organ
enclosed inside and attached to the human via an umbilical cable donated from
a new born
child and enclosed in a protective tube.
DRAWINGS-Reference Numerals
- Cryoprotectant Solution
12 - One or more cells
14 - Preparation of cells for preservation
16 Bio-paper
18 - Cryo-prepared cells assembled into self assembling tissue spheroids/bio-
ink
- Other materials
22 - Dispensing system
24 - Output from dispensing system containing spheroid shaped cryo-prepared
cellular
compositions situated for the process of self assembly
26 - Tissue Construct #1
28 - Tissue Construct #2
- Tissue Construct #3
32 - Vat
34 - Means of cooling a tissue selection
36 - Means of storage and transport
38 - Means of warming a tissue selection
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40 - Means of transferring a tissue selection into a molding system
42 - Section. or layer of a tissue selection to be assembled into a larger
structure.
44 - Large tissue structure fabricated from smaller portions
46 - One pin of a pin mold
48 - Pin mold pin holding portion
50 - Protrective holding vessel for tissue selection(s)
52 - Holes for nutrient, blood and supply delivery
54 - Vasculature
56 - Organism with circulatory system that will. supply nutrient delivery and
waste removal to
a tissue selection.
58 - Transmedic style holding vessel
60 - Protective tube that holds umbilical cord
DETAILED DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow chart showing cryoprotectant solutions 1.0 and one or more
cells 12 coming
together wherein they are provided with a means of being prepared for
preservation 14. The
prepared cells of 14 are assembled into self-assembling tissue spheroids or
what is known in
the art as bio-ink IS. Loaded into a dispensing system 22 are the bio-ink 18,
the bio-paper 16
and other materials 20 which may include other cryoprotectant solution, matrix
materials,
scaffolds and gels. From the dispensing system 22 we get an output containing
spheroid
shaped cryo-prepared cellular compositions situated for the process of self-
assembly 24 into a
desired shape, pattern or three dimensional structure.
Fig. 2 is a flow chart showing a number of different tissues 26, 27, 28, which
will be
loaded into a vat 32 with a shape complementary to the shape of the printed
tissue selections.
A means of cooling 34 will be provided and when cooled to a desired
temperature the tissues
will be stored and/or transported 36. When the tissues reach their location or
it is desired to
remove them from their cryopreserved state a means of warming 3 8 will be
provided so as to
enable transfer to a pin molding system 40 or for other uses.
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Fig. 3 is a diagram of a heart valve printed in layers or sections 42. Each
section 42 was
printed as a separate unit with a specific shape. At this stage of the process
we can see how
when the layers are placed together that they will form. the shape of a heart
valve.
Fig. 4 is a diagram of our layers 42 stacked together to form a structure 44
that will be
coaxed into self assembly and form the shape of a heart valve.
Fig. 5 shows what separate sections of a heart valve could look like once
fully assembled
into a finished structure 46.
Fig. 6 provides a visual example of what a pin molding system capable of
adjusting to
different shapes for compensating tissue compaction, maturation and movement
could look
like. 42 shows a holding vessel that has a means to provide a protective
environment for the
tissue selections. In the preferred embodiment it will be a membrane like
encasing made from
a material that is flexible, malleable and capable of a variety of shapes so
that when self
assembly, fusion, maturation, compaction or change in shape occurs to the
construct our pins
46 can move to compensate for these changes and in most instances prevent
unwanted areas of
the structure from moving into undesirable locations. 48 is a stand for
holding the pins of our
mold. 46 shows pins that when moved together can create a desired shape or
mold that can
change over time for allowing the same casing or holding vessel the ability to
provide
structural support during changes to the structure. Holes 52 are provided to
the holing vessel
to allow transport of materials to and from the tissue selection(s) from an
outside source.
Vasculature 54 is shown passing through the holes. The vertical three-
dimensional image
screen described in patent number: 4654989 Filing date: Aug 16, 1.985 Issue
date: Apr 7, 1987
descibes a screen with ability to mold to many objects. United States Patent
6,625,088 to
Mah; Pat Y. and Tinier; Robert Bruce issued on September 23, 2003 describes a
similar pin
display device that has an electronic mechanisms for moving its pins into
different shapes.
Fig. 7 shows a human being or patient 56 perfusing a tissue structure or organ
by means of
attachment to their circulatory system. The holding vessel is a Transmedic
device 58 with the
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organ enclosed inside and attached to the human via an u#iobilical cable
donated from a new
born child and enclosed in a protective tube 60.
