NZ796375A - Tissue container systems - Google Patents
Tissue container systemsInfo
- Publication number
- NZ796375A NZ796375A NZ796375A NZ79637518A NZ796375A NZ 796375 A NZ796375 A NZ 796375A NZ 796375 A NZ796375 A NZ 796375A NZ 79637518 A NZ79637518 A NZ 79637518A NZ 796375 A NZ796375 A NZ 796375A
- Authority
- NZ
- New Zealand
- Prior art keywords
- kit
- tissue
- perimeter wall
- container
- sterile
- Prior art date
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Abstract
The present invention relates generally to tissue container systems that find use in the transport of tissues and methods of using the tissue container systems. In particular the present invention relates to systems that support the transport, thawing and use of cryopreserved human skin equivalents, and methods of their use by a health care provider.
Description
The present invention relates generally to tissue ner s that find use in the transport
of tissues and methods of using the tissue container systems. In particular the present invention
relates to systems that support the transport, thawing and use of cryopreserved human skin
equivalents, and methods of their use by a health care provider.
NZ 796375
TISSUE CONTAINER SYSTEMS
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a onal application from New
Zealand Patent Application 755045, which is hereby incorporated by reference in its
entirety. This application claims the benefit of U.S. provisional ation number
62/451,379, filed January 27, 2017, which is hereby incorporated by reference in its
entirety.
FIELD OF THE ION
The present invention relates generally to tissue container systems
that find use in the ort of tissues and methods of using the tissue container
s. In particular the present invention relates to systems that support the
transport, g and use of cryopreserved human skin equivalents, and methods of
their use by a health care provider.
BACKGROUND OF THE INVENTION
A major impediment to the acceptance of engineered tissues by
medical practitioners, healthcare providers, and second party payers is the lack of a
means to effectively and efficiently preserve and store engineered tissues. The nature
of living cells and tissue products makes development of long-term storage challenging.
Current engineered tissues must often be stored and shipped under carefully controlled
conditions to maintain viability and function. Typically, engineered tissue products take
weeks or months to produce but must be used within hours or days after manufacture.
As a result, tissue engineering companies must ually operate with their production
facilities at top capacity and absorb the costs of unsold product which must be
ded. As one specific example, APLIGRAF requires about four weeks to
cture, is usable for only 15 days and must be maintained between 20 and 23°C
until used. As another e, EPICEL is transported by a nurse from Genzyme
Biosurgery's production facility in Cambridge, MA to the point of use in a portable
tor and is used immediately upon arrival. Such constraints represent significant
challenges to developing convenient and cost-effective products.
Cryopreservation has been explored as a solution to the e
problem, but it is known to induce tissue damage through ice formation, chilling injury,
and osmotic imbalance. Besides AF, the only other approved full-thickness
living skin equivalent, ORCEL, has been evaluated as a frozen product but had the
ck that it must be maintained at temperatures below -100°C prior to use. This
requires specialized product delivery and storage conditions, ing use of liquid
nitrogen for storage, which is expensive and not readily available in rural clinics and
field hospitals.
Accordingly, what is needed in the art are improved methods of
cryopreserving viable engineered tissues and cells for storage under ions that are
routinely available at the point of use.
SUMMARY OF THE ION
The present invention relates generally to tissue container systems
that find use in the transport of tissues and their subsequent use by a health care
provider, and in particular to systems that support the transport, thawing and use of
cryopreserved human skin lents.
Accordingly, in some embodiments, the present invention provides
tissue containers sing: a perimeter wall and a substantially planar bottom surface
defining a dish, the perimeter wall having a male end and a female end, the male end of
the perimeter wall having projecting therefrom a ridge having a length and width,
wherein the female end of the perimeter wall defines a space corresponding to the
length and width of the ridge so that when an identical tissue container is placed on top
of the tissue container the female end of the tissue container releasably receives the
ridge extending from the male end of the identical tissue container, and the bottom
surface having a ter and comprising a perimeter ledge extending around the
perimeter to provide a oir defined by the perimeter ledge and the bottom surface.
In some ments, the perimeter wall has a flange extending therefrom. In some
embodiments, the flange comprises one or more tabs extending from the male end of
the perimeter wall. In some embodiments, the flange comprises one or more tabs
ing from the female end of the perimeter wall. In some embodiments, the ridge
has a proximal end and the proximal end of the ridge has one or more indents therein.
In some embodiments, the present invention provides tissue
container lies comprising: substantially identical top and bottom tissue
containers, each of the top and bottom tissue containers comprising a ter wall
and a substantially planar bottom surface defining a dish, the bottom surface having a
perimeter and comprising a perimeter ledge extending around the perimeter to provide
a reservoir d by the perimeter ledge and the bottom surface, and the perimeter
wall having a male end and a female end, the male end of the perimeter wall having
projecting therefrom a ridge having a length and width, wherein the female end of the
perimeter wall defines a space corresponding to the length and width of the ridge so
that when the top tissue ner is placed on the bottom tissue ner the female
end of the bottom tissue container releasably receives the ridge extending from the
male end of the top tissue container. In some embodiments, the perimeter wall of the
top tissue container has a top flange extending therefrom and the perimeter wall of the
bottom tissue container has a bottom flange extending rom so that when the top
and bottom tissue containers are assembled the top and bottom s contact one
another. In some embodiments, the top flange comprises one or more tabs extending
from the male end of the perimeter wall and one or more tabs extending from the female
end of the perimeter wall. In some embodiments, the bottom flange comprises one or
more tabs extending from the male end of the perimeter wall and one or more tabs
extending from the female end of the perimeter wall. In some embodiments, the bottom
flange comprises one or more tabs extending from the male end of the perimeter wall
and one or more tabs extending from the female end of the perimeter wall and the
wherein the top flange comprises one or more tabs extending from the male end of the
perimeter wall and one or more tabs extending from the female end of the perimeter
wall so that when the top and bottom tissue ners are assembled the tabs are
offset.
In some embodiments, the present ion provides tissue
container systems comprising: substantially identical top and bottom tissue containers
and a tray sing a porous bottom surface, each of the top and bottom tissue
containers comprising a perimeter wall and a substantially planar reservoir bottom
surface defining a dish, the reservoir bottom surface having a perimeter and comprising
a perimeter ledge extending around the perimeter to provide a reservoir defined by the
perimeter ledge and the reservoir bottom surface, wherein the tray is sized to be
supported by the ledge and above the reservoir bottom surface when ed into the
tissue container, and the perimeter wall having a male end and a female end, the male
end of the perimeter wall having projecting therefrom a ridge having a length and width,
wherein the female end of the perimeter wall defines a space corresponding to the
length and width of the ridge so that when the top tissue container is placed on the
bottom tissue container the female end of the bottom tissue container releasably
es the ridge extending from the male end of the top tissue container. In some
embodiments, the ter wall of the top tissue container has a top flange extending
therefrom and the perimeter wall of the bottom tissue container has a bottom flange
extending therefrom so that when the top and bottom tissue containers are assembled
the top and bottom flanges contact one another. In some embodiments, the top flange
comprises one or more tabs extending from the male end of the perimeter wall and one
or more tabs ing from the female end of the perimeter wall. In some
embodiments, the bottom flange comprises one or more tabs extending from the male
end of the ter wall and one or more tabs extending from the female end of the
perimeter wall. In some embodiments, the bottom flange ses one or more tabs
extending from the male end of the perimeter wall and one or more tabs extending from
the female end of the perimeter wall and the wherein the top flange comprises one or
more tabs extending from the male end of the perimeter wall and one or more tabs
extending from the female end of the perimeter wall so that when the top and bottom
tissue containers are assembled the tabs are offset. In some embodiments, the porous
bottom surface of the tray is a porous membrane. In some embodiments, the ridge has
a proximal end and the proximal end of the ridge has one or more indents therein and
the tray has one or more tray tabs so that when the tray is inserted into the bottom
tissue container the one or more tabs are ed into the one or more s. In some
embodiments, the s further comprise a tissue supported on the porous bottom
e of the tray. In some embodiments, the tissue is cryopreserved. In some
embodiments, the tissue is an organotypic skin substitute. In some embodiments, the
systems further se a sterile package containing the tissue container system. The
tissue container system can be provided as a kit with one or more absorbent medium
and/or one or more liquid media, such as a tissue compatible solution.
