U.S. patent application number 13/030507 was filed with the patent office on 2011-09-01 for immunocompatible chorionic membrane products.
Invention is credited to Alla Danilkovitch, Timothy Jansen, Jin-Qiang Kuang, Jennifer Michelle Marconi, Samson Tom, Dana Yoo.
Application Number | 20110212158 13/030507 |
Document ID | / |
Family ID | 44476694 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212158 |
Kind Code |
A1 |
Tom; Samson ; et
al. |
September 1, 2011 |
IMMUNOCOMPATIBLE CHORIONIC MEMBRANE PRODUCTS
Abstract
Provided herein is a placental product comprising an
immunocompatible chorionic membrane. Such placental products can be
cryopreserved and contain viable therapeutic cells after thawing.
The placental product of the present invention is useful in
treating a patient with a tissue injury (e.g. wound or burn) by
applying the placental product to the injury. Similar application
is useful with ligament and tendon repair and for engraftment
procedures such as bone engraftment.
Inventors: |
Tom; Samson; (Baltimore,
MD) ; Danilkovitch; Alla; (Columbia, MD) ;
Yoo; Dana; (Sykesville, MD) ; Jansen; Timothy;
(Baltimore, MD) ; Kuang; Jin-Qiang; (Woodstock,
MD) ; Marconi; Jennifer Michelle; (Glen Burnie,
MD) |
Family ID: |
44476694 |
Appl. No.: |
13/030507 |
Filed: |
February 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61338464 |
Feb 18, 2010 |
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61338489 |
Feb 18, 2010 |
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61369562 |
Jul 30, 2010 |
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Current U.S.
Class: |
424/443 ;
424/400; 424/93.7 |
Current CPC
Class: |
A61K 38/57 20130101;
A61P 43/00 20180101; A61P 17/00 20180101; C12N 2502/025 20130101;
C12N 2500/02 20130101; C12N 2501/115 20130101; A61K 38/1841
20130101; A61K 38/39 20130101; A61P 17/02 20180101; A61K 35/50
20130101; A01N 1/0221 20130101; A61K 38/1825 20130101; A61K 35/28
20130101; C12N 5/0605 20130101 |
Class at
Publication: |
424/443 ;
424/93.7; 424/400 |
International
Class: |
A61K 9/70 20060101
A61K009/70; A61K 35/50 20060101 A61K035/50; A61K 9/00 20060101
A61K009/00; A61P 43/00 20060101 A61P043/00; A61P 17/02 20060101
A61P017/02 |
Claims
1-121. (canceled)
122. A placental product comprising a cryopreservation medium and a
chorionic membrane, wherein: a. the chorionic membrane comprises
viable therapeutic cells and is substantially free of trophoblasts,
CD14+ macrophages, or both trophoblasts and CD14+ macrophage; and
b. the cryopreservation medium comprises a cryopreserving amount of
a cryopreservative; and c. optionally, the chorionic membrane is
substantially free of vascularized tissue or vascularized
tissue-derived immunogenic cells.
123. The placental product of claim 122, wherein the viable
therapeutic cells are native to the chorionic membrane and comprise
MSCs, fibroblasts; stromal cells, or a combination thereof.
124. The placental product of claim 122, wherein the
cryopreservative comprises a cell-permeating cryopreservative, a
non-cell-permeating cryopreservative, or a combination thereof,
optionally wherein the cell-permeating cryopreservative comprises
DMSO in amount of about 2% to about 20%
125. The placental product of claim 122, wherein the viable
therapeutic cells comprise stromal cells in an amount of about
5,000 to about 50,000 per cm.sup.2 of the chorionic membrane.
126. The placental product of any claims 122, wherein the chorionic
membrane comprises fibroblasts in an amount of about 50% to about
90% of the total cells, wherein at least about 40% of the
fibroblasts are viable after one freeze-thaw cycle, and wherein the
fibroblasts are in an amount of at least about 7,000
cells/cm.sup.2
127. The placental product of claim 122, wherein the chorionic
membrane comprises CD14+ macrophages in an amount of less than
about 5%.
128. The placental product of claim 122, wherein the chorionic
membrane comprises functional macrophages in an amount of less than
about 3,000 cells/cm.sup.2.
129. The placental product of claim 122, wherein the chorionic
membrane is substantially free of maternal dendritic cells,
maternal leukocytes, or immunogenic maternal cells.
130. The placental product of claim 122, comprising about 5,000 to
about 50,000 stromal cells per cm.sup.2, wherein the viability of
said stromal cells after one freeze-thaw cycle is about 70% to
about 95% relative to fresh placental or chorionic membrane.
131. The placental product of claim 122, wherein after one
freeze-thaw cycle, the placental product or chorionic membrane
releases TNF-.alpha. in an amount up to about 420 pg/mL into a
tissue culture medium upon placing a 2 cm.times.2 cm piece of the
placental product in a tissue culture medium and exposing the piece
to a bacterial lipopolysaccharide for about 20 to about 24
hours.
132. The placental product of claim 122, wherein the chorionic
membrane further comprises one or more native factors selected from
the group consisting of IGFBP1, adiponectin,
.alpha.2-macroglobulin, bFGF, EGF, MMP-9 and TIMP1, wherein the one
or more native factors are present in an amount, wherein the amount
is substantially greater per gram of chorionic membrane than that
of a fresh unprocessed chorionic membrane.
133. The placental product of claim 122, further comprising an
amniotic membrane wherein the amniotic membrane comprises a layer
of amniotic epithelial cells.
134. The placental product of claim 122, wherein the chorionic
membrane comprises a basement membrane, a reticular layer, or a
combination thereof.
135. The placental product of claim 122, wherein the chorionic
membrane has a thickness of about 40 .mu.m to about 400 .mu.m.
136. The placental product of claim 122, wherein the maternal side
of the chorionic membrane comprises fragments of extracellular
matrix proteins in a concentration substantially greater than that
of a native, unprocessed chorion.
137. The placental product of claim 122, wherein the chorionic
membrane has been treated with a protease, optionally wherein the
protease is a dispase.
138. The placental product of claim 122, wherein the chorionic
membrane comprises a basement membrane, a reticular layer, or a
combination thereof.
139. The placental product of claim 122, wherein the chorionic
membrane comprises MSCs, wherein: a. the MSCs are present in an
amount of about 5% to about 30% relative to the total number of
cells in the chorionic membrane; b. at least about 40% of the MSCs
are viable after one freeze-thaw cycle; and c. the MSCs are present
in an amount of at least about 1,500 cells/cm.sup.2.
140. The method of claim 139, wherein: a. the viable therapeutic
cells are native to the chorionic membrane and comprise MSCs; b.
the cryopreservative comprises DMSO in amount of about 2% to about
20%; c. the viable therapeutic cells comprise stromal cells in an
amount of about 5,000 to about 50,000 per cm.sup.2 of the chorionic
membrane, wherein the viability of said stromal cells after one
freeze-thaw cycle is about 70% to about 95% relative to fresh
placental or chorionic membrane; d. the viable therapeutic cells
comprise fibroblasts in an amount of about 50% to about 90% of the
total cells, wherein the viability of the fibroblasts is at least
about 40% after one freeze-thaw cycle, and wherein the fibroblasts
are in an amount of at least about 7,000 cells/cm.sup.2; e. the
chorionic membrane comprises CD14+ macrophages in an amount of less
than about 5% of the total viable cells and less than about 3,000
cells/cm.sup.2; f. the chorionic membrane is substantially free of
maternal dendritic cells, maternal leukocytes, and immunogenic
maternal cells; g. after one freeze-thaw cycle, the placental
product or chorionic membrane release TNF-.alpha. up to about 420
pg/mL into a tissue culture medium upon placing a 2 cm.times.2 cm
piece of the placental product in the tissue culture medium and
exposing the piece to a bacterial lipopolysaccharide for about 20
to about 24 hours; h. the chorionic membrane further comprises one
or more native factors selected from the group consisting of
IGFBP1, adiponectin, .alpha.2-macroglobulin, bFGF, EGF, MMP-9 and
TIMP1; and i. the chorionic membrane has a thickness of about 40
.mu.m to about 400 .mu.m.
141. A method of treating a wound of a patient comprising
administering the placental product of claim 122 to the wound.
Description
RELATED APPLICATIONS
[0001] This application claims priority to:
[0002] U.S. Provisional Application Ser. No. 61/338,464 entitled
"Selectively Immunodepleted Chorionic Membranes", filed on Feb. 18,
2010 bearing Docket No. 22924US01,
[0003] U.S. Provisional Application Ser. No. 61/338,489 entitled
"Selectively Immunodepleted Amniotic Membranes", filed on Feb. 18,
2010 bearing Docket No. 22925US01, and
[0004] U.S. Provisional Application Ser. No. 61/369,562 entitled
"Therapeutic Products Comprising Vitalized Placental Dispersions
filed on Jul. 30, 2010 bearing Docket No 23498US01, the contents of
which are hereby incorporated by reference in their entireties.
[0005] This application is being co-filed on Feb. 18, 2011 with,
and incorporates by reference, applications entitled:
[0006] "Methods of Manufacture of Immunocompatible Chorionic
Membrane Products",
[0007] "Immunocompatible Amniotic Membrane Products",
[0008] "Methods of Manufacture of Immunocompatible Amniotic
Membrane Products",
[0009] "Therapeutic Products Comprising Vitalized Placental
Dispersions", and
[0010] "Methods of Manufacture of Therapeutic Products Comprising
Vitalized Placental Dispersions"
FIELD OF THE INVENTION
[0011] The present technology relates to products to facilitate
wound healing such as placenta membrane-derived products and
biologic skin substitutes. The present technology relates to
products to protect injured or damaged tissue, or as a covering to
prevent adhesions, to exclude bacteria, to inhibit bacterial
activity, or to promote healing or growth of tissue. The field also
relates to methods of manufacturing and methods of use of such
membrane-derived products.
BACKGROUND OF THE INVENTION
[0012] Fresh or decellularized placental membranes have been used
topically in surgical applications since at least 1910 when Johns
Hopkins Hospital reported the use of placental membrane for dermal
applications. Subsequently unseparated amnion and chorion were used
as skin substitutes to treat burned or ulcerated surfaces. During
the 1950's and 60's Troensegaard-Hansen applied boiled amniotic
membranes to chronic leg ulcers.
[0013] The human chorionic membrane (CM) is one of the membranes
that exists during pregnancy between the developing fetus and
mother. It is formed by extraembryonic mesoderm and the two layers
of trophoblast and surrounds the embryo and other membranes. The
chorionic villi emerge from the chorion, invade the endometrium,
and allow transfer of nutrients from maternal blood to fetal
blood.
[0014] Both fresh and frozen CMs have been used for wound healing
therapy. When fresh CM is used, there is increased risk of disease
transmission. According to published reports, fresh placental
tissue, for example, chorionic tissue exhibits cell viability of
100%, however within 28 days of storage above 0.degree. C.
diminished cell viability to 15 to 35%. Freezing over a time of 3
weeks reduced cell viability to 13 to 18%, regardless of the
temperature or medium. As the CM is believed to be immunogenic, it
has not been used in commercial wound healing products.
[0015] Two placental tissue graft products containing living cells,
Apligraf and Dermagraft, are currently commercially available. Both
Apligraf and Dermagraft comprise ex vivo cultured cells. Neither
Apligraf nor Dermagraft comprise stem cells. Furthermore, neither
Apligraf nor Dermagraft comprise Insulin-like Growth Factor Binding
Protein-1 (IGFBP-1) and adiponectin, which are key factors in the
natural wound healing process. In addition, neither Apligraf nor
Dermagraft exhibit a protease-to-protease inhibitor ratio favorable
for wound healing. As wound healing is a multi-factorial biological
process, many factors are needed to properly treat a wound;
products having non-native cellular populations are less capable of
healing wounds relative to a product having an optimal population
of cells representing the native array. It would represent an
advance in the art to provide a chorion-derived biologic skin
substitute comprising a population of cells representing the native
array of factors, including, for example, growth factors and
cytokines.
[0016] Apligraf is a living, bi-layered skin substitute
manufactured using neonatal foreskin keratinocytes and fibroblasts
with bovine Type I collagen. As used in this application, Apligraf
refers to the product available for commercial sale in November
2009.
[0017] Dermagraft is cryopreserved human fibroblasts derived from
newborn foreskin tissue seeded on extracellular matrix. According
to its product literature, Dermagraft requires three washing steps
before use which limits the practical implementation of Dermagraft
as a skin substitute relative to products that require less than
three washing steps. As used in this application, Dermagraft refers
to the product available for commercial sale in November 2009.
[0018] Engineered skin substitutes such as Apligraf and Dermagraft
do not provide the best potential for wound healing because they
comprise sub-optimal cellular compositions and therefore do not
provide proper wound healing. For example, neither Apligraf nor
Dermagraft comprises stem cells and, as a result, the ratio between
different factors secreted by cells does not enable efficient wound
healing. Additionally, some factors that are important for wound
healing, including EGF, IGFBP1, and adiponectin are absent from
both Apligraf and Dermagraft. Additionally, some factors, including
MMPs and TIMPs, are present in proportions that differ greatly from
the proportions found in the natural wound healing process; this
difference significantly alters, among other things, upstream
inflammatory cytokine pathways which in turn allows for sub-optimal
micro-environments at the wound site. The present inventors have
identified a need for the development of chorionic membrane
products that more closely resemble natural tissue.
[0019] Paquet-Fifield et al. report that mesenchymal stem cells and
fibroblasts are important for wound healing (J Clin Invest, 2009,
119: 2795). No product has yet been described that comprise
mesenchymal stem cells and fibroblasts.
[0020] Both MMPs and TIMPs are among the factors that are important
for wound healing. However, expression of these proteins must be
highly regulated and coordinated. Excess of MMPs versus TIMPs is a
marker of poor chronic wound healing (Liu et al, Diabetes Care,
2009, 32: 117; Mwaura et al, Eur J Vasc Endovasc Surg, 2006, 31:
306; Trengove et al, Wound Rep Reg, 1999, 7: 442; Vaalamo et al,
Hum Pathol, 1999, 30: 795).
[0021] .alpha.2-macroglobulin is known as a plasma protein that
inactivates proteinases from all 4 mechanistic classes: serine
proteinases, cysteine proteinases, aspartic proteinases, and
metalloproteinases (Borth et al., FASEB J, 1992, 6: 3345; Baker et
al., J Cell Sci, 2002, 115:3719). Another important function of
this protein is to serve as a reservoir for cytokines and growth
factors, examples of which include TGF, PDGF, and FGF (Asplin et
al, Blood, 2001, 97: 3450; Huang et al, J Biol Chem, 1988; 263:
1535). In chronic wounds like diabetic ulcers or venous ulcers, the
presence of high amount of proteases leads to rapid degradation of
growth factors and delays in wound healing. Thus, a placental
membrane skin substitute comprising .alpha.2-macroglobulin would
constitute an advance in the art.
[0022] bFGF modulates a variety of cellular processes including
angiogenesis, tissue repair, and wound healing (Presta et al.,
2005, Reuss et al., 2003, and Su et al., 2008). In wound healing
models, bFGF has been shown to increase wound closure and enhance
vessel formation at the site of the wound (Greenhalgh et al.,
1990).
[0023] An in vitro cell migration assay is important for assessing
the wound healing potential of a skin substitute. The process of
wound healing is highly complex and involves a series of structured
events controlled by growth factors (Goldman, Adv Skin Wound Care,
2004, 1:24). These events include increased vascularization,
infiltration by inflammatory immune cells, and increases in cell
proliferation. The beginning stages of wound healing revolve around
the ability of individual cells to polarize towards the wound and
migrate into the wounded area in order to close the wound area and
rebuild the surrounding tissue. Keratinocytes are the primary cell
type of the epithelial layer. Upon proper stimulation, they are
implicated in the wound healing process (Pastar et al, 2008 and
Bannasch et al., 2000). Specifically, they proliferate and migrate
into the wound area to promote healing. An assay capable of
evaluating the wound healing potential of skin substitutes by
examining the correlation between cell migration and wound healing
would represent an advance in the art.
SUMMARY OF THE INVENTION
[0024] The present invention provides a pharmaceutically acceptable
placental product.
[0025] A placental product according to the present invention
comprises an immunocompatible chorionic membrane in a
cryopreservation medium (optionally cryopreserved) and viable
native therapeutic cells and native therapeutic factors.
[0026] In some embodiments, the placental product further comprises
an amniotic membrane that is selectively devitalized.
[0027] There is now provided a placental product that is
selectively depleted of substantially all immunogenic cells.
[0028] There is now provided a placental product that does not
contain ex vivo cultured cells.
[0029] There is now provided a placental product that comprises at
least one of Epidermal Growth Factor, IGFBP1, and Adiponectin.
[0030] Optionally, the therapeutic factors include one or more of
IGFBP1, adiponectin, .alpha.2-macroglobulin, bFGF, and EGF.
Optionally, the therapeutic factors include MMP-9 and TIMP1,
wherein the ratio of MMP-9:TIMP1 is from about 7 to about 10.
Optionally, the therapeutic factors include IGFBP1, adiponectin,
.alpha.2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally,
the therapeutic factors include IGFBP1, adiponectin,
.alpha.2-macroglobulin, bFGF, MMP-9, and TIMP1, wherein the ratio
of MMP-9:TIMP1 is from about 7 to about 10. Optionally, the
therapeutic factor is present in a substantial amount in comparison
to the equivalent unprocessed human placental membrane. Optionally,
each placental product embodiment optionally is devoid of ex-vivo
expanded cultured cells.
[0031] The present invention also provides a method of
manufacturing a placental product comprising: obtaining a placenta,
wherein the placenta comprises a chorionic membrane, selectively
depleting the placenta of immunogenicity, and cryopreserving the
placenta, thereby providing a placental product. According to the
present invention, the selective depletion step comprises removing
immunogenic cells (e.g. CD14+ macrophages and/or trophoblasts)
and/or immunogenic factors (e.g. TNF.alpha.). Optionally, the
selective depletion step comprises selectively immunodepleting the
placenta, whereby the placental product is purified from
immunogenic cells and/or immunogenic factors. Optionally, the
selective depletion step comprises removing a layer of
trophoblasts, for example, by treatment with a digestive enzyme
and/or mechanical removal. Optionally, the selective depletion step
comprises removing CD14+ macrophages by a cryoprocess wherein the
placental product is incubated for a period of time (e.g. about
30-60 mins.) at a temperature above freezing (e.g. at 2-8.degree.