DETAILED DESCRIPTION OF PREFERRED EMBODNTS
Reference will now be made in detail to various embodiments of the invention,
one or
more examples of which are set forth below. Each embodiment is provided by way
of
explanation of the invention, not limitation, of the invention. In, fact, it
will be apparent to those
skilled in the art that various modifications and variations may be made in
the present
invention without departing from the scope or spirit of the invention. For
instance, features
illustrated or described as part of one embodiment, may b used in another
embodiment to
yield a still further embodiment. Thus, it is intended that the present
invention cover such
modifications and variations as come within the scope of line appended claims
and their
equivalents.
In the preferred embodiments the present invention describes a number of steps
for the
fabrication, preservation and perfusion of a tissue constru t.
The process begins by immersing cells in varying level) s of cryoprotectant
solutions. The
cells are then aggregated into self-assembling tissue spheroids, and dispensed
into a desired
shape.
The innovative method comprises ink jet printing a cei composition onto a
substrate
wherein the cells within the composition have been prepar~d for
cryopreservation, cooling,
freezing or vitrification. A great example of Ink jet printing of viable cells
is US Patent
7,051,654 Boland; Thomas (Suwanee, GA), Wilson, Jr.; illiam Crisp (Easley,
SC), Xu; Tao
(Clemson, SC), which, is hereby incorporated by reference in its entirety. It
describes a
method for forming an array of viable cells. In one embodi' cnt, the method
comprises ink jet
printing a cellular composition containing cells onto a substrate. Upon
printing, at least about
25% of the cells remain viable after incubation for 24 hour at 37° C.
in a 5%
CO2/95% O2 environment.
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In the preferred embodiment the cultured cells that are included in the
cellular composition
to be printed are prepared with. varying levels of cryoprotectant solutions. A
variety of
solutions can be used to generate various levels of results and successes.
Examples of some
potential methods that may be used in whole or in part include, but are not
limited to "Method
of cryopreservation of tissues by vitrification" (Khirabadi; Bijan S., Song;
Ying C.,
Brockbank; Kelvin G. M. "Method of cryopreservation of tissues by
vitrification.", Organ
Recovery Systems, Inc. US 7,157,222, 2007),
The cryogenically prepared cells will form a. bio ink that will be loaded into
a three
dimensional fabrication device. A great example of a bio ink is US Patent
Application
20080070304 to Forgacs; Gabor; (Columbia, MO) ; Jakab; Karoly; (Columbia, MO)
; Neagu;
Adrian; (Columbia, MO) ; Mironov; Vladimir; (Mount Pleasant, SC) "Self
Assembling Cell,
Aggregates and. Methods of Making Engineered Tissue Using the Same", which is
hereby
incorporated by reference in its entirety and explains bio ink and bio paper.
No prior art reference provides a description of a process incorporating the
use of
cryogenic preparation of cells or cell aggregates for the purpose of being
loaded into a printer.
This is one of the novel features of the present invention. With prior methods
of applying
cryoprotectant solutions to some tissue constructs, (especially into think
constructs or organs)
it has been found difficult if not impossible to get the cryoprotectant
solutions to the desired
locations. The present invention provides a remedy for this problem.
After being dispensed from an ink jet printer the cellular spheroids or
aggregates will be
preserved by methods of freezing or vitrification. The construct will be
stored and transported
for cell therapies, drug tests, and in the preferred embodiment oftbe present
invention used, as
a section to be fused with other similar sections to create a larger
construct.
The prepared cells are then placed into a vat or similar cooling device, next
to one or more
other cellular constructs that have been prepared in a similar manner. The
constructs are then.
vitrified or frozen. The cells may now be banked or transported for drug
testing or cell
CA 02685964 2011-02-10
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therapies and once taken out of their preserved state they will be coaxed into
self=assembly
and fused together to create a larger structure.
One of the reasons the present inventor feels it is pertinent to create and
preserve the
tissues in sections is so that they can be easily stored at a later time when
they are needed, and
so that the bioprinting system is freed up for use. Another reason for
printing the constructs in.
sections is that some bioprinters will be designed and/or set up for the
creation of specific
tissue structures. As an example; bioprinter I would be programmed to print
section A of a
kidney with specific materials and preservation solutions and thus the
technician is trained to
know exactly what is required each time, while bioprinter 2 is programmed to
print section B
of the same kidney with their different but specific materials and
preservation solutions. The
process continues with bioprinter 3 doing section C and so on., until all the
required parts are
constructed. By printing the sections simultaneously we can decrease the time
it takes to
complete the more elaborate structure.