In some embodiments, the present ion provides methods of
providing a tissue for use by a health care provider comprising packaging a tissue in the
tissue ner system of the preceding paragraph and ing the ed tissue
to a health care provider in need thereof. In some embodiments, the present invention
provides methods of thawing a cryopreserved tissue comprising: providing a
cryopreserved tissue in the tissue container system as described above, removing the
top tissue container to expose the cryopreserved tissue, optionally transferring the
cryopreserved tissue to a new ner system, and filling the reservoir in the bottom
tissue container with a liquid medium under conditions that the cryopreserved tissue
thaws to provide a thawed tissue. In some embodiments, the cryopreserved tissue is an
organotypic human skin substitute. In some embodiments, the methods further
comprise applying or grafting the organotypic human skin substitute to a burn or a
wound on a patient in need thereof.
In some embodiments, the present invention provides a tissue
container 100 shown in Figure 1 that comprises a perimeter wall 105 and a substantially
planar bottom surface 110 defining a dish. The ter wall 105 has a male end 115
and a female end 120. The male end 115 of the perimeter wall 105 has a ridge 125
extending rom that has a length and a width. The female end 120 of the perimeter
wall 105 defines a space 130 corresponding to the length and width of the ridge 125 so
that when an identical tissue container is placed on top of the tissue container 100 the
space 130 provided in said female end 120 of the tissue container can releasably
e the ridge 125 extending from the male end of the identical tissue container as
shown in more detail below. The bottom surface 110 comprises a perimeter ledge 135
extending around the perimeter of the bottom surface 110. The perimeter ledge 135
forms a reservoir 140 on the bottom of the container that is preferably about 0.50 to 1.5
mm deep, and most preferably about 0.75 mm deep and which can be filled with a liquid
medium. The perimeter wall 105 preferably has a flange 145 extending rom. In
some embodiments, the tissue container 100 further ses (a) a flange 145
comprising one or more tabs 150 extending the male end 115 and female end 120 of
the perimeter wall, (b) a ridge 125 that has one more s 155 therein that are
configured to receive tabs on a tray, (c) a perimeter wall 105 sing a plurality of
grip tions 160, preferably positioned on the male end 115 of the perimeter wall
105, or (d) any combination thereof. The present invention also provides a tissue
container assembly comprising substantially identical bottom and top containers,
wherein the bottom and top containers are a tissue container described in this
paragraph. The present invention also provides a tissue container system shown in
Figure 4 comprising a tissue container assembly of this paragraph and a tray 410. The
tray is sized so that it rests on top of the perimeter ledge on the bottom surface of the
bottom ner as described above. The tray 410 comprises sidewalls 415. Tabs 420
extend from the sidewalls 415 so that they engage and are inserted into indents 425 in
the ridge 430 on the male end 435 of the bottom container 405. The tray has a porous
bottom surface 440, which is optionally a porous membrane. An identical top container
can be placed on the bottom container and closed, without interference from the
contained tray. The tissue container system can be ally sealed, preferably heat
sealed, in a e bag to provide a primary e. The primary package can be
optionally sealed inside a secondary bag. The tissue container system or package
containing the tissue container system can be provided as a kit with one or more
absorbent medium and/or one or more liquid media, such as a tissue compatible
solution.
In some embodiments, the present invention provides methods of
providing a tissue for use by a health care er comprising packaging a tissue in the
tissue container system as described in the ing paragraph and providing the
packaged tissue to a health care provider in need thereof. In some embodiments, the
present invention provides methods of ing a tissue for use to treat a wound or a
burn comprising packaging a tissue in the tissue container system as described in the
preceding paragraph and providing the packaged tissue to a health care provider for
use to treat wound or a burn. In some embodiments, the present invention provides a
method of g a cryopreserved skin equivalent prior to application to a subject. The
method ses providing a cryopreserved tissue, preferably an organotypically
ed skin equivalent, in a tissue container system as described in the preceding
paragraph, removing the top tissue container to expose the cryopreserved tissue, and
filling the reservoir in the bottom tissue container with a liquid medium under conditions
that the cryopreserved tissue thaws to provide a thawed , where the
cryoprotectant contained within the tissue is diluted into the liquid medium, leaving a
tissue that is substantially free of cryoprotectant. In other embodiments, the method
comprises ng a primary or secondary package containing a tissue container
system comprising a cryopreserved tissue from a freezer or shipping container,
removing the tissue container system from the package(s), removing the top tissue
container to expose the cryopreserved tissue, and transferring the tray with the
cryopreserved skin equivalent from the first tissue container into a second tissue
container that is sterile and staged in the sterile field and contains a liquid medium in the
container reservoir, such that the transferred cryopreserved tissue thaws to provide a
thawed tissue and the cryoprotectant contained within the tissue is diluted into the liquid
medium. In some of the above embodiments, the liquid medium is a tissue compatible
on, preferably a buffered solution. In still other ments, the tray with the
cryopreserved skin equivalent is removed from the tissue container and placed on an
absorbent medium to remove thawed otectant solution from the skin equivalent.
The absorbent medium may be in any suitable, preferably sterile, vessel (e.g., a culture
vessel or a fresh tissue container ly). The present invention is not limited to the
use of a particular absorbent medium. The absorbent medium preferably comprises a
tissue-compatible solution.
Other aspects and iterations of the invention are described more
ghly below.
BRIEF DESCRIPTION OF THE FIGURES
is a perspective view of a tissue container in accordance
with one embodiment.
is disassembled perspective view of a tissue container
assembly according to one embodiment.
is a perspective view of an assembled tissue ner
assembly accordingly to one embodiment.
is a perspective view of a tissue ner with an inserted
tray according to one embodiment.
is a graph of tissue viability after 1-day re-culture. Data are
mean ± stdev of 15 samples per group (5 samples/tissue x 3 tissues/condition in each
batch).
is a graph of post-thaw VEGF secretion during 1-day re-
culture. Data are mean ± stdev of 3 tissues per condition in each batch.
and are graphs of post-thaw tissue barrier function
after 1-day re-culture with initial DPM () and DPM change (). Data are
mean ± stdev of 12 reads per group (4 samples/tissue x 3 tissues/condition in each
batch).
ED PTION
The present ion relates generally to tissue container systems
that find use in the transport of tissues and their subsequent use by a health care
provider, and in particular to systems that support the transport, thawing and use of
cryopreserved human skin equivalents.
As used herein, the terms "skin equivalent," "human skin
equivalent," "human skin substitute," and "organotypic human skin equivalent" are used
interchangeably to refer to an in vitro derived culture of keratinocytes that has stratified
into squamous lia. Typically, the skin equivalents are produced by organotypic
culture and include a dermal layer in addition to a keratinocyte layer.
As used , the term "sterile" refers to a skin equivalent that is
essentially or tely free of able microbial or fungal contamination.
As used herein, the term "NIKS cells" refers to cells having the
characteristics of the cells deposited as cell line ATCC CRL-12191. “NIKS” stands for
near-diploid immortalized keratinocytes and is a registered trademark.
As used , the term e" when used in reference to a skin
equivalent refers to the viability of cells in the skin equivalent following cryopreservation.
In preferred embodiments, a "viable" skin has an A550 of at least 50%, 60%, 70%, 80%
or 90% of a control non-cryopreserved tissue as measured by an MTT assay or at least
50%, 60%, 70%, 80% or 90% of the readout value of a similar viability assay.
As used herein, the term "culture vessel" refers to any vessel of the
type commonly used to culture cells or tissues and includes circular, rectangular, and
square dishes formed from a suitable material such as tissue culture c,
polystyrene, polymers, cs, glass, etc. The term “culture vessel” and “growth
chamber” are used interchangeably. Tissue containers of the present disclosure are not
culture s, as used herein, at least because the tissue containers of the present
disclosure are not of a suitable size for long-term culture.
