Note: Descriptions are shown in the official language in which they were submitted.
208933 i
METHOD FOR PROCESSING AND PRESERVING COLLAGEN-BASED
TISSUES FOR TRANSPLANTATION
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to methods for procuring decellularizing and further
processing and dry preserving collagen-based tissues derived from humans and
animals
for transplantation into humans or other animals. These methods produce a
tissue
product that consists of a selectively preserved extracellular protein matrix
that is devoid
of certain viable cells which normally express major histocompatibility
complex antigenic
determinants and other antigens which would be recognized as foreign by the
recipient.
1o This extracellular protein matrix is made up of collagen and other proteins
and provides
a structural template which may be repopulated with new viable cells that
would not be
rejecxed by the host. These viable cells may be derived from the host
(autologous cells)
before or after transplantation or from an alternative human source including
foreskin,
umbilical cord or aborted fetal tissues. More particularly, this invention
relates to the
procurement and processing of collagen-based tissues such that complications
following
implantation (including but not limited to immunorejection, contracture,
calcification,
occlusion, and infection) are significantly reduced relative to current
implant procedures
and materials.
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208933
Description of the Related Art
Tissue and organ transplantation is a rapidly growing therapeutic field as a
result
of improvements in surgical procedures, advancements in immunosuppressive
drugs and
S increased knowledge of graft/host interaction. Despite major advancements in
this field,
modern tissue transplantation remains associated with complications including
inflammation, degradation, scarring, contracture, calcification (hardening),
occlusion and
rejection. There are numerous investigations underway directed toward the
engineering
of improved transplantable tissue grafts, however, it is generally believed in
the industry
that ideal implants have yet to be produced.
Autologous or self-derived human tissue is often used for transplant
procedures.
These procedures include coronary and peripheral vascular bypass surgeries,
where a
blood vessel, usually a vein, is harvested from some other area of the body
and
transplanted to correct obstructed blood flow through one or more critical
arteries.
Another application of autologous tissue is in the treatment of third degree
burns and
other full-thickness skin injury. This treatment involves grafting of healthy
skin from
uninjured body sites to the site of the wound, a process called split-skin
grafting.
Additional applications of autologous tissue transplantation include bone,
cartilage and
fascia grafting, used for reconstructive procedures.
The motive for using autologous tissue for transplantation is based upon the
concept that complications of immunorejection will be eliminated, resulting in
enhanced
conditions for graft survival. Unfortunately, however, other complications can
ensue with
autologous transplants. For example, significant damage can occur to several
tissue
components of transplanted veins during harvesting and prior to implantation.
This
damage can include mechanical contraction of the smooth muscle cells in the
vein wall
leading to loss of endothelium and smooth muscle cell hypoxia and death.
Hypoxic
damage can result in the release of cellular lysosomes, enzymes which can
cause
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2~8933~
significant damage to the extracellular matrix. Following implantation, such
damage can
lead to increased platelet adhesion, leucocyte and macrophage infiltration and
subsequently further damage to the vessel wall. The end result of such damage
is
thrombosis and occlusion in the early post implant period. Even in the absence
of such
S damage, transplanted autologous veins typically undergo thickening of the
vessel wall and
advancing atherosclerosis leading to late occlusion. The exact cause of this
phenomena
is uncertain but may relate to compliance mismatch of the vein in an arterial
position of
high blood pressure and flow rate. This phenomena may be augmented and
accelerated
by any initial smooth muscle cell and matrix damage occurring during
procurement.
Occlusion of transplanted veins can necessitate repeat bypass procedures, with
subsequent re-harvesting of additional autologous veins, or replacement with
synthetic
conduits or non-autologous vessels.
Another example of complications resulting from autologous tissue
transplantation
is the scarring and contracture that can occur with split-skin grafts for full-
thickness
wound repair. Split-skin grafts are typically mechanically expanded by the use
of a
meshing instrument, which introduces a pattern of small slits in the skin. The
split-skin
graft is then stretched to cover a larger wound area. Dividing epidermal cells
will
ultimately grow into and cover the areas of the slits, however, the underlying
dermal
support matrix does not readily expand into these areas. The dermal matrix,
composed
primarily of collagen, other extracellular protein matrix proteins, and
basement
membrane complex, is responsible for the tensile, flexible nature of skin.
Absence of a
dermal matrix results in scarring and contracture in the area of the slits.
This
contracture can be severe and in cases of massively burned patients that
undergo
extensive split-skin grafting, can necessitate subsequent release surgical
procedures to
restore joint movement.
When the supply of transplantable autologous tissues is depleted, or when
there is
no suitable autologous tissue available for transplant (e.g., heart valve
replacement), then
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_208933
substitutes may be used, including man-made synthetic materials, animal-
derived tissues
and tissue products, or allogeneic human tissues donated from another
individual
(usually derived from cadavers). Man-made implant materials include synthetic
polymers
(e.g. (PTFE) polytetrafluroethylene, Dacron and Goretex) sometimes formed into
a
tubular shape and used as a blood flow conduit for some peripheral arterial
bypass
procedures. Additionally, man-made synthetics (polyurethanes) and
hydrocolloids or gels
may be used as temporary wound dressings prior to split-skin grafting.
Other man-made materials include plastics and carbonized metals, fashioned
into
a prosthetic heart valve, utilized for aortic heart valve replacement
procedures. Synthetic
materials can be made with low immunogenicity but are subject to other
limitations. In
the case of mechanical heart valves, their hemodynamic characteristics
necessitate
life-long anticoagulant therapy. Synthetic vascular conduits, often used in
above-the-knee
peripheral vascular bypass procedures, are subjected to an even higher
incidence of
occlusion than autologous grafts. In many cases, a preference is made for a
biological
implant which can be a processed animal tissue or a fresh or cryopreserved
allogeneic
human tissue.
Animal tissues (bovine or porcine) chemically treated are commonly used as
replacements for defective human heart valves, and have been used in the past
for
vascular conduits. The concept in the chemical processing is to stabilize the
structural
protein and collagen matrix by cross-linking with glutaraldehyde or a similar
cross-linking
agent. This treatment also masks the antigenic determinants, such that the
human host
will not recognize the implant as foreign and precludes an immunorejection
response.
Glutaraldehyde-treated tissues, however, will not allow in-migration of host
cells which
are necessary for remodeling, and will gradually harden as a result of
calcification. For
this reason, glutaraldehyde-treated tissues generally require replacement in 5-
7 years.
Glutaraldehyde-treated bovine veins have been used in the past for vascular
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2~8~3~~
bypass procedures, however, their use has been discontinued due to the
unacceptable
incidence of aneurysm formation and occlusion.
The use of allogeneic transplant tissues has been applied to heart valve
replacement procedures, arterial bypass procedures, bone, cartilage, and
ligament
replacement procedures and to full-thickness wound treatment as a temporary
dressing.
The allogeneic tissue is used fresh, or may be cryopreserved with the use of
DMSO
and/or glycerol, to maintain viability of cellular components. It is thought
that the
cellular components contain histocompatibility antigens, and are capable of
eliciting an
immune response from the host. In many cases, the patient receiving the
allogeneic
transplant undergoes immunosuppressive therapy. Despite this therapy, many
allogeneic
transplants, including heart valves and blood vessels, undergo an inflammatory
response,
and fail within 5-10 years. Allogeneic skin is typically rejected within 1-5
weeks of
application, and has never been demonstrated to be permanently accepted by the
host,
even with the use of immunosuppressive drugs.
Alternative processing methods have been developed by others that are intended
to address the limitations of allogeneic and animal-derived transplant
tissues.
Freeze-drying is used routinely in the processing of allogeneic bone for
transplantation.
It has been found that the freeze drying process results in a graft which
elicits no
significant rejection response as compared to fresh or cryopreserved
allogeneic bone.
The freeze-dried bone following implant acts as a template, which is
subsequently
remodelled by the host. When the freeze-drying process has been applied to
more
complex tissues such as heart valves, the results have been mined but overall
unsatisfactory. A study was conducted in which 15 allogeneic heart valves were
processed by freeze-drying prior to transplantation. Most of the freeze-dried
valves
failed due to mechanical causes in the early post-graft interval. Those freeze-
dried
valves which did not fail, however, demonstrated prolonged functionality (up
to 15 years).
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2~8~3~
Enzymes and detergent processing has also been used to remove antigenic cells
from collagen-based transplantable tissues. Organic solvents and detergent
treatments
have been used successfully with relatively simple tissues such as dura mater
used in
reconstructive surgical procedures. Chemical processing of more complex
structures such
S as heart valves, vascular conduits and skin, however, has had only limited
success in
clinical applications.
The invention of this patent is a comprehensive processing technique that
addresses potential damaging events in the preparation of complex collagen-
based tissues
for transplantation. The technology combines both biochemical and physical
processing
steps to achieve the ideal features of template function such that the tissue
graft can be
remodeled for long-term maintenance by the host.
BRIEF SUMMARY OF THE INVENTION
In its preferred form, the method of this invention includes the steps of
processing
biological tissues including treatment with a stabilizing solution to reduce
procurement
damage, treatment with a processing solution to remove cells and other
antigenic tissue
components, treatment with a cryoprotectant solution, freezing and storage
under specific
conditions to avoid functionally significant damaging ice crystal formation,
drying under
conditions to prevent damaging ice recrystallization, storage in the dry state
at above
freezing temperatures, rehydration under specific conditions and with a
rehydration
solution to minimize surface tension damage and further augment the selective
preservation of the matrix, and reconstitution with viable cells that will not
be rejected
by the host.
2~9336
According to the invention in one broad aspect, there is provided a method for
processing collagen-based tissue for transplantation. The method comprises
procuring
collagen-based tissue and placing it into a stabilizing solution to prevent
osmotic,
hypoxic, autolytic and proteolytic degradation and to protect against
microbial
contamination. The collagen-based tissue is then incubated in a processing
solution to
produce processed tissue, the processing solution extracting viable cells from
the
structural protein and collagen matrix of the collagen-based tissue. The
processed tissue
is cryoprepared by incubation in a cryoprotective solution and freezing at
cooling rates
such that minimal functional damage occurs to the structural protein and
collagen matrix
of the processed tissue to produce cryoprepared, processed tissue. The
cryoprepared,
processed tissue is then dryed under temperature and pressure conditions that
permit
removal of water without substantial ice recrystallization or ultrastructural
damage, the
drying resulting in a residual moisture content of the cryoprepared, processed
tissue that
permits both storage and rehydration of the tissue, to produce dried,
cryoprepared,
processed tissue. The dried cryoprepared, processed tissue is then incubated
in a
rehydration solution, the rehydration solution preventing osmotic, hypoxic,
autolytic, or
proteolytic damage, microbial contamination and ultrastructural damage and to
result in
a rehydrated tissue having a final water content of 20% to 70%. The method
concludes
with inoculation of the rehydrated tissue with viable cells selected from the
group
consisting of autogeneic cells, allogeneic cells or combinations thereof or
allowing
viable cells to repopulate the collagen matrix following transplantation.