C.), and then freezing, whereby CD14+ macrophages are selectively
killed relative to therapeutic cells.
[0032] The present invention also provides a method of screening a
placental product for therapy comprising assaying the placental
product for immunogenicity and/or therapeutic value. Optionally,
the step of assaying the placental product for immunogenicity
comprises a Mixed Lymphocyte Reaction (MLR) and/or
Lipopolysaccharide (LPS)-induced Tumor Necrosis Factor
(TNF)-.alpha. secretion. Optionally, the step of assaying the
placental product for therapeutic value comprises assaying the
placental product for cell migration induction.
[0033] The present invention also provides a method of treating a
subject comprising administering a placental product to the
subject. Optionally, the step of administering comprises applying
the placental product to a wound, for example, topically applying
the placental product to a skin wound. In one embodiment, a
placental product is used in a tendon or ligament surgery to
promote healing of a tendon or ligament.
[0034] The present inventors have identified a need for the
development of chorionic membrane products comprising at least one
of IGFBP1, and adiponectin, providing superior wound healing
properties.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 depicts freezing rates of various freezing
methods.
[0036] FIG. 2 depicts process cell recovery as a function of cryo
volume.
[0037] FIG. 3 depicts process cell recovery as a function of
refrigeration time.
[0038] FIG. 4 shows representative images of the live/dead staining
of the epithelial layer of fresh amniotic membrane.
[0039] FIG. 5 depicts IL-2sR concentrations of various
manufacturing intermediates.
[0040] FIG. 6 depicts IL-2sR concentrations of various
manufacturing intermediates.
[0041] FIG. 7 depicts TNF a concentrations from LPS-induced
secretion by placental tissues.
[0042] FIG. 8 shows representative images of the live/dead staining
of the epithelial layer of fresh amniotic membrane.
[0043] FIG. 9 depicts a correlation between IL-2sR release and the
number of CD45+ cells.
[0044] FIG. 10 depicts a correlation between the amount of CD45+
cells present in amnion-derived cell suspensions and immunogenicity
in MLR in vitro.
[0045] FIG. 11 depicts expression of EGF (A), IGFBP1 (B), and
Adiponectin (C) in amniotic and/or chorionic membranes.
[0046] FIG. 12 depicts expression of IFN-2.alpha. and TGF-.beta.3
in amniotic membrane homogenates.
[0047] FIG. 13 depicts expression of BMP-2, BMP-4, PLAB, PIGF (A),
and IGF-1 (B) in amniotic membrane homogenates.
[0048] FIG. 14 depicts the ratio of MMPs to TIMPs in various
membrane products.
[0049] FIG. 15 depicts bFGF levels in amniotic and chorionic
membranes (CM).
[0050] FIG. 16 depicts representative expression of bFGF in
chorionic tissue samples derived from two separate placenta
donors.
[0051] FIG. 17 depicts a schematic of the cell migration assay.
[0052] FIG. 18 depicts the results of cell migration assay of
various membrane preparations.
[0053] FIG. 19 depicts growth factor and adiponectin expression in
protein extracts of various membrane preparations.
DETAILED DESCRIPTION OF THE INVENTION
[0054] As used herein, the following definitions apply:
[0055] "Examplary" (or "e.g." or "by example") means a non-limiting
example.
[0056] "hCMSCs: means human chorionic membrane stromal cells.
hCMSCs are generally positive for CD73, CD70, CD90, CD105, and
CD166; negative for CD45 and CD34. hCMSCs differentiate to
mesodermal lineages (osteogenic, chondrogenic, and adipogenic).
[0057] "Selective depletion of immunogenicity" or "selective
depletion of immunogenic cells or factors" or "selective depletion"
means a placental product that retains live therapeutic cells
and/or retains therapeutic efficacy for the treatment of tissue
injury yet is free, substantially free, or depleted of at least one
of immune cell type (e.g. CD14+ macrophages, trophoblasts, and/or
vascular-tissue derived cells) and/or immunogenic factor that are
otherwise present in a native placenta or chorionic membrane.
[0058] "MSC" means mesenchymal stem cells and include fetal,
neonatal, adult, or post-natal. "MSCs" include chorionic MSCs
(CMSCs). MSCs generally express one or more of CD73, CD90, CD105,
and CD166.
[0059] "Native cells" means cells that are native, resident, or
endogenous to the placental membrane, i.e. cells that are not
exogenously added to the placental membrane.
[0060] "Native factors" means placental membrane factors that are
native, resident, or endogenous to the placental membrane, i.e.
factors that are not exogenously added to the placental
membrane.
[0061] "Placental products" means the instant placental products
disclosed herein.
[0062] "Substantially free" means present in only a negligible
amount or not present at all. For example, when a cell is abundant
at least than about 20% or less than about 10% or less than about
1% of the amount in an unprocessed sample.
[0063] "Substantial amount" of an element of the present invention,
e.g. native factors, therapeutic factors, or selective depletion,
means a value at least about 2% or at least 10% in comparison to an
unprocessed, not cryopreserved, fresh membrane sample. A
substantial amount can optionally be at least about 50%.
[0064] "Therapeutic cells" or "beneficial cells" means stromal
cells, MSCs, and/or fibroblasts.
[0065] "Therapeutic factors" means placenta- or chorionic
membrane-derived factors that promote wound healing. Examples
include IGFBP1, adiponectin, .alpha.2-macroglobulin, and/or bFGF.
Other examples include MMP-9 and TIMP1.
[0066] "Stromal cells" refers to a mixed population of cells
present (optionally in native proportions) composed of neonatal
mesenchymal stem cells and neonatal fibroblasts. Both neonatal
mesenchymal stem cells and neonatal fibroblasts are
immunoprivileged; neither express surface proteins present on
immunogenic cell types.
[0067] In some embodiments, the present technology discloses
placental products for clinical use, including use in wound healing
such as diabetic foot ulcers, venous leg ulcers, and burns. The
manufacturing process optionally eliminates essentially all
potentially immunogenic cells from the placental membrane while
preserving of specific cells that play an important role in wound
healing.
[0068] In some embodiments, the present technology discloses a
placental product that is selectively devitalized. There is now
provided a placental product that is selectively depleted of
substantially all immunogenic cells. There is now provided a
placental product that does not contain ex vivo cultured cells.
There is now provided a placental product that comprises at least
one of IGFBP1, and adiponectin. There is now provided a placental
product that comprises IGFBP1. There is now provided a placental
product that comprises adiponectin.
[0069] In some embodiments, the present technology discloses a
method of cyropreserving a placental product that preserves the
viability of specific beneficial cells that are the primary source
of factors for the promotion of healing to the wound healing
process while selectively depleting immunogenic cells (e.g. killing
or rendering non-immunogenic) from the chorionic membranes.
[0070] In some embodiments, the present technology discloses a
bioassay to test immunogenicity of manufactured placental
products.
[0071] In some embodiments, the present technology discloses a
placental product exhibiting a ratio of MMP:TIMP comparable to that
exhibited in vivo. The present inventors have identified a need for
the development of placental products exhibiting a ratio of MMP-9
and TIMP1 of about 7-10 to one.
[0072] In some embodiments, the present technology discloses a
placental product that comprises .alpha.2-macroglobulin.
[0073] The present inventors have identified a need for the
development of placental products that comprise
.alpha.2-macroglobulin.
[0074] There is now provided a placental product that inactivates
substantially all serine proteinases, cysteine proteinases,
aspartic proteinases, and metalloproteinases present in the
chorionic membrane. There is now provided a placental product that
inactivates substantially all serine proteinases present in the
chorionic membrane. There is now provided a placental product that
inactivates substantially all cysteine proteinases present in the
chorionic membrane. There is now provided a placental product that
inactivates substantially all aspartic proteinases present in the
chorionic membrane. There is now provided a placental product that
inactivates substantially all metalloproteinases present in the
chorionic membrane.
[0075] In some embodiments, the present technology discloses a
placental product that comprises bFGF, optionally in a substantial
amount.
[0076] In some embodiments, the present technology discloses a
placental product exhibiting a protease-to-protease inhibitor ratio
favorable for wound healing, optionally in a substantial
amount.
[0077] In some embodiments, the present technology discloses a cell
migration assay capable of evaluating the wound-healing potential
of a placental product.
[0078] IGFBP1 and adiponectin are among the factors that are
important for wound healing. Evaluation of proteins derived from
placental products prepared according to the presently disclosed
technology reveal that bFGF is one of the major factors secreted in
substantial higher quantities by the chorionic membrane.
Additionally, the importance of EGF for wound healing together with
high levels of bFGF detected in the presently disclosed chorionic
membranes support selection of bFGF as a potency marker for
evaluation of membrane products manufactured for clinical use
pursuant to the present disclosure.
[0079] The present technology discloses a cryopreservation
procedure for a placental products that selectively depletes
immunogenic cells from the a chorionic membranes and preserves the
viability of other beneficial cells (including at least one of
mesenchymal stem cells, and fibroblasts in some embodiments and all
of mesenchymal stem cells and fibroblasts in some embodiments) that
are the primary source of factors for the promotion of healing.
During the development of cryopreservation methodology for
chorionic membranes, the inventors of the present application
evaluated key parameters of cryopreservation including volume of
cryopreservative solution, effect of tissue equilibration prior to
freezing, and cooling rates for a freezing procedures.
[0080] Placental products, their usefulness, and their
immunocompatability are surprisingly enhanced by depletion of
maternal trophoblast and selective elimination of CD14+ fetal
macrophages. Immunocompatability can be demonstrated by any means
commonly known by the skilled artisan, such demonstration can be
performed by the mixed Lymphocyte Reaction (MLR) and by
lipopolysaccharide (LPS)-induced Tumor Necrosis Factor
(TNF)-.alpha. secretion.
[0081] The instant placental products contain bFGF, optionally at a
substantial concentration.
[0082] The instant placental products optionally secrete factors
that stimulate cell migration and/or wound healing. The presence of
such factors can be demonstrated by any commonly recognized method.
Optionally, the factors are in a substantial amount.
[0083] For example, commercially available wound healing assays
exist (Cell Biolabs) and cell migration can be assessed by cell
line (HMVEC, Lonza Inc.). In one embodiment, conditioned medium
from the present placental products enhance cell migration.
[0084] The placental products disclosed herein are useful in
treating a number of wounds including: tendon repair, cartilage
repair (e.g. femoral condyle, tibial plateau), ACL replacement at
the tunnel/bone interface, dental tissue augmentation, fistulas
(e.g. Crohn's disease, G-tube, tracheoesophogeal), missing tissue
at adhesion barriers (e.g. nasal septum repair, vaginal wall
repair, abdominal wall repair, tumor resection), dermal wounds
(e.g. partial thickness burns, toxic epidermal necrolysis,
epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g. diabetic
ulcers (e.g. foot), venous leg ulcers), surgical wounds, hernia
repair, tendon repair, bladder repair, periosteum replacement,
keloids, organ lacerations, epithelial defects, and repair or
replacement of a tympanic membrane.
[0085] The placental products disclosed herein exhibit one or more
of the following properties beneficial to the wound healing
process: [0086] a. approximate number of cells per cm.sup.2 being
about 20,000 to about 200,000, [0087] b. thickness of about 40 to
about 400 .mu.m, [0088] c. a thin basement membrane, [0089] d. low
immunogenicity, [0090] e. cryopreserved/cryopreserveable, and
[0091] f. human Chorionic Membrane Stromal Cells (hCMSC).
[0092] The present inventors have now identified a need for the
development of placental products that do not contain ex vivo
cultured cells.
[0093] The present inventors have now identified a need for the
development of placental products comprising IGFBP1.
[0094] The present inventors have now identified a need for the
development of placental products comprising adiponectin.
[0095] The present inventors have now identified a need for the
development of placental products exhibiting a protease-to-protease
inhibitor ratio favorable for wound healing.
[0096] The present inventors have now identified a need for the
development of a method of cyropreserving placental products that
preserves the viability of specific cells that are other beneficial
cells that are the primary source of factors for the promotion of
healing to the wound healing process while selectively depleting
immunogenic cells from chorionic membranes.
[0097] The present inventors have now identified a need for the
development of a bioassay to test immunogenicity of manufactured
placental products.
[0098] The present inventors have now identified a need for the
development of placental products exhibiting a ratio of MMP to TIMP
comparable to that exhibited in vivo. The present inventors have
now identified a need for the development of placental products
exhibiting a ratio of MMP-9 and TIMP1 of about 7-10 to one.
[0099] The present inventors have now identified a need for the
development of placental products that comprise
.alpha.2-macroglobulin.
[0100] The present inventors have now identified a need for the
development of placental products that inactivate serine
proteinases, cysteine proteinases, aspartic proteinases, and
metalloproteinases. The present inventors have now identified a
need for the development of placental products that inactivate
serine proteinases. The present inventors have now identified a
need for the development of placental products that inactivate
cysteine proteinases. The present inventors have now identified a
need for the development of placental products that inactivate
aspartic proteinases. The present inventors have now identified a
need for the development of placental products that inactivate
metalloproteinases.
[0101] The present inventors have now identified a need for the
development of placental products that comprise bFGF.
[0102] The present inventors have now identified a need for the
development of a cell migration assay to evaluate the potential of
placental membrane products.
[0103] The present inventors have now identified a need for the
development of a placental product for wound healing that comprises
mesenchymal stem cells and fibroblasts.
[0104] Placental Product
[0105] Overview
[0106] One embodiment of the present invention provides a placental
product comprising a cryopreservation medium and a chorionic
membrane, wherein the chorionic membrane comprises viable
therapeutic native cells and native therapeutic factors, and
wherein the cryopreservation medium comprises a cryopreserving
amount of a cryopreservative. According to this embodiment, the
chorionic membrane is substantially free of at least one at least
one or 2 or 3 immunogenic cell types such as: trophoblasts, CD14+
macrophages, and vascularized tissue-derived cells.
[0107] In one embodiment, the chorionic membrane comprises one or
more layers which exhibit the architecture of the native chorionic
membrane (e.g. has not been homogenized or treated with
collagenase).
[0108] In one embodiment, the placental product is suitable for
dermal application to a wound.
[0109] With the teachings provided herein, the skilled artisan can
now produce the present placental products. The present disclosure
provides methods of manufacture that produce the technical features
of the present placental products. Accordingly, in one embodiment,
the placental product is manufactured by steps taught herein. The
present placental products are not limited to products manufactured
by the methods taught here. For example, products of the present
invention could be produced through methods that rely on screening
steps; e.g. steps to screen for preparations with the described
technical features and superior properties.
[0110] The present placental products comprises one or more of the
following technical features: [0111] a. the viable therapeutic
native cells are capable of differentiating into cells of more than
one lineage (e.g. osteogenic, adipogenic and/or chonodrogenic
lineages), [0112] b. the native therapeutic factors include IGFBP1,
optionally present in a substantial amount, [0113] c. the native
therapeutic factors include adiponectin, optionally present in a
substantial amount, [0114] d. the native therapeutic factors
include .alpha.2-macroglobulin, optionally present in a substantial
amount, [0115] e. the native therapeutic factors include bFGF,
optionally present in a substantial amount, [0116] f. the native
therapeutic factors include EGF, optionally present in a
substantial amount, [0117] g. the native therapeutic factors
include MMP-9 and TIMP1, optionally present in a substantial
amount, [0118] h. the native therapeutic factors include MMP-9 and
TIMP1 in a ratio of about 7 to about 10, [0119] i. the placental
product does not comprise ex-vivo cultured cells, [0120] j. the
cryopreservative medium is present in an amount of greater than
about 20 ml or greater than about 50 ml, [0121] k. the
cryopreservative comprises DMSO, [0122] l. cryopreservative
comprises DMSO in a majority amount, [0123] m. the cryopreservation
medium further comprises albumin, optionally wherein the albumin is
HSA, [0124] n. the cryopreservative comprises DMSO and albumin
(e.g. HSA), [0125] o. the chorionic membrane comprises about 5,000
to about 240,000 cells/cm2 or about 20,000 to about 60,000
cells/cm2, [0126] p. the chorionic membrane comprises 20,000 to
about 200,000 cells/cm2, with a cell viability of at least about
70%, [0127] q. comprises at least: about 7,400 or about 15,000 or
about 23,217, or about 35,000, or about 40,000 or about 47,800 of
stromal cells per cm2 of the chorionic membrane, [0128] r.
comprises about 5,000 to about 50,000 of stromal cells per cm2 of
the chorionic membrane, [0129] s. comprises about 4% to about 46%
of viable non-culturally expanded fibroblasts per cm2 of the
placental product, [0130] t. comprises stromal cells wherein at
least: about 40%, or about 50%, or about 60%, or about 70%, or
about 74.3%, or about 83.4 or about 90%, or about 92.5% of the
stromal cells are viable after a freeze-thaw cycle, [0131] u.
comprises stromal cells wherein about 40% to about 92.5% of the
stromal cells are viable after a freeze-thaw cycle, [0132] v. the
chorionic membrane has a thickness of about 40 .mu.m to about 400
.mu.m, [0133] w. secretes less than about any of: 420 pg/mL, 350
pg/mL, or 280 pg/mL TNF-.alpha. into a tissue culture medium upon
placing a 2 cm.times.2 cm piece of the tissue product in a tissue
culture medium and exposing the tissue product to a bacterial
lipopolysaccharide for about 20 to about 24 hours, [0134] x.
cryopreservation and thawing, secretes less than about any of: 420
pg/mL, 350 pg/mL, or 280 pg/mL TNF-.alpha. into a tissue culture
medium upon placing a 2 cm.times.2 cm piece of the tissue product
in a tissue culture medium and exposing the tissue product to a
bacterial lipopolysaccharide for about 20 to about 24 hours, [0135]
y. after refrigeration, cryopreservation and thawing, secretes less
than about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-.alpha.
into a tissue culture medium upon placing a 2 cm.times.2 cm piece
of the tissue product in a tissue culture medium and exposing the
tissue product to a bacterial lipopolysaccharide for about 20 to
about 24 hours, [0136] z. the maternal side of the chorionic
membrane comprises fragments of extracellular matrix proteins in a
concentration substantially greater than that of a native,
unprocessed chorion, optionally wherein the chorionic membrane has
been treated with Dispase II or wherein a substantial portion of
the protein fragments comprises terminal leucine or phenylalanine,
[0137] aa. further comprises an amniotic membrane, [0138] bb.
further comprises an amniotic membrane, wherein the amniotic
membrane comprises a layer of amniotic epithelial cells, [0139] cc.
further comprises an amniotic membrane, wherein the amniotic
membrane and the chorionic membrane are associated to one another
in the native configuration, [0140] dd. further comprises an
amniotic membrane, wherein the amniotic membrane and the chorionic
membrane are not attached to one another in the native
configuration, [0141] ee. further comprises an amniotic membrane
wherein the chorionic membrane comprises about 2 to about 4 times
more stromal cells relative to the amniotic membrane, [0142] ff.
does not comprise an amniotic membrane, [0143] gg. the chorionic
membrane comprises about 2 to about 4 times more stromal cells
relative to an amniotic membrane of the same area derived from the
same placenta, and [0144] hh. is suitable for dermal application to
a wound.