The tissue containers of the instant invention make efficient use of
freezer and surgical suite space as they are approximately 60% smaller than previously
utilized ners. The tissue containers are compatible with a tray that includes a
porous membrane as bottom surface upon which a tissue (e.g., an organotypic skin
substitute) can be supported. The other surfaces of the tray are preferably clear or
translucent cs produced by a thermoforming process from a plastic sheet, injection
molding, or other methods known in the art to manipulate plastics. Suitable plastics
include medical grade plastics, for e, polyethylene thlate glycol-modified
(PETG), polystyrene, etc. In some preferred embodiments, the tray is a preferably a tray
as described in paragraph [0030] herein. The tissue containers include a reservoir that
can be filled with media to thaw the tissue in the container and remove cryoprotectant
when the tissue has been frozen. This provides an age over previous systems
used for thawing tissues where the tissue had to be removed from the container in the
surgical sterile field and then placed on a Telfa® pad. The tissue containers of the
present invention preferably include a top and bottom which are mirror images of one
r. The top and bottom pieces of the container ly are substantially identical
and can be snapped together to form an ed container. The use of a top and
bottom which are substantially identical means that both the top and bottom piece can
be produced from the same molds, which creates efficiencies during the production of
the top and bottom pieces. The top and bottom pieces are preferably clear and
produced by a thermoforming process from a c sheet. Suitable plastics include
medical grade thermoformable plastics, for example, polyethylene terephthlate modified
(PETG). Accordingly, the present invention provides improved tissue
containers and tissue container systems which will be described in more detail below.
Figure 1 shows a tissue container 100. In some embodiments, the
tissue container 100 preferably comprises a perimeter wall 105 and a substantially
planar bottom e 110 defining a dish. The perimeter wall 105 has a male end 115
and a female end 120. The male end 115 of the perimeter wall 105 has a ridge 125
extending therefrom that has a length and a width. The female end 120 of the perimeter
wall 105 defines a space 130 corresponding to the length and width of the ridge 125 so
that when an identical tissue container is placed on top of the tissue container 100 the
space 130 provided in said female end 120 of the tissue container can releasably
receive the ridge 125 extending from the male end of the identical tissue container as
shown in more detail below. The bottom surface 110 comprises a perimeter ledge 135
extending around the perimeter of the bottom e 110. The perimeter ledge 135
forms a reservoir 140 on the bottom of the ner that is preferably about 0.50 to 1.5
mm deep, and most preferably about 0.75 mm deep and which can be filled with a liquid
medium. The perimeter wall 105 preferably has a flange 145 extending therefrom. In
some embodiments, the flange 145 comprises one or more tabs 150 extending the male
end 115 and female end 120 of the perimeter wall. In some ments, the ridge 125
has one or more s 155 therein that are configured to receive tabs on a tray, which
is shown in more detail below. In some further embodiments the perimeter wall 105
preferably comprises a ity of grip projections 160, preferably positioned on the
male end 115 of the perimeter wall 105.
Figure 2 shows an expanded view of a tissue container assembly
200 of the instant invention. The tissue container assembly 200 preferably comprises
substantially identical bottom and top containers 205 and 210. Each of the bottom and
top containers 205 and 210 comprise a perimeter wall 215 and 220 and have a bottom
surface 225 in the case of the bottom container 205 and a top surface 230 in the case of
the top container 210. The bottom surface 225 ses a perimeter ledge 235
ing around the perimeter of the bottom surface 225. The perimeter ledge 235
forms a reservoir 240 on the bottom of the bottom container 205 that is ably about
0.50 to 1.5 mm deep, and most preferably about 0.75 mm deep and which can be filled
with a liquid medium. Each of the bottom and top containers 205 and 210 comprise
male and female ends 245 and 250. The male ends 245 have a ridge 255 extending
therefrom that has a length and a width. The female ends 250 define a space 260
corresponding to the length and width of the ridges 255 so that when the top ner
210 is placed on the bottom container 205 along the alignment shown by dashed lines
265 the space 260 provided in said female ends 250 of the bottom and top tissue
containers 205 and 210 can releasably receive the ridges 255 so that the bottom and
top containers 205 and 210 can be releasably snapped together. The perimeter walls
215 and 220 preferably have flanges 270 and 275 extending therefrom. In some
embodiments, the flanges comprise one or more tabs 280 extending the male and
female ends 245 and 250. In some embodiments, the ridges 255 have one or more
indents 285 therein that are configured to receive tabs on a tray, which is shown in more
detail below. In some further embodiments the perimeter walls preferably comprises a
plurality of grip projections 290, preferably positioned on the male ends 245. Figure 3
shows a container assembly 300 of the present invention where the bottom container
305 and top container 310 are fully engaged to form an ed container.
The present ion r provides a tissue container system
comprising the bottom and top containers described above along with a tray. Figure 4
shows a bottom container of the present invention into which a tray 410 has been
inserted. The tray 410 is sized so that it rests on top of the perimeter ledge on the
bottom surface of the bottom container as described above. The tray 410 ses
sidewalls 415. Tabs 420 extend from the sidewalls 415 so that they engage and are
inserted into s 425 in the ridge 430 on the male end 435 of the bottom container
405. Sidewalls 415 and tabs 420 are preferably clear or translucent plastics produced
by a thermoforming process from a plastic sheet, injection molding, or other methods
known in the art to manipulate cs. Preferred plastics are medical grade
thermoformable plastics including, but not limited to, polyethylene terephthlate glycolmodified
(PETG) and polystyrene. In some preferred embodiments, the plastic used for
sidewalls 415 and tabs 420 is polystyrene. The tray preferably has a porous bottom
surface 440. In some preferred ments, the porous bottom surface is a porous
membrane, preferably a semi-permeable polymer film, more preferably a semipermeable
track-etched polymer film. The membrane can be tissue culture treated (e.g.,
plasma d) to improve cell attachment. In further embodiments, the membrane has
a nominal thickness of at least 5 microns, in some example, about 5 microns to about
microns, preferably about 10 microns to about 20 microns, more ably about 10
microns to about 15 microns. In other examples, the membrane has a nominal
thickness of about 10 microns. Suitable membrane materials are known in the art and
e, but are not d to, polyethylene terephthalate, polyester, rbonate, or
any other membrane material used in commercially available, tissue-culture treated
inserts (e.g., Transwell®, Snapwell™, etc.) with a multiplicity of open pores
therethrough. Preferably the pores have a nominal pore size of about 0.1 micron to
about 10 microns, preferably about 0.1 micron to about 0.8 micron, more preferably
about 0.2 micron to about 0.8 micron, even more preferably about 0.4 micron, about 0.5
micron, or about 0.6 micron. The membrane preferably has a nominal pore density
between about 1x108 and about 4x108 pores per square eter, though a wider
range is also able. Most preferably, a membrane is formed from polycarbonate
having pores with a nominal size of about 0.4 micron and a l pore density about
1x108 pores per square centimeter. The membrane may be attached to sidewalls 415
by any suitable method known in the art, for e by heat sealing, sonic welding,
solvent g, adhesive bonding and the like.
The present ion may be used to cryopreserve, store and/or
transport a variety of tissues. The tissues are preferably ted on the porous
bottom surface of the tray and are enclosed with a container assembly of the present
invention comprising bottom and top containers. In some preferred embodiments, the
tissues are cryopreserved. In some embodiments, the tissues are skin tissues, for
example, cadaver skin or organotypic skin equivalents. In some exemplary
embodiments, the tissues are organotypic skin equivalents or cryopreserved
organotypic skin equivalents.
The present invention is not limited to any particular organotypic
skin equivalent. Indeed, the present invention contemplates the use of a variety of cell
lines and sources that can differentiate into squamous epithelia, including both primary
and immortalized nocytes. Sources of cells include keratinocytes and dermal
fibroblasts biopsied from humans and cavaderic donors (Auger et al, In Vitro Cell. Dev.
Biol. — Animal 36:96-103; U.S. Pat. Nos. 5,968,546 and 5,693,332, each of which is
incorporated herein by nce), neonatal ins (Asbill et al., Pharm. Research
17(9): 1092-97 ; Meana et al., Burns 24:621-30 (1998); U.S. Pat. Nos. 4,485,096;
6,039,760; and 5,536,656, each of which is orated herein by reference), and
immortalized keratinocytes cell lines such as NM1 cells (Baden, In Vitro Cell. Dev. Biol.