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zo~9~~~
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a method for processing and preserving
collagen-based biological tissues for transplantation, through steps of
chemical
S pretreatment and cell removal, cryopreparation, dry stabilization, drying,
rehydration and
cellular reconstitution. The processing and preservation method is designed to
generate
a transplantable biological tissue graft that specifically meets the following
criteria:
(a) provides an extracellular protein and collagen matrix which can be
remodelled and repaired by the host,
(b) provides an intact basement membrane for secure reattachment of viable
endothelial or epithelial cells,
(c) does not elicit an immune response by the host,
(d) does not calcify, and
(e) can be easily stored and transported at ambient temperatures.
In the preferred embodiment, the biological tissue to be processed is first
procured or harvested from a human cadaver or animal donor and immediately
placed in
a stabilizing transportation solution which arrests and prevents osmotic,
hypoxic, autolytic
and proteolytic degradation, protects against bacterial contamination and
reduces
mechanical damage that can occur with tissues that contain smooth muscle
components
(e.g. blood vessels). The stabilizing solution generally contains an
appropriate buffer,
one or more antioxidants, one or more oncotic agents, an antibiotic, one or
more
protease inhibitors, and in some cases, a smooth muscle relaxant.
In the preferred embodiment, the tissue is then incubated in a processing
solution
to remove viable antigenic cells (including epithelial cells, endothelial
cells, smooth
muscle cells and fibroblasts) from the structural matrix without damaging the
basement
membrane complex or the structural integrity of the collagen matrix. The
processing
solution generally contains an appropriate buffer, salt, an antibiotic, one or
more
_g_
20893
detergents, one or more protease inhibitors, and/or one or more enzymes.
Treatment of
the tissue with this processing solution must be at a concentration for a time
duration
such that degradation of the basement membrane complex is avoided and the
structural
integrity of the matrix is maintained including collagen fibers and elastin.
After the tissue is decellularized, it is preferably incubated in a
cryopreservation
solution. In the preferred embodiment, this solution generally contains one or
more
cryoprotectants to minimize ice crystal damage to the structural matrix that
could occur
during freezing, and one or more dry-protective components, to minimize
structural
damage alteration during drying and may include a combination of an organic
solvent
and water which undergoes neither expansion or contraction during freezing. As
an
alternate method, the decellularized tissue matrix can be fixed with a
crosslinking agent
such as glutaraldehyde and stored prior to transplantation. Following
incubation in this
cryopreservation solution, the tissue is packaged inside a sterile container,
such as a glass
vial or a pouch, which is permeable to water vapor yet impermeable to
bacteria.
In the preferred embodiment, one side of this pouch consists of medical grade
porous Tyvek membrane, a trademarked product of DuPont Company of Wilmington,
Delaware. This membrane is porous to water vapor and impervious to bacteria
and dust.
The Tyvek membrane is heat sealed to a 2.5 millimeter impermeable polythylene
laminate sheet, leaving one side open, thus forming a two-sided pouch. The
open pouch
is sterilized by gamma radiation prior to use. The tissue is aseptically
placed through
this opening into the sterile pouch. The open side is then aseptically heat
sealed to close
the pouch. The packaged tissue is henceforth protected from microbial
contamination
throughout subsequent processing steps.
In the preferred embodiment, the packaged tissue is cooled to a low
temperature
at a specified rate which is compatible with the specific cryoprotectant to
minimize
damaging hexagonal ice and to generate the less stable ice forms of amorphous
and
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CA 02089336 1999-OS-19
cubic phases. The tissue is then dried at a low temperature under vacuum
conditions, such
that water vapor is removed sequentially from each ice crystal phase without
ice
recrystallization. Such drying is achieved either by conventional freeze
drying or by using a
previously patented molecular distillation dryer. Suitable molecular
distillation dryers can
be obtained from LifeCell Corporation in Woodlands, Texas and are disclosed in
U.S.
Patent Nos. 4,567,847 and 4,799,361 which may be referred to for further
details.
At the completion of the drying cycle of samples dried in a pouch, the vacuum
of
the freeze drying apparatus is reversed with a dry inert gas such as nitrogen,
helium or
argon. While being maintained in the same gaseous environment, the semi-
permeable
pouch is placed inside an impervious pouch which is further heat or pressure
sealed. The
final configuration of the d:ry sample is therefore in an inert gaseous
atmosphere,
hermetically sealed in an impermeable pouch.
At the completion of the drying cycle of samples dried in a glass vial, the
vial is
sealed under vacuum with an appropriate inert stopper and the vacuum of the
drying
apparatus reversed with an inert gas prior to unloading.
In the preferred embodiment, the packaged dried tissue may be stored for
extended time periods under ambient conditions. Transportation may be
accomplished via
standard carriers and under standard conditions relative to normal temperature
exposure and
delivery times.
In the preferred embodiment, the dried tissue is rehydrated prior to
transplantation. It is important to minimize osmotic forces and surface
tension effects
during rehydration. The aim in rehydration is to augment the selective
preservation of
the extracellular support matrix while removing any residual antigenic cells
and
other potentially antigenic components. Appropriate rehydration may be
accomplished by
an initial incubation of the dried tissue in an environment of about 100%
relative humidity,
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298938
followed by immersion in a suitable rehydration solution. Alternatively, the
dried tissue
may be directly immersed in the rehydration solution without prior incubation
in a high
humidity environment. Rehydration should not cause osmotic damage to the
sample.
Vapor rehydration should ideally achieve a residual moisture level of at least
15% and
fluid rehydration should result in a tissue moisture level of between 20% and
70%.
Depending on the tissue to be rehydrated, the rehydration solution may be
simply
normal saline, Ringer's lactate or a standard cell culture medium. Where the
tissue is
subject to the action of endogenous collagenases, elastases or residual
autolytic activity
from previously removed cells, additives to the rehydration solution are made
and
include protease inhibitors. Where residual free radical activity is present,
agents to
protect against hypoxia are used including antioxidants, enzymatic agents
which protect
against free radical damage and agents which minimize the disturbance of
biochemical
pathways which result from hypoxic damage. Antibiotics may also be included to
inhibit
bacterial contamination. Oncotic agents being in the form of proteoglycans,
dextran
and/or amino acids may also be included to prevent osmotic damage to the
matrix
during rehydration. Rehydration of a dry sample is especially suited to this
process as it
allows rapid and uniform distribution of the components of the rehydration
solution. In
addition, the rehydration solution may contain specific components not used
previously,
for example diphosphonates to inhibit alkaline phosphatase and prevent
subsequent
calcification. Agents may also be included in the rehydration solution to
stimulate
neovascularization and host cell infiltration following transplantation of the
rehydrated
extracellular matrix. Alternatively, rehydration may be performed in a
solution
containing a crosslinking agent such as glutaraldehyde
Immunotolerable viable cells must be restored to the rehydrated structural
matrix
to produce a permanently accepted graft that may be remodeled by the host. In
the
preferred embodiment, immunotolerable viable cells may be reconstituted by in
vitro cell
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_208~3~~
culturing techniques prior to transplantation, or by in vivo repopulation
following
transplantation.
In the preferred embodiment the cell types used for in vitro reconstitution
will
depend on the nature of the transplantable graft. The primary requirement for
reconstitution of full-thickness skin from processed and rehydrated dermis is
the
restoration of epidermal cells or keratinocytes. These cells may be derived
from the
intended recipient patient, in the form of a small meshed split-skin graft or
as isolated
keratinocytes expanded to sheets under cell culture conditions. Alternatively,
allogeneic
keratinocytes derived from foreskin or fetal origin, may be used to culture
and
reconstitute the epidermis.
The important cell for reconstitution of heart valves and vascular conduits is
the
endothelial cell, which lines the inner surface of the tissue. Endothelial
cells may also be
expanded in culture, and may be derived directly from the intended recipient
patient or
from umbilical arteries or veins.
Following drying, or following drying and rehydration, or following drying,
rehydration and reconstitution, the processed tissue graft will be transported
to the
appropriate hospital or treatment facility. The choice of the final
composition of the
product will be dependent on the specific intended clinical application.
In the practice of this invention, it is fundamental that suitable tissues are
obtained prior to processing. Human cadaver tissues are obtainable through
approximately 100 tissue banks throughout the nation. Additionally, human
tissues are
obtainable directly from hospitals. A signed informed consent document is
required
from the donor's family to allow harvesting of tissues for transplantation.
Animal tissues
are obtainable from a number of meat processing companies and from suppliers
of
laboratory animals. The particular type of tissue harvested is not limiting on
the method
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~089~~~;
of this invention. However, processing of the tissue is enhanced by the use of
specific
procurement procedures, and treatment with a stabilizing solution to prevent
certain
mechanical and biochemical damaging events.
The harvested tissues can undergo a variety of mechanical and biochemical
damaging events during procurement. Both the cellular components and the
extracellular matrix can be injured during these events. Damage to the
extracellular
matrix occurs primarily as a result of destabilization of the cellular
component. The
intent of this invention is to ultimately remove this cellular component and
to optimally
preserve the extracellular matrix, therefore the stabilizing solution is
formulated to
minimize the initial cellular and subsequently the extracellular matrix
damage. The
extracellular protein and collagen matrix comprises a native three dimensional
lattice
that includes various proteins such as Type I collagen, Type II collagen, Type
III
collagen, Type IV collagen, elastin, laminin, teninsin and actinin, and
proteoglycans.
The initiating event in cellular damage is hypoxia (deficiency of oxygen
reaching
tissues of the body) and a lack of nutrient supply required for the cell to
maintain
metabolism and energy production. Hypoxia and especially hypoxia and
reperfusion
results in the generation of free radicals such as hydrogen peroxide, an
oxidizing species
that reacts with cellular components including membranes and proteins. The
subsequent
changes of lipid peroxidation and crosslinking result in structural and
functional
derangement of the cell and initiate release of autolytic enzymes (normally
contained in
lysosomes) into the extracellular matrix. The damage to the matrix is two-
fold, oxidant
damage and enzymatic degradation. A lack of nutrient supply amplifies these
events in
that the cell can no longer provide the energy requirements necessary to
maintain its
defense mechanisms against hypoxic damage. In minimizing these events, several
approaches are possible. These include the use of enzymes (superoxide
dismutase and
catalase) to neutralize the superoxide anion and hydrogen peroxide or
compounds which
can directly react with and neutralize other free-radical species. These
compounds
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X089336
referred to as antioxidants include tertiary butylhydroquinone (BHT), alpha
tocopherol,
mannitol, hydroxyurea, glutathione, ascorbate, ethylenediaminetetraacetic acid
(EDTA)
and the amino acids histidine, proline and cysteine. In addition to
antioxidants, the
stabilizing solution generally contains agents to inhibit hypoxic alteration
to normal
biochemical pathways, for example, allopurinol to inhibit xanthine
dehydrogenase,
lipoxygenase inhibitors, calcium channel blocking drugs, calcium binding
agents, iron
binding agents, metabolic intermediaries and substrates of adenosine
triphosphate (ATP)
generation.