[0145] Cells
[0146] According to the present invention, a placental product
comprises native therapeutic cells of the chorionic membrane. The
cells comprise one or more of stromal cells, MSCs, and
fibroblasts.
[0147] In one embodiment, the native therapeutic cells comprise
viable stromal cells.
[0148] In one embodiment, the native therapeutic cells comprise
viable MSCs.
[0149] In one embodiment, the native therapeutic cells comprise
viable fibroblasts.
[0150] In one embodiment, the native therapeutic cells comprise
viable MSCs and viable fibroblasts.
[0151] In one embodiment, the native therapeutic cells comprise
viable MSCs and viable fibroblasts.
[0152] In one embodiment, the native therapeutic cells comprise
viable stromal cells and viable epithelial cells.
[0153] In one embodiment, the therapeutic native cells are viable
cells capable of differentiating into cells of more than one
lineage (e.g. osteogenic, adipogenic and/or chonodrogenic
lineages).
[0154] In one embodiment, the chorionic membrane comprises about
10,000 to about 360,000 cells/cm.sup.2 or about 40,000 to about
90,000 cells/cm.sup.2.
[0155] In one embodiment, the chorionic membrane comprises at
least: about 7,400 or about 15,000 or about 23,217, or about
35,000, or about 40,000 or about 47,800 of stromal cells per
cm.sup.2 of the chorionic membrane.
[0156] In one embodiment, the chorionic membrane comprises about
5,000 to about 50,000 of stromal cells per cm.sup.2 of the
chorionic membrane.
[0157] In one embodiment, the chorionic membrane comprises stromal
cells wherein at least: about 40%, or about 50%, or about 60%, or
about 70%, or about 74.3%, or about 83.4 or about 90%, or about
92.5% of the stromal cells are viable after a freeze-thaw
cycle.
[0158] In one embodiment, the chorionic membrane comprises stromal
cells wherein about 40% to about 92.5% of the stromal cells are
viable after a freeze-thaw cycle.
[0159] In one embodiment, the chorionic membrane (of the placental
product) comprises fibroblasts in about 50% to about 90% of the
total cells.
[0160] In one embodiment, the chorionic membrane comprises CD14+
macrophage in an amount of less than about 5% or less than about 1%
or less than about 0.5%, optionally as demonstrated by a
substantial decrease in LPS stimulation of TNF.alpha. release.
[0161] In one embodiment, the placental product comprises about 2
to about 4 times more stromal cells relative to an amniotic
membrane of the same area derived from the same placenta.
[0162] In one embodiment, the placental product further comprises
an amniotic membrane, wherein the placental product contains about
2 to about 4 times more stromal cells relative to the amniotic
membrane.
[0163] In one embodiment, the placental product further comprises
an amniotic membrane, wherein the amniotic membrane comprises a
layer of amniotic epithelial cells.
[0164] In one embodiment, the placental product is substantially
free of trophoblasts.
[0165] In one embodiment, the placental product is substantially
free of functional CD14+ macrophages.
[0166] In one embodiment, the placental product is substantially
free of vascularized tissue-derived cells.
[0167] In one embodiment, the placental product is substantially
free of trophoblasts, functional CD14+ macrophages, and
vascularized tissue-derived cells. Optionally, the placental
product comprises viable stromal cells. Optionally, the placental
product comprises viable MSCs. Optionally, the placental product
comprises viable fibroblasts. Optionally, the placental product
comprises viable MSCs and viable fibroblasts.
[0168] In one embodiment, the placental product is substantially
free of maternal decidual cells.
[0169] In one embodiment, the placental product is substantially
free of maternal leukocytes and/or trophoblast cells.
[0170] In one embodiment, the chorionic membrane (of the placental
product) comprises MSCs in an amount of about 5% to about 30%,
about 5% to about 25%, about 5% to about 20%, about 5% to about
15%, about 3% to about 12%, at least about 5%, at least about 10%,
or at least about 15%, relative to the total number of cells in the
chorionic membrane. Optionally, at least: about 40%, about 50%,
about 60%, or about 70% of the MSCs are viable after a freeze-thaw
cycle.
[0171] In one embodiment, the chorionic membrane (of the placental
product) comprises fibroblasts in an amount of about 50% to about
95%, about 60% to about 90%, or about 70% to about 85%, relative to
the total number of cells in the chorionic membrane. Optionally, at
least: about 40%, about 50%, about 60%, or about 70% of the
fibroblasts are viable after a freeze-thaw cycle.
[0172] In one embodiment, the chorionic membrane (of the placental
product) comprises functional macrophages in an amount of less than
about any of: 5%, 4%, 3%, 2%, 1%, or 0.1%.
[0173] In one embodiment, the chorionic membrane (of the placental
product) comprises MSCs and functional macrophages in a ratio of
greater than about any of: 3:1, 4:1, 5:1, 7:1, 10:1, 12:1, or
15:1.
[0174] In one embodiment, the chorionic membrane comprises
fibroblasts and functional macrophages in a ratio of greater than
about any of: 14:1, 15:1, 16:1, 17:1, 28:1, 30:1, 35:1, 45:1, or
50:1.
[0175] In one embodiment, the chorionic membrane (of the placental
product) comprises fibroblasts and MSCs in a ratio of: about 9:2 to
about 17:3.
[0176] In one embodiment, the chorionic membrane (of the placental
product) comprises MSCs in an amount of at least about 1,500
cells/cm.sup.2, at least about 3,000 cells/cm.sup.2, about 15,000
to about 9,000 cells/cm.sup.2, or about 3,000 to about 9,000
cells/cm.sup.2. Optionally, at least: about 40%, about 50%, about
60%, or about 70% of the MSCs are viable after a freeze-thaw
cycle.
[0177] In one embodiment, the chorionic membrane (of the placental
product) comprises fibroblasts in an amount of at least about 7,000
cells/cm.sup.2, at least about 14,000 cells/cm.sup.2, about 7,000
to about 51,000 cells/cm.sup.2, or about 14,000 to about 51,000
cells/cm.sup.2. Optionally, at least: about 40%, about 50%, about
60%, or about 70% of the fibroblasts are viable after a freeze-thaw
cycle.
[0178] In one embodiment, the chorionic membrane (of the placental
product) comprises functional macrophages in an amount of less than
about 3,000 cells/cm.sup.2, or less than about 1,000
cells/cm.sup.2.
[0179] In one embodiment, the placental product is substantially
free of ex-vivo cultured cells.
[0180] Placental Factors
[0181] According to the present invention, a placental product
comprises native therapeutic factors of the chorionic membrane.
[0182] In one embodiment, the factors include one or more of:
IGFBP1, adiponectin, .alpha.2-macroglobulin, bFGF, EGF, MMP-9, and
TIMP1. Optionally, the factors are present in amounts/cm.sup.2 that
are substantially similar to that of a native chorionic membrane or
layer thereof (e.g. .+-.10% or 20%).
[0183] In one embodiment, the factors include IGFBP1, adiponectin,
.alpha.2-macroglobulin, bFGF, EGF, MMP-9, and TIMP1. Optionally,
the factors are present in ratios that are substantially similar to
that of a native chorionic membrane or layer thereof. Optionally,
the factors are present in amounts/cm.sup.2 that are substantially
similar to that of a native chorionic membrane or layer thereof
(e.g. .+-.10% or 20%).
[0184] In one embodiment, the factors include MMP-9 and TIMP1 in a
ratio of about 7 to about 10 (e.g about 7). Optionally, the factors
are present in amounts/cm.sup.2 that are substantially similar to
that of a native chorionic membrane or layer thereof (e.g. .+-.10%
or 20%).
[0185] In one embodiment, the factors include one or more (e.g. a
majority or all) of the factors listed in Table 15. Optionally, the
factors are present in ratios that are substantially similar to
that of a native chorionic membrane or layer thereof. Optionally,
the factors are present in amounts/cm.sup.2 that are substantially
similar to that of a native chorionic membrane or layer thereof
(e.g. .+-.10% or 20%).
[0186] In one embodiment, the placental product secretes
substantially less TNF-.alpha./cm.sup.2 than a native, unprocessed
chorionic membrane.
[0187] In one embodiment, the placental product secretes
substantially less TNF-.alpha./cm.sup.2 than a native placental
product upon stimulation by LPS or CT.
[0188] In one embodiment, the placental product secretes less than
about any of: 420 pg/mL, 350 pg/mL, or 280 pg/mL TNF-.alpha. into a
tissue culture medium upon placing a 2 cm.times.2 cm piece of the
tissue product in a tissue culture medium and exposing the tissue
product to a bacterial lipopolysaccharide for about 20 to about 24
hours.
[0189] In one embodiment, after cryopreservation and thawing, the
placental product secretes less than about any of: 420 pg/mL, 350
pg/mL, or 280 pg/mL TNF-.alpha. into a tissue culture medium upon
placing a 2 cm.times.2 cm piece of the tissue product in a tissue
culture medium and exposing the tissue product to a bacterial
lipopolysaccharide for about 20 to about 24 hours.
[0190] In one embodiment, after refrigeration, cryopreservation and
thawing, the placental product secretes less than about any of: 420
pg/mL, 350 pg/mL, or 280 pg/mL TNF-.alpha. into a tissue culture
medium upon placing a 2 cm.times.2 cm piece of the tissue product
in a tissue culture medium and exposing the tissue product to a
bacterial lipopolysaccharide for about 20 to about 24 hours.
[0191] In one embodiment, the placental product further comprises
an exogenously added inhibitor of TNF-.alpha.. Optionally, the
inhibitor of TNF-.alpha. is IL-10.
[0192] In one embodiment, the product has been treated with an
antibiotic
[0193] Architecture
[0194] A placental product of the present invention comprises one
or more non-trophoblast layers which exhibit the architecture of
the native chorionic membrane. With the teachings provided herein,
the skilled artisan will recognize placental layers that exhibit
native architecture, for example, layers that have not been
homogenized or treated with collagenase or other enzyme that
substantially disrupts the layer.
[0195] In one embodiment, the placental product comprises a stromal
layer with native architecture.
[0196] In one embodiment, the placental product comprises a
basement membrane with native architecture.
[0197] In one embodiment, the placental product comprises a
reticular layer with native architecture.
[0198] In one embodiment, the placental product comprises a
reticular layer and a basement layer with native architecture.
[0199] In one embodiment, the placental product comprises a stromal
layer, a basement layer, and a reticular layer with native
architecture.
[0200] In one embodiment, the placental product is substantially
free of trophoblasts. In one embodiment, the placental product
comprises a basement membrane with native architecture and the
chorionic membrane is substantially free of trophoblasts.
Optionally, the maternal side of the placental product comprises
fragments of extracellular matrix proteins in a concentration
substantially greater than that of a native chorionic membrane.
Optionally, the placental product has been treated with Dispase
(e.g. Dispase II) and/or a substantial portion of the extracellular
matrix protein fragments comprises terminal leucine or
phenylalanine.
[0201] In one embodiment, the placental product has a thickness of
about 40 .mu.m to about 400 .mu.m.
[0202] In one embodiment, the placental product further comprises
an amniotic membrane. Optionally, the amniotic membrane and the
chorionic membrane in the placental product are associated to one
another in the native configuration. Alternatively, the amniotic
membrane and the chorionic membrane are not attached to one another
in the native configuration.
[0203] In one embodiment, the placental product does not comprise
an amniotic membrane.
[0204] Formulation
[0205] According to the present invention, the placental product
can be formulated with a cryopreservation medium.
[0206] In one embodiment, the cryopreservation medium comprising
one or more cell-permeating cryopreservatives, one or more non
cell-permeating cryopreservatives, or a combination thereof.
[0207] Optionally, the cryopreservation medium comprises one or
more cell-permeating cryopreservatives selected from DMSO, a
glycerol, a glycol, a propylene glycol, an ethylene glycol, or a
combination thereof.
[0208] Optionally, the cryopreservation medium comprises one or
more non cell-permeating cryopreservatives selected from
polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a
monosaccharides, a sugar alcohol, an alginate, a trehalose, a
raffinose, a dextran, or a combination thereof.
[0209] Other examples of useful cryopreservatives are described in
"Cryopreservation" (BioFiles Volume 5 Number 4--Sigma-Aldrich.RTM.
datasheet).
[0210] In one embodiment, the cryopreservation medium comprises a
cell-permeating cryopreservative, wherein the majority of the
cell-permeating cryopreservative is DMSO. Optionally, the
cryopreservation medium does not comprise a substantial amount of
glycerol.
[0211] In one embodiment, the cryopreservation medium comprises
DMSO. Optionally, the cryopreservation medium does not comprise
glycerol in a majority amount. Optionally, the cryopreservation
medium does not comprise a substantial amount of glycerol.
[0212] In one embodiment, the cryopreservation medium comprises
additional components such as albumin (e.g. HSA or BSA), an
electrolyte solution (e.g. Plasma-Lyte), or a combination
thereof.
[0213] In one embodiment, the cryopreservation medium comprises 1%
to about 15% albumin by weight and about 5% to about 20%
cryopreservative by volume (e.g. about 10%). Optionally, the
cryopreservative comprises DMSO (e.g. in a majority amount).
[0214] In one embodiment, the placental product is formulated in
greater than about 20 ml or greater than about 50 ml of
cryopreservation medium. Optionally, the cryopreservative comprises
DMSO (e.g. in a majority amount). Optionally, the cryopreservation
medium does not comprise a substantial amount of glycerol.
[0215] In one embodiment, the placental product is placed on
nitrocellulose paper.
[0216] In one embodiment, the placenta is cut into a plurality of
sections. Optionally, the sections are less than about 10
cm.times.10 cm. Optionally, the sections are between about 2
cm.times.2 cm and 5 cm.times.5 cm.
[0217] Manufacture
[0218] Overview
[0219] A placental product of the present invention can
manufactured from a placenta in any suitable manner that provides
the technical features taught herein. According to the present
invention, a placental product comprises at least an
immunocompatible chorionic membrane.
[0220] In one embodiment, a placental product is manufactured by a
method comprising:
[0221] a. obtaining a placenta,
[0222] b. selectively depleting the placenta of immunogenicity;
and
[0223] c. cryopreserving the placenta.
[0224] In one embodiment, a placental product is manufactured by a
method comprising:
[0225] a. obtaining a placenta;
[0226] b. removing a substantial portion of trophoblasts from the
placenta; and
[0227] c. cryopreserving the placenta.
[0228] Optionally, the method comprises a step of removing the
amniotic membrane or portion thereof. Can amniotic membrane') from
the placenta. Optionally, the method comprises a step of removing
an amniotic membrane from the placenta without removing a
substantial portion of amniotic epithelial cells from the
placenta.
[0229] Optionally, the step of removing a substantial portion of
trophoblasts from the placenta comprises treating the placenta with
a digestive enzyme such as a protease (e.g. dispase or dispase II),
mechanically removing trophoblasts from the placenta (e.g. by
scraping), or a combination thereof.
[0230] Optionally, the method comprises a step of removing
vascularized tissue from the placenta, for example, by lysing red
blood cells, by removing blood clots, or a combination thereof.
[0231] Optionally, the method comprises a step of treating the
placenta with one or more antibiotics.
[0232] Optionally, the method comprises a step of selective
depletion of CD14+ macrophages.
[0233] Optionally, the step of cryopreserving the placenta
comprises freezing the placenta in a cryopreservation medium which
comprises one or more cell-permeating cryopreservatives, one or
more non cell-permeating cryopreservatives, or a combination
thereof.
[0234] Optionally, the step of cryopreserving the placenta
comprises refrigerating for a period of time and then freezing,
thereby selectively depleting CD14+ macrophages.
[0235] An exemplary placental product of the present invention can
be manufactured or provided with a bandage or skin substitute.
[0236] Immunocompatability and Selective Depletion
[0237] In one embodiment, the invention the placental product is
immunocompatible. Immunocompatability can be accomplished by any
selective depletion step that removes immunogenic cells or factors
or immunogenicity from the placenta (or chorionic membrane
thereof).
[0238] In one embodiment, the placental product is made
immunocompatible by selectively depleting it of functional
immunogenic cells. A placenta can be made immunocompatible by
selectively removing immunogenic cells from the placenta (or
chorionic membrane thereof) relative to therapeutic cells. For
example, immunogenic cells can be removed by killing the
immunogenic cells or by purification of the placenta there
from.
[0239] In one embodiment, the placenta is made immunocompatible by
selectively depleting trophoblasts, for example, by removal of the
trophoblast layer.
[0240] In one embodiment, the placenta is made immunocompatible by
selective depletion of functional CD14+ macrophages, optionally
resulting in depletion of TNF.alpha. upon stimulation, or a
combination thereof.
[0241] In one embodiment, the placenta is made immunocompatible by
selective depletion of vascularized tissue-derived cells.
[0242] In one embodiment, the placenta is made immunocompatible by
selective depletion of functional CD14+ macrophages, trophoblasts,
and vascularized tissue-derived cells.
[0243] In one embodiment, the placenta product is made
immunocompatible by selective depletion of trophoblasts and/or
CD14+ macrophages, optionally resulting in depletion of TNF.alpha.
upon stimulation.
[0244] Trophoblast Removal
[0245] In one embodiment, trophoblasts are depleted or removed from
the placental product. Surprisingly, such a placental product has
one or more of the following superior features:
[0246] a. is substantially non-immunogenic;
[0247] b. provides remarkable healing time; and
[0248] c. provides enhanced therapeutic efficacy.