23(3):205-213 (1987)), HaCaT cells (Boucamp et al., J. cell. Boil. 106:761-771 (1988));
and NIKS® cells (Cell line BCEp/SL; U.S. Pat. No. 5,989,837, incorporated herein by
reference; ATCC CRL-12191). Each of the mentioned cell lines can be cultured or
genetically modified in order to produce a cell line capable of expressing or coexpressing
the desired protein(s). In particularly preferred ments, NIKS® cells
are utilized. The discovery of the novel NIKS® human keratinocyte cell line provides an
opportunity to genetically engineer human keratinocytes with non-viral vectors. A unique
advantage of the NIKS® cells is that they are a consistent source of genetically-uniform,
pathogen-free human keratinocytes. For this reason, they are useful for the application
of c engineering and genomic gene expression approaches to provide human
skin lents with enhanced properties over currently available skin equivalents.
NIKS® cells, identified and characterized at the University of sin, are
nontumorigenic, karyotypically stable, and exhibit normal growth and differentiation both
in monolayer and organotypic culture. NIKS® cells form fully stratified skin lents
in culture. These cultures are indistinguishable by all criteria tested thus far from
organotypic cultures formed from y human keratinocytes. Unlike primary cells
however, NIKS® cells exhibit an ed lifespan in monolayer culture. This provides
an opportunity to genetically manipulate the cells and isolate new clones of cells with
new useful properties -Hoffmann et al., J. Invest. Dermatol., 114(3): 444-455
(2000)).
The NIKS® cells arose from the BCEp strain of human neonatal
foreskin keratinocytes isolated from an apparently normal male infant. In early
passages, the BCEp cells exhibited no logical or growth characteristics that
were atypical for cultured normal human keratinocytes. Cultivated BCEp cells
exhibited stratification as well as features of programmed cell death. To determine
replicative lifespan, the p cells were serially cultivated to ence in standard
keratinocyte growth medium at a density of 3 x 105 cells per 100-mm dish and
passaged at weekly intervals (approximately a 1:25 split). By passage 15, most
keratinocytes in the tion appeared senescent as judged by the presence of
us abortive colonies which exhibited large, flat cells. However, at passage 16,
nocytes exhibiting a small cell size were evident. By passage 17, only the smallsized
keratinocytes were present in the culture and no large, senescent keratinocytes
were t. The resulting population of small keratinocytes that survived this putative
crisis period appeared morphologically uniform and produced colonies of keratinocytes
exhibiting typical keratinocyte characteristics including cell-cell adhesion and apparent
squame production. The keratinocytes that survived senescence were serially cultivated
at a density of 3 x 105 cells per 100-mm dish. Typically the cultures reached a cell
y of approximately 8 x 106 cells within 7 days. This stable rate of cell growth was
maintained through at least 59 passages, demonstrating that the cells had achieved
immortality. The keratinocytes that emerged from the original senescencing population
are now termed NIKS®. The NIKS® cell line has been screened for the presence of
proviral DNA sequences for HIV-1, HIV-2, EBV, CMV, HTLV-1, HTLV-2, HBV, HCV, B-
19 parvovirus, HPV-16, SV40, HHV-6, HHV-7, HPV-18 and HPV-31 using either PCR or
Southern analysis. None of these viruses were detected.
Chromosomal analysis was performed on the parental p
cells at e 3 and NIKS® cells at passages 31 and 54. The parental BCEp cells
have a normal chromosomal ment of 46, XY. At e 31, all NIKS cells
contained 47 chromosomes with an extra isochromosome of the long arm of
chromosome 8. No other gross chromosomal abnormalities or marker chromosomes
were detected. The ype of the NIKS® cells has been shown to be stable to at
least passage 54.
The DNA fingerprints for the NIKS® cell line and the BCEp
keratinocytes are identical at all twelve loci analyzed demonstrating that the NIKS® cells
arose from the parental BCEp population. The odds of the NIKS® cell line having the
parental BCEp DNA fingerprint by random chance is 4 x 10-16. The DNA fingerprints
from three ent sources of human keratinocytes, EDEp, SCC4 and SCC13y are
different from the BCEp pattern. This data also shows that keratinocytes isolated
from other humans, EDEp, SCC4, and SCC13y, are unrelated to the BCEp cells
or each other. The NIKS® DNA fingerprint data provides an unequivocal way to identify
the NIKS® cell line.
Loss of p53 function is associated with an enhanced proliferative
ial and increased frequency of ality in cultured cells. The sequence of p53
in the NIKS® cells is identical to published p53 ces (GenBank accession
number: M14695). In , p53 exists in two predominant polymorphic forms
distinguished by the amino acid at codon 72. Both alleles of p53 in the NIKS® cells are
wild-type and have the sequence CGC at codon 72, which codes for an arginine. The
other common form of p53 has a proline at this position. The entire sequence of p53 in
the NIKS® cells is identical to the BCEp progenitor cells. Rb was also found to be
wild-type in NIKS® cells.
Anchorage-independent growth is highly correlated to
tumorigenicity in vivo. For this reason, the anchorage-independent growth
characteristics of NIKS® cells in agar or methylcellulose-containing medium were
investigated. NIKS® cells remained as single cells after 4 weeks in either agar- or
methylcellulose-containing . The assays were continued for a total of 8 weeks to
detect slow growing ts of the NIKS® cells. None were observed.
To determine the tumorigenicity of the parental BCEp
keratinocytes and the immortal NIKS® keratinocyte cell line, cells were injected into the
flanks of athymic nude mice. The human squamous cell carcinoma cell line, SCC4, was
used as a positive control for tumor production in these animals. The injection of
samples was designed such that s received SCC4 cells in one flank and either
the parental p keratinocytes or the NIKS® cells in the opposite flank. This
injection gy eliminated animal to animal variation in tumor production and
confirmed that the mice would support vigorous growth of tumorigenic cells. Neither the
al BCEp keratinocytes (passage 6) nor the NIKS® keratinocytes (passage 35)
produced tumors in athymic nude mice.
NIKS® cells were analyzed for the ability to undergo differentiation
in both submerged culture and organotypic culture. ques for organotypic culture
are bed in detail in the examples. In particularly preferred embodiments, the
organotypically cultured skin equivalents of the present invention comprise a dermal
equivalent formed from collagen or a similar material and fibroblasts. The keratinocytes,
for example NIKS® cells or a combination of NIKS® cells and cells from a patient are
seeded onto the dermal equivalent and form an epidermal layer characterized by
squamous differentiation following the organotypic culture process.
For cells in submerged culture, the formation of cornified envelopes
was monitored as a marker of squamous differentiation. In cultured human
keratinocytes, early stages of cornified envelope assembly results in the ion of an
immature structure composed of involucrin, cystatin-a and other ns, which
represent the innermost third of the mature cornified envelope. Less than 2% of the
keratinocytes from the nt BCEp cells or the NIKS® cell line produce cornified
envelopes. This finding is consistent with previous studies demonstrating that actively
growing, subconfluent keratinocytes produce less than 5% cornified envelopes. To
determine whether the NIKS® cell line is e of producing cornified envelopes
when induced to entiate, the cells were removed from adherent culture and
suspended for 24 hours in medium made semi-solid with methylcellulose. Many aspects
of terminal differentiation, ing differential expression of keratins and cornified
envelope formation can be triggered in vitro by loss of keratinocyte cell-cell and bstratum
adhesion. The NIKS® keratinocytes produced as many as and usually more
ied pes than the parental keratinocytes. These findings demonstrate that
the NIKS® keratinocytes are not defective in their ability to initiate the formation of this
cell type-specific differentiation structure.
To confirm that the NIKS® keratinocytes can undergo squamous
differentiation, the cells were ated in organotypic culture. nocyte cultures
grown on plastic substrata and submerged in medium replicate but exhibit limited
differentiation. Specifically, human nocytes become confluent and undergo limited
stratification producing a sheet consisting of 3 or more layers of keratinocytes. By light
and electron microscopy there are ng ences between the architecture of the
multilayered sheets formed in submerged culture and intact human skin. In st,
organotypic culturing techniques allow for keratinocyte growth and differentiation under
in vivo-like conditions. Specifically, the cells adhere to a physiological substratum
consisting of dermal fibroblasts embedded within a fibrillar collagen base. The
organotypic culture is maintained at the air-medium interface. In this way, cells in the
upper sheets are air-exposed while the proliferating basal cells remain closest to the
gradient of nutrients provided by diffusion h the collagen gel. Under these
conditions, correct tissue architecture is formed. Several characteristics of a normal
differentiating epidermis are evident. In both the parental cells and the NIKS® cell line a
single layer of cuboidal basal cells rests at the junction of the epidermis and the dermal
equivalent. The d morphology and high nuclear to cytoplasmic ratio is indicative
of an actively dividing population of keratinocytes. In normal human epidermis, as the
basal cells divide they give rise to er cells that migrate upwards into the
differentiating layers of the tissue. The daughter cells increase in size and become
flattened and squamous. ally these cells enucleate and form ied,
keratinized structures. This normal entiation process is evident in the upper layers
of both the parental cells and the NIKS® cells. The appearance of ned squamous
cells is evident in the upper epidermal layers and demonstrates that stratification has
occurred in the organotypic cultures. In the uppermost part of the organotypic cultures
the enucleated squames peel off the top of the culture. To date, no histological
ences in differentiation at the light microscope level between the parental
keratinocytes and the NIKS® keratinocyte cell line grown in organotypic culture have
been observed.