In the preferred embodiment the printers will be programmed to complete the
sections at
the same time so that they can be placed into a vessel for preservation. Once
they are
preserved they can be transported to the geographic location in which they are
needed.
A viable method of transportation is also a. very beneficial, outcome of the
present
invention. Organs and tissue assays do not last long if not protected
properly.
Bioprinting labs are very expensive and require expertise in operation. As an.
example
Inventec sells their bioprinters for $250,000.00 a pretty steep price for any
lab. These labs
and expertise are only located in a few geographic locations, but with the
described
preservation methods, preserved tissues fabricated with computer and
roboticially targeted.
precision will make it much more viable for the transportation of these
products for their use
in cell therapy products, tissues, organs and tissue engineered constructs.
The cryogenically prepared cells are printed in layers, and as the layers are
completed they
are mechanically lowered into a vat, and put into a vitrified or frozen state.
The layers are
organized so that they fit together in a desired shape or pattern that will
allow the proper
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portions to fuse in the correct areas when taken out of their vitrified or
frozen states. An
example of this is to position a vascular network such that when cellular
adhesion (self
assembly) occurs it will become one unit and thus when taken out of a
vitrified or frozen state
the sections will fuse together in the correct and desired locations, and be
ready to be placed. in
a device capable of providing the required supply of nutrients and materials.
Vasculature has
been bioprinted in the labs of Anthony Atala without cryopresevation included
to a limited
degree. It has also been successfully attached to a perfused bioreactor.
In a preferred embodiment self-assembly may occur after preservation, however
in
alternative embodiments it will occur prior to preservation.
When the individual sections are placed into a vat, the vat will be shaped as
to support the
dimensions of the larger structure to be fabricated or preserved. At this
point the structures
will have been persevered or will undergo a preservation process. The
construct is now
transported to its required location.
When the structure reaches its destination it is taken out of its state of
preservation. At this
point the structure can be carefully removed from its vat into a holding
vessel with
electronically programmable and movable pin molding capabilities for
supporting the
structures and for also providing support for a protective membrane that will
encase the
structure in a protective biological environment. This type of protective pin
molding
biological environment will provide support for the structure yet allow for
changes to occur
during post processing fusion, retraction, remodeling and compaction. Another
object of the
holding vessel is its temperature specific environment.
When the structures are taken out of their vitrified state they will be coaxed
into self
assembly as is described in US Patent Application 20080070304. The self
assembling tissue
spheroids of each section may have aggregated into a larger tissue structure
prior to
preservation and thus these fused tissue sections will aggregate to form yet
an even larger
structure. In another embodiment the smaller sections will also require time
for self assembly.
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14
There are a number of instances where the present methods of preservation will
be very
useful. if the tissue constructs are not needed for some time after printing
or if they are
required to be transported the present methods will assist with preventing
damage and cell
death from occurring. Often tissues are transported long distances for drug
testing, cell
therapies and if there are no bio-printing laboratories near an area where
bioprinted structures
are wanted or needed, the aggregation methods explained herein may be a very
necessary
requirement.
Another reason that the described method is practical is the high cost and
low'success rate
of many other alternatives. Time is of the essence when printing tissue
constructs, as they can
only be maintained for relatively short periods of time after, printing,
before damage occurs.
The present sectioning method can be used without the use of cryogenic
solutions
integrated into the construction process, but only if a company has a number
of bioprinters
printing sections of a predetermined structure at the same time. This type of
lab would then
need to stack the sections into a holding vessel. and perfuse them with
nutrients as soon as
possible. This method is not practical if the construct is to be transported.
The structures will be provided with nutrients and waste removal using
standard methods
found in the art, such as bio reactors, perfused bioreactors or solutions used
in systems for ex
vivo care at new physiologic conditions, however once a vascular structure or
vascular
system, such as a bioprinted intraorgan branched vascular system has been
assembled and
becomes mature and functional enough for initiation of intravascular perfusion
it will be
attached to an umbilical cable. The umbilical cable may be fabricated from
human cells or
may be one donated by a suitably matched new born baby and then attached to a
human
circulatory system. This would likely be the circulatory system of the future
recipient of the
structure.