The stabilizing solution also generally contains one or more antibiotics,
antifungal
agents, protease inhibitors, proteoglycans, and an appropriate buffer.
Antibiotics are
necessary to inhibit or prevent bacterial growth and subsequent tissue
infection.
Antibiotics may be selected from the group of penicillin, streptomycin,
gentamicin
kanamycin, neomycin, bacitracin, and vancomycin. Additionally, anti-fungal
agents may
be employed, including amphotericin-B, nystatin and polymyxin.
Protease inhibitors are included in the stabilizing solution to inhibit
endogenous
proteolytic enzymes which, when released, can cause irreversible degradation
of the
extracellular matrix, as well as the release of chemoattractant factors. These
chemoattractants solicit the involvement of polymorphonuclear leukocytes,
macrophages
and other killer cells which generate a nonspecific immune response that can
further
damage the extracellular matrix. Protease inhibitors are selected from the
group
consisting of N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF),
ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethyl
ether)-
N,N,N',N'-tetraacetic acid (EGTA), leupeptin, ammonium chloride, elevated pH
and
apoprotinin.
Proteoglycans are included in the stabilizing solution to provide a colloid
osmotic
balance between the solution and the tissue, thereby preventing the diffusion
of
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....,_"~..
2089336
endogenous proteoglycans from the tissue to the solution. Endogenous
proteoglycans
serve a variety of functions in collagen-based connective tissue physiology.
They may be
involved in the regulation of cell growth and differentiation (e.g. heparin
sulfate and
smooth muscle cells) or, alternatively, they are important in preventing
pathological
calcification (as with heart valves). Proteoglycans are also involved in the
complex
regulation of collagen and elastin synthesis and remodelling, which is
fundamental to
connective tissue function. Proteoglycans are selected from the group of
chondroitin
sulfate, heparin sulfate, and dermatan sulfate. Non-proteoglycan osmotic
agents which
may also be included are polymers such as dextran and polyvinyl ~yrrolidone.
(PVP) and
amino acids such as glycine and proline.
The stabilizing solution also generally contains an appropriate buffer. The
nature
of the buffer is important in several aspects of the processing technique.
Crystalloid, low
osmotic strength buffers have been associated with damage occurring during
saphenous
vein procurement and with corneal storage. Optimum pH and buffering capacity
against
the products of hypoxia damage (described below), is essential. In this
context the
organic and bicarbonate buffers have distinct advantages. (In red cell
storage,
acetate-citrate buffers with glycine and glucose have been shown to be
effective in
prolonging shelf-life and maintaining cellular integrity.) The inventors
prefer to use an
organic buffer selected from the group consisting of 2-(N-
morpholino)ethanesulfonic acid
(MES), 3-(N-morpholine)propanesulfonic acid (MPOS) and N-2-
hydroxyethylpiperazine-
N'-2-ethane-sulfonic acid (HEPES). Alternatively, a low salt or physiological
buffer,
including phosphate, bicarbonate and acetate-citrate, may be more appropriate
in certain
applications.
In another preferred embodiment, components of the stabilizing solution
address
one or more of the events that occur during the harvesting of vascular
tissues, such as
spasm, hypoxia, hypoxia reperfusion, lysosomal enzyme release, platelet
adhesion, sterility
and buffering conditions. Involuntary contraction of the smooth muscle lining
of a blood
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r.~...
;~;.
zos~~~~
vessel wall can result from mechanical stretching or distension, as well as
from the
chemical action of certain endothelial cell derived contraction factors,
typically released
under hypoxic (low oxygen) conditions. This involuntary contraction results in
irreversible damage to the muscle itself, the endothelial cells and the
surrounding
extracellular matrix. For this reason, the stabilizing solution for blood
vessels includes
one or more smooth muscle relaxants, selected from the group of calcitonin
gene related
peptide (CGRP), papaverine, sodium nitroprusside (NaNP), H7 (a protein Kinase
C
inhibitor) calcium channel blockers, calcium chelators, isoproterenol,
phentolamine,
pinacidil, isobutylmethylxanthine (IBMX), nifedepine and flurazine. The
harvested tissue
is immediately placed into this stabilizing solution and is maintained at
4°C during
transportation and any storage prior to further processing.
In the practice of this invention, it is essential that the harvested tissue
be
processed to remove antigenic cellular components.
Decellularization can be accomplished using a number of chemical treatments,
including incubation in certain salts, detergents or enzymes. The use of the
detergent
Triton X-100, a trademarked product of Rohm and Haas Company of Philadelphia,
PA,
has been demonstrated to remove cellular membranes, as detailed in U.S. Patent
No.
4,801,299. Other acceptable decellularizing detergents include polyoxyethylene
(20)
sorbitan mono-oleate and polyoxyethylene (80) sorbitan mono-oleate (Tween 20
and 80),
sodium deoxycholate, 3-((3-chloramidopropyl)-dimethylammino]-1-propane-
sulfonate,
octyl-glucoside and sodium dodecyl sulfate.
Alternatively, enzymes may be used to accomplish decellularization, including
but
not limited to dispase II, trypsin, and thermolysin. These enzymes react with
different
components of collagen and intercellular connections in achieving their
effects. Dispase
II attacks Type IV collagen, which is a component of the lamina densa and
anchoring
fibrils of the basement membrane. Thermolysin attacks the bulbous phemphigoid
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2U8~~~~
antigen in the hemidesmosome of the basal layer of keratinocytes. Trypsin
attacks the
desmosome complex between cells. Due to the proteolytic nature of these
enzymes, care
must be taken that cellular removal occurs without significant damage to the
extracellular matrix, including the basement membrane complex. This is a
function of
concentration, time and temperature. If used for too long a time or at too
high a
concentration, dispase II for example can completely remove the basement
membrane
complex from the dermis.
For example, with human cadaver skin Dispase II at 1.0 units/ml for 90 minutes
at 37°C will remove all heratinocytes except the basal layer, while
some damage is
already occurring to the basement membrane complex. Thermolysin at 200 ug/ml
for 30
minutes at 4°C will essentially remove all keratinocytes without damage
to the basement
membrane complex on some occasions, but this varies from donor to donor with
evidence of basement membrane damage being seen in some donors. Incubation of
skin
in 1 molar sodium chloride for 16 hours for human skin and 48 hours for
porcine skin
will routinely allow clean separation of the epidermis and dermis without
damage to the
basement membrane complex.
In addition to salts, detergents and enzymes, the processing solution also
contains
certain protease inhibitors, to prevent degradation of the extracellular
matrix.
Collagen-based connective tissues contain proteases and collagenases as
endogenous
enzymes in the extracellular protein matrix. Additionally, certain cell types
including
smooth muscle cells, fibroblasts and endothelial cells contain a number of
these enzymes
inside vesicles called lysosomes. When these cells are damaged by events such
as
hypoxia, the lysosomes are ruptured and their contents released. As a result,
the
extracellular matrix can undergo severe damage from protein, proteoglycan and
collagen
breakdown. This damage may be severe, as evidenced in clinical cases of
cardiac
ischemia where a reduction in oxygen which is insufficient to cause cell death
results in
pronounced damage to the collagen matrix. Additionally, a consequence of
extracellular
-17-
2089336
breakdown is the release of chemoattractants, which solicit inflammatory
cells, including
polymorphonuclear leukocytes and macrophages, to the graft, which are intended
to
remove dead or damaged tissue. These cells also, however, perpetuate the
extracellular
matrix destruction through a nonspecific inflammatory response. Accordingly,
the
S processing solution contains one or more protease inhibitors selected from
the group of
N-ethylmaleimide (NEM), phenylmethylsulfonylfluoride (PMSF), ethylenediamine
tetraacetic acie (EDTA), ethylene glycol-bis-(2-aminoethyl e.ther)NNN'N'-
tetraacetic acid,
ammoniurin chloride, elevated pH, apoprotinin and leupeptin to prevent such
damage.
In addition to salts, detergents, enzymes and protease inhibitors, the
processing
solution generally contains an appropriate buffer. This may involve one of
many
different organic buffers which are described above. The inventors prefer to
use an
organic buffer selected from the group consisting of 2-(N-
morpholino)ethanesulfonic
acid (MES), Tris (hydroxymethyl)amionomethane (TRIS) and (N-(2-
hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES). Alternatively, a
low salt or
physiological buffer including phosphate bicarbonate acetate citrate glutamate
with or -
without glycine, may be more appropriate in certain applications. Low salt or
physiological buffers are more able to support the infiltration of the graft
with viable
cells and hence are mole relevant when cellular infiltration including
neovascularization
is essential to early survival of the graft as in transplated dermal matrix.
As the processing solution may contain chemicals that would be irritating or
inflammatory on transplantation, it is important to the practice of this
invention that the
processing solution-be thoroughly rinsed from the tissue. In the preferred
embodiment,
this washing occurs by rinsing in sufficient changes of appropriate buffer,
until residues
of the processing solution are reduced to levels compatible with
transplantation.
Alternatively, components of the processing solution may be neutralized by
specific
inhibitors, e.g., dispase II by ethylenediaminetetraacetic acid (EDTA) or
trypsin by
serum.
-18-
".
>~,3 i
.,..;,,y ,.,.rn~~~:."~~s
2Q8~~~ ~
The cryopreparation or freezing of the tissue takes place following thorough
washing. Biological materials generally undergo siguficant deterioration
during freezing
and thawing or following freeze-drying by conventional means. Accordingly,
these steps
should be avoided prior to incubation of the processed, decellularized tissue
in a
cryoprotectant solution by the method described in this application.
The initial steps of cryopreserving the decellularized tissue includes
incubating the
tissue in a cryosolution prior to the freezing step. The cryosolution
comprises an
appropriate buffer, one or more cryoprotectants and/or dry protectants with or
without
an organic solvent which in combination with water undergoes neither expansion
or
contraction.
An appropriate buffer may involve any of the previously described buffers
utilized
in procurement or decellularization processing of the harvested tissue.
In addition to an appropriate buffer, the cryosolution generally contains a
cryoprotectant. Cryoprotectants raise the glass transition temperature range
of the tissue
thereby allowing optimum stabilization of the tissue in the frozen state. By
raising this
range, the tissue can be dried at a faster rate. The cryoprotectant also
decreases ice
formation for a given cooling rate allowing to some degree vitrification
(absence of a
crystalline lattice), but to_a greater extent, the formation of cubic ice.
With current
methods of ultra-rapid cooling in the absence of cryoprotectants,
vitrification is only
achieved with very small samples, and only to a depth of a few microns. Cubic
and
hexagonal ice are then encountered. Vitrified water and cubic ice are less
damaging to
extracellular matrix components than is hexagonal ice. In some cases, however,
it is
permissible to allow hexagonal ice to occur (e.g., the processing of skin).