To observe more detailed characteristics of the parental (passage
) and NIKS® (passage 38) organotypic cultures and to confirm the histological
observations, s were analyzed using electron microscopy. Parental cells and the
immortalized NIKS® human keratinocyte cell line were ted after 15 days in
organotypic culture and sectioned perpendicular to the basal layer to show the extent of
stratification. Both the parental cells and the NIKS® cell line undergo extensive
stratification in organotypic culture and form structures that are characteristic of normal
human epidermis. Abundant desmosomes are formed in organotypic cultures of
parental cells and the NIKS® cell line. The formation of a basal lamina and associated
hemidesmosomes in the basal keratinocyte layers of both the parental cells and the cell
line was also noted.
Hemidesmosomes are lized structures that increase
adhesion of the keratinocytes to the basal lamina and help maintain the ity and
strength of the tissue. The presence of these structures was especially evident in areas
where the parental cells or the NIKS® cells had attached directly to the porous support.
These findings are tent with earlier ultrastructural findings using human foreskin
keratinocytes cultured on a fibroblast- ning porous support. Analysis at both the
light and on microscopic levels demonstrate that the NIKS® cell line in organotypic
e can stratify, differentiate, and form structures such as desmosomes, basal
lamina, and hemidesmosomes found in normal human epidermis.
In some embodiments, the tissues that are supported on the porous
membrane and enclosed with the container assembly are cryopreserved. Where this
tissue is a skin equivalent, the cryopreserved skin equivalents are preferably storable at
imately -50C, -60C, -70C, -80C or colder for an ed period of time such as
r than 1, 2, 3, 4, 5 or 6 months and up to 12 or 24 months without a substantial
loss of viability.
In preferred embodiments, all steps of the cryopreservation process
prior to product packaging are performed aseptically inside a Class 100 biosafety
cabinet in a Class 10,000 oom. In some embodiments, the cryopreservation
process comprises treating an organotypically cultured skin equivalent in a
cryoprotectant solution. The organotypically cultured skin equivalent is supported on a
porous membrane of a tray of the t disclosure, and the tray is placed in a suitable
vessel, such as a culture vessel or a tissue container assembly of the present
disclosure. A suitable volume of cryoprotectant solution is added to the vessel to be in
contact with the porous membrane, but not submerge the tissue, allowing
cryoprotectant er into the tissue through its base. Certain embodiments of the
present invention are not limited to the use of any particular cryoprotectant. In some
red embodiments, the otectant is glycerol. The cryoprotectant may be
provided in different concentrations in the cryoprotectant solution. In some
embodiments, the cryoprotectant is provided in a solution sing about 20% or 21%
to about 70% of the solution by volume, and more ably about 20% or 21% to
about 45% of the solution by volume or 37.5% to 62.5% of the solution by volume, or
most preferably from about 25% to 40% of the on by volume or 42.5% to 57.5% of
the solution by volume, depending on the temperature. In some embodiments, the
cryoprotectant solution preferably comprises about 32.5% v/v or about 50% v/v
cryoprotectant (e.g., glycerol). In some embodiments, the cryoprotectant is provided in a
base medium solution. Suitable base medium solutions include, but are not limited to,
DMEM, Ham's F-10, Ham's F-12, DMEM/F-12, Medium 199, MEM and RPMI. In some
embodiments, the base medium forms the der of the solution volume. In some
embodiments, the cryoprotectant solution is buffered. Suitable buffers include, but are
not limited to, HEPES, Tris, MOPS, and Trizma buffers. Buffering agents may be
included at an amount to provide a buffered system in the range of pH 7.0 to 7.4. In
some preferred embodiments, the cryoprotectant solution is buffered with from about 5
mM to 15 mM HEPES, most preferably about 10 mM HEPES to a pH of about 7.0 to
In some particularly preferred embodiments, treatment with the
cryoprotectant solution is conducted in a single step. By "single step" it is meant that the
cryoprotectant solution is not ged during the equilibration procedure as is
common in the art. For example, the treatment step is performed using a cryoprotectant
solution with a defined tration of cryoprotectant as d to a stepwise
bration procedure where several media changes with increasing concentrations of
cryoprotectant at each step. In some embodiments, the treatment step is conducted at a
reduced temperature. In preferred embodiments, the treatment step is conducted at
from about 2C to 8C, while in other embodiments, the treatment step is conducted at
room temperature, for example from about 15C to 30C. In some embodiments, the skin
equivalent is incubated in the cryoprotectant solution for about 10 to 60 minutes,
preferably from about 20 to 30 minutes.
In some embodiments, the skin equivalent ted on the porous
membrane of the tray is frozen following treatment with the cryoprotectant on,
ably after excess otectant solution is removed from the skin equivalent, for
example by aspirating the solution or moving the treated skin equivalent to a fresh
vessel (e.g., a sterile culture vessel or a sterile tissue container assembly of the present
sure). Accordingly, in some embodiments, the treated skin equivalent supported
on the porous membrane of the tray is frozen by re to temperatures ranging from
about -50C to -100C, and most preferably at about -80C. In some preferred
ments the tray with the treated skin lent is simply placed in a bag or other
vessel (e.g., a sterile culture vessel or a sterile tissue container assembly of the present
disclosure) and placed in a freezing unit such as a low temperature (e.g., -80°C freezer)
freezing unit. In contrast, it is common in the art to control the rate of freezing either by
controlling the temperature in the freezing unit or by placing the tissue to be frozen in a
container that allows control of the rate of decrease in temperature.
In some embodiments, the cryopreserved skin equivalent is
packaged for long term storage. In some preferred embodiments, the skin equivalent, in
its tray, is enclosed with the bottom and top containers as described in detail above. Is
some embodiments, the assembly containing the human skin equivalent is sealed,
preferably heat sealed in a sterile bag (e.g., a c or polymer bag) to provide a
primary package. The primary package is then sealed inside a secondary bag, for
example a secondary plastic, foil, or Mylar bag. The cryopreserved tissues of the
present invention may preferably be stored at low temperature, from about -50C to
about -100C or lower, preferably about -80C. The skin equivalents may be ably
stored from about 1, 2, 3, 4, 5 or 6 months and up to 12 or 24 months without a
substantial loss of viability.
In a preferred embodiment, an organotypically cultured skin
equivalent in its tray, which is ed into a sterile bottom container of the present
disclosure, is treated with a cryoprotectant solution as described above. Excess
cryoprotectant solution is removed from the skin equivalent prior to freezing by
ting the cryoprotectant solution from the bottom container. The d skin
equivalent in its tray is then enclosed with a sterile top container of the present
disclosure, thereby forming a tissue container system. Alternatively, excess
cryoprotectant on is removed from the skin lent prior to freezing by moving
the tray with the treated skin equivalent to a second, sterile bottom container of the
present disclosure and then enclosing the tray with a sterile top container of the present
disclosure, thereby forming a tissue container system. The tissue container system
containing the treated human skin equivalent is then sealed, preferably heat sealed in a
sterile bag (e.g., a plastic or polymer bag) to provide a y package. The primary
package may be sealed inside a ary bag, for example a secondary plastic, foil,
or Mylar bag. The primary or secondary bag is then stored at low temperature, from
about -50C to about -100C, preferably about -80C. The skin equivalents may be stored
from about 1, 2, 3, 4, 5 or 6 months and up to 12 or 24 months without a ntial loss
of viability.