A group from South Carolina as well as a group led by Gabor Forgacs' have
recently
demonstrated that building a branching intraorgan vascular tree is a realistic
and achievable
goal. This issue was also addressed by Peter Wu (University of Oregon, USA)
who presented
CA 02685964 2011-02-10
applications of LAB in fabricating branch/stem, structures with human
endothelial cells and T
Boland who presented results on thermal inkjet printing ofbiomaterials and
cells for capillary
constructs. (Cui X and Boland T 2009 Human microvasculature fabrication using
thermal
ink jet printing technology Biomateri.als 30 6221-7)
When the structure has fused into a single unit it will remain in its holding
vessel where it
will continue to receive nutrients and blood from the human circulatory
system. This system.
will also provide the structure with the ability to remove waste.
One of the great benefits of the structure being located outside of the body
is that it may be
tended to by doctors, engineers and other professionals for other additional
procedures, tests or
substance delivery that may be beneficial to the survival and maintenance of
the structure.
The structure may when required also receive external, electrical stimuli.
Other great benefits
of the structure being perfused by the patient's own circulatory system, yet
essentially being
located outside the body is that it can be accessed, repaired, manipulated and
supplied with
additional substances or therapies.
Current methods of perfusing a tissue structure are limited, due to time
constraints. This is
seen in cases of organ donation. When a donated organ is matched with a
recipient, it is
imperative that the organ reaches the recipient in as short of time as
possible. Even with our
advanced technologies, helicopters and database matching systems organs are
often lost, due
to a variety of reasons that include injuries during brain-death, ischemia,
cell death and other
causes.
Currently there are a number of systems that are perfusing organs such as
Transm.edics,
"Organ Care System", Organ Recovery Systems "LifePort" technologies and the
Toronto
XVIVO Lung Perfusion System. The Lung Perfusion Systern is being worked on by
Dr. Shaf
Keshavjee in the Lung Transplant Program at Toronto General Hospital (TGIF).
They have
developed an "ex vivo" or outside the body technique capable of continuously
perfusing or
pumping a bloodless solution containing oxygen, proteins and nutrients into
injured donor
CA 02685964 2011-02-10
16
lungs. This technique allows the surgeons the opportunity to assess and treat
injured donor
lungs, while they are outside the body, to make them suitable for
transplantation.
These methods of perfusion are great advances in medical technologies, but
still have their
limitations. The present invention describes that at first seems odd, but is
actually the most
natural method of perfusing either a transplanted organ or a tissue engineered
construct. If we
think of how a fetus is perfused in the womb we have a fetus attached to an
umbilical cord,
which, is attached to its mother. Both the fetus and the umbilical are in a
protective solution.
In the present invention we create something very similar. Our fetus is out
tissue engineered
construct and our mother is the person who will be having the construct or
organ implanted
into them. In the preferred embodiment the ex vivo perfusion module will be
attached via
existing or fabricated umbilical cables to the construct or organ to be
perfused. The construct
or organ will be located outside of the body and housed in a protective
temperature specific
environment, likely at 37 degrees C and may include a protective solution for,
surrounding the
construct/organ. The tube attaching to the recipients circulatory system via
an umbilical cable
will be housed in a tube containing a protective solution, which may contain
Wharton's Jelly
or a suitable substitute, nutrient composition, or liquid that may assist in
sustaining the cord
during perfusion of the construct. Connection of this cord will require
surgical attachment.
In placental mammals, the umbilical cord (also called the birth cord or
funiculus
uznbilicalis) is the connecting cord from the developing embryo or fetus to
the placenta.
During prenatal development, the umbilical cord comes from the same zygote as
the fetus and
(in humans) normally contains two arteries (the umbilical arteries) and one
vein (the umbilical
vein), buried within Wharton's jelly. The umbilical vein supplies the fetus
with oxygenated,
nutrient-rich blood from the placenta. Conversely, the umbilical arteries
return the
deoxygenated, nutrient-depleted blood.
Successful perfusion of an extra organ using a similar procedure in vivo has
been
accomplished in the art by what is known as hetcrotopic surgery. In this
medical procedure
the patient's own heart is not removed before implanting a donor heart. The
donor heart is
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positioned so that the chambers and blood vessels of both hearts can be
connected to form
what is effectively a'double heart'.
Another example of in vivo perfusion of an extra organ is that of a kidney
transplant. In
many kidney transplants the original but likely damaged kidneys are left in
the recipient.