[0249] Trophoblasts can be removed in any suitable manner which
substantially diminishes the trophoblast content of the placental
product. Optionally, the trophoblasts are selectively removed or
otherwise removed without eliminating a substantial portion of one
or more therapeutic components from the placenta (e.g. MSCs,
placental factors, etc). Optionally, a majority (e.g. substantially
all) of the trophoblasts are removed.
[0250] One method of removing trophoblasts comprises treating the
placenta (e.g. chorion or amnio-chorion) with a digestive enzyme
such as dispase (e.g. dispase II) and separating the trophoblasts
from the placenta. Optionally, the step of separating comprises
mechanical separation such as peeling or scraping. Optionally,
scraping comprises scraping with a soft instrument such as a
finger.
[0251] One method of removing trophoblasts comprises treating the
chorionic membrane with dispase for about 30 to about 45 minutes
separating the trophoblasts from the placenta. Optionally, the
dispase is provided in a solution of about less than about 1% (e.g.
about 0.5%). Optionally, the step of separating comprises
mechanical separation such as peeling or scraping. Optionally,
scraping comprises scraping with a soft instrument such as a
finger.
[0252] Useful methods of removing trophoblasts from a placenta
(e.g. chorion) are described by Portmann-Lanz et al. ("Placental
mesenchymal stem cells as potential autologous graft for pre- and
perinatal neuroregeneration"; American Journal of Obstetrics and
Gynecology (2006) 194, 664-73), ("Isolation and characterization of
mesenchymal cells from human fetal membranes"; Journal Of Tissue
Engineering And Regenerative Medicine 2007; 1: 296-305.), and
(Concise Review: Isolation and Characterization of Cells from Human
Term Placenta: Outcome of the First International Workshop on
Placenta Derived Stem Cells").
[0253] In one embodiment, trophoblasts are removed before
cryopreservation.
[0254] Macrophage Removal
[0255] In one embodiment, functional macrophages are depleted or
removed from the placental product. Surprisingly, such a placental
product has one or more of the following superior features:
[0256] a. is substantially non-immunogenic;
[0257] b. provides remarkable healing time; and
[0258] c. provides enhanced therapeutic efficacy.
[0259] Functional macrophages can be removed in any suitable manner
which substantially diminishes the macrophage content of the
placental product. Optionally, the macrophages are selectively
removed or otherwise removed without eliminating a substantial
portion of one or more therapeutic components from the placenta
(e.g. MSCs, placental factors, etc). Optionally, a majority (e.g.
substantially all) of the macrophages are removed.
[0260] One method of removing immune cells such as macrophages
comprises killing the immune cells by rapid freezing rates such as
60-100.degree. C./min.
[0261] Although immune cells can be eliminated by rapid freezing
rates, such a method can also be detrimental to therapeutic cells
such as stromal cells (e.g. MSCs). The present inventors have
discovered a method of selectively killing CD14+ macrophages can be
selectively killed by refrigerating the placenta for a period of
time (e.g. for at least about 10 min such as for about 30-60 mins)
at a temperature above freezing (e.g. incubating at 2-8.degree. C.)
and then freezing the placenta (e.g. incubating at -80.degree.
C..+-.5.degree. C.). Optionally, the step of freezing comprises
freezing at a rate of less than 10.degree./min (e.g. less than
about 5.degree./min such as at about 1.degree./min).
[0262] In one embodiment, the step of refrigerating comprises
soaking the placenta in a cryopreservation medium (e.g. containing
DMSO) for a period of time sufficient to allow the cryopreservation
medium to penetrate (e.g. equilibrate with) the placental tissues.
Optionally, the step of freezing comprises reducing the temperature
at a rate of about 1.degree./min. Optionally, the step of freezing
comprises freezing at a rate of less than 10.degree./min (e.g. less
than about 5.degree./min such as at about 1.degree./min).
[0263] In one embodiment, the step of refrigerating comprises
soaking the placenta in a cryopreservation medium (e.g. containing
DMSO) at a temperature of about -10-15.degree. C. (e.g. at
2-8.degree. C.) for at least about any of: 10 min, 20 min, 30 min,
40 min, or 50 min. In another embodiment, the step of refrigerating
comprises soaking the placenta in a cryopreservation medium (e.g.
containing DMSO) at a temperature of about -10-15.degree. C. (e.g.
at 2-8.degree. C.) for about any of: 10-120, 20-90 min, or 30-60
min. Optionally, the step of freezing comprises freezing at a rate
of less than 10.degree./min (e.g. less than about 5.degree./min
such as at about 1.degree./min).
[0264] Removal of Vascularized Tissue-Derived Cells
[0265] In one embodiment, vascularized tissue-derived cells (or
vascularied tissue) are depleted or removed from the placental
product. Surprisingly, such a placental product has one or more of
the following superior features:
[0266] a. is substantially non-immunogenic;
[0267] b. provides remarkable healing time; and
[0268] c. provides enhanced therapeutic efficacy.
[0269] Vascularized tissue-derived cells can be removed in any
suitable manner which substantially diminishes such cell content of
the placental product. Optionally, the vascularized tissue-derived
cells are selectively removed or otherwise removed without
eliminating a substantial portion of one or more therapeutic
components from the placenta (e.g. MSCs, placental factors,
etc).
[0270] In one embodiment, removal of vascularized tissue-derived
cells comprises separating the chorion from the placenta by cutting
around the placental skirt on the side opposite of the umbilical
cord. The chorion on the umbilical side of the placenta is not
removed due to the vascularization on this side.
[0271] In one embodiment, removal of vascularized tissue-derived
cells comprises rinsing the chorionic membrane (e.g. with buffer
such as PBS) to remove gross blood clots and any excess blood
cells.
[0272] In one embodiment, removal of vascularized tissue-derived
cells comprises treating the chorionic membrane with an
anticoagulant (e.g. citrate dextrose solution).
[0273] In one embodiment, removal of vascularized tissue-derived
cells comprises separating the chorion from the placenta by cutting
around the placental skirt on the side opposite of the umbilical
cord and rinsing the chorionic membrane (e.g. with buffer such as
PBS) to remove gross blood clots and any excess blood cells.
[0274] In one embodiment, removal of vascularized tissue-derived
cells comprises separating the chorion from the placenta by cutting
around the placental skirt on the side opposite of the umbilical
cord and treating the chorionic membrane with an anticoagulant
(e.g. citrate dextrose solution).
[0275] In one embodiment, removal of vascularized tissue-derived
cells comprises separating the chorion from the placenta by cutting
around the placental skirt on the side opposite of the umbilical
cord, rinsing the chorionic membrane (e.g. with buffer such as PBS)
to remove gross blood clots and any excess blood cells, and
treating the chorionic membrane with an anticoagulant (e.g. citrate
dextrose solution).
[0276] Selective Depletion of Immunogenicity as Demonstrated by a
Substantial Decrease in LPS Stimulation of TNF.alpha. Release.
[0277] In one embodiment, the placental product is selectively
depleted of immunogenicity as demonstrated by a reduction in LPS
stimulated TNF-.alpha. release. depletion d of TNF-.alpha. depleted
or removed from the placental product.
[0278] In one embodiment, TNF-.alpha. is depleted by killing or
removal of macrophages.
[0279] In one embodiment, TNF-.alpha. is depleted by treatment with
an anti-TNF-.alpha. antibody.
[0280] In one embodiment, TNF-.alpha. is functionally depleted by
treatment with IL-10, which suppresses TNF-.alpha. secretion.
[0281] Preservation
[0282] A placental product of the present invention may be used
fresh or may be preserved for a period of time. Surprisingly,
cryopreservation results in immunocompatible placental
products.
[0283] In one embodiment, a placental product is cryopreserved. A
placental product may be cryopreserved by incubation at freezing
temperatures (e.g. a -80.degree. C..+-.5.degree. C.) in a
cryopreservative medium.
[0284] Cryopreservation can comprise, for example, incubating the
placental product at 4.degree. C. for 30-60 min, and then
incubating at -80.degree. C. until use. The placental product may
then be thawed for use. Optionally, the placental product is
cryopreserved in a manner such that cell viability is retained
surprisingly well after a freeze-thaw cycle.
[0285] In one embodiment, cryopreservation comprises storage in a
cryopreservation medium comprising one or more cell-permeating
cryopreservatives, one or more non cell-permeating
cryopreservatives, or a combination thereof. Optionally, the
cryopreservation medium comprises one or more cell-permeating
cryopreservatives selected from DMSO, a glycerol, a glycol, a
propylene glycol, an ethylene glycol, or a combination thereof.
Optionally, the cryopreservation medium comprises one or more non
cell-permeating cryopreservatives selected from
polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a
monosaccharides, a sugar alcohol, an alginate, a trehalose, a
raffinose, a dextran, or a combination thereof. Other examples of
useful cryopreservatives are described in "Cryopreservation"
(BioFiles Volume 5 Number 4--Sigma-Aldrich.RTM. datasheet).
[0286] In one embodiment, the cryopreservation medium comprises a
cell-permeating cryopreservative, wherein the majority of the
cell-permeating cryopreservative is DMSO. Optionally, the
cryopreservation medium does not comprise a substantial amount of
glycerol.
[0287] In one embodiment, the cryopreservation medium comprises
DMSO. Optionally, the cryopreservation medium does not comprise
glycerol in a majority amount. Optionally, the cryopreservation
medium does not comprise a substantial amount of glycerol.
[0288] In one embodiment, the cryopreservation medium comprises
additional components such as albumin (e.g. HSA or BSA), an
electrolyte solution (e.g. Plasma-Lyte), or a combination
thereof.
[0289] In one embodiment, the cryopreservation medium comprises 1%
to about 15% albumin by weight and about 5% to about 20%
cryopreservative by volume (e.g. about 10%). Optionally, the
cryopreservative comprises DMSO (e.g. in a majority amount).
[0290] In one embodiment, cryopreservation comprises placing the
placenta on nitrocellulose paper.
[0291] In one embodiment, the placenta is cut into a plurality of
sections before cryopreservation. Optionally, the sections are
placed on nitrocellulose paper before refrigeration.
[0292] Methods of Use
[0293] The placental products (e.g. derived from chorionic tissue)
of the present invention may be used to treat any tissue injury. A
method of treatment may be provided, for example, by administering
to a subject in need thereof, a placental product of the present
invention.
[0294] A typical administration method of the present invention is
topical administration. Administering the present invention can
optionally involve administration to an internal tissue where
access is gained by a surgical procedure.
[0295] Placental products can be administered autologously,
allogeneically or xenogeneically.
[0296] In one embodiment, a present placental product is
administered to a subject to treat a wound. Optionally, the wound
is a laceration, scrape, thermal or chemical burn, incision,
puncture, or wound caused by a projectile. Optionally, the wound is
an epidermal wound, skin wound, chronic wound, acute wound,
external wound, internal wounds, congenital wound, ulcer, or
pressure ulcer. Such wounds may be accidental or deliberate, e.g.,
wounds caused during or as an adjunct to a surgical procedure.
Optionally, the wound is closed surgically prior to
administration.
[0297] In one embodiment, a present placental product is
administered to a subject to treat a burn. Optionally, the burn is
a first-degree burn, second-degree burn (partial thickness burns),
third degree burn (full thickness burns), infection of burn wound,
infection of excised and unexcised burn wound, loss of epithelium
from a previously grafted or healed burn, or burn wound
impetigo.
[0298] In one embodiment, a present placental product is
administered to a subject to treat an ulcer, for example, a
diabetic ulcer (e.g. foot ulcer).
[0299] In one embodiment, a placental product is administered by
placing the placental product directly over the skin of the
subject, e.g., on the stratum corneum, on the site of the wound, so
that the wound is covered, for example, using an adhesive tape.
Additionally or alternatively, the placental product may be
administered as an implant, e.g., as a subcutaneous implant.
[0300] In one embodiment, a placental product is administered to
the epidermis to reduce rhtids or other features of aging skin.
Such treatment is also usefully combined with so-called cosmetic
surgery (e.g. rhinoplasty, rhytidectomy, etc.).
[0301] In one embodiment, a placental product is administered to
the epidermis to accelerate healing associated with a dermal
ablation procedure or a dermal abrasion procedure (e.g. including
laser ablation, thermal ablation, electric ablation, deep dermal
ablation, sub-dermal ablation, fractional ablation, and microdermal
abrasion).
[0302] Other pathologies that may be treated with placental
products of the present invention include traumatic wounds (e.g.
civilian and military wounds), surgical scars and wounds, spinal
fusions, spinal cord injury, avascular necrosis, reconstructive
surgeries, ablations, and ischemia.
[0303] In one embodiment, a placental product of the present
invention is used in a tissue graft procedure. Optionally, the
placental product is applied to a portion of the graft which is
then attached to a biological substrate (e.g. to promote healing
and/or attachment to the substrate). By way of non-limiting
example, tissues such as skin, cartilage, ligament, tendon,
periosteum, perichondrium, synovium, fascia, mesenter and sinew can
be used as tissue graft.
[0304] In one embodiment, a placental product is used in a tendon
or ligament surgery to promote healing of a tendon or ligament.
Optionally, the placental product is applied to portion of a tendon
or ligament which is attached to a bone. The surgery can be any
tendon or ligament surgery, including, e.g. knee surgery, shoulder,
leg surgery, arm surgery, elbow surgery, finger surgery, hand
surgery, wrist surgery, toe surgery, foot surgery, ankle surgery,
and the like. For example, the placental product can be applied to
a tendon or ligament in a grafting or reconstruction procedure to
promote fixation of the tendon or ligament to a bone.
[0305] Through the insight of the inventors, it has surprisingly
been discovered that placental products of the present invention
provide superior treatment (e.g. healing time and/or healing
strength) for tendon and ligament surgeries. Tendon and ligament
surgeries can involve the fixation of the tendon or ligament to
bone. Without being bound by theory, the present inventors believe
that osteogenic and/or chondrogenic potential of MSCs in the
present placental products promotes healing process and healing
strength of tendons or ligaments. The present inventors believe
that the present placental products provide an alternative or
adjunctive treatment to periosteum-based therapies. For example,
useful periosteum based treatments are described in Chen et al.
("Enveloping the tendon graft with periosteum to enhance
tendon-bone healing in a bone tunnel: A biomechanical and
histologic study in rabbits"; Arthroscopy. 2003 March;
19(3):290-6), Chen et al. ("Enveloping of periosteum on the
hamstring tendon graft in anterior cruciate ligament
reconstruction"; Arthroscopy. 2002 May-June; 18(5):27E), Chang et
al. ("Rotator cuff repair with periosteum for enhancing tendon-bone
healing: a biomechanical and histological study in rabbits"; Knee
Surgery, Sports Traumatology, Arthroscopy Volume 17, Number 12,
1447-1453), each of which are incorporated by reference.
[0306] As non-limiting example of a method of tendon or ligament
surgery, a tendon is sutured to and/or wrapped or enveloped in a
placental membrane and the tendon is attached to a bone.
Optionally, the tendon is placed into a bone tunnel before attached
to the bone.
[0307] In one embodiment, the tendon or ligament surgery is a graft
procedure, wherein the placental product is applied to the graft.
Optionally, the graft is an allograft, xenograft, or an autologous
graft.
[0308] In one embodiment, the tendon or ligament surgery is repair
of a torn ligament or tendon, wherein the placental product is
applied to the torn ligament or tendon.
[0309] Non-limiting examples of tendons to which a placental
product can be applied include a digitorum extensor tendon, a
hamstring tendon, a bicep tendon, an Achilles Tendon, an extensor
tendon, and a rotator cuff tendon.
[0310] In one embodiment, a placental product of the present
invention is used to reduce fibrosis by applying the placental
product to a wound site.
[0311] In one embodiment, a placental product of the present
invention is used as an anti-adhesion wound barrier, wherein the
placental product is applied to a wound site, for example, to
reduce fibrosis (e.g. postoperative fibrosis).
[0312] Non-limiting examples of wound sites to which the placental
product can be applied include those that are surgically induced or
associated with surgery involving the spine, laminectomy, knee,
shoulder, or child birth, trauma related wounds or injuries,
cardiovascular procedures, angiogenesis stimulation,
brain/neurological procedures, burn and wound care, and ophthalmic
procedures. For example, optionally, the wound site is associated
with surgery of the spine and the stromal side of the placental
product is applied to the dura (e.g. the stromal side facing the
dura). Direction for such procedures, including the selection of
wound sites and/or methodologies, can be found, for example, in WO
2009/132186 and US 2010/0098743, which are hereby incorporated by
reference.
[0313] A placental product of the present invention can optionally
be used to reduce adhesion or fibrosis of a wound. Postoperative
fibrosis is a natural consequence of all surgical wound healing. By
example, postoperative peridural adhesion results in tethering,
traction, and compression of the thecal sac and nerve roots, which
cause a recurrence of hyperesthesia that typically manifests a few
months after laminectomy surgery. Repeated surgery for removal of
scar tissue is associated with poor outcome and increased risk of
injury because of the difficulty of identifying neural structures
that are surrounded by scar tissue. Therefore, experimental and
clinical studies have primarily focused on preventing the adhesion
of scar tissue to the dura matter and nerve roots. Spinal adhesions
have been implicated as a major contributing factor in failure of
spine surgery. Fibrotic scar tissue can cause compression and
tethering of nerve roots, which can be associated with recurrent
pain and physical impairment.
[0314] Without being bound by theory, the present inventors believe
that placental products taught herein are useful to reduce adhesion
or fibrosis of a wound, at least in part, because the placental
products can perform the very critical function in-situ of
providing a immunoprivileged environment (i.e. relatively high
resistance against immune responses) in the human development
process. One advantage of the wound dressings and processes of the
present invention is that an anti-adhesion barrier is provided
which can be used to prevent adhesions following surgery, and in
particular following back surgery.
[0315] In the preceding paragraphs, use of the singular may include
the plural except where specifically indicated. As used herein, the
words "a," "an," and "the" mean "one or more," unless otherwise
specified. In addition, where aspects of the present technology are
described with reference to lists of alternatives, the technology
includes any individual member or subgroup of the list of
alternatives and any combinations of one or more thereof.
[0316] The disclosures of all patents and publications, including
published patent applications, are hereby incorporated by reference
in their entireties to the same extent as if each patent and
publication were specifically and individually incorporated by
reference.
[0317] It is to be understood that the scope of the present
technology is not to be limited to the specific embodiments
described above. The present technology may be practiced other than
as particularly described and still be within the scope of the
accompanying claims.