In another preferred embodiment, an organotypically cultured skin
equivalent in its tray, which is placed in a culture vessel, is treated with a cryoprotectant
solution as described above. Excess cryoprotectant solution is removed from the skin
equivalent prior to freezing by moving the tray with the d skin equivalent to a
sterile bottom container of the present disclosure and then enclosing the tray with a
sterile top container of the present disclosure, thereby forming a tissue container
system. The tissue container system containing the treated human skin equivalent is
then sealed, preferably heat sealed in a sterile bag (e.g., a c or polymer bag) to
e a primary package. The primary package may be sealed inside a secondary
bag, for example a secondary plastic, foil, or Mylar bag, to produce a secondary
package. The primary or secondary package is then stored at low temperature, from
about -50C to about -100C, preferably about -80C. The skin equivalents may be stored
from about 1, 2, 3, 4, 5 or 6 months and up to 12 or 24 months without a ntial loss
of viability.
In some embodiments, the present invention provides a method of
g a cryopreserved skin equivalent prior to application to a subject, comprising
providing a cryopreserved tissue in the tissue container system as described above,
removing the top tissue container to expose the cryopreserved tissue, and filling the
oir in the bottom tissue container with a liquid medium under conditions that the
cryopreserved tissue thaws to provide a thawed tissue, where the cryoprotectant
contained within the tissue is diluted into the liquid , leaving a tissue that is
substantially free of otectant. In other ments, the method comprises
removing a primary or secondary package containing a tissue container system
comprising a cryopreserved tissue from a freezer or shipping container, removing the
tissue container system from the package(s), removing the top tissue container to
expose the cryopreserved tissue, and transferring the tray with the cryopreserved skin
equivalent from the first tissue container into a second tissue container that is sterile
and staged in the sterile field and contains a liquid medium in the container reservoir,
such that the erred cryopreserved tissue thaws to provide a thawed tissue and the
cryoprotectant contained within the tissue is diluted into the liquid medium. In some of
the above embodiments, the liquid medium is a tissue compatible solution, preferably a
buffered solution. Suitable tissue compatible solutions include, but are not d to,
DMEM, Ham's F-10, Ham's F-12, DMEM/F-12, Medium 199, MEM and RPMI. Suitable
buffers include, but are not limited to, HEPES, Tris, MOPS, and Trizma s.
Buffering agents may be included at an amount to provide a ed system in the
range of pH 7.0 to 7.4. In still other embodiments, the tray with the cryopreserved skin
equivalent is removed from the tissue container and placed on an absorbent medium to
remove thawed cryoprotectant solution from the skin equivalent. The absorbent medium
may be in any suitable, preferably sterile, vessel (e.g., a culture vessel or a fresh tissue
container assembly). The present invention is not limited to the use of a particular
absorbent medium. Suitable absorbent media include, but are not limited to, Telfa®
pads, cellulosic pads (e.g., Whatman 1003-090 filter pads and Pall 70010 filter pads),
gauze pads, and foam pads (e.g., Covidien 55544 hydrophilic foam pad). In some
preferred embodiments, the absorbent medium is a Telfa® pad. In some embodiments,
the absorbent medium further comprises a tissue-compatible on. In some
embodiments, the tissue ible on is a buffered solution. Suitable tissue
compatible ons include, but are not limited to, DMEM, Ham's F-10, Ham's F-12,
DMEM/F-12, Medium 199, MEM and RPMI. Suitable buffers include, but are not limited
to, HEPES, Tris, MOPS, and Trizma buffers. Buffering agents may be included at an
amount to e a buffered system in the range of pH 7.0 to 7.4.
It is plated that the cryopreserved skin equivalents of the
present ion may be used therapeutically after thawing. In some embodiments, the
cryopreserved skin substitute is used after thawing in wound closure and burn treatment
applications. The use of afts and afts for the treatment of burns and wound
closure is described in Myers et al., A. J. Surg. 170(1):75-83 (1995) and U.S. Pat. Nos.
,693,332; 5,658,331; and 6,039,760, each of which is incorporated herein by
nce. In some embodiments, the skin equivalents may be used in conjunction with
dermal replacements such as DERMAGRAFT or INTEGRA. Accordingly, the present
invention provides methods for wound closure, including ulcers or wounds caused by
burns, comprising providing a cryopreserved skin equivalent in a tissue container
system of the present disclosure, thawing the skin equivalent, and treating a patient
suffering from a wound with the thawed skin equivalent under ions such that the
wound is closed.
In some embodiments, the skin equivalents are utilized to treat
chronic skin wounds. Chronic skin wounds (e.g., venous ulcers, diabetic ulcers,
pressure ulcers) are a serious problem. The healing of such a wound often takes well
over a year of treatment. Treatment options currently include dressings and
debridement (use of chemicals or surgery to clear away necrotic tissue), and/or
antibiotics in the case of infection. These treatment options take extended periods of
time and high levels of t compliance. As such, a therapy that can se a
practitioner's success in healing c wounds and rate the rate of wound
healing would meet an unmet need in the field. Accordingly, the present ion
contemplates treatment of skin wounds with cryopreserved skin equivalents. In some
embodiments, skin equivalents are topically applied to wounds after thawing. In other
embodiments, cryopreserved skin equivalents are used for application to partial
thickness wounds after thawing. In other embodiments, cryopreserved skin equivalents
are used to treat full thickness wounds after thawing. In other embodiments,
cryopreserved skin equivalents are used to treat numerous types of internal wounds
after thawing, including, but not limited to, al wounds of the mucous membranes
that line the gastrointestinal tract, ulcerative colitis, and mation of mucous
membranes that may be caused by cancer therapies. In still other embodiments, skin
equivalents expressing host defense peptides or pro-angiogenic factors are used as a
temporary or permanent wound ng after thawing.
In still r embodiments, the cells are engineered to provide
additional therapeutic agents to a subject. The present invention is not limited to the
delivery of any particular therapeutic agent. Indeed, it is contemplated that a variety of
therapeutic agents may be delivered to the subject, including, but not limited to,
enzymes, peptides, peptide hormones, other proteins, ribosomal RNA, ribozymes, small
interfering RNA (siRNA) micro RNA (miRNA), and antisense RNA. In preferred
embodiments, the agents are host defense peptides such as human beta-defensin 1, 2,
or 3 or icidin or other proteins such as VEGF and HIF-1 a, see, e.g., U.S. Pat.
Nos. 7,674,291; 7,807,148; 7,915,042; 7,988,959; and 8,092,531; each of which is
incorporated herein by reference in its entirety. These therapeutic agents may be
delivered for a variety of purposes, including but not limited to the purpose of correcting
genetic defects. In some particular preferred embodiments, the therapeutic agent is
delivered for the purpose of detoxifying a patient with an inherited inborn error of
metabolism (e.g., aminoacidopathesis) in which the skin equivalent serves as wild-type
. It is contemplated that delivery of the therapeutic agent corrects the defect. In
some embodiments, the cells are transfected with a DNA construct encoding a
eutic agent (e.g., insulin, ng factor IX, erythropoietin, etc.) and skin
lents prepared from transfected cells are administered to the subject. The
therapeutic agent is then delivered to the patient's bloodstream or other tissues from the
graft. In preferred embodiments, the nucleic acid encoding the therapeutic agent is
operably linked to a le promoter. The present ion is not limited to the use of
any ular promoter. Indeed, the use of a variety of promoters is plated,
including, but not limited to, inducible, constitutive, tissue-specific, and nocytespecific
promoters. In some embodiments, the nucleic acid encoding the therapeutic
agent is introduced directly into the keratinocytes (i.e., by electroporation, calcium
ate co-precipitation, or liposome transfection). In other preferred embodiments,
the nucleic acid encoding the therapeutic agent is provided as a vector and the vector is
introduced into the keratinocytes by methods known in the art. In some embodiments,
the vector is an episomal vector such as a replicating d. In other embodiments,
the vector ates into the genome of the keratinocytes. Examples of integrating
vectors include, but are not limited to, retroviral vectors, adeno-associated virus vectors,
non-replicating plasmid vectors and transposon vectors
EXAMPLES
The following examples are provided in order to demonstrate and
further illustrate certain preferred embodiments and aspects of the present invention
and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); mM (millimolar); µM (micromolar); N
l); mol (moles); mmol (millimoles); µmol moles); nmol (nanomoles); g
(grams); mg (milligrams); lig (micrograms); ng rams); 1 or L (liters); ml or mL
(milliliters); µ1 or µL (microliters); cm (centimeters); mm (millimeters); pm meters);
nm (nanometers); C (degrees Centigrade); U (units), mU (milliunits); min. (minutes);
sec. (seconds); % (percent); kb (kilobase); bp (base pair); PCR (polymerise chain
reaction); BSA (bovine serum n); CFU (colony forming units); kGy (kiloGray);
PVDF (polyvinylidine fluoride); BCA (bicinchoninic acid); SDS-PAGE (sodium l
e polyacrylamide gel electrophoresis).