An example of ex vivo perfusion is that of babies who are occasionally born
with organs
outside their body and often survive this way for many months prior to having
the organs
transferred inside their body.
Where the present invention differs from these procedures is that the
heterotropic
procedure takes place inside an. organism not via an, ex vivo attachment.
Another difference is
that in the present invention the organism that the construct or organ is
first attached to after
being printed acts as a temporary lobby area or location and once the organ
has been matured
it will be attached to the recipient
The tissue constructs of the present invention include portions of, or whole
tissues (i.e.,
bone, cartilage, blood vessels, bladder, etc.) The tissue harvested may
consist of any
biological material and may include materials that have been manipulated
and/or changed
from their original state, such as geneticially altered materials or stem cell
cultivations.
The dispensing systems of the present invention include computer aided design,
manufacturing, assembly and/or printing systems. These systems make use of
computer
technology to aid in the design, manufacturir, assembly and/or printing of a
product.
Examples of such systems include, Direct Digital Manufacturing, Rapid
Prototyping, Three
Dimensional Printing, Bio-printing, (CAD/CAM), Stereolithography, Solid
Freeform
Fabrication, Self-Replicating Machines, 3D Microfabrication, Digital
Fabrication and Desktop
Manufacturing Systems, and the methods and technologies involved, developed
and
understood by those skilled in the art.
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The Bio-printing systems of the present invention will include the use of what
is known in
the art as bio-paper and bio-ink.
In the preferred embodiment the holding vessel will include a pin molding
system capable
of providing structural support that can be manipulated. This molding method
will be
beneficial in post processing fusion, retraction, remodeling and compaction of
printed soft
tissue constructs because for a printed tissue construct or organ to be
fabricated to the desired
mature size and shape it will initially be larger and in many instances have a
slightly different
shape.
When the construct is matured to a desired state it will be removed from its
umbilical and
surgically implanted into a recipient.
ALTERNATIVE EMBOBIMENTS
In one alternative embodiment the methods described will be used to create
products
consisting of biological materials integrated with non-organic materials such
as electronic
devices and computer components. The methods described in the present
invention being a
means of storing and/or transporting the integrated organic/electronic
materials.
In another alternative embodiment the present invention's ex vivo human
perfusion
methods could assist with donor organ care. As an example if patient A lives
in California and
needs a kidney and patient B lives in Boston and needs a kidney, we could have
the following
scenario. Donor organs become available, but Organ # 1, in California is a
poor match for
Patient A and Organ #2 in Boston is a poor match for Patient B. Patient A has
a family
member or friend that is willing to perfuse the kidney while traveling to
Boston. Patient B has
a family member or friend that is willing to peruse the kidney while traveling
to California.
Both patients receive kidneys that may have otherwise gone to waste, been
damaged due to
ischeniia poor preservation or any other number of reasons.
It does seem like a lot to ask of a friend or family member, but it seems like
a more
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practical scenario than asking a living friend or family member to go into
surgery and give up
one of their kidneys forever, which is a. relatively frequent procedure.
In another alternative embodiment the present invention will utilize
genetically altered
animals for assistance with the maturation of tissue constructs or for
perfusing tissue
selections or organs.
When organs are transplanted between species, immune attack is swift and
severe. Pigs for
example and other animals have a specific sugar not present in humans and old-
world
primates. So when a pig organ is transplanted into a baboon, for example,
antibodies
circulating in the baboon's blood immediately swarm and attack the pig tissue,
leading to the
death of the organ.
As one example, scientists (particularly David Sachs, the director of the
Transplantation
Biology Research Center at MGH) made a major advance in overcoming this immune
barrier
in 2002 by creating genetically engineered pigs that lack the enzyme that
attaches the sugar to
the surface of, pig cells. In a paper published in Nature Medicine, Sachs
showed that baboons
given kidneys from these genetically modified pigs lived for up to 83 days,
far longer than the
average 30-day survival time for animals receiving regular pig kidneys.
The tissue selection is attached to a swine designed to lack an immune system
in a surgical
process. The tissue selection remains in a system for ex-vivo organ care at
near-physiologic
conditions, but is also attached to a swine by means of an umbilical cable.
This procedure
allows for many beneficial outcomes, such as providing a preferred environment
for organ
repair, maturation, transport and the use of an animal rather than a human for
the perfusion of
the tissue selection or organ.