[0318] Likewise, the following examples are presented in order to
more fully illustrate the present technology. They should in no way
be construed, however, as limiting the broad scope of the
technology disclosed herein.
[0319] The presently described technology and its advantages will
be better understood by reference to the following examples. These
examples are provided to describe specific embodiments of the
present technology. By providing these specific examples, it is not
intended limit the scope and spirit of the present technology. It
will be understood by those skilled in the art that the full scope
of the presently described technology encompasses the subject
matter defined by the claims appending this specification, and any
alterations, modifications, or equivalents of those claims.
EXAMPLES
[0320] Other features and embodiments of the present technology
will become apparent from the following examples which are given
for illustration of the present technology rather than for limiting
its intended scope.
Example 1
Characterization of Placental Membranes
[0321] Characterization of cells in placental membranes by
Fluorescence Activated Cell Sorting (FACS) demonstrated the
presence of stromal cells (Mesenchymal Stem Cell-like cells) in
addition to fetal epithelial cells and fibroblasts in amniotic
and/or chorionic membranes.
[0322] One unique characteristic of the presently disclosed
placental products is the presence of MSCs, which have been shown
to be one of three types of cells (in addition to epithelial cells
and fibroblasts) that are important for wound healing. Placental
membranes secrete a variety of factors involved in wound healing
such as angiogenic factors, factors supporting proliferation and
migration of epithelial cells and fibroblasts, factors attracting
endothelial stem cells from blood circulation to the wound site,
antibacterial factors, and others.
[0323] Evaluation of proteins secreted by exemplary placental
products of the invention in comparison to Apligraf and Dermagraft
demonstrated a number of growth factors present in the tested
products that are involved in wound healing. Examples are Vascular
Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor
(PDGF), Transforming Growth Factor (TGF) and others. However,
several unique factors including Epidermal Growth Factor (EGF),
which is one of the key factors for wound healing, are present in
placental membranes and absent in Apligraf and Dermagraft. Also,
placental membranes have a favorable protease-to-protease inhibitor
ratio for wound healing. In an in vitro model of wound healing
(cell migration assay, disclosed herein), the present inventors
have demonstrated that placental membranes secrete factors
promoting cell migration that will support wound closure.
Example 2
Exemplary Manufacturing Process of a Placental Product
[0324] In one embodiment, the present application discloses a
procedure for manufacturing chorionic membranes from placenta post
partum.
Example 2.1
Exemplary Manufacturing Process of Chorionic Membrane Product
[0325] One method of manufacturing a placental product comprising a
chorionic membrane according to the presently disclosed
manufacturing procedure is as follows: [0326] a. Remove umbilical
cord close to placental surface, [0327] b. Blunt dissect of the
amnion to placental skirt, [0328] c. Flip placenta over and
completely remove amnion, [0329] d. Remove chorion by cutting
around placental skirt, [0330] e. Rinse the chorionic membrane in
PBS to remove red blood cells, [0331] f. Rinse the chorionic
membrane once with 11% ACD-A solution to assist in red blood cell
removal, [0332] g. Rinse the chorionic membrane PBS to remove ACD-A
solution, [0333] h. Treat chorion in 0.5% dispase solution at
37.degree. C..+-.2.degree. C. for 30-45 minutes, optionally, during
dispase incubation period, use PBS to remove any remaining blood
from the amnion, [0334] i. When dispase treatment is complete,
rinse chorion with PBS to remove dispase solution, [0335] j. Gently
remove trophoblast layer from the chorion, for example, by scraping
(e.g. with finger), [0336] k. Place chorion into a bottle
containing antibiotic solution and incubate at 37.degree.
C..+-.2.degree. C. for 24-28 hrs, [0337] l. Remove bottle from the
incubator and rinse each membrane with PBS to remove antibiotic
solution, [0338] m. Mount chorion on reinforced nitrocellulose
paper and cut to size, [0339] n. Place each piece into an FP-90
cryobag and heat seal, [0340] o. Add 50 mL cryopreservation
solution to the bag through a syringe and remove any air trapped
within the bag with the syringe, [0341] p. Tube seal the solution
line on the FP-90 bag, [0342] q. Place filled bag into secondary
bag and heat seal, [0343] r. Place unit into packaging carton,
[0344] s. Refrigerate at 2-8.degree. C. for 30-60 minutes, Freeze
at -80.degree. C..+-.5.degree. C. inside a Styrofoam container.
Example 2.2
Exemplary Manufacturing Process of Product Comprising Chorionic
Membrane and Amniotic Membrane
[0345] One method of manufacturing a placental product comprising a
chorionic membrane and an amniotic membrane according to the
presently disclosed manufacturing procedure is as follows: [0346]
a. Remove umbilical cord close to placental surface, [0347] b.
Blunt dissect of the amnion to placental skirt, [0348] c. Flip
placenta over and completely remove amnion, [0349] d. Remove
chorion by cutting around placental skirt, [0350] e. Rinse both
membranes in PBS to remove red blood cells, [0351] f. Rinse both
membranes once with 11% ACD-A solution to assist in red blood cell
removal, [0352] g. Rinse both membranes with PBS to remove ACD-A
solution, [0353] h. Treat chorion in 0.5% dispase solution at
37.degree. C..+-.2.degree. C. for 30-45 minutes, optionally, during
dispase incubation period, use PBS to remove any remaining blood
from the amnion, [0354] i. Gently remove the connective tissue
layer from the amnion, [0355] j. Place the amnion in PBS and set
aside, [0356] k. When dispase treatment is complete, rinse chorion
with PBS to remove dispase solution, [0357] l. Gently remove
trophoblast layer from the chorion, [0358] m. Place the amnion and
chorion each into a bottle containing antibiotic solution and
incubate at 37.degree. C..+-.2.degree. C. for 24-28 hrs, [0359] n.
Remove bottles from the incubator and rinse each membrane with PBS
to remove antibiotic solution, [0360] o. Mount amnion (epithelial
side up) or chorion on reinforced nitrocellulose paper and cut to
size, [0361] p. Place each piece into an FP-90 cryobag and heat
seal, [0362] q. Add 50 mL cryopreservation solution to the bag
through a syringe and remove any air trapped within the bag with
the syringe, [0363] r. Tube seal the solution line on the FP-90
bag, [0364] s. Place filled bag into secondary bag and heat seal,
[0365] t. Place unit into packaging carton, [0366] u. Refrigerate
at 2-8.degree. C. for 30-60 minutes, Freeze at -80.degree.
C..+-.5.degree. C. inside a Styrofoam container.
Example 2.3
Exemplary Placental Product Manufacturing Process
[0367] One method manufacturing a placental product comprising a
chorionic membrane according to the presently disclosed
manufacturing procedure was as follows:
[0368] The placenta was processed inside a biological safety
cabinet. The umbilical cord was first removed, and the amniotic
membrane was peeled from the underlying chorionic membrane using
blunt dissection. Subsequently, the chorion was removed by cutting
around the placental skirt on the side opposite of the umbilical
cord. The chorion on the umbilical side of the placenta was not
removed due to the vascularization on this side. The chorionic
membrane was rinsed with phosphate buffered saline (PBS) (Gibco
Invitrogen, Grand Island, N.Y.) to remove gross blood clots and any
excess blood cells. The membrane was then washed with 11%
anticoagulant citrate dextrose solution (USP) formula A (ACD-A)
(Baxter Healthcare Corp., Deerfield, Ill.) in saline (Baxter
Healthcare Corp., Deerfield, Ill.) to remove remaining blood
cells.
[0369] The chorion was then incubated in 200 mL of a 0.5% dispase
(BD Biosciences, Bedford, Mass.) solution in Dulbecco's Modified
Eagles media (DMEM) (Lonza, Walkersville, Md.) at 37.degree.
C..+-.2.degree. C. for 30-45 minutes to digest the connective
tissue layer between the chorion and adjacent trophoblast layer.
Once the chorion incubation period was complete, the chorion was
rinsed with PBS to remove the dispase solution. Subsequently, the
trophoblast layer was removed by gently peeling or scraping away
these maternal decidual cells.
[0370] The chorion was then disinfected in 500 mL of antibiotic
solution consisting of gentamicin sulfate (50 .mu.g/mL) (Abraxis
Pharmaceutical Products, Schaumburg, Ill.), vancomycin HCl (50
.mu.g/mL) (Hospira Inc., Lake Forest, Ill.), and amphotericin B
(2.5 .mu.g/mL) (Sigma Aldrich, St. Louis, Mo.) in DMEM at
37.degree. C..+-.2.degree. C. for 24-28 hours. Vented caps were
used with the incubation flasks. After the incubation period, the
membrane was washed with PBS to remove any residual antibiotic
solution.
[0371] The membrane was mounted on Optitran BA-S 85 reinforced
nitrocellulose paper (Whatman, Dassel, Germany) and cut to the
appropriate size prior to packaging into an FP-90 cryobag (Charter
Medical Ltd., Winston-Salem, N.C.). Once the membrane unit was
placed into the FP-90 cryobag and the cryobag was heat sealed, 50
mL of a cryopreservation solution containing 10% dimethyl sulfoxide
(DMSO) (Bioniche Teo. Inverin Co., Galway, Ireland) and 5% human
serum albumin (HSA) (Baxter, West Lake Village, Calif.) in
PlasmaLyte-A (Baxter Healthcare Corp., Deerfield, Ill.) were added
through the center tubing line. Any excess air was removed, and the
tubing line was subsequently sealed.
[0372] The FP-90 cryobag was placed into a mangar bag (10
in..times.6 in.) (Mangar Industries, New Britain, Pa.), which was
then heat sealed. The mangar bag was placed into a packaging carton
(10.5 in..times.6.5 in..times.0.6 in.) (Diamond Packaging,
Rochester, N.Y.). All cartons were refrigerated at 2-8.degree. C.
for 30-60 minutes prior to freezing at -80.degree. C..+-.5.degree.
C. inside a Styrofoam container.
Example 2.4
Exemplary Manufacturing Process of a Placental Product Comprising
Chorionic Membrane and Amniotic Membrane
[0373] One method of manufacturing a placental product comprising a
chorionic membrane product and an amniotic membrane product
according to the presently disclosed manufacturing procedure was as
follows:
[0374] The placenta was processed inside a biological safety
cabinet. The umbilical cord was first removed, and the amniotic
membrane was peeled from the underlying chorionic membrane using
blunt dissection. Subsequently, the chorion was removed by cutting
around the placental skirt on the side opposite of the umbilical
cord. The chorion on the umbilical side of the placenta was not
removed due to the vascularization on this side. Both membranes
were rinsed with phosphate buffered saline (PBS) (Gibco Invitrogen,
Grand Island, N.Y.) to remove gross blood clots and any excess
blood cells. The membranes were then washed with 11% anticoagulant
citrate dextrose solution (USP) formula A (ACD-A) (Baxter
Healthcare Corp., Deerfield, Ill.) in saline (Baxter Healthcare
Corp., Deerfield, Ill.) to remove remaining blood cells.
[0375] The chorion was then incubated in 200 mL of a 0.5% dispase
(BD Biosciences, Bedford, Mass.) solution in Dulbecco's modified
eagles media (DMEM) (Lonza, Walkersville, Md.) at 37.degree.
C..+-.2.degree. C. for 30-45 minutes to digest the connective
tissue layer between the chorion and adjacent trophoblast layer.
During this incubation period, the stromal side of the amnion was
cleaned by gently scraping away any remaining connective tissue.
Once the chorion incubation period was complete, the chorion was
rinsed with PBS to remove the dispase solution. Subsequently, the
trophoblast layer was removed by gently peeling or scraping away
these maternal decidual cells.
[0376] The amnion and chorion were then each disinfected in 500 mL
of antibiotic solution consisting of gentamicin sulfate (50
.mu.g/mL) (Abraxis Pharmaceutical Products, Schaumburg, Ill.),
vancomycin HCl (50 .mu.g/mL) (Hospira Inc., Lake Forest, IL), and
amphotericin B (2.5 .mu.g/mL) (Sigma Aldrich, St. Louis, Mo.) in
DMEM at 37.degree. C..+-.2.degree. C. for 24-28 hours. Vented caps
were used with the incubation flasks. After the incubation period,
the membranes were washed with PBS to remove any residual
antibiotic solution.
[0377] The membranes were mounted on Optitran BA-S 85 reinforced
nitrocellulose paper (Whatman, Dassel, Germany) and cut to the
appropriate size prior to packaging into an FP-90 cryobag (Charter
Medical Ltd., Winston-Salem, N.C.). For the amnion, the stromal
side was mounted towards the nitrocellulose paper. Once a membrane
unit was placed into the FP-90 cryobag and the cryobag was heat
sealed, 50 mL of a cryopreservation solution containing 10%
dimethyl sulfoxide (DMSO) (Bioniche Teo. Inverin Co., Galway,
Ireland) and 5% human serum albumin (HSA) (Baxter, West Lake
Village, Calif.) in PlasmaLyte-A (Baxter Healthcare Corp.,
Deerfield, Ill.) were added through the center tubing line. Any
excess air was removed, and the tubing line was subsequently
sealed.
[0378] The FP-90 cryobag was placed into a mangar bag (10
in..times.6 in.) (Mangar Industries, New Britain, Pa.), which was
then heat sealed. The mangar bag was placed into a packaging carton
(10.5 in..times.6.5 in..times.0.6 in.) (Diamond Packaging,
Rochester, N.Y.). All cartons were refrigerated at 2-8.degree. C.
for 30-60 minutes prior to freezing at -80.degree. C..+-.5.degree.
C. inside a Styrofoam container.
Example 3
Quantitative Evaluation of Cell Number and Cell Viability after
Enzymatic Digestion of Placental Membranes
[0379] Amnion and chorion membranes and present placental products
(from above) were evaluated for cell number and cell viability
throughout the process. These analyses were performed on fresh
placental tissue (prior to the antibiotic treatment step),
placental tissue post antibiotic treatment, and product units post
thaw. Cells were isolated from the placental membranes using
enzymatic digestion. For the frozen product units, the FP-90
cryobags were first removed from the packaging cartons and mangar
bags. Then the FP-90 cryobags were thawed for 2-3 minutes in a room
temperature water bath. Early experiments involved the use of a
37.degree. C..+-.2.degree. C. water bath. After thaw, the placental
membranes were removed from the FP-90 cryobag and placed into a
reservoir containing saline (Baxter Healthcare Corp., Deerfield,
Ill.) for a minimum of 1 minute and a maximum of 60 minutes. Each
membrane was detached from the reinforced nitrocellulose paper
prior to digestion.
[0380] Amniotic membranes were digested with 40 mL of 0.75%
collagenase (Worthington Biochemical Corp., Lakewood, N.J.)
solution at 37.degree. C..+-.2.degree. C. for 20-40 minutes on a
rocker. After collagenase digestion, the samples were centrifuged
at 2000 rpm for 10 minutes. The supernatant was removed, and 40 mL
of 0.05% trypsin-EDTA (Lonza, Walkersville, Md.) were added and
incubated at 37.degree. C..+-.2.degree. C. for an additional 5-15
minutes on a rocker. The trypsin was warmed to 37.degree.
C..+-.2.degree. C. in a water bath prior to use. After trypsin
digestion, the suspension was filtered through a 100 .mu.m cell
strainer nylon filter to remove any debris. Centrifugation at 2000
rpm for 10 minutes was performed, and supernatant was removed. Cell
pellets were reconstituted with a volume of PlasmaLyte-A that was
proportional to the pellet size, and 20 .mu.L of the resuspended
cell suspension were mixed with 80 .mu.L of trypan blue (Sigma
Aldrich, St. Louis, Mo.) for counting. The cell count sample was
placed into a hemocytometer and evaluated using a microscope.
[0381] Chorionic membranes were digested with 25 mL of 0.75%
collagenase solution at 37.degree. C..+-.2.degree. C. for 20-40
minutes on a rocker. After collagenase digestion, the suspension
was filtered through a 100 .mu.m cell strainer nylon filter to
remove any debris. Centrifugation at 2000 rpm for 10 minutes was
performed, and supernatant was removed. Cell pellets were
reconstituted with a volume of PlasmaLyte-A that was proportional
to the pellet size, and 20 .mu.L of the resuspended cell suspension
were mixed with 80 .mu.L of trypan blue for counting. The cell
count sample was placed into a hemocytometer and evaluated using a
microscope.
[0382] Placenta membranes were analyzed prior to any processing to
determine the initial characteristics of the membranes. Table 1
contains the average cell count per cm.sup.2 and cell viability
values for the amniotic and chorionic membranes from 32 placenta
lots.
[0383] The average cell count per cm.sup.2 for the amniotic
membrane was 91,381 cells with a corresponding average cell
viability of 84.5%. For the chorionic membrane, the average cell
count per cm.sup.2 was 51,614 cells with a corresponding cell
viability of 86.0%.
[0384] These data illustrate cell numbers that are useful with
certain embodiments of the present invention; e.g. a placental
product comprising a chorionic membrane containing about 20,000 to
about 200,000 cells/cm.sup.2.
[0385] Since the amniotic membrane consists of epithelial cells and
stromal cells, experiments were conducted to determine the ratio of
epithelial cells to stromal cells. Amniotic membranes from 3
placenta lots were analyzed. First, a 5 cm.times.5 cm piece of
amniotic membrane was digested with approximately 25 mL of 0.05%
Trypsin-EDTA (Lonza, Walkersville, Md.) at 37.degree.
C..+-.2.degree. C. in a water bath for 30 minutes. After the
incubation step, epithelial cells were removed by gently scraping
the cells from the membrane. After rinsing with PBS (Gibco
Invitrogen, Grand Island, N.Y.), the membrane was subsequently
digested in the same manner as chorionic membrane (described
above). In addition, another intact 5 cm.times.5 cm piece of
amniotic membrane was digested using the standard procedure
(described above) to determine the total number of cells. The
percentage of stromal cells was then determined by dividing the
cell count from the amniotic membrane with the epithelial cells
removed with the cell count from the intact membrane.
[0386] Results indicate that 19% of the total cells were stromal
cells. Therefore, approximately 17,362 stromal cells were present
in amniotic membrane with approximately 74,019 epithelial cells.
These data indicated that there are approximately 3 times more
stromal cells in chorionic membranes as compared to amniotic
membranes. This ratio is consistent with certain embodiments of the
present invention that provide a placental product comprising a
chorionic membrane and an amniotic membrane, wherein the chorionic
membrane comprises about 2 to about 4 times more stromal cells
relative to the amniotic membrane.