Example 1
StrataGraft® skin tissue is a living, full-thickness, allogeneic
human skin substitute that reproduces many of the structural and biological properties
of normal human skin. StrataGraft® skin tissue contains both a viable, fully-stratified
epidermal layer derived from NIKS® cells, which are a consistent and wellcharacterized
source of pathogen-free human keratinocyte progenitors, and a dermal
layer containing normal human dermal fibroblasts (NHDF) embedded in a collagen-rich
matrix. Graft® skin tissue possesses ent tensile strength and handling
characteristics that enable it to be meshed, stapled, and sutured similarly to human skin
grafts. Graft® also exhibits barrier function comparable to that of intact human
skin and is capable of delivering bioactive molecules for wound bed conditioning and
tissue regeneration. The physical and biological characteristics of StrataGraft® skin
tissue make it ideal for the treatment of a variety of skin wounds.
The manufacturing process for StrataGraft® skin tissue
asses three tial cell and tissue culture processes. In Stage I of the
cturing process, NIKS® keratinocytes are expanded in monolayer cell culture.
rent with the NIKS® keratinocyte culture in Stage I, NHDF are expanded in
monolayer culture and combined with purified type I collagen and culture medium and
allowed to gel to form the cellularized dermal equivalent (DE). Alternatively, NHDF are
seeded into Transwell® inserts (Corning) and allowed to proliferate and secrete and
assemble extracellular matrix molecules into a simplified dermal equivalent. In Stage II,
NIKS® keratinocytes are seeded onto the e of the DE and cultured under
submerged ions for two days to promote complete epithelialization of the DE
surface. The tissue is then lifted to the quid interface in Stage III, where it is
maintained for 18 days in a controlled, low humidity environment to promote tissue
maturation. The skin equivalents are generally prepared as described in U.S. Pat. Nos.
7,674,291; 7,807,148; 7,915,042; 7,988,959; 8,092,531; and U.S. Pat. Publ.
20140271583; each of which is incorporated herein by reference in its entirety.
Example 2
This example describes improved cryopreservation methods for
human skin equivalents utilizing a pre-freeze treatment step with cryopreservation
solutions containing 32.5% or 50% glycerol at room temperature and is described in copending
U.S. Pat. Publ. 20140271583, which is incorporated by reference herein in its
entirety. The general production s is unchanged from the current method
described usly. At the end of the production process, the tissues are d and
cryopreserved as follows.
ter Operating Range
Cryoprotectant formulation 32.5% (v/v) glycerol
DMEM (1X)
mM HEPES (pH 7.0 to 7.4); or
50% (v/v) glycerol
DMEM (1X)
mM HEPES (pH 7.0 to 7.4)
Pre-freeze cryoprotectant Room temperature
incubation ature
Pre-freeze cryoprotectant incubation time 15-45 minutes
Freeze method Direct transfer to -80C freezer
e temperature -70 to -90C
Shipping conditions Overnight delivery on dry ice
All steps of the eservation process prior to the final product
packaging step are med aseptically inside a Class 100 biosafety cabinet in a
Class 10,000 cleanroom. The specific volumes and dishes described in this example
are applicable to tissues generated in the previous ar, 44 cm2 format, not the
larger rectangular format of the current disclosure.
Step 1- Dispense 20 ml of otectant solution to 100 mm
culture dishes.
Step 2- Transfer Transwell® inserts containing Graft®
tissues into individual dishes containing cryoprotectant solution. Incubate tissues 15-45
minutes in cryoprotectant solution.
Step 3- er ell® inserts containing treated
StrataGraft® tissues to new sterile 100 mm culture dishes ning final product label
so that the tissue rests on the bottom of the culture dish. Excess cryoprotectant is
allowed to drain from the skin equivalent to provide a d skin equivalent that is
substantially free of excess cryoprotectant on the exterior surfaces of the skin
equivalent.
Step 4- Heat-seal 100 mm culture dishes in clear, sterile bags.
Place primary package into secondary Mylar bag and heat-seal.
Step 5- Remove the packaged StrataGraft® tissues from
cleanroom and transfer tissues to an ultralow freezer (-70°C to -90°C). Place s in
a pre-cooled rack in the freezer that allows unrestricted airflow to the top and bottom of
the packaged tissues to ensure uniform and rapid cooling. Leave tissues undisturbed
overnight during the freezing process.
Cryopreserved s were thawed at room temperature for 10
minutes, transferred to a hold chamber containing Telfa® pads saturated with 40 ml of
HEPES-buffered culture medium that had been warmed to room ature (RT), and
held at RT for 15 to 20 minutes. Tissues were transferred to a culture dish containing 90
ml of SMO1 medium and returned to culture overnight. Tissues were analyzed for
viability after overnight re-culture. Tissues treated with 32.5% glycerol at room
temperature for 15 to 45 minutes had acceptable post-thaw viability. Tissues treated
with 50% glycerol at room ature for 15 minutes also had acceptable viability;
however, tissues treated with 50% glycerol at room temperature for 45 minutes had
unacceptable viability.
Example 3
This study was performed to evaluate the performance of product
packaging plasticware, which is a tissue container assembly of the present disclosure,
for use as packaging for cryopreserved StrataGraft® tissues. The study evaluated three
independent lots of rectangular, 100 cm2 StrataGraft® tissues comparing tissues
packaged in the ell® growth chamber and those packaged in the tissue
ners described herein. For each batch, post-thaw ties of s packaged
in the tissue containers of the instant invention were evaluated following ent hold
conditions and compared to those of control tissues using current ing and
thaw/hold procedures. The results of this study demonstrated that tissue containers of
the instant invention are suitable for use in transporting and thawing cryopreserved
StrataGraft® tissues and that acceptable thawing can be achieved in the sterile field
without use of a Telfa® pad.
StrataGraft® skin tissues are produced in batches of 100 cm2
Graft® skin tissues. This larger tissue format and increase in batch sizes put an
added emphasis on efficient storage and shipment of the skin tissues. To address that
issue, plasticware tissue containers were designed which reduce the volume of the final
packaged product by 60% compared to packaging in the Transwell® growth chamber
as disclosed in copending U.S. Pat. Publ. 20140271583. In this example, this packaging
is introduced into the process following cryoprotectant treatment, immediately before the
product is sealed in the foil pouch and transferred to an old r for long-term
storage. The tissue containers of the instant invention were designed with a 0.75 mm
deep reservoir below tissue that can be flooded with hold solution. This design allows
the packaging to be used as a haw hold container, which simplifies the
preparation of StrataGraft® tissue for clinical use by eliminating the need for a separate
hold basin.
This experiment evaluated the post-thaw properties of StrataGraft®
skin tissues from three batches, and frozen in either a Transwell® growth chamber or in
the tissue containers of the instant invention. In addition, this study evaluated post-thaw
hold procedures performed in the tissues containers of the instant invention without the
use of Telfa® pads, compared to l hold conditions performed in basins ning
Telfa® pads.
Pre-Freeze Thaw Hold Hold
Group Packaging Hold Solution
Treatment Condition Chamber ion
Transwell®
1 Growth DeRoyal 250 mL Hold
Chamber Basin Solution
37.5% (2-Telfa) Warmed to 35-39 °C
glycerol 10 min at 15-20 min
Tissue RT at RT
min at RT Tissue
container container 15 mL Hold Solution
3 assembly assembly (no Warmed to 35-39 °C
Telfa®)
Batches of 20 rectangular, 100 cm2 StrataGraft® skin tissues were
produced using Stratatech's standard processes. y, NIKS® cells and normal
human dermal fibroblasts (NHDF) were expanded in monolayer culture. NHDF were
thawed and expanded in monolayer. Following expansion, the NHDF cells were
harvested and mixed into a type I collagen on, dispensed to 100 cm2 gular
trays of the present disclosure (tissue-culture treated rbonate ne, nominal
thickness of about 10 microns, nominal pore size of about 0.4 microns), and gelled to
create the dermal equivalent layer (DE). After gelling, the DE was submerged in media
in a growth r and cultured for five days prior to the NIKS® seed. NIKS® were
thawed, expanded, and then harvested and seeded onto DE surfaces. Tissues were
maintained in submerged culture for two days to allow for attachment and eration
of NIKS® over the DE surface and then cultured at the air-liquid interface for 18 days to
enable complete epidermal differentiation. Transfers of media, NHDF/collagen mixture,
and NIKS® sion to the trays and Transwell® growth chambers were performed
using peristaltic pumps.