Some degree
of hexagonal ice formation is permissible when it does not result in
impairment of the
functional characteristics of the tissue. Heart valves following implantation
are subject to
repetitive stress and hence will tolerate less ice crystal damage than, for
example, dermis.
-19-
Various cryoprotectants can be used in the present invention. These include:
dimethylsulfoxide (DMSO), dextran, sucrose, 1,2 propanediol, glycerol,
sorbitol, fructose,
trehalose, raffinose, propylene glycol, 2-3 butane diol, hydroxyethyl starch,
polyvinylpyrrolidone (PVP), proline (or other protein stabilizers), human
serum albumin
and combinations thereof. Suitable cryoprotectants structure water molecules
such that
the freezing point is reduced and/or the rate of cooling necessary to achieve
the vitreous
phase is reduced. They also raise the glass transition temperature range of
the vitreous
state.
The cryosolution may also include exposing the biological tissue to one or
more
dry protectant compounds. Dry protectants, by definition, stabilize samples in
the dry
state. Some cryoprotectants also act as dry protectants. Some compounds
possess
variable amounts of each activity, e.g., trehalose is predominantly a dry
protectant and a
weaker cryoprotectant, whereas sucrose is predominantly a cryoprotectant and a
weaker
dry protectant. For example, trehalose and polyhydroxyl carbohydrates bind to
and
stabilize macromolecules such as proteins. Various dry protectants can be used
in the
present invention: sucrose, raffinose, trehalose, zinc, proline (or other
protein stabilizers),
myristic acid, spermine (a polyanionic compound) and combinations thereof.
The combination of 0.5 Molar dimethyl sulfoxide, 0.5 M propylene glycol 0.25 M
2-3 butanediol, 1.0 M proline, 2.5% raffinose 15% polyvinylpyrrolidone and 15%
dextran
(MWT 70,000) in combination with cooling rates of the order of -
2500°C/second has
been shown to be effective in maintaining the structural integrity of human
saphenous
veins following both freezing and drying. The inventors have also demonstrated
that
when this solution of cryoprotectants, dry protectants and buffer are used
with a larger
tissue sample, such as a heart valve, then the tissue can undergo cracking
following
freezing and/or drying. This phenomena can be overcome by replacing a percent
of the
water with an organic solvent such as formamide. The percent (50%) is
determined as
that combination of solvent, water, cryoprotectants and dry protectants which
will not
-20-
2os~~~~
expand or contract during freezing. Formamide (HCONHZ) is a one carbon,
hydrophilic,
organic solvent which dissolves carbohydrate based cryoprotectants. It may be
substituted with other organic solvents with similar properties such as
dimethylformamide, dimethylsulfoxide (DMSO), glycerol, proplyene glycol,
ethylene
glycol, and pyridine.
The biological samples are incubated in the cryosolutions for a period of a
few
minutes to a few hours before they are rapidly cooled. In general,
cryopreservation is
performed as a continuous sequence of events. The tissue is first incubated in
the
cryosolution for a defined period (0.5 to 2 hours) until complete penetration
of the
components of the cryosolution is achieved and the sample is then frozen to a
temperature at which it is stable, usually less than -20°C.
The inventors have been involved in the development of cryofixation and
ultralow
temperature molecular distillation drying as a method for preparing biological
samples
for electron microscopic analysis. To validate this approach, they
investigated the
relationship between drying characteristics and ice phases present within
frozen samples.
Sample preparation for electron microscopy by purely physical or dry
processing
techniques has theoretical appeal, especially when the ultimate aim is the
analysis of
both ultrastructure and biochemistry. Since the earliest days of electron
microscopy,
several attempts have been made to refine and develop freezing and vacuum
drying or
the freeze-drying (FD) process for cell and tissue samples.
Despite the conceptual advantages and the progress made, freeze-drying for
electron microscopy has yet to achieve the status of a routine, broadly
applicable
technique. Several reasons account for this. First, the ultrastructural
preservation is
often inferior when compared to conventional chemical, or wet processing
techniques or
hybrid techniques such as freeze substitution. Second, there are numerous
practical
-21-
20~93~fi
problems with sample manipulation, temperature control, vacuum parameters, and
end
processing protocols. Third, and perhaps most fundamentally, is a belief that
drying at
temperatures below -123°C to avoid ultrastructural damage is either
impossible or
impractical. As a result of these practical and theoretical obstacles, only
sporadic
S investigation of low temperature freeze-drying has been reported.
The basis of this theoretical barrier comes from application of the kinetic
gas
theory and the predicted sublimation rates as expressed by the Knudsen
equation:
o.s
Js = NPs ~ 2,~QTJ
where Js = sublimation rate
N - coefficient of evaporation
Ps = saturation vapor pressure
Q - universal gas constant
T - absolute temperature of
the sample
M - molecular weight of water.
For theoretically ideal drying conditions, this equation states that the
sublimation
rate is directly proportional to the saturation vapor pressure of water within
the sample
and inversely proportional to the absolute temperature of the sample. Although
the
temperature of the sample is clearly definable, saturation vapor pressure is a
more
complex parameter.
Prior applications of this equation have used saturation vapor pressures which
were theoretically determined. These theoretical vapor pressures, however,
include the
latent heat of fusion, and hence, are applicable only to hexagonal ice.
Calculations
based on these theoretical values have led to conclusions such as "at 150K it
would take
3.5 years until an ice layer of 1 mm thickness is completely removed by freeze
drying. It
is therefore unrealistic to attempt freeze drying at temperatures below 170K."
-22-
20893~~
Several phases of ice other than hexagonal, however, can coexist within a
sample
depending upon the mode of cooling and the use of cryoprotectants. These
different
phases can be achieved by several methods including; vapor condensation,
hyperbaric
application and ultrarapid quench cooling.
The major phases of ice now recognized are amorphous, cubic, and hexagonal.
These ice phases exhibit different stabilities, which would suggest that the
saturation
vapor pressures would also be different. It has been determined that for vapor
condensed water at temperatures where both phases can coexist, the saturation
vapor
pressure of amorphous ice is one to two logs higher than that of cubic ice.
Application of these experimentally determined saturation vapor Pressures in
the
Knudsen equation reduces the drying time at 150K from 3.5 years to 0.035
years, or 12.7
days, for 1 mm of amorphous ice. Because quench cooling techniques of
biological
samples achieve approximately 5 ~cm of this phase, the drying time of this
component,
based solely on the Knudsen equation, would be of the order of 1.5 hours.
Hence, in
terms of practical drying times, the theoretical barrier to drying at ultralow
temperatures
can be overcome.
Drying, however, is not a static, but a rate-dependent process. In addition to
saturation vapor pressure of the different ice phases, one must also account
for the rate
of transition from one phase to another with increasing temperature. For
electron
microscopy sample preparation, drying should ideally occur without any such
transition
or devitrification. Information as to the rate of these transitions is
limited. It has been
found that the amorphous to cubic transition occurred as an irreversible
process strongly
dependent upon temperature in the range of -160°C to -130°C and
expressed by
t = 2.04 x 10~ x exp (-0.465T)
-23-
208936
The cubic to hexagonal transition was less temperature-dependent, occurring in
the range
of -120°C to -65°C, and expressed by
t = 2.58 x 1012 x exp (-0.126T)
S
Interestingly, when the sample temperature was increased at a rate of
S°C/minute, the
amorphous to cubic transition occurred as a sudden event near -130°C.
Based upon the above data, the transition rate, as well as the saturation
vapor
pressure, determine the depth to which a particular ice phase can be dried at
a specific
temperature. For amorphous ice at -160°C, the transition time is 205
days. Based upon
extrapolation of experimentally determined saturation vapor pressures and the
Knudsen
equation, this would allow drying of 26 microns. At -140°C, transition
time is 28 minutes
and would allow drying of 0.8 ~cm under ideal conditions. Below -160°C,
i.e., prior to the
onset of the transition, one could predict little, if any, translational
kinetic energy of the
water molecules and hence little, if any, drying.
Based upon these considerations, one can postulate the hypothesis of
transitional
drying, i.e., that for a sample containing multiple phases of ice, it is
possible to dry each
phase sequentially during its transition. The amount of each phase dried will
obviously
be dependent upon multiple parameters including efficiency of drying
apparatus, rate of
heating, and impedance of the dry shell.
Cryoyreservation
Cryopreservation is the preservation of cell or tissue structure against
injury
associated with freezing events. Natural cryoprotection can result from
adaptive
metabolism of the organism, with changes in cellular structure, composition
and
metabolic balance giving an enhanced tolerance of freezing. In laboratory
experiments
when cell viability or tissue ultrastructure are to be preserved following
cooling, two
-24-
20~~~~~
methods are available. The first is to ultrarapidly cool the sample, resulting
in the tissue
fluids being vitrified, i.e., absence of ice crystals. The second is to
incorporate chemical
additives to confer a degree of cryoprotection. The chemicals range from
naturally
occurring cryoprotectants such as glycerol, proline, sugars, and alcohols to
organic
S solvents such as dimethylsulfoxide (DMSO) to high molecular weight polymers
such as
polyvinylpyrrolidone (PVP), dextran and hydroxyethyl starch (HES).
Vitrification of cells and tissues is limited by the rate at which the sample
can be
cooled and the insulating properties of the tissue itself. Due to physical
limitations, one
can only achieve vitrification of a thin layer of tissues using state of the
art techniques.
This makes the idea of chemical additives for cryoprotection and manipulating
the
cooling rate very appealing in attempts to cool and store biological samples
without
causing structural and functional damage.
Injury to biological samples due to freezing is subject to fundamental
physical and
biological principles, some long known, but others only recently being
understood.
Serious investigations into the mechanisms of freezing injury in biological
samples did
not begin until the second quarter of this century. These early studies were
dominated
by the belief that physical damage by ice crystals was the principal cause of
freeze injury.
The effects of dehydration and a correlation between the concentration of
extracellular
solutes and cell and tissue damage has been demonstrated. A "two factor"
hypothesis for
cell freezing injury proposed that cell injury was the result of either the
concentration of
solutes by extracellular ice or the formation of intracellular ice which
caused mechanical
injury.
The action of glycerol and other small polar compounds has been interpreted as
penetrating and exerting colligative action within the cells. In the
proportion that the
colligative action of the penetrating compounds maintains water in the liquid
state at
temperatures below 0°C, an increased volume of cellular solution is
maintained. This
-25-
208930
avoids an excessive concentration of toxic electrolytes in the nonfrozen
cellular solution.
A similar influence also takes place in the external solution. In this
context, colligative
action is referred to as action by an extraneous solute, in lowering the
freezing point of
the solution in contact with ice. If enough protective compound is present,
the salt
S concentration does not rise to a critically damaging level until the
temperature becomes
so low that the damaging reactions are slow enough to be tolerated by the
cells. Similar
concepts of damage to tissue matrix by both mechanical growth of ice crystals
and
chemical damage due to concentration of solute and changes in pH can also be
applied.