TABLE-US-00001 TABLE 1 Cell count per cm.sup.2 and cell viability
values from fresh placental tissue from 32 donors. Membrane
Statistics Cell Count per cm.sup.2 Cell Viability Amnion Average
91,381 84.5% SD 49,597 3.7% Chorion Average 51,614 86.0% SD 25,478
4.7%
[0387] The second point in the manufacturing process where cell
count and cell viability values were assessed was after the
antibiotic treatment step. Table 2 provides the results from these
analyses. Cell recoveries from this step for the amniotic membrane
and the chorionic membrane were 87.7% and 70.3%, respectively.
TABLE-US-00002 TABLE 2 Cell count per cm.sup.2, cell viability, and
process (antibiotic treatment) cell recovery values for post
antibiotic placental tissue from 28 donors. Cell Count per Process
Cell Membrane Statistics cm.sup.2 Cell Viability Recovery Amnion
Average 75,230 84.4% 87.7% SD 46,890 4.2% 49.4% Chorion Average
33,028 85.6% 70.3% SD 18,595 4.4% 31.1%
Example 4
Development of a Placental Product Cryopreservation Procedure
[0388] Cryopreservation is a method that provides a source of
tissues and living cells. A main objective of cryopreservation is
to minimize damage to biological materials during low temperature
freezing and storage. Although general cryopreservation rules are
applicable to all cells, tissues, and organs, optimization of the
cryopreservation procedure is required for each type of biological
material. The present application discloses a cryopreservation
procedure for placental membrane products that can selectively
deplete immunogenic cells from the placental membranes; and
preserve viability of other beneficial cells that are the primary
source of factors for the promotion of healing.
[0389] During cryopreservation method development for placental
membranes, the present inventors evaluated key parameters of
cryopreservation including volume of cryopreservative solution,
effect of tissue equilibration prior to freezing, and cooling rates
for a freezing procedures.
[0390] Acceptance of tissue allografts in the absence of
immunosuppression will depend on the number of satellite immune
cells present in the tissue. Cryopreservation is an approach which
can be utilized to reduce tissue immunogenicity. This approach is
based on differential susceptibility of different cell types to
freezing injury in the presence of DMSO; leukocytes are sensitive
to fast cooling rates. The freezing rate of 1.degree. C./min is
considered optimal for cells and tissues including immune cells.
Rapid freezing rates such as 60-100.degree. C./min eliminate immune
cells. However, this type of procedure is harmful to other tissue
cells, which are desirable for preservation according to the
present invention. The developed cryopreservation procedure
utilized a cryopreservation medium containing 10% DMSO, which is a
key component protecting cells from destruction when water forms
crystals at low temperatures. The second step of cryopreservation
was full equilibration of placental membrane in the
cryopreservation medium, which was achieved by soaking membranes in
the cryopreservation medium for 30-60 min at 4.degree. C. This step
allowed DMSO to penetrate the placental tissues. Although there are
data in the literature showing that tissue equilibration prior to
freezing affects survival of immune cells (Taylor & Bank,
Cryobiology, 1988, 25:1), it was an unexpected finding that 30-60
min placental membrane equilibration in a DMSO-containing solution
at 2-8.degree. C. selectively increases sensitivity of immune cells
to freezing (in comparison to therapeutic cells) so that these type
of cells are selectively depleted during the freezing process (e.g.
1.degree. C./min freezing rate).
[0391] For example, CD14+ macrophages are selectively killed
relative to therapeutic cells such as hMSCs and/or fibroblasts.
[0392] Temperature mapping experiments were performed to analyze
the temperature profiles of potential cryopreservation conditions
for the membrane products. These results are illustrated in FIG. 1.
Eight (8) FP-90 cryobags were filled with either 20 mL or 50 mL of
cryopreservation solution, and temperature probes were placed
inside each cryobag. The first set of parameters (conditions 1
through 4 of FIG. 1a through FIG. 1d, respectively) involved a
30-minute refrigeration (2-8.degree. C.) step prior to freezing
(-80.degree. C..+-.5.degree. C.). In addition, the analysis
involved freezing of the cryobags either inside a Styrofoam
container or on the freezer shelf. The second set of parameters
(conditions 5 through 8 of FIG. 1e through FIG. 1h, respectively)
involved direct freezing (-80.degree. C..+-.5.degree. C.) of the
cryobags either inside a Styrofoam container or on the freezer
shelf. The results indicated that condition 6 and condition 2
exhibited the most gradual temperature decreases. Gradual
temperature decreases are typically desired in order to preserve
cell viability. The difference between condition 6 and condition 2
was that condition 2 included a 30-minute refrigeration step.
Therefore, the decrease in temperature from the start of freezing
to -4.degree. C., where latent heat evolution upon freezing occurs,
was examined further. For condition 6, the rate of cooling was
approximately -1.degree. C./minute during this period. The rate of
cooling for condition 2 was approximately -0.4.degree. C./minute
during the same timeframe. Therefore, condition 2 was selected for
incorporation into a non-limiting cryopreservation process since
slower rates of cooling are generally desired to maintain optimal
cell viability.
[0393] FIG. 2 depicts the effects of cryopreservation solution
volume on process (cryopreservation) cell recovery for the
chorionic membrane. The analysis of the 10 mL cryopreservation
solution volume involved 5 placenta lots, and the analysis of the
20 mL cryopreservation solution volume included 3 lots. For the 50
mL cryopreservation solution volume, 16 placenta lots were
analyzed.
[0394] As depicted in FIG. 2, the 50 mL volume of cryopreservation
solution volume provided superior cell recovery compared to that of
the 10 ml and 20 ml. These data indicate that a cryopreservation
medium volume of greater than 20 mL such as about 50 mL or more can
provide superior placental product according to the present
invention.
[0395] Experiments were conducted to evaluate different potential
freezing conditions to maximize cell recovery after the
cryopreservation process.
[0396] FIG. 3 includes these results, depicting the effects of
refrigeration time and freezing parameters on process
(cryopreservation) cell recovery for the chorionic membrane. Three
conditions were analyzed. These conditions were also linked to the
temperature mapping studies. The first condition involved directly
freezing the product unit on a shelf within the freezer
(-80.degree. C..+-.5.degree. C.). The second condition also
contained a direct freeze, but the product unit was placed into a
Styrofoam container within the freezer. The third condition
included a refrigeration (2-8.degree. C.) period of 30 minutes
prior to the freezing step. For the amniotic membrane, 3 placenta
lots were evaluated. Two (2) placenta lots were analyzed for the
chorionic membrane. Results indicated that the third condition was
optimal for both membrane types. As depicted in
[0397] FIG. 3, a refrigeration period at least about 30 min
provided the best cell recovery.
[0398] Cryopreservation parameters are assessed for the amniotic
and chorionic membranes and summarized in Table 3 and Table 4. The
evaluation of the cell recoveries and cell viabilities from these
experiments resulted in the selection of the final parameters for
the manufacturing process. In addition, all average cell viability
values were .gtoreq.70%.
TABLE-US-00003 TABLE 3 Post thaw cell count per cm.sup.2, cell
viability, and process (cryopreservation) cell recovery values for
the chorionic membrane. Cell Count Process Condition per Cell Cell
Parameter Tested Statistics cm.sup.2 Viability Recovery Refrigerate
All Average 23,217 87.3% 102.8% at 2-8.degree. C. for conditions SD
9,155 4.1% 65.5% 30-60 min N 27 27 27 and freeze at -80.degree. C.
.+-. 5.degree. C. Dispase 30 min Average 22,354 85.7% 81.1% No
decrease treatment SD 9,505 5.1% 32.4% in process cell N 24 24 24
recovery for 45 min Average 27,125 90.6% 172.6% the 45 min SD 7,963
2.2% 101.2% treatment. A N 6 6 6 30-45 min range was established.
Refrigeration 30 min Average 23,815 86.8% 102.2% The process time
interval SD 9,681 5.2% 68.8% recovery value N 25 25 25 was >80%
for 60 min Average 20,773 85.8% 84.9% the 60 min SD 7,356 4.7%
14.4% time interval. N 5 5 5 A 30-60 min range was established.
Thawing 37.degree. C. .+-. Average 33,360 85.9% 114.7% No
significant temperature 2.degree. C. water SD 8,497 4.0% 38.1%
difference bath N 5 5 5 found in Room Average 21,298 86.8% 96.3%
process cell temp SD 8,189 5.3% 67.2% recovery. The water bath N 25
25 25 room temp condition was selected for logistical reasons.
Holding 1-15 min Average 23,733 86.6% 100.6% No significant period
after SD 9,674 5.1% 67.0% difference transfer into N 26 26 26 found
in saline 1 hr Average 20,550 87.0% 91.4% process cell SD 6,575
4.8% 32.0% recovery. N 4 4 4 Membranes can be held in saline for up
to 1 hr. Tissue size 5 cm .times. 5 cm Average 23,391 86.1% 99.6%
No decrease SD 8,865 5.0% 58.7% in process cell N 23 23 23 recovery
from 2 cm .times. 2 cm Average 23,036 88.4% 98.7% the 5 cm .times.
5 cm SD 11,362 5.0% 81.3% product to N 7 7 7 the 2 cm .times. 2 cm
product. Both sizes were acceptable for use. Notes: cm =
centimeter; min = minutes; temp = temperature; hr = hour, SD =
standard deviation; N = number
TABLE-US-00004 TABLE 4 Post thaw cell count per cm.sup.2, cell
viability, and process (cryopreservation) cell recovery values for
the amniotic membrane Cell Count Process Condition per Cell Cell
Comments/ Parameter Tested Statistics cm.sup.2 Viability Recovery
Conclusions Refrigerate All Average 55,709 83.4% 64.2% Overall at
2-8.degree. C. for conditions SD 45,210 4.4% 22.5% assessment 30-60
min N 32 32 32 and freeze at -80.degree. C. .+-. 10.degree. C.
Refrigeration 30 min Average 52,173 83.1% 63.7% No significant time
interval SD 39,750 4.5% 21.4% difference N 26 26 26 found in 60 min
Average 71,033 85.0% 66.5% process cell SD 66,525 3.9% 29.3%
recovery. A N 6 6 6 30-60 min range was established. Thawing
37.degree. C. .+-. Average 48,524 83.3% 64.0% No significant
temperature 2.degree. C. water SD 27,804 1.7% 34.4% difference bath
N 7 7 7 found in Room Average 57,721 83.5% 64.3% process cell temp
SD 49,271 4.9% 19.0% recovery. The water bath N 25 25 25 room temp
condition was selected for logistical reasons. Holding 1-15 min
Average 50,873 83.1% 65.0% No significant period after SD 38,969
3.9% 24.2% difference transfer into N 26 26 26 found in saline 1 hr
Average 76,667 85.1% 61.0% process cell SD 66,565 6.2% 14.3%
recovery. N 6 6 6 Membranes can be held in saline for up to 1 hr.
Tissue size 5 cm .times. 5 cm Average 58,431 83.3% 62.8% No
decrease SD 47,603 4.5% 21.7% in process cell N 28 28 28 recovery
from 2 cm .times. 2 cm Average 36,656 84.4% 73.9% the 5 cm .times.
5 cm SD 13,175 3.4% 29.5% product to N 4 4 4 the 2 cm .times. 2 cm
product. Both sizes were acceptable for use.
[0399] These data are consistent with certain embodiments of the
present invention that provide a placental product comprising a
chorionic membrane containing about 20,000 to about 60,000 or to
about 200,000 cells/cm.sup.2.
Example 5
Qualitative Evaluation of Cell Viability by Tissue Staining
[0400] The amniotic and chorionic membranes were stained using a
LIVE/DEAD.RTM. Viability/Cytotoxicity kit (Molecular Probes Inc.,
Eugene, Oreg.) to qualitatively assess cell viability. Staining was
performed as per the manufacturer's protocol. Membrane segments of
approximately 0.5 cm.times.0.5 cm were used. Evaluation of stained
membranes was performed using a fluorescent microscope. An intense
uniform green fluorescence indicated the presence of live cells,
and a bright red fluorescence indicated the presence of dead cells.
Images of fresh amniotic and chorionic membranes as well as
cryopreserved amniotic and chorionic membranes demonstrated that
the manufacturing process did not alter the phenotypic
characteristics of the membranes and the proportion of viable cell
types (epithelial and stromal cells) in the membranes post
thaw.
[0401] FIG. 4 shows representative images of the live/dead staining
of the epithelial layer of fresh amniotic membrane (A); epithelial
layer of cryopreserved amniotic membrane (B); stromal layer of
fresh amniotic membrane (C); stromal layer of cryopreserved
amniotic membrane (D); fresh chorionic membrane (E); and
cryopreserved chorionic membrane (F). Live cells are green, and
dead cells are red.
Example 6
Placental Tissue Immunogenicity Testing
[0402] One unique feature of the human chorion is the absence of
fetal blood vessels that prevent mobilization of leukocytes from
fetal circulation. On the fetal side, macrophages resident in the
chorioamniotic mesodermal layer represent the only population of
immune cells. Thus, fetal macrophages present in the chorion are a
major source of tissue immunogenicity, as such the chorion is
considered immunogenic. In a study where the amnion was used
together with the chorion for plastic repair of conjunctival
defects, the success rate was low (De Roth Arch Ophthalmol, 1940,
23: 522). Without being bound by theory, the present inventors
believe that removal of CD14+ cells from placental membranes
eliminates activation of lymphocytes in vitro. In addition to the
presence of fetal macrophages, the present inventors believe that
immunogenicity of chorion can be mediated by contamination of blood
cells coming from the maternal trophoblast, which contains blood
vessels. Thus, the processing of placental membrane for clinical
use can be enhanced by purification of the chorion from maternal
trophoblasts and selective elimination of all CD14+ fetal
macrophages. Immunogenicity testing can be used to characterize a
chorion-derived product as safe clinical therapeutics. For example,
two bioassays can be used to test immunogenicity of manufactured
placental products: Mixed Lymphocyte Reaction (MLR) and
Lipopolysaccharide (LPS)-induced Tumor Necrosis Factor
(TNF)-.alpha. secretion.
Example 7
Mixed Lymphocyte Reaction (MLR)
[0403] An MLR is a widely used in vitro assay to test cell and
tissue immunogenicity. The assay is based on the ability of immune
cells (responders) derived from one individual to recognize
allogeneic Human Leukocyte Antigen (HLA) and other antigenic
molecules expressed on the surface of allogeneic cells and tissues
(stimulators) derived from another individual when mixed together
in a well of an experimental tissue culture plate. The response of
immune cells to stimulation by allogeneic cells and tissues can be
measured using a variety of methods such as secretion of particular
cytokines (e.g., Interleukin (IL-2), expression of certain
receptors (e.g., IL-2R), or cell proliferation, all of which are
characteristics of activated immune cells.
[0404] Placental tissue samples representing different steps of the
presently disclosed manufacturing process were used for
immunogenicity testing. These samples included amnion with chorion
and trophoblast as a starting material and separated
choriotrophoblast, chorion, trophoblast, and amnion. Both freshly
purified and cryopreserved (final products) tissues were
tested.
[0405] For the MLR assay, cells from placental tissues were
isolated using 280 U/mL of collagenase type II (Worthington, Cat
No. 4202). Tissues were treated with enzyme for 60-90 min at
37.degree. C..+-.2.degree. C., and the resulting cell suspension
was filtered through a 100 .mu.m filter to remove tissue debris.
Single cell suspensions were then centrifuged using a Beckman, TJ-6
at 2000 rpm for 10 min and washed twice with DPBS. Supernatant was
discarded after each wash, and cells were resuspended in 2 mL of
DMEM (Invitrogen, Cat No. 11885) and evaluated for cell number and
cell viability by counting cells in the presence of Trypan blue dye
(Invitrogen, Cat No. 15250-061). For the MLR, placental-derived
cells were mixed with allogeneic hPBMCs at a 1:5 ratio in 24-well
culture plates in DMEM supplemented with 5% fetal bovine serum
(FBS) and incubated for 4 days in the incubator containing 5%
CO.sub.2, 95% humidity at 37.degree. C..+-.2.degree. C. Human
Peripheral Blood Mononuclear Cells (hPBMCs) alone were used as a
negative control, and a mixture of two sets of hPBMCs derived from
two different donors was used as a positive MLR control. After 4
days of incubation, cells were collected from wells, lysed using a
lysis buffer (Sigma, Cat No. C2978) supplemented with protease
inhibitor cocktail (Roche, Cat No. 11836153001), and IL-2R was
measured in cell lysates using the sIL-2R ELISA kit (R&D
Systems, Cat No. SR2A00) generally following the manufacturer's
protocol. The level of IL-2R is a measure of activation of T-cells
in response to immunogenic molecules expressed by allogeneic cells.
Results of 2 out of 12 representative experiments are shown in FIG.
5 and FIG. 6. Results presented in these figures demonstrated that
the present application discloses a process for manufacturing of
placental membranes that result in low immunogenicity of the final
chorionic membrane products.
[0406] As depicted in FIG. 5, the manufacturing process serially
reduces immunogenicity of the placental product. Samples
representing different steps of the manufacturing process
Chorion+Trophoblast (CT), Trophoblast (T), Amnion (AM), and Chorion
(CM) were co-cultured with hPBMCs for 4 days. IL-2sR was measured
in cell lysates as a marker of T-cell activation. Negative control
shows a basal level of immune cell activation: PBMCs derived from
one donor were cultured alone. Positive control: a mixture of PBMCs
derived from 2 different donors.
[0407] As depicted in FIG. 6, selective depletion of immunogenicity
results from the present cryopreservation process of producing the
present placental products, as evidenced by the significant
decrease in immunogenicity upon cryopreservation.
Example 8
LPS-Induced TNF-.alpha. Secretion by Placental Membrane Cells
[0408] As described herein, fetal macrophages present in the amnion
and chorion are a major source of tissue immunogenicity. Without
being bound by theory, the present inventors believe that removal
of CD14+ cells from placental membrane eliminates activation of
lymphocytes and that depletion of allogeneic donor tissue
macrophages decreases the level of inflammatory cytokine secretion
and tissue immunogenicity. The inventors also believe that
reduction of tissue immunogenicity can also be reached by depletion
of TNF-.alpha. with anti-TNF-.alpha. antibodies or suppression of
TNF-.alpha. secretion by IL-10. Macrophages in fetal placental
membranes respond to bacteria by secretion of inflammatory
cytokines. The secretion of TNF-.alpha. by fresh placental
membranes in vitro in response to bacterial LPS is significantly
higher in the chorionic membrane. Thus, the present inventors
believe that immunogenicity of placental membranes is mediated by
macrophages, the amount and/or activity of which is higher in the
chorionic membrane.