At the end of the production process, culture media was aspirated
and tissues were treated in the Transwell® growth chamber with 50 mL of
cryopreservation solution containing 37.5% ol for 20 minutes at room temperature
(RT) whilst still supported on the membrane of the tray. At the end of treatment, the
trays containing the nine s designated for this experiment were removed from the
excess cryopreservation solution and ed into one of two packaging
configurations: 1) three tissues were kept in the ell® growth r in the high
position and sealed inside of 7.875" x 12" foil pouches (Group 1); and 2) six tissues
were transferred to sterile tissue containers of the t invention and sealed inside
6.75" x 10.25" foil peel pouches (Group 2 and Group 3, n=3 per group). At the end of
packaging, all packaged tissues were transferred to an ultracold freezer and stored at -
70 to -90 °C until analysis.
Group 1 and Group 2 tissues were then thawed using previously
established procedures that utilized an absorbent medium (e.g., Telfa® pad). Group 3
tissues were thawed using a simplified hold procedure. Briefly, Group 3 cryopreserved
tissues were thawed at room temperature for 10 minutes in the tissue container in which
the tissue was frozen, the bottom tissue containers were then flooded with hold solution
(15 ml of HEPES-buffered e medium that had been warmed to 35-39C) and held
at room temperature for 15 to 20 minutes. Following the post-thaw hold, s from all
groups were transferred to new rectangular growth chambers ning SM01 and recultured
for 22 to 26 hours
Tissues were evaluated for appearance, barrier function, viability,
histology, and VEGF secretion in the conditioned media. In addition, whole tissue MTT
staining was performed to evaluate uniformity of the tissue viability. The results of
tissues frozen in the tissue ners of the t invention (groups 2 and 3) were
compared to those of the control group.
The results of this study demonstrate that use of the tissue
containers of the instant invention does not affect the properties of cryopreserved
StrataGraft® s. Tissues packaged in the two configurations and thawed/held using
the previously established procedures (Groups 1 and 2) had comparable appearance,
histology, viability, and barrier function, and VEGF secretion. The tissue containers of
the instant invention also showed promising results for use in a simplified hold
procedure. Tissues packaged and kept in tissue containers of the instant invention for
the post-thaw hold (Group 3) had r properties to both other groups. Tissue
appearance, histology, VEGF secretion, and barrier function were not significantly
different than control tissues (Group 1); ity showed a modest (-10%), but
statistically significant (p<0.05), reduction compared to controls, while still easily
exceeding the established lot release criterion. MTT staining patterns of tissues from all
groups were comparable, with qualitatively consistent staining across the tissue
surfaces. See Figures 5, 6 and 7.
All publications and patents mentioned in the above specification
are herein incorporated by reference. Various modifications and variations of the
described method and system of the invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention. Although the invention has
been described in connection with specific preferred embodiments, it should be
tood that the invention as claimed should not be unduly d to such specific
embodiments. Indeed, various modifications of the described modes for carrying out the
invention that are obvious to those skilled in tissue culture, molecular biology,
biochemistry, or related fields are intended to be within the scope of the following
claims.
Claims (29)
1. A kit comprising: a e tissue container system comprising: substantially identical top and bottom sterile tissue containers; a tray comprising a porous bottom surface; and an organotypic skin substitute supported on the porous bottom surface of the tray; a sterile package containing the sterile tissue container system; and a tissue compatible solution.
2. The kit of claim 1, wherein each of the top and bottom sterile tissue containers comprise: a ter wall comprising a flange extending therefrom; a substantially planar bottom surface, the perimeter wall surrounding the bottom surface defining a dish having a dish length and a dish width; at least one ridge projecting from the perimeter wall above the flange, the at least one ridge having a ridge length and a ridge width; and at least one space recessed below the flange on the perimeter wall, the recessed space having a recess length and a recess width.
3. The kit of claim 2, wherein the bottom surface has a perimeter defined by the perimeter wall and comprising a perimeter ledge extending around the perimeter to provide a reservoir defined by the perimeter ledge and the bottom surface.
4. The kit of claim 2, wherein the ter wall has a male end and a female end opposite the male end along the dish , wherein the at least one ridge is located at the male end of the perimeter wall and the at least one space is d at the female end of the perimeter wall.
5. The kit of claim 4, wherein the flange comprises one or more tabs extending from the male end of the perimeter wall.
6. The kit of claim 5, n the flange ses one or more tabs extending from the female end of the perimeter wall.
7. The kit of claim 4, wherein the ridge has a proximal end and the proximal end of the ridge has one or more indents therein.
8. The kit of claim 4, n when the top and bottom sterile tissue containers are assembled the flanges of the top and bottom containers contact one another.
9. The kit of claim 8, wherein the flange comprises one or more tabs extending from the male end of the perimeter wall and one or more tabs extending from the female end of the perimeter wall.
10. The kit of claim 9, wherein when the top and bottom sterile tissue containers are assembled, the one or more tabs extending from the male end of the perimeter wall of the bottom sterile tissue container do not overlap with the one or more tabs extending from the female end of the perimeter wall of the top sterile tissue container.
11. The kit of claim 4, wherein when the top sterile tissue ner is rotated and placed facing the bottom sterile tissue container, the at least one space on the female end of the bottom sterile tissue container releasably receives the at least one ridge on the male end of the top sterile tissue container, thereby forming an enclosed container.
12. The kit of claim 1, wherein the sterile tissue container is formed from a medical grade plastic.
13. The kit of claim 1, wherein the porous bottom surface of the tray is a porous membrane.
14. The kit of claim 13, n the porous membrane comprises a semi-permeable polymer film or a semi-permeable track-etched polymer film.
15. The kit of claim 14, wherein the porous membrane comprises polyethylene terephthalate, ter, rbonate, or combinations thereof.
16. The kit of claim 13, wherein the porous membrane is plasma treated to improve cell attachment.
17. The kit of claim 13, wherein the porous membrane has a thickness of about 5 microns to about 20 microns.
18. The kit of claim 13, wherein the porous membrane comprises pores with a pore size of about 0.1 micron to about 10 microns.
19. The kit of claim 13, wherein the porous membrane has a pore density of about 1×105 and about 1×108 pores per square centimeter.
20. The kit of claim 1, wherein the tray further comprises one or more tray tabs.
21. The kit of claim 1, wherein the typic skin substitute is cryopreserved.
22. The kit of claim 1, further comprising an absorbent medium.
23. The kit of claim 22, n the absorbent medium is selected from the group consisting of Telfa pads, cellulosic pads, gauze pads, and foam pads.
24. The kit of claim 1, wherein the tissue compatible solution is a buffered solution in the range of pH 7.0 to 7.4.
25. The kit of claim 24, wherein the tissue compatible solution comprises DMEM, Ham's F-10, Ham's F-12, DMEM/F-12, Medium 199, MEM, RPMI, HEPES, Tris, MOPS, Trizma buffer, or combinations thereof.
26. The kit of claim 1, wherein the sterile package comprises a heat sealed sterile
27. The kit of claim 26, n the sterile bag is a plastic or r bag.
28. The kit of claim 1, wherein the sterile e is sealed inside a secondary bag.
29. The kit of claim 28, wherein the secondary bag comprises plastic, foil, or Mylar. WO 40755 WO 40755 WO 40755 .0.”— WO 40755 .0.“— FEG. 5 >2x .J Y\E. a,(noLS“ is: {kmy}.it .( n...«k SKxx.m,A ‘fHaki i33 i;in i3 8asM
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62/451,379 | 2017-01-27 |
Publications (1)
Publication Number | Publication Date |
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NZ796375A true NZ796375A (en) | 2023-01-27 |
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