The nonpenetrating cryoprotectants vary in size from sucrose to large
polymeric
substances such as PVP, HES and dextran. It has been suggested that
nonpenetrating
substances act by some other means than that in the colligative mechanism
described
above. The role of larger molecules is believed to be dehydrative by osmotic
action.
When a large proportion of water is withdrawn from the cells by means of an
osmotic
differential, less free water is available for intracellular ice
crystallization which is often
identified as a lethal factor. In tissues, polymeric substances may act by
binding and
structuring water molecules.
The cooling rate in the presence of cryoprotective compounds is a very
important
factor in freezing injury. Normally for cells, slow cooling is better than
elevated cooling
rates since the latter promotes intracellular ice formation. This occurs
because there is
insufficient time for water to escape from the cells before the contained cell
water
freezes. With slow rate cooling, extracellular ice forms first, resulting in
dehydration of
the cell which, together with the presence of the cryoprotectant, prevents
intracellular ice
formation. For tissue matrix samples there is a more direct correlation to the
overall
reduction in the degree of total ice crystal formation.
Penetrating compounds were thought to act by not allowing an excessive
transport
of water from the cells too early in the freezing process while nonpenetrating
compounds
-26-
2~g~336
have a dehydrative effect on cells along with a colligative effect of diluting
the solution
surrounding the cell. Neither of these descriptions, however, tells the whole
story.
Solutes such as HES and PVP are totally nonpenetrating, water-withdrawing
S compounds of merely larger molecular weight than nonpenetrating sucrose. The
larger
molecular weight should render such compounds less osmotically and
colligatively
effective, when considered on a weight basis. Yet in concentrated solutions,
the
compounds' colligative action has been shown to be far greater than would be
expected
based on merely a linear relationship to concentration.
A source of damage to frozen tissue, other than freezing itself, is the
osmotic and
toxic effects of many of the cryoprotective agents. When used in mixtures,
some
cryoprotective compounds may counteract the toxicity of other cryoprotectants,
as was
demonstrated by the addition of polyethylene glycol (PEG) to a mixture of DMSO
and
glycerol. The inventors have developed several vitrification solutions (VS).
The toxicity of the individual components of these solutions were tested. In
the mixtures,
the toxic effects were lower than when an equivalent concentration of any one
component was
used alone. The resulting solutions are nontoxic to cell cultures and remains
glass like and
optically clear (i.e., no visible ice crystal is formed) when plunged into
liquid nitrogen.
Vitrification Solution 1
Dimethylsulfoxide (DMSO) - O.SM
Propylene glycol - 0.5 M
2-3 butanediol - 0.25M
Proline - 1.OM
Raffinose - 2.5% (w/v)
Polyvinylpyrrolidone (PVP) - 15% (w/v) (Ave. M.W. ~ 40,000)
Dextran - 15% (w/v) (Ave. M.W. ~ 40,000-
70,000)
-27-
....,r..._
,~-»,.~"M.yw,-.-. .. "r
24893
A modified vitrification solution (VSZ) has also been developed which
comprises a
mixture of:
DMSO - O.SM
Propylene glycol - O.SM
2-3 butanediol - 0.25M
Raffinose - 10% (w/v)
Trehalose - 6% (w/v)
Sucrose - 6% (w/v)
PVP - 12% (w/v) (Ave. M.W. ~ 40,000)
Dextran - 12% (w/v) (Ave. M.W. ~ 40,000-70,000)
Another modified vitrification solution (VS3) which has been developed
comprises
a mixture of:
DMSO - O.SM
Propylene glycol - O.SM
2-3 butanediol - 0.25M
Raffinose - 2.5% (w/v)
Sucrose - 12% (w/v)
pVp - 15% (w/v) (Ave. M.W. ~ 40,000)
Dextran - 15% (w/v) (Ave. M.W. ~ 40,000-70,000)
A fourth modified solution (VS4) has been developed. This solution differs in
that it contains 50% formamide, an organic solvent. This mixture neither
expands nor
contracts with freezing and hence does not cause cracking when freezing larger
tissue
samples. It comprises a mixture of:
Formamide - 50% (w/v)
70K Dextran - 15% (w/v)
Raffinose - 2.5 % (w/v)
40K PVP - 15% (w/v)
Sucrose - 12% (w/v)
-28-
208935
In summary, the factors affecting the cryoprotective nature of compounds are
(a)
chemical composition, (b) low toxicity, (c) molecular size and penetrating
ability, and (d)
interaction with other compounds in the mixture.
S The physicochemical effects of cryoprotectants are (a) depression of the
equilibrium freezing point of substrate and cytoplasm on a colligative basis,
(b)
depression of homogeneous ice nucleation temperature, (c) reduced rate of ice
crystal
growth due to change in the viscosity and thermal diffusivity of the solution,
and (d)
dehydrative effects on cells by osmotic action.
Cooling Parameters
For purposes of cryopreparation of the biological tissues of this invention,
it is
essential to note that a variety of cooling processes can be used. In a
preferred
embodiment of this invention, rapid cooling is considered essential to obtain
the proper
ice crystal blend. In the most preferred embodiment of this invention, a
vitrification
procedure is used which results in the formation of a substantial proportion
of
amorphous water in the biological sample. As will be disclosed hereinafter,
regardless of
the form of cooling that is used, it is believed that amorphous phase water,
cubic ice
crystals and hexagonal ice crystals are present in the final product. The
method of
cooling has a distinct bearing on the distribution of ice crystal types found
in the cooled
cryosolution.
DrXin~ Parameters
The aim of controlled drying of a frozen biological tissue by molecular
distillation
drying is to remove water from the sample without further mechanical or
chemical
damage occurring during the drying process. This involves avoiding, by use of
appropriate drying conditions, two fundamental damaging events. The first is
to remove
water from ice crystalline phases without transition to larger more stable and
more
destructive crystals. The second is to remove water from solid but
noncrystalline water
-29-
2089~~~
or water-solute mixtures without melting or crystallization of these solid
phases. This
second component refers to water present in the amorphous condition, water
together
with solute in the eutectic or water together with a compound which binds and
structures
water and hence, prevents its crystallization during the freezing process.
Hence, vitreous
water can be of low energy and stability, as in ultrarapidly-cooled pure
water, or high
energy and stability, as that achieved with cryoprotective agents with
intermediate rates
of cooling.
Many of the features required of controlled drying to avoid the occurrence of
these events are overlapping. The reason for this is that each form of water
will have a
particular energy state, whether in a crystal or bound to a cryoprotective
compound, and
it is this energy state, rather than its configuration, which determines the
requirements
for drying. Consider for example, (1) a sample of cubic ice achieved by
cooling pure
water at an intermediate cooling rate and (2) vitrified water achieved by
mixing water
with glycerol to 45% vol:vol and cooling at an intermediate rate. The first
sample will
be crystalline and the aim of drying is to remove water from this state
without transition
to hexagonal ice. The second sample is an amorphous solid and the aim of
drying is to
remove water from this phase without melting of the glass to a liquid with
subsequent
boiling. For cubic ice, the onset of its transition is -130°C and the
rate of transition is
temperature dependent being very slow at -130°C and very rapid at -
90°C. For 45%
glycerol-water, the glass transition temperature is -120°C and
represents the onset of
melting. The melting process is very slow at -120°C and is temperature
dependent,
becoming very rapid at -90°C.
Prior to the onset of the cubic to hexagonal transition or the glass
transition of
45% glycerol-water, the saturation vapor pressure of water in these phases is
extremely
low and drying would occur at extremely slow rates. The aim of controlled
drying,
therefore, is to remove water from the cubic ice phase during its transition
and in a time
less than is required for any significant transition to hexagonal ice and from
the 45%
-30-
20893~~
glycerol-water phase during its transition to a liquid but in less time than
is required for
any appreciable liquid to form.
This argument can be applied repetitively to all forms of water present
whether it
be crystalline in the form of cubic or hexagonal or noncrystalline as
amorphous or bound
to any molecule, be it cryoprotectant, protein, carbohydrate, or lipid. To
simplify this
concept, water in a frozen biological sample can be described as having a
specific energy
level E. In a frozen biological sample, there will be water forms of multiple
definable
energy levels:
El EZ E3 __-_ En
The mode of preparation, the nature of the sample, the use of cryoprotectants
or other
additives, and the cooling rate used will determine the relative proportions
of these
different water forms. Each energy level will determine the onset temperature
of its
transition or melting and the temperature dependence of the rate of the
transition or
melt.
Controlled drying processes must be able to remove each of these different
states
of water during the transition and in less time than is required to complete
the
transition. This mode of drying, therefore, requires that several conditions
be met.
First, the frozen sample must be loaded into the dryer without temperature
elevation above its lowest transition temperature. If elevation of temperature
does
occur, this must be over a short period of time such that no appreciable
transition
occurs. Ideally, loading occurs under liquid nitrogen at -190°C, well
below the lowest
discernible transition of -160°C for pure, ultrarapidly-cooled
amorphous water. If,
however, the sample is predominantly cubic ice or a mixture of water and
cryoprotectants with a glass transition of the order of -100°C to -
130°C, a closed circuit
refrigeration system may be sufficient to enable maintenance of the sample
temperature
below the onset of transition.
-31-
208936
Once loaded, the sample must be exposed to vacuum and be in direct line of
sight
of the condenser surfaces. The criteria for these are again determined by the
nature of
the water phases present in the sample. The following objectives must be
attained. The
vacuum within the chamber during the drying of a particular phase must create
a partial
pressure of water at least equivalent to or less than the saturation vapor
pressure of
water in the phase to be removed. This saturation vapor pressure is dependent
on the
nature of the water phase and its temperature. Hence, for pure amorphous water
in the
transition range of -160°C to -130°C, the approximate saturation
vapor pressures are 6 x
10-12 mbar (-160°C) and 5 x 10-~ mbar (-130°C), respectively. As
the transition times of
amorphous to cubic ice in this same temperature range, -160°C to -
130°C, vary from S x
105 minutes to S minutes, drying will be very slow until temperatures of the
order of -
150°C to -140°C are reached requiring a vacuum of 5 x 10-
1° to 2 x 10~ mbar. This
represents one extreme.
For cubic ice, little if any drying will occur below its onset of transition
at -130°C
as its saturation vapor pressure will be of the order of one log lower than
for amorphous
water. In the transition range, -130°C to -100°C, the saturation
vapor pressure of cubic
ice is approximately 5 x 10$ to 9 x 10-5 mbar. The transition times of cubic
to hexagonal
are 700 minutes and 109 minutes respectively. The saturation vapor pressure,
therefore,
determines the vacuum requirements for drying and can be applied to all water
phases
present. It is important to note that the same vacuum criteria are not
applicable to all
phases, but rather are phase-dependent.