[0409] According to the present invention, selective depletion of
macrophages is an optional approach to selectively deplete
immunogenicity of the amniotic and chorionic membranes, allowing
the use of both allogeneic membranes for clinical applications. The
assay of functional macrophages in a placental product is used here
as an assay for immunogenicity testing (e.g. in production or prior
to clinical use) based on the facts that: macrophages are the
source of immunogenicity in chorionic membranes. Macrophages in
placenta-derived membranes respond to bacterial LPS by secretion of
high levels of TNF-.alpha.; and TNF-.alpha. is a critical cytokine
involved in immune response and allograft tissue rejection.
Therefore, secretion of TNF-.alpha. by placenta-derived membranes
in response to LPS is used here to characterize tissue
immunogenicity and for pre-use screening.
Example 9
Establishment of Allowed LPS-Induced TNF-.alpha. Secretion Level by
Chorionic Membranes
[0410] Data from published reports regarding the level of
TNF-.alpha., which is associated with the absence or an
insignificant immune response in a variety of experimental systems,
are presented in Table 5. These data indicate that a TNF-.alpha.
level below 100 pg/mL correlates with a low immune response. The
ability of amniotic and chorionic membranes to produce TNF-.alpha.
spontaneously and in response to bacteria or bacterial LPS in vitro
has been shown by a number of investigators. Table 6 summarizes
such data. The lowest spontaneous TNF-.alpha. secretion by amniotic
membrane of about 70 pg/cm.sup.2 of the membrane was reported by
Fortunato et al. (Am J Reprod Immunol, 1994, 32:184). All reports
also showed that fresh placental membranes secrete large amounts of
TNF-.alpha. in response to bacteria or bacterial LPS (Table 6),
which is attributed to the presence of viable functional
macrophages.
TABLE-US-00005 TABLE 5 TNF-.alpha. levels associated with the
Description of absence/reduction experimental system of immune
response Comments References IL-10-induced inhibition Mean 260
pg/mL Wang et al., of MLR in vitro. Transplantation, TNF was
measured in 2002, 74: 772 tissue culture supernatant by ELISA. MLR
using skin tissue Mean 100 pg/mL explants (0.02 cm.sup.2 per well)
as stimulators in the presence or absence of IL-10 (skin explant
assay). Skin tissue destruction was assessed microscopically, and
severity was assigned based on histopathological tissue damage.
Endogeneous TNF ~0.04 U/mL for the TNF activity Shalaby et al., J
production in MLR in the negative control and per mg is not
Immunol, 1988, presence or absence of MLR in the presence provided.
141: 499 anti-TNF antibodies. TNF of anti-TNF levels were assessed
antibodies, which using the WEHI-164 correlated with no or
cytotoxicity assay. significant inhibition of lymphocyte
proliferation TNF levels in BAL fluid of Isograft: below Unmodified
Sekine et al., J lung isografts, unmodified detection; allograft:
~45 pg/mL Immunol, 1997, allograft, and alveolar AM-depleted
allograft: (immunogenic) 159: 4084 macrophages (AM) ~15 pg/mL of
BAL depleted allograft in rats. (total 75 pg/5 ml of BAL) TNF
levels in MLR after ~<200 pg/mL TNF Ohashi et al, 48 hours in
the presence correlated with a Clin Immunol, or absence of advanced
complete inhibition of 2010, 134: 345 glycation end products MLR
(MLR inhibitors). TNF levels in MLR. <100 pg/mL TNF in Toungouz
et al., MLR with HLA- Hum Immunol, matched donors 1993, 38: 221
(control, no stimulation) TNF activity in MLR when Negative control
~20 Unit of activity Lomas et al., pieces of cryopreserved U of TNF
activity; was calculated Cell Tissue skin allografts (~0.2
cm.sup.2) MLR with skin as TNF in Bank, 2004, were incubated with
explants: 0-40 U; ng/mL divided 5: 23. hPBMCs for 24 hours.
Positive control: 600 U by OD at 570 nm Positive control: for the
hPBMC + LPS; negative: same hPBMC alone. experimental well Cytokine
time course in Optimal TNF Jordan & Ritter, MLR, including TNF.
after 24 hours: J Immunol ~150 pg/mL Meth, 2002, 260: 1 MLR using
skin tissue For no skin Recalculation Dickinson et al., explants
(0.02 cm.sup.2 per destruction: 0.5-1.1 pg/mL per 1 cm.sup.2 of
Cytokine, 1994, well) as stimulators in the for HLA skin tissue: 6:
141 presence or absence of compatible lowest TNF anti-TNF
antibodies (skin responders, and 2.6-1376 pg/mL non- explant
assay). Skin for immunogenic tissue destruction was unmatched MLR
level is 100 pg/cm.sup.2 assessed microscopically, and severity was
assigned based on histopathological tissue damage.
TABLE-US-00006 TABLE 6 TNF levels Comments/ secreted by fresh
recalculations placental of the lowest Description of membranes in
TNF levels per experimental system culture cm.sup.2 References TNF
secretion by Chorion: basal Lowest TNF Zaga et al., Biol "fresh"
amnion and 3.3 .+-. 0.46 ng/cm.sup.2, level for amnion Reprod,
2004, chorion tissues (1.44 cm.sup.2) LPS-induced: 150-250
ng/cm.sup.2 is 1200 pg/cm.sup.2 71: 1296 incubated for 24 Amnion:
basal hours in the presence 2.5 .+-. 1.3 ng/cm.sup.2, or absence of
LPS LPS-induced: ~50 ng/cm.sup.2 (500 ng/mL). TNF secretion by
Basal ~1-2.5 pg/.mu.g Lowest TNF Zaga-Clavellina "fresh" amnion and
total protein in the level for amnion et al., Reprod chorion
tissues (1.8 cm medium for both is 800 pg/cm.sup.2 Biol Endocrinol,
diameter disks: 2.5 cm.sup.2) amnion and 2007, 5: 46 incubated for
24 chorion; hours in the presence E. Coli-induced: or absence of E.
Coli amnion .fwdarw. 29.2 in 1 mL medium. (14.5-35.3) pg and
chorion .fwdarw. 53.15 (40-94.2) pg per .mu.g total protein TNF
secretion by Basal: ~ 2-64 U/mL 1 unit = ~100-200 pg/mL; Paradowska
et "fresh" amnion and or 8-10 mg chorion; Lowest TNF al., Placenta,
chorion tissues <1 U/mL for 5-7 mg level for amnion 1997, 18:
441 (chorion 8-10 mg amnion; is <100 pg/mL tissue/mL; amnion 5-7
mg/mL, LPS-induced: >100 corresponding to 0.02-0.04 cm.sup.2)
U/10 mg for chorion <2500 pg/cm.sup.2 incubated for 20 hours and
~15-17 U/10 mg in the presence or for amnion absence of LPS (5
.mu.g/mL). TNF secretion by Amnion: Basal .fwdarw. Lowest TNF
Fortunato et al., "fresh" amnion (0.57 cm.sup.2) 40 pg/mL, level
for fresh Am J Obstet in 0.8 mL LPS-induced .fwdarw. amnion is ~70
pg/cm.sup.2 Gynecol, 1996, incubated for 24 hours 410 pg/mL 174:
1855 in the presence or absence of LPS (50 ng/mL). TNF secretion by
Basal: Amnion ~7-13 ng/mL/g Amnion is 5-7 mg Thiex et al., "fresh"
amnion and tissue); corresponds Reprod Biol chorion tissues (4
cm.sup.2) Chorion ~18 ng/mL/g ~0.02-0.04 cm.sup.2; Endocrinol,
incubated for 24 tissue 1 g is ~6 cm.sup.2; 2009, 7: 117 hours in
the presence LPS-induced (1000 ng/mL): Lowest TNF or absence of LPS
(1-1000 ng/mL) Amnion level for amnion ~14 ng/mL/g), is ~1000
pg/cm.sup.2 Chorion ~27 ng/mL/g
Example 10
LPS-Induced TNF-.alpha. Secretion Immunogenicity Assay
[0411] 2 cm.times.2 cm pieces of placental derived membranes
representing production intermediates and final placental products
were placed in tissue culture medium and exposed to bacterial LPS
(1 .mu.g/mL) for 20-24 hr. After 24 hours, tissue culture
supernatant were collected and tested for the presence of
TNF-.alpha. using a TNF-.alpha. ELISA kit (R&D Systems)
according to the manufacturer's protocol. Human hPBMCs (SeraCare)
known to contain monocytes responding to LPS by secretion of high
levels of TNF-.alpha. were used as a positive control in the assay.
hPBMCs and placental tissues without LPS were also included as
controls in the analysis. In this assay, TNF detected in the
culture medium from greater than 70 pg/cm.sup.2 (corresponding to
280 pg/mL) for both spontaneous and LPS-induced TNF-.alpha.
secretion was considered immunogenic.
[0412] The low levels of TNF-.alpha. and the absence of the
response to LPS by AM and CM indicates the absence of viable
functional macrophages that are the major source of immunogenicity
for amniotic and chorionic membranes. Results of this assay showed
a correlation with the MLR data: tissues that produce high levels
of TNF-.alpha. in response to LPS are immunogenic in the MLR assay
(FIG. 7A and FIG. 7B for TNF-.alpha. secretion; FIG. 9, C-MLR).
[0413] As depicted in FIG. 7A and FIG. 7B, the manufacturing
process serially reduces immunogenicity of the placental product.
Samples representing different steps of the manufacturing process
(Amnion+Chorion+Trophoblast (ACT), Chorion+Trophoblast (CT), Amnion
(AM), and Chorion (CM)) were incubated in the presence of LPS for
24 hr, and after that tissue culture supernatants were tested for
the TNF-.alpha. by ELISA. Tissues cultured in medium without LPS
show the basal level of TNF a secretion. PBMCs, which are known to
secrete high levels of TNF, were used as a positive control.
[0414] Choriotrophoblast (CT), which secreted high levels of
TNF-.alpha. (FIG. 7 B), was tested in MLR against two different
PBMC donors. CT cells were co-cultured with PBMCs for 4 days.
IL-2.alpha.R was measured in cell lysates as a marker of T-cell
activation. Positive control: a mixture of PBMCs derived from 2
different donors.
[0415] FIG. 7C shows that preparations producing high levels of
TNF-.alpha. are immunogenic. Choriotrophoblast (CT), which secreted
high levels of TNF-.alpha. (FIG. 7, B), was tested in MLR against
two different PBMC donors. CT cells were co-cultured with PBMCs for
4 days. IL-2.alpha.R was measured in cell lysates as a marker of
T-cell activation. Positive control: a mixture of PBMCs derived
from 2 different donors.
Example 11
Analysis of Placental Cells by FACS
[0416] Knowing the cellular composition of chorionic membranes is
important for developing a thorough understanding of potential
functional roles in wound healing and immunogenicity. Previous
reports demonstrated that the chorion contains multiple cell types.
In addition to fibroblasts, stromal cells were identified in the
chorion. Although there are no fetal blood vessels within the
chorionic membranes, it comprises resident fetal macrophages. The
close proximity to maternal blood circulation and decidua provide a
potential source of immunogenic cells (maternal leukocytes and
trophoblast cells) and therefore are a potential source of
immunogenicity. To investigate the cellular composition of the
chorion, FACS analysis was performed.
Example 11.1
FACS Procedure: Single Cell Suspension Preparation
[0417] Purified chorionic membranes were used for cellular
phenotypic analysis via FACS. Cells from chorion were isolated
using 280 U/mL collagenase type II (Worthington, Cat No. 4202).
Tissues were treated with enzyme for 60-90 min at 37.degree.
C..+-.2.degree. C., and the resulting cell suspension was filtered
through a 100 .mu.m filter to remove tissue debris. Single cell
suspensions were then centrifuged using a Beckman TJ-6 at 2000 rpm
for 10 min and washed twice with DPBS. Supernatant was discarded
after each wash, and cells were resuspended in 2 mL of FACS
staining buffer (DPBS+0.09% NaN.sub.3+1% FBS).
Example 11.2
Immunolabeling Cells for Specific Cellular Markers
[0418] Once the single cell suspension was prepared according to
Example 10.1, a minimum of 1.times.10.sup.5 cells in 100 .mu.L of
FACS staining buffer was treated with antibodies labeled with
fluorescent dye. Table 7 provides descriptions of the antibodies
and the amounts used. For cell surface markers, cells were
incubated for 30 min at room temperature in the dark with
antibodies followed by washing twice with FACS staining buffer by
centrifugation at 1300 rpm for 5 min using a Beckman TJ-6
centrifuge. Cells were then resuspended in 400 .mu.L of FACS
staining buffer and analyzed using a BD FACSCalibur flow cytometer.
To assess cell viability, 10 .mu.L of 7-AAD regent (BD, Cat No.
559925) was added just after the initial FACS analysis and analyzed
again. For intracellular staining, cells were permeabilized and
labeled following the manufacturer's recommendations (BD
Cytofix/Cytoperm, Cat No. 554714) and analyzed using a BD
FACSCalibur flow cytometer.
TABLE-US-00007 TABLE 7 Description of reagents used for placental
cell characterization by FACS. Cell marker Volume of antibody and
antibody Cell marker Cell marker label type Cat No. solution used
type specificity IgG1 isotype- BD 559320 5 .mu.L Cell surface
Isotype control PE CD105-PE Caltag 20 .mu.L Cell surface MSC marker
MHCD10504 CD166-PE BD 559263 80 .mu.L Cell surface MSC marker
CD45-PE BD 555483 10 .mu.L Cell surface Hematopoietic cell marker
IgG2a isotype- BD 555574 2 .mu.L Cell surface Isotype control PE
CD14-PE BD 555398 20 .mu.L Cell surface Monocyte marker HLA-DR-PE
BD 556644 20 .mu.L Cell surface HLA class II specific for antigen-
presenting cells IgG1 isotype- BD555748 5 .mu.L Cell surface
Isotype control FITC CD86-FITC BD 557343 20 .mu.L Cell surface
Immune co- stimulatory marker CD40-FITC BD 556624 20 .mu.L Cell
surface Immune co- stimulatory marker IgG1 isotype- Dako X0931 10
.mu.L Intracellular Isotype control unlabeled Cytokeratin 7- Dako
M7018 2 .mu.L Intracellular Trophoblast unlabeled marker Rabbit
anti- Dako F0261 5 .mu.L Intracellular Secondary mouse FITC
antibody
Example 12
Phenotypic Analysis of Placental Cells
[0419] FACS analysis of single cell suspensions of chorionic
membranes demonstrates that both membranes contain cells expressing
markers specific for mesenchymal stem cells (refer to Table 8),
implicating the presence of stromal cells. In addition, several
immunogenic markers, which are more likely expressed on CD14+
placental macrophages, were detected. The % ranges for different
markers are wide. It can be explained by: 1) high variability in
cell number between placenta donors; and 2) technical issues, which
include the presence of the high and variable cellular and tissue
debris in the cellular suspension. Although debris can be gated
out, debris particles that are comparable with cells by size will
affect the accuracy of the calculated % for each tested marker. In
addition, Table 9 provides a FACS analysis of cells from the
chorionic membranes that were cultured in 10% FBS in DMEM at
37.degree. C..+-.2.degree. C. until confluency (passage 0 cells).
These data demonstrated that cells derived from chorionic membranes
retained a phenotype similar to MSCs after culturing. In
conclusion, the presence of stromal cells in placental tissues was
confirmed by FACS analysis.
[0420] These data are consistent with certain embodiments of the
present invention that provide a placental product comprising a
chorionic membrane containing MSCs.
TABLE-US-00008 TABLE 8 Characterization of the cellular composition
of placental membranes based on selective CD markers. Marker
Chorion (% range) MSC Markers CD105 6.4-78.5 CD166 4.8-51.5
Hematopoietic Cell CD14 0.9-6.1 Markers CD45 4.6-14.7 Immune
co-stimulatory HLA-DR 0-14.7 markers CD86 4.9-22.5 CD40 2-5.8
Trophoblast marker Cytokeratin-7 2.71-23.07
TABLE-US-00009 TABLE 9 FACS analysis of cultured cells (passage 0)
from placenta lot D16. Cell Surface Marker Chorion (%) CD45 0.53
CD166 82.62 CD105 86.73 CD49a 92.26 CD73 94.57 CD41a -0.05 CD34
-0.25 HLA-DR -0.19 CD19 -0.22 CD14 -0.27 CD90 98.00
Example 13
Differentiation Capacity of Cells Derived from the Chorionic
Membrane
[0421] Therapeutic cells, in optional embodiments of the present
invention, are adherent, express specific cellular markers such as
CD105 and lack expression of other markers such as CD45, and
demonstrate the ability to differentiate into adipocytes,
osteoblasts, and chondroblasts.
[0422] The expression of specific cellular markers has already been
described in Example 12. To determine if the cells within the
placental product derived from the chorionic membrane can adhere to
plastic and differentiate into one of the lineages, cells were
isolated from the placental product derived from the chorion as
described in this invention and cultured at 37.degree.
C..+-.2.degree. C. and expanded.
[0423] FIG. 8-A shows a representative image of passage 2 cells,
demonstrating the ability of the cells to adhere to tissue culture
plastic. As a comparison, a representative image of MSCs isolated
and expanded from human bone marrow aspirate is shown in FIG.
8-B.
[0424] Osteogenic differentiation capacity was demonstrated by
staining the cultured cells with alkaline phosphatase labeling
following the manufacturer's recommendations (BCIP/NBT Alkaline
Phosphatase Substrate Kit IV, Vector Laboratories Cat. No.
SK-5400). Alkaline phosphatase is an enzyme involved in bone
mineralization (Allori et al., Tissue Engineering: Part B, 2008,
8:275), and its expression within cells is indicative of
osteo-precursor cells (Majors et al., J Orthopaedic Res, 1997,
15:546). Staining for alkaline phosphatase is carried out through
an enzymatic reaction with Bromo-4-Chloro-3'-Indolylphosphate
p-Toluidine Salt (BCIP) and Nitro-Blue Tetrazolium Chloride (NTP).