A second criteria of the vacuum is that the mean free path be in excess of the
distance between the sample and the condenser surface. Ideally, this should be
a tenfold
excess. The condenser surface must be a lower temperature than the onset
transition
temperature of the phase of water being removed from the sample so that the
saturation
vapor pressure of water condensed on this surface during drying is
considerably lower
than that of the water phase within the sample. Ideally, this should be three
orders of
-32-
2089336
magnitude lower. For a sample containing multiple water phases, the
temperature of the
condenser surface must remain below the onset of transition of the least
stable ice phase
remaining to be removed. Ideally, the condenser should also be in line of
sight of the
sample.
S
Once the sample has been loaded and exposed to vacuum and the condenser
surfaces, the sample and sample holder must be heated so as to increase the
mobility of
water molecules and hence, cause their escape. This is the essential and
critical
component in the drying of a sample containing multiple phases or energy
levels of
water. The temperature of the sample must be accurately known. The control of
temperature and the rate of sample heating must be accurately controlled. This
is
necessary to ensure that the drying of each phase of water in the sample is
sequential.
Hence, for a sample containing multiple phases of water of energy level E1,
and E2 ---- En where El is the least stable, then heating must occur at such a
rate that El
is removed prior to its transition to E2, E2 prior to its transition to E3 and
so on. This
requires nonequilibrium drying conditions and heating at a continuous rate or
by holding
at a constant temperature level such that sublimation occurs as determined by:
o.s
Js = NPs
( 2nQT~
where Js - sublimation rate in g cm 1 sec 1
N - coefficient of evaporation
Ps - saturation vapor pressure
M - molecular weight of water
Q - universal gas constant
T - absolute temperature of the sample.
-33-
208~3~~
This is consistent with the transition rate for the particular phase being
removed.
For example, the rate of the amorphous to cubic transition is given by:
E = 2.04 x 10'~ x exp (-0.465T)
Alternatively, if the transition window is Tl to T2, the sublimation rate and
the
transition rate will vary with temperature during this interval. The rate of
heating during
this window Tl to TZ must be such that sublimation occurs throughout the
dimensions of
the sample before transition at any particular temperature is completed.
In this way, the aim of controlled drying is achieved, i.e., the sequential
removal
of each phase of water under conditions appropriate to the properties of each
phase
without appreciable ice crystal growth, formation or melting of the particular
phase.
Once dry, the sample must be physically or mechanically isolated from water on
the
condenser surface or any other source and stored in a closed container either
under
vacuum or dry inert gas.
In a preferred embodiment, samples are cooled by an appropriate method such
that ice crystal formation is below the degree that would cause damage to the
sample.
Once frozen, the sample is then stored below the transition temperature of the
most
unstable ice form. For amorphous ice, this is preferentially below -
160°C. The sample is
then loaded into a sample holder, precooled to -196°C and transferred
into a molecular
distillation dryer. The dryer chamber is then closed and sealed for vacuum
integrity. To
avoid recrystallization, the hydrated sample must remain below the transition
temperature of the most unstable ice form throughout all manipulations.
Once the sample is loaded, high vacuum ( 10-g to 10~ mbar) is generated inside
the chamber. The sample is placed considerably closer to the condenser surface
(liquid
nitrogen cooled chamber walls) than the mean free path within the chamber. The
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2089336
condenser temperature must always be below that of the sample. For an
amorphous
sample, the condenser is preferentially -196°C.
The sample holder is then heated via a programmable heater microprocessor
S thermocouple loop. Heating programs are determined according to the ice
composition
of the sample. A typical program for a sample containing amorphous, cubic and
hexagonal ice is 10°C per hour from -180°C to -150°C,
1°C per hour from -150°C to -
70°C, and 10°C per hour from -70°C to +20°C.
Once the sample has reached 20°C, it can be sealed inside an
appropriate
container within the vacuum chamber and unloaded for subsequent storage. In
one
configuration, the sample is contained within a glass vial and sealed with a
butyl rubber
lyophilization stopper at the end of cycle. More specific details of the
operation of the
molecular distillation dryer are given in U.S. Patent No. 4,865,871.
Reconstitution
The freezing and drying of biological tissues impart great physical stress
upon the
bonding forces which normally stabilize macromolecular conformation.
Contributing to
this destabilizing effect is the increase in concentration of electrolytes and
possible pH
changes as the solution freezes. As a consequence, modifications to the
sample,
including the inactivation of certain enzymes, and the denaturation of
proteins, may
result.
Studies with lactic dehydrogenase have shown that freezing and thawing cause
dissociation of the tetrameric enzyme into subunits which is accompanied by a
change in
biological activity. The dissociation was found to be dependent on the ionic
strength and
pH during freezing.
-35-
P.~yr..I
.A~(/.~'1~~ 3:<
20~93~~
Other studies investigating the quaternary structure of L-asparaginase
demonstrated that this enzyme dissociated from the active tetramer to inactive
monomers when freeze-dried. This monomeric state was found to be stabilized by
reconstitution of the dried enzyme with bLffers of high pH and high ionic
strength.
However, the dissociation was shown to be completely reversible on
reconstitution at
neutral pH and low ionic strength. The effect of pH on the other hand may
induce
changes in the three dimensional structure resulting in subunits
conformationally
restrained from reassociation.
These studies indicate the importance of determining optimal pH and ionic
strength conditions of not only the formulation used in the cryopreservation
protocol, but
also the reconstitution solution. In this way, maximal sample activity and
stability may
be obtained.
Other variables of reconstitution such as vapor phase rehydration or
temperature
may also be important to the retention of activity following freezing and
drying. Other
workers in the field have demonstrated a marked difference in proliferative
response to
lectins depending on the temperature of rehydration or whether samples were
reconstituted by vapor phase. Improved responses to lectins were noted when
the
freeze-dried lymphocytes were rehydrated at dry ice temperatures and then
allowed to
warm. This gradual method of reconstitution reduced the osmotic stress induced
by
sudden rehydration.
In the processing of biological tissues, the rehydration step can also be used
to
augment the processing and stabilization compounds used in the procurement and
processing steps. These include components to minimize the effects of hypoxia
and free
radical generation, agents to inhibit enzymes, oncotic agents including
proteoglycans,
dextran and amino acids to prevent osmotic damage.
-36-
20~93~~
In addition, the rehydration of certain tissues, e.g., the vascular conduits
and heart
valves, may require specific agents to inhibit plaletet aggregation during the
early post
implant period. Where the biological tissue is to be crosslinked, rehydration
directly in
the fixative has the additional advantage of immediate and uniform
distribution of the
fixative throughout the tissue.
Storage Considerations
Sublimation of water from a frozen sample is one method for preserving the
active components of biological material. However, the optimal preservation of
activity
with long-term stability requires critical control of the drying process and
storage
conditions. Following the removal of free or unbound water, the process of
secondary
drying proceeds, during which structurally bound water is removed. Bound water
is
intimately associated with the maintenance of protein conformation. Thus, the
amount
of water remaining in the dried sample, known as the residual moisture
content, is a
significant variable in the drying process. The final residual moisture
content affects both
the survival and stability of the sample.
Residual moisture content is expressed as the "percentage residual moisture"
and
is equated to the weight (gm) of residual water per unit weight (gm) of
original sample.
It is generally agreed that biological materials dried by vacuum sublimation
of ice
show increased stabilization when dried to optimum contents of residual
moisture.
Materials which have been under or overdried, i.e., to moisture contents that
are above
or below the optimum, will show increased deterioration.
Although the optimal residual moisture content will vary depending on the
particular dried sample, certain stability problems can be expected when the
levels of
moisture are suboptimal. Overdrying a sample, i.e., residual moisture contents
less than
1-2% without using a dry stabilizer, generally results in removal of nearly
all structured
-37-
_ 2Q$
water allowing modification or blocking of exposed hydrophilic sites of
proteins by
oxidation. This oxidation causes degradation with a corresponding decrease in
the
biological activity. On the other hand, residual moisture contents of greater
than S%
generally are indicative of underdrying where sufficient amounts of "free
water" remain
in the sample which could contribute to transconformation of the protein. The
resulting
rearrangements of the polypeptide chains shift from the typical ordered
arrangement of
the native protein to a more disordered arrangement. These protein
perturbations can
result in poor long-term stability of the dried product.
Successful long-term storage requires sample drying to optimal levels of
residual
moisture. Inadequate drying of biological samples and its consequences have
been
shown in the literature. Maximal stability of suspensions of influenza virus
dried by
sublimation of water in vacuo occurred at a residual moisture content of
approximately
1.7%. Under or over drying to non-optimal water content resulted in the
degradation of
the virus suggesting that varying amounts of free and bound water in a dried
sample
have an effect upon protein structure and activity.
To maximize sample stability and satisfy regulatory requirements for the
preparation of dried pharmaceuticals or reagents, it is essential that the
residual moisture
content be determined following sample drying.
Several methods are available to measure residual moisture contents;
1. Gravimetric (Heating Method) - A known quantity of dried product is
heated and the weight loss can be equated with water content.
2. Chemical Assay - This method is based on the reaction between water and
free iodine in a mixture of pyridine, sulphur dioxide and methanol. The
endpoint is detected coulometrically when free iodine is present. H20 + IZ
+ SOZ + ROH + 3RN -~ 2RNHI + RN + HS04R
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2089336
3. Gas Chromatography
Each of the methods has limitations and therefore, it is wise to not rely on
any
single method of moisture determination. Rather, multiple methods should be
employed
to validate the results.
Once dried to optimal residual moisture contents, the sample is still
considered
unstable when removed from the vacuum due to its hygroscopic nature and
susceptibility
to oxidation. Measures must be taken during storage to protect the sample from
atmospheric rehydration and minimize exposure to oxygen. Such protection is
essential
to the maintenance of the sample's long-term stability.
Evidence in the literature indicates that the gaseous condition under which
the
samples are sealed, as well as the storage temperature, effects the long-term
stability of
the sample. It has been demonstrated in a study comparing different gases and
storage
temperatures, that maximum stability of influenza virus was obtained when
samples were
stored under helium or hydrogen gas at low temperature (-20°C). Sealing
under other
gases or vacuum at different storage temperatures resulted in varying levels
of stability.
The inventors postulate that those conditions which most effectively limit
oxygen contact
with the sample, markedly improve biological activity by reducing oxidation of
exposed
hydrophilic sites at the protein surface. Appropriate storage parameters,
i.e.,
temperature, and sealing under gas or vacuum are important to obtain long-term
sample
stability.
-39-
2D$93~G
EXAMPLE 1
PROCESSING AND STORAGE OF TRANSPLANTABLE SKIN
Human donor skin is routinely harvested from cadavers and stored under
refrigerated or frozen conditions at a number of tissue banks throughout the
nation.
This skin is used as a temporary dressing for burn victims that are undergoing
extensive
autografting. Porcine skin is also harvested under similar conditions and used
as a
temporary burn dressing. In its unprocessed condition, the allogeneic skin and
porcine
skin are ultimately rejected by the patient. This same skin is also available
for
processing by the methods described below.
Donor skin is harvested under aseptic conditions with a dermatome, and
maintained at 4°C in RPMI 1640 tissue culture media containing
penicillin and
streptomycin solution for no more than 7 days prior to further processing.
Transportation to LifeCell's tissue processing center is via overnight
delivery, on wet ice,
in the same media. On arrival at the processing center, the temperature of the
tissue
container is verified to be at least 4°, or the skin discarded.
Following verification of
container temperature, donor identification and test screening data, the skin
is
transferred to a laminar-flow hood for further processing.
The donor skin is removed from the transportation container and placed with
its
reticular side down on a piece of sizing support being a low density
polyethylene. An
appropriately sized piece of gauze is added to the epidermal side of the skin
which is
then cut into a rectangular piece as large as possible, not to exceed a 4 x 4
inch square
and no smaller than 2 x 3 inches. The skin is then placed reticular side down,
in a petri
dish, to which SOmI of De-epidermizing Solution consisting of 1M NaCI is
added. The
petri dish is then transferred to an incubator and incubated at 37°~
2°C for 18 to 32
hours for human skin and 35 to 55 hours for porcine skin.
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2089336
After incubation, the petri dish containing the skin is transferred to a
laminar flow
hood for deepidermization. The gauze is first removed and discarded. The
epidermis is
then gently grasped with forceps and pulled away from dermis as a sheet. The
excess
Deepiderizing Solution is then aspirated. A slit approximately one centimeter
long is
S then made in the lower left corner of the dermis to identify the upper and
lower
surfaces.
The dermis is next rinsed in the same petri dish by the addition of SO ml
Tissue
Wash Solution, consisting of sterile Hanks balanced salt solution. The petri
dish is then
placed on a rotator at 40~S RPM for 5 minutes at room temperature (20°-
26°C). The
petri dish is then returned to the laminar flow hood and the lid from the
petri dish is
removed in order to aspirate the Tissue Wash Solution. This procedure is
repeated a
further two times.
The dermis is then treated with 50 ml. of De-Cellularizing solution and the
petri
dish is placed on a rotator at 40~S RPM for 1 hour at room temperature
(20°-26°C).
The decellarizing solution for human skin consists of 0.5% sodium dodecyl
sulfate in
Hanks' balanced salt solution and for porcine skin contains 1 mM disodium
ethylenediamine tetraacetic acid (EDTA). The De-cellularizing solution is
removed by
aspiration. The dermis is then washed with SOml of Tissue Wash Solution. The
petri
dish is then placed on a rotator at 40~5 RPM for S minutes at room temperature
(20°-
26°C). The Tissue Wash Solution is removed by aspiration. The washing
procedure is
repeated (2) times. After the dermis has been washed a total of 3 times SOmI
of Pre-
freezing Solution is added to the petri dish. The dish is then placed on a
rotator at
40~5 RPM for 30 minutes at room temperature (20°-26°C). The
prefreezing solution for
human skin consists of 7% dextran (70,000 MWT), 6% sucrose, 6% raffinose and 1
mM
disodium ethylenediamine tetraacetic acid in Hanks' balanced salt solution.
The
prefeezing solution for porcine skin consists of 7.5% dextran (70,000 MWT), 6%
sucrose,
-41-
_..
2089336
7.5% polyvinylpyrrolidone (MWT 40,000), 1.25% raffinose and 1 mM disodium
ethylenediamine tetraacetic acid made up in Hanks' balanced salt solution.
A new piece of gauze is then placed or_ the papillary side of the dermis and
the
dermis is turned over so that the reticular side faces up. The backing from
the reticular
side of the piece of dermis is discarded into a biohazard waste container. An
approximately 0.5 to 1.0 cm wide strip of backing and dermis is then cut from
the
original sample. This strip is then cut into two satellite pieces, each
approximately 1.0
cm long. All necessary quality assurance is ultimately performed on these
satellite
samples, including microbiology and structural analysis.
The tissues are then transferred into individual Tyvek bags. The tissues are
positioned in the bag backing side up with the white vent side down. The
Tyvek~ bag is
then heat sealed.
The sealed Freeze-dry Bag is transferred to a freeze-dryer which has a minimum
shelf temperature of -70°C and a minimum condenser temperature of -
85°C. The tissue
is then frozen on the freeze-dryer shelf by ramping the shelf temperature at a
rate of
-2.5°C/minute to -35°C, and held for at least 10 minutes.
The drying cycle is such that the final residual moisture content of the
sample is
less than 6% and optimally 2%. In this example, the frozen dermis is dried by
the
following program:
1. The shelf temperature is ramped at a rate of -2.5°C/minute to -
35°C, and
held for 10 minutes, with vacuum set to 2000mT.
2. The shelf temperature is then ramped at a rate of 1.5°C/minute to -
23°C,
and held for 36 hours with vacuum set to 2000mT.
-42-
,~~
~o~~~~~
3. The temperature is then ramped at rate of 1.5°C/minute to a shelf
temperature of -15°C, and held for 180 minutes with vacuum set to
2000mT.
4. The temperature is then ramped at a rate of 1.5°C/W mute to a shelf
temperature of -5°C and held for -180 nunutes with vacuum set to
2000mT.
5. The temperature is finally ramped at a rate of 1.5°C/minute to a
shelf
temperature of 20°C and held for 180 minutes with the vacuum set to
OmT.
Following drying, the Freeze-dry Bag containing the dried dermis is unloaded
under an atmosphere of dry nitrogen gas, placed in a second predried
impervious pouch
and heat sealed under the same inert environment.
(During the processing procedure and prior to sealing for freeze drying, a
satellite
sample is cut from the main sample and further processed under identical
conditions to
the main sample. Prior to use of the main sample in transplantation, all
necessary
quality assurance is performed on the satellite sample, including microbiology
and
structural analysis.)
Following drying, the sample is stored at above freezing temperatures,
optimally
4°C in a light. protected environment.
Prior to use, the sample is removed from the sealed pouch under aseptic
conditions and rehydrated by immersion in balanced salt solution at 20°
to 37°C.
Rehydration is complete after 30 minutes of incubation in this rehydration
solution.
Analysis of the end product by light and electron microscopy has demonstrated
it
to be structurally intact with normal collagen banding and the presence of
collagen
bundles in the matrix of the dermis and with structural preservation of the
lamina densa
and anchoring fibrils of basement membrane complex.
-43-
2089~3~
The reticular aspect of processed dermis has been demonstrated to provide a
substratum for the outgrowth of keratinocytes from a foreskin explant in a
laboratory by
cell culture methods. The processed dermis has also been demonstrated to
support the
growth of isolated keratinocytes. In this circumstance, when cultured at an
air liquid
interface, keratinocytes differentiate to all identifiable layers of normal
skin and interact
with the processed dermis through the basement membrane complex. Processed
porcine
skin has also been demonstrated to support the growth of keratinocytes from
human
foreskin explants.
The processed dermis, either in combination with a meshed, ultra thin or
epidermal autologous graft or reconstituted with cultured keratinocytes, has a
number of
clinical applications in full thickness skin injury. These include, but are
not limited to,
burn patients; patients suffering from venous, diabetic, or pressure ulcers,
and patients
who undergo reconstructive surgery, or skin replacement following excision of
skin
lesions.
Processed human and porcine skin have been shown to undergo fibroblast
infiltration and neovascularization in human burns patients and in surgically
induced full
thickness skin injury in pigs.
EXAMPLE 2
VASCULAR CONDUIT: HUMAN DONOR SAPHENOUS VEINS
Saphenous veins are harvested from cadaver donors and made available by tissue
banks across the U.S. Tissue banks have established procurement guidelines,
published
by the American Association of Tissue Banks. These guidelines include
instructions for
patient selection, completion of consent forms and a caution to avoid
mechanical
distention or other mechanical damage to the vein during the dissection
process.
-44-
20893~~
Harvesting begins with flushing and distension of the vein with Vein Flushing
Solution, consisting of 1000 cc PlasmaLyte Solution for injection, amended
with 5000
units of Heparin and 120 mg of Papaverine ( 1 liter per vein). The veins are
carefully
removed under sterile conditions with as many tributaries maintained intact as
possible,
S with a length of at least 5 mm. These tributaries are ligated with 3-0 silk.
The
surrounding fatty tissue is also maintained with wide margins around the vein.
Once the
vein is removed, it is rinsed again with Vein Flushing Solution, packaged in
500 cc of
cold (4°C) Vein Transport Medium, consisting of 500 cc RPMI 1640 Tissue
Culture
Medium amended with 60 mg Papaverine and shipped by overnight delivery to a
tissue
bank for further processing.
At the tissue bank, all tributaries are suture ligated and the subcutaneous
fat/soft
tissue removed using standard surgical procedures. Following dissection, the
vein is
disinfected of any surface contaminants by placing it in a tissue culture
medium amended
with Cefoxitin (240mcg/ml), Lincomycin ( 120mcg/ml), Polymyxin B Sulfate (
100mcg/ml)
and Vancomycin (SOmcg/ml). The vein is maintained in the antibiotic mixture at
4°C for
24 hours. The disinfected vein is placed in SOOcc of cold (4°C)
Transport Medium,
consisting of SOOcc RPMI 1640 Tissue Culture Medium and transported on wet ice
to
LifeCell's Tissue Processing Center by overnight delivery.
On arrival, the container temperature is verified to be at least 4°C.
Following
verification, the vein is placed into a container containing Cryosolution and
incubated for
one hour at room temperature. The Cryosolution consists of the following:
O.SM Dimethyl Sulfoxide (DMSO)
O.SM Propylene Glycol
0.25M 2-3 Butanediol
2.5 % (w/v) Raffinose
12.0% (w/v) Sucrose
15.0% (w/v) Polyvinylpyrrolidone (PVP)
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208933~i
15.0% Dextran.
After incubation, the vein is then placed into an inert plastic bag containing
a porous
vent which allows water vapor to pass out, but prevents bacteria from passing
in and is heat
sealed. The bag and vein is then frozen by plunging into liquid nitrogen. The
frozen vein
is stored at temperatures below -160°C.
For drying, the frozen vein within the bag is transferred under liquid
nitrogen to a
molecular distillation dryer, and dried by methods described in U.S. Patent
No. 4,865,871.
For saphenous veins processed in the above described cryosolution and rapidly
frozen, the
optimum range for drying is -130°C to -70°C with a heating rate
of 1°C per minute during
the drying phase. Once dry, the vein is sealed in the container under dry
inept nitrogen gas
and stored at refrigerated temperatures (2-4°C) until needed for
transplantation.
The vein is rehydrated in a vapor phase, by opening the plastic pouch
container and
placing the vein in a 37°C humidified incubator. The vein is maintained
in this incubator
for one hour, after which it is removed and placed in a container with
phosphate buffered
saline (PBS). The vein is then rinsed with 3 changes of PBS.
Analysis of the processed veins show them to possess an intact extracellular
matrix
both by light and electron microscopy. Protease digestion indicates no
increased
susceptibility of collagen to degradation. Stress testing on a dynamic loop
with an artificial
heart has demonstrated them to withstand supraphysiological pressures without
compromise
of their leak barrier function to either liquid or gas.
-46-
X089336
EXAMPLE 3
VASCULAR CONDUIT PROCESSING FOR ANIMAL STUDY
Procurement
Twenty to thirty kilogram mongrel dogs of either sex are induced via sodium
pentathol, intubated, and prepped and draped in a sterile fashion. Anaeshesia
is maintained
with oxygen, nitrogen, and Halothane. A midline incision is made in the neck
whereupon
the external jugular veins and internal carotid arteries are exposed,
isolated, and freed of
surrounding fascia. During this procedure, a flushing solution comprised of
5000 units of
heparin. and 120 mg of Papavarine in 1000 cc sterile Hanks' Buffered Saline
Solution
(HBSS) of pH 7.4 is sprayed on the vessels via a needle and syringe. The
proximal and
distal ends of the vessel are then clamped with atraumatic vascular clamps
whereupon the
vessel is rapidly excised. Immediately the vessel is flushed through and
through with the
above mentioned flushing solution and placed in 4°C flushing solution
for transport.
Alternatively, the vessel may be placed in the below mentioned
Decellularization Solution A
for incubation during transport.
Decellularization
After the trimming of any excess fascia, the vessel is placed in
Decellularization
Solution A (DSA). DSA is comprised of 25 mM EDTA, 1 M NaCI, and 8 mM CHAPS or
similar zwitterionic detergent in a sterile PBS base at 7.5 pH. After a 30
minute to one
hour incubation, the vessel is given two ten minute washes in PBS and then
placed in
Decellularization Solution B (DSB). DSB is comprised of 25 mM EDTA, 1 M NaCI,
and
1.8 mM Sodium Dodecylsulfate (SDS) or similar anionic or nonionic detergent in
a sterile
PBS base at 7.5 pH. After a 30 minute to one hour incubation, the vessel is
given two ten
minute washes in PBS.
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20~93~~
Vitrification
After decellularization, the vessel is placed in Vitrification Solution Fifty-
fifty (VSFF)
for one to five hours. VSFF is comprised of 2.5% raffinose, 15%
polyvinylpyrrolidone
(PVP) of 40,000 molecular weight, 15% Dextran of 70,000 molecular weight, and
12%
S sucrose in a 50/50 (by volume) water-formamide solution. The vessel is then
rapidly
submerged in liquid nitrogen (LN2) until frozen as evidenced by the cessation
of boiling.
The vessel may then be stored in LNZ or LN2 vapor, or immediately dried.
After vitrification, the vessel is transfered in a nitrogen gas atmosphere to
a special
Molecular Distillation Dryer sample holder which has been pre-cooled to -
196°C. The
sample holder is then rapidly transferred under nitrogen gas atmosphere to the
Molecular
Distillation Dryer. The dryer is then evacuated and run according to a
protocol developed
specifically for VSFF. Under a vacuum less than 1 X 10~ mbar, the sample
holder is
warmed according to the following protocol:
-196°C -> -150°C over 10 hours
-150°C -> -70°C over 80 hours
-70°C - > 20°C over 10 hours
The dryer is then opened and the vessel is transferred to a sealed sterile
glass vial
under nitrogen gas atmosphere. The vessel is then stored at 4°C until
needed.
Rehydration
Twenty-four hours prior to use, the glass vial is opened in a 100% humidity,
37°C
atmosphere. The vessel is allowed to vapor rehydrate in this manner for one to
two hours.
The vessel is then submerged in sterile PBS at 4°C for two hours. The
PBS is then
exchanged with fresh solution whereupon the vessel is stored at 4°C
overnight. The vessel
is ready for use the following day.
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2089336
EXAMPLE 4
PORCINE HEART VALVE LEAFLETS
S Porcine heart valves were obtained from isolated hearts immediately
following
slaughter at an abattoir. Discs from the leaflets of the intact valve were
obtained by punch
biopsy under aseptic conditions and transferred to a transportation solution
comprising
Dulbecco's PBS with 5.6 mM glucose, 0.33 mM sodium pyruvate with added anti-
oxidants
comprising 0.025 mg/I alpha-tocopherol phosphate, SO mg/1 ascorbic acid and 10
mg/1
glutathione (monosodium) at 4°C.
Upon receipt of tissue, the discs were transferred to a cryosolution
comprising 0.5 M
DMSO, 0.5 M propylene glycol, 0.25 M 2-3 butanediol, 2-5% raffinose, 15%
polyvinyl
pyrrolidone, 15% Dextran and 12% sucrose and incubated at 20°C for 60
minutes with
moderate agitation.
Tissue samples were then placed on thin copper substrates matching the size of
the
tissue sample and cooled by immersion in liquid nitrogen.
The frozen samples were then stored at below -160°C until further
processing.
Prior to drying, the samples were transferred under liquid nitrogen to a
sample
holder equipped with thermocouple and heater. The sample holder was precooled
to liquid
nitrogen temperature, and the transfer was completed under liquid nitrogen.
The frozen samples were then loaded into a molecular distillation dryer and
dried
by molecular distillation drying employing the method described in United
States Patent No.
4,865,871. The drying cycle employed was -180°C to -150°C in 3
hours, -150°C to -70°C in
80 hours and -70°C to +20°C in 9 hours. Following drying, the
vacuum in the drying
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2089336
chamber was reversed with ultrapure nitrogen gas and the discs maintained in
this
atmosphere until processing.
Rehydration of the dry samples first consisted of exposure of samples to 100%
S humidity at 37°C for 60 minutes. Samples were then rehydrated in a
rehydration solution
which consisted of one of the following:
a. 0.06 M HEPES buffer
b. 0.06 M~HEPES buffer + 0.06 M MgCl2
c. 0.06 M HEPES buffer + 1% SDS
d. 0.06 M HEPES buffer + 0.5 mM PMSF
Samples were incubated with agitation for at least four hours.
Following rehydration, samples were assessed under the following criteria:
a. Structure was assessed by both light and electron microscopy and the valve
matrix was found to be indistinguishable from that of fresh unprocessed
samples.
b. Protease digestion was found to be equivalent to fresh sample.
c. Stress testing (static) was found to be able to withstand greater stress
load
than control samples.
d. Subcutaneous animal implant model with subsequent explant at 7 or 21
days.
Explanted samples demonstrated:
i. Decreased capsule formation relative to fresh or cryopreserved controls
ii. Decreased calcification relative to glutaraldehyde treated controls
iii. Variable inflammatory cell infiltration depending on the nature of the
rehydration
solution as follows:
-S 0-
:..f y 3
s
2089336
Treatment: 0.06 M MgCl2 in 0.06 M HEPESbuffer
Clearly demarcated disc with well defined normal valve morphology. Sample
incompletely surrounded by thin capsule with minimal inflammatory cell
infiltration near
disc periphery.
S
Treatment: 1% SDS in 0.06 M~HEPESbuffer
Clearly demarcated disc with well defined normal valve morphology. Sample
completely surrounded by a slightly thicker capsule than that observed in
MgCl2 treated
sample. Minimal inflammatory cell infiltration.
Treatment: O.S mM PMSF in 0.06 HEPESbuffer
Well defined normal valve morphology. Capsule formation nearly absent.
Minimal inflammatory cell infiltration.
1S Treatment: Control - 0.06 M HEPES buffer
Poorly defined valve structure. Massive inflammatory cell infiltration, but
little
evidence of capsule formation.
EXAMPLE S
INTACT PORCINE HEART VALVES
Procurement
Porcine heart valves are obtained from isolated hearts immediately following
2S slaughter at an abattoir. The aortic valve and at least one inch or more of
ascending
aorta is then carefully excised with pre-sterilized instruments.
-S 1-
........"~"~
208J3~~
The valve is washed twice in sterile phosphate buffer solution (PBS) and then
placed in sterile, 10°C PBS for transport. Within three hours of
procurement, the valve
is brought to the LifeCell facility where it is further trimmed and processed.
S Decellularization
After trimming, the intact valve is placed in Decellularization Solution A
(DSA).
DSA is comprised of 25 mM EDTA, 1 M NaCI, and 8 mM CHAPS or similar
zwitterionic detergent in a sterile PBS base at 7.5 pH. After a 30 minute to
one hour
incubation, the valve is given two ten minute washes in PBS and then placed in
Decellularization Solution B (DSB). DSB is comprised of 25 mM EDTA, 1 M NaCI,
and 1.8 mM Sodium Dodecylsulfate (SDS) or similar anionic or nonionic
detergent in a
sterile PBS base at 7.5 pH. After a 30 minute to one hour incubation, the
valve is given
two ten minute washes in PBS.
Vitrification
After decellularization, the valve is placed in Vitrification Solution Fifty-
fifty
(VSFF) for one to five hours. VSFF is comprised of 2.5% raffinose, 15%
polyvinylpyrrolidone (PVP) of 40,000 molecular weight, 15% Dextran of 70,000
molecular weight, and 12% sucrose in a 50/50 (by volume) water-formamide
solution.
The valve is then rapidly submerged in liquid nitrogen (LNZ) until frozen as
evidenced by
the cessation of boiling. The valve may then be stored in LNZ or LN2 vapor
prior to
drying.
Drying
After vitrification, the valve is transfered in a nitrogen gas atmosphere to a
special
Molecular Distillation Dryer sample holder which has been pre-cooled to -
196°C. The
sample holder is then rapidly transferred under nitrogen gas atmosphere to the
Molecular Distillation Dryer. The dryer is then evacuated and a heating cycle
initiated
-52-
~o~~~~s
which has been optimized specifically for dehydration of VSFF. Under a vacuum
less
than 1 X 10~ mbar, the sample holder is warmed according to the following
protocol:
-196°C - > -150°C over 10 hours
-150°C - > -70°C over 80 hours
-70°C - > 20°C over 10 hours
The dryer is then opened and the valve transferred to a sealed sterile glass
vial
under nitrogen gas atmosphere. The valve is then stored at 4°C until
required for
transplantation.
Re ~dration
Twenty-four hours prior to use, the glass vial is opened in a 100% humidity,
37°C
atmosphere. The valve is allowed to vapor rehydrate in this manner for one to
two
hours. The valve is then submerged in sterile PBS at 4°C for two hours.
The PBS is
then exchanged with fresh solution whereupon the valve is stored at 4°C
overnight. The
valve is ready for use the following day.
While the invention has been described in terms of the preferred embodiments,
it
will be apparent to those of skill in the art that variations and
modifications may be
applied to the compositions, methods and in the steps or in the sequence of
steps of the
methods described herein without departing from the concept, spirit and scope
of the
invention. Such substitutes and modifications are considered to be within the
scope of
the invention as defined by the appended claims.
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