BCIP is hydrolyzed by alkaline phosphatase to form an intermediate
that undergoes dimerization to produce an indigo dye. The NBT is
reduced to the NBT-formazan by the two reducing equivalents
generated by the dimerization. Together these reactions produce an
intense, insoluble black-purple precipitate when reacted with
alkaline phosphatase. FIG. 8-C shows a representative image of
passage 2 cells staining positively for alkaline phosphatase.
Example 14
Live CD45+FACS Analysis
[0425] As CD45 is a general marker for hematopoietic cells and
therefore a marker for the presence immunogenic cells, the presence
of CD45+ cells may correlate well with how immunogenic a tissue may
be. An initial study indeed showed a correlation between amount of
immunogenicity as measured via an in vitro MLR assay of placental
tissue at various stages within the manufacturing process (as
described previously), and the amount of CD45+ cells was determined
via FACS analysis. As FIG. 9 demonstrates, membranes that trigger
the expression of high levels of IL-2sR on hPBMC responders in MLR
also contained a high percentage of CD45+ cells, indicating that
immunogenicity of placental membranes can be correlated with the
number of CD45+ cells. Further studies revealed, however, that
quantifying CD45+ cells via FACS alone showed high variability that
did not allow for the establishment of a safety threshold for CD45+
cells in placental membranes. Accordingly, the inventors evaluated
whether or not viability of CD45+ cells is correlated with
immunogenicity.
[0426] To eliminate some of the variability in CD45+ measurements
via FACS, viability of CD45+ cells was assessed, as dead CD45+
cells do not contribute to immunogenicity. To ensure an accurate
assessment of live CD45+ cells, a pilot experiment was conducted in
which a single cell suspension of amnion membrane was spiked in
with a known concentration of live CD45+ cells (hPBMCs) ranging
from a theoretical 1.25% to 20% (0.75-12%--actual % of the spiked
cells) of the total cell concentration in suspension. Cells were
stained with CD45-PE antibody at determined concentrations (refer
to Table 10), incubated with 7-AAD cell viability test reagent, and
analyzed using a BD FACSCalibur. Table 10 demonstrates that
recovery of known amounts of CD45+ cells was not correct (4th
column in the table). For example, although 12% of PBMCs was spiked
into a single-cell suspension of amnion membrane, only 4.26% of
CD45+ cells were recovered according to FACS analysis (>60%
difference from the actual spike). To correlate with
immunogenicity, MLR was also performed in parallel. Briefly, single
cell suspensions of amniotic membrane spiked with various amounts
of live hPBMCs were co-cultured with another donor of PBMCs in the
MLR. FIG. 10 depicts a correlation between the amount of CD45+
cells present in amnion-derived cell suspensions and immunogenicity
in MLR in vitro. Table 10 and FIG. 10 show that the suspensions
spiked with higher amounts of live CD45+ cells resulted in higher
immunogenicity as measured by IL-2sR expression on the hPBMC
responder donor.
TABLE-US-00010 TABLE 10 % CD45+ recovery experiments. Cell
suspension Sample immunogenicity Description % CD45+ Actual spike
(tested in MLR (in % of cell cells (%, based on 60% % Difference
and expressed types in the (detected CD45+ cells in this from
actual as IL-2R in mixture) by FACS) hPBMC batch) spike pg/mL) 100%
amnion 0.65 N/A N/A 20.23 0% PBMC N/A N/A N/A 15.6 (negative
control) 100% PBMC 61.51 N/A N/A 86.31 (positive control) 20% PBMC
+ 4.26 12 64.5% 24.38 80% Amnion 10% PBMC + 2.24 6 62.7% 21.17 90%
Amnion 5% PBMC + 1.7 3 43.3% 16.75 95% Amnion 2.5% PBMC + 1.36 1.5
Not 15.9 97.5% calculated* Amnion 1.25% PBMC + 1.06 0.75 Not 12.27
98.75% calculated* Amnion Notes: N/A--not applicable; *Not
calculated - values are close to the method detection limits.
Example 15
Protein Array Analyses
[0427] The protein profiles of amniotic and chorionic membranes
were investigated using a SearchLight Multiplex chemiluminescence
array. The presence of proteins in tissue membrane extracts and
secreted by tissues in culture medium was investigated. For
comparison, two commercially available products containing living
cells, Apligraf and Dermagraft, were assayed.
Example 15.1
Dermagraft
[0428] Dermagraft membrane was thawed and washed according to the
manufacturer's instructions. Dermagraft membrane was cut into 7.5
cm.sup.2 pieces. For tissue lysates, one 7.5 cm.sup.2 piece of
membrane was snap frozen in liquid nitrogen followed by
pulverization using a mortar and pestle. Crushed tissue was
transferred to a 1.5 mL microcentrifuge tube and 500 .mu.L of Lysis
buffer (Cell Signaling Technologies, Cat No. 9803) with protease
inhibitor (Roche, Cat No. 11836153001) was added and incubated on
ice for 30 min with frequent vortexing. The sample was then
centrifuged at 16000 g for 10 min. The supernatant was collected
and sent for protein array analysis by Aushon Biosystems. For
tissue culture, one 7.5 cm.sup.2 piece of membrane was plated onto
a well of a 12-well dish and 2 mL of DMEM+1%
HSA+antibiotic/antimycotic were added and incubated at 37.degree.
C..+-.2.degree. C. for 3, 7, or 14 days. After incubation, tissue
and culture media were transferred to a 15 mL conical tube and
centrifuged at 2000 rpm for 5 min. Culture supernatant was
collected and sent for protein array analysis by Aushon
Biosystems.
Example 15.2
Apligraf
[0429] Apligraf membrane was cut into 7.3 cm.sup.2 pieces. For
tissue lysates, one 7.3 cm.sup.2 piece of membrane was snap frozen
in liquid nitrogen followed by pulverization using a mortar and
pestle. Crushed tissue was transferred to a 1.5 mL microcentrifuge
tube and 500 .mu.L of Lysis buffer (Cell Signaling Technologies,
Cat No. 9803) with protease inhibitor (Roche, Cat No. 11836153001)
was added and incubated on ice for 30 min with frequent vortexing.
The sample was then centrifuged at 16000 g for 10 min. The
supernatant was collected and sent for protein array analysis by
Aushon Biosystems. For tissue culture, one 7.3 cm2 piece of
membrane was plated onto a well of a 12-well dish and 2 mL of
DMEM+1% HSA+antibiotic/antimycotic were added and incubated at
37.degree. C..+-.2.degree. C. for 3, 7, or 14 days. After
incubation, tissue and culture media were transferred to a 15 mL
conical tube and centrifuged at 2000 rpm for 5 min. Culture
supernatant was collected and sent for protein array analysis by
Aushon Biosystems.
Example 15.3
Chorionic Membranes
[0430] Chorionic membranes were isolated and packaged at
-80.degree. C..+-.5.degree. C. according to the manufacturing
protocols disclosed herein in Example 2. Packaged membranes were
then thawed in a 37.degree. C..+-.2.degree. C. water bath and
washed 3 times with DPBS. Membranes were cut into 8 cm.sup.2
pieces. For tissue lysates, one 8 cm.sup.2 piece of membrane was
snap frozen in liquid nitrogen followed by pulverization using a
mortar and pestle. Crushed tissue was transferred to a 1.5 mL
microcentrifuge tube and 500 .mu.L of Lysis buffer (Cell Signaling
Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No.
11836153001) was added and incubated on ice for 30 min with
frequent vortexing. Tissue lysate was then centrifuged at 16000 g
for 10 min. The supernatant was collected and sent for protein
array analysis by Aushon Biosystems. For tissue culture, one 8
cm.sup.2 piece of membrane was plated onto a well of a 12-well dish
and 2 mL of DMEM+1% HSA+antibiotic/antimycotic were added and
incubated at 37.degree. C..+-.2.degree. C. for 3, 7, or 14 days.
After incubation, tissue and culture media were transferred to a 15
mL conical tube and centrifuged at 2000 rpm for 5 min. Culture
supernatant was collected and sent for protein array analysis by
Aushon Biosystems.
[0431] Initial testing consisted of an analysis of 36 proteins that
are important for wound healing. The list of identified proteins is
described in Table 11.
TABLE-US-00011 TABLE 11 List of selected proteins for analysis.
Protein Group Based on Functionality Comments Metalloproteases
Matrix Metalloproteinase 1 Matrix and growth factor (MMP1),
MMP2,3,7,8,9,10,13 degradation; facilitate cell migration. MMP
Inhibitors Tissue Inhibitors of MMPs Have angiogenic activity;
(TIMP1 and 2) can be placed in the "angiogenic factors" group.
Angiogenic Factors Angiotensin-2 (Ang-2); basic Majority of these
factors Fibroblast Growth Factor also have growth and (bFGF);
heparin-bound migration stimulatory Epidermal Growth Factor
activities and can be (HB-EGF); EGF; FGF-7 (also placed in a group
of known as Keratinocyte growth factors. Growth Factor-KGF);
Platelet derived Growth Factors (PDGF) AA, AB, and BB; Vascular
Endothelial Growth Factor (VEGF), VEGF-C and VEGF-D; Neutrophil
gelatinase-associated lipocalin (NGAL); Hepatocyte Growth Factor
(HGF); Placenta Growth Factor (PIGF); Pigment Epithelium Derived
Factor (PEGF); Thrombopoetin (TPO) Protease Inhibitor/Protein
Alpha-2-macroglobulin Inhibit protease activity; Carrier regulate
growth factor activity. Growth Factors See "angiogenic factors" +
See "angiogenic factors." Transforming Growth Factor alpha (TGF-a)
Cytokines Adiponectin (Acrp-30) Affect keratinocyte functions.
Granulocyte Colony- Protection from infections. Stimulating Factor
(G-CSF) Interleukin1 Receptor Regulate activity of Antagonist
(IL-1RA) inflammatory cytokine IL- 1. Leukemia Inhibitory Factor
Support angiogenic (LIF) growth factors. Chemokines SDF-1beta
Attracts endothelial and other stem cells from circulation to wound
site. Regulators of IGF Insulin-like growth factor Regulate IGF
activity. binding protein (IGFBP1,2,3)
Example 15.4
Protein Expression in Present Placental Products
[0432] Preliminary protein array data analyses showed that the
majority of selected testing factors (see Table 11) were expressed
in amniotic membrane, chorionic membrane, Apligraf, and
Dermagraft.
[0433] Three proteins were identified as unique for the chorionic
membrane which are undetectable in Apligraf and Dermagraft. These
proteins are EGF, IGFBP1, and Adiponectin). FIG. 11 depicts
expression of EGF (A), IGFBP1 (B), and Adiponectin (C) in amniotic
or chorionic membranes. CM75 and CM 78 are placental products of
the present invention (e.g. cryopreserved), AM75 and AM78 are
cryopreserved amniotic membrane products. These proteins are
believed by the inventors to facilitate the therapeutic efficacy of
the present placental products for wound healing.
[0434] These data are consistent with certain embodiments of the
present invention that provide a placental product comprising a
chorionic membrane containing EGF, IGFBP1, and/or adiponectin.
Example 16
Wound Healing Proteins are Secreted for a Minimum of 14 Days
[0435] Placental products of the present invention demonstrate a
durable effect, desirable for wound healing treatments. The
extracellular matrix and presence of viable cells within the
amniotic membrane described in this invention allow for a cocktail
of proteins that are known to be important for wound healing to be
present for at least 14 days. Amniotic membranes were thawed and
plated onto tissue culture wells and incubated at 37.degree.
C..+-.2.degree. C. for 3, 7, and 14 days. At each time point, a
sample of the culture supernatant was collected and measured
through protein array analysis as described in Example 15. Table 12
illustrates the level of various secreted factors in tissue culture
supernatants from two donors of chorionic membranes at 3, 7 and 14
days as measured through protein array analysis.
TABLE-US-00012 TABLE 12 Levels of proteins secreted in chorion
tissue culture supernatants at different time points (pg/ml). Day 3
Day 7 hACRP30 298.4 614.3 hAlpha2Macroglobulin 34,480.5 6,952.5
hANG2 0.0 2.0 hEGF 0.7 0.4 hFGF 84.3 13.5 hFibronectin 37,510.9
41,871.4 hHBEGF 102.6 40.0 hHGF 1,382.9 1,715.4 hIGFBP1 201.6 201.0
hIGFBP2 62.7 172.9 hIGFBP3 778.1 812.4 hIL1ra 30,037.4 556.1 hKGF
4.2 2.4 hMMP1 32,388.5 67,665.6 hMMP10 4,016.4 4,140.1 hMMP13 13.3
0.0 hMMP2 768.8 1,230.5 hMMP3 1,294.7 2,646.0 hMMP7 14.7 43.7 hMMP8
95.9 249.4 hMMP9 10,034.6 29,201.5 hNGAL 1,968.1 2,608.9 hPDGFAA
18.6 21.8 hPDGFAB 6.2 55.5 hPDGFBB 15.1 5.2 hPEDF 9,216.2 576,962.0
hSDF1b 85.9 15.3 hTGFa 0.0 0.0 hTGFb1 377.5 410.9 hTGFb2 11.2 20.7
hTIMP1 12,279.0 15,562.7 hTIMP2 216.7 419.6 hTSP1 223.1 0.0 hTSP2
42.7 210.7 hVEGF 53.1 45.9 hVEGFC 197.4 182.7
Example 17
Interferon 2.alpha. (IFN-2.alpha.) and Transforming Growth
Factor-.beta.3 (TGF-.beta.3)
[0436] Placental products described in this invention have been
analyzed for the presence of IFN-2.alpha. and TGF-.beta.3. Briefly,
after thawing, the membranes were homogenized and centrifuged at
16,000 g to collect the resulting supernatants. Supernatants were
analyzed on a commercially available ELISA kit from MabTech
(IFN-2.alpha.) and R&D Systems (TGF-.beta.3).
[0437] FIG. 12 shows significant expression of IFN-2.alpha. (A) and
TGF-.beta.3 (B) in cellular chorionic membrane homogenates.
[0438] Without being bound by theory, interferon-2.alpha. and
TGF-.beta.3 may aid in the prevention of scar and contracture
formation. IFN-2.alpha. may serve a role to decrease collagen and
fibronectin synthesis and fibroblast-mediated wound
contracture.
Example 18
Tissue Reparative Proteins in Chorionic Membranes
[0439] Chorionic membrane homogenates were analyzed for the
presence of proteins that are important in tissue repair.
[0440] Chorionic membranes described in this invention have been
analyzed for the presence of tissue reparative proteins. Briefly,
amniotic membranes were incubated in DMEM+10% FBS for 72 hrs. The
membranes were then homogenized in a bead homogenizer with the
culture media. The homogenates were centrifuged, and the
supernatants were analyzed on commercially available ELISA kits
from R&D Systems. Error! Not a valid bookmark self-reference.
shows significant expression of BMP-2, BMP-4, PLAB, PIGF, and IGF-1
in several donors of chorionic membranes.
[0441] Without being bound by theory, the inventors believe that
efficacy of the present placental products for wound repair are
due, in part, to the role of BMPs, IGF-1, and PIGF in the
development and homeostasis of various tissues by regulating key
cellular processes. BMP-2 and BMP-4 may stimulate differentiation
of MSCs to osteoblasts in addition to promote cell growth;
placental BMP or PLAB is a novel member of the BMP family that is
suggested to mediate embryonic development. Insulin-like growth
factor 1 (IGF-1) may promotes proliferation and differentiation of
osteoprogenitor cells. Placental derived growth factor (PIGF) may
acts as a mitogen for osteoblasts.
Example 19
MMPs and TIMPs
[0442] Both MMPs and TIMPs are among the factors that are important
for wound healing. However, expression of these proteins must be
highly regulated and coordinated. Excess of MMPs versus TIMPS is a
marker of poor chronic wound healing. We investigated expression of
MMPs and TIMPs and its ratio in amniotic membrane and chorionic
membrane and compared it to the expression profile in Apligraf and
Dermagraft.
[0443] Results showed that all membranes express MMPs and TIMPs;
the ratio in the thawed placental products and amniotic membranes
is significantly lower. Therefore, the placental products
(optionally including chorionic membranes) will be more beneficial
for wound healing.
[0444] Accumulated data indicate that the MMP to TIMP ratio is
higher in cases of non-healing wounds. For example, the ratio
between MMP-9 and TIMP1 is approximately 7-10 to one or good
healing and 18-20 to one or higher for poor healing.
[0445] As shown in FIG. 14, analysis of the ratio between MMPs and
TIMPs secreted by placental tissues, Apligraf, and Dermagraft
showed that the chorionic membrane products contain MMPs and TIMPs
at an approximate ratio of 7, which is favorable for wound healing.
In contrast, Dermagraft had a ratio >20, and Apligraf had a
ratio >200.
[0446] These data are consistent with certain embodiments of the
present invention that provide a placental product comprising a
chorionic membrane containing MMP-9 and TIMP1 at a ratio of about
7-10 to one.
Example 20
.alpha.2-Macroglobulin
[0447] .alpha.2-macroglobulin is known as a plasma protein that
inactivates proteinases from all 4 mechanistic classes. Another
important function of this protein is to serve as a reservoir for
cytokines and growth factors, examples of which include TGF, PDGF,
and FGF. In the chronic wounds like diabetic ulcers or venous
ulcers, the presence of high amount of proteases leads to rapid
degradation of growth factors and delays in wound healing. Thus,
the presence of .alpha.2-macroglobulin in products designed for
chronic wound healing will be beneficial. Results of the protein
array analysis showed that amniotic and chorionic membranes contain
.alpha.2-macroglobulin (Table 13).
[0448] These data are consistent with certain embodiments of the
present invention that provide a placental product comprising a
chorionic membrane containing .alpha.2-macroglobulin.
TABLE-US-00013 TABLE 13 Expression of .alpha.2-macroglobulin in
placental tissue protein extracts. .alpha.2-macroglobulin Sample
(pg/mL/cm.sup.2) AM75 7 CM75 790 AM78 53042 CM78 1014
Example 21
Establishment of bFGF as a Marker for Chorionic Tissue Potency
[0449] bFGF modulates a variety of cellular processes including
angiogenesis, tissue repair, and wound healing (Presta et al.,
2005, Reuss et al., 2003, and Su et al., 2008). In wound healing
models, bFGF has been shown to increase wound closure and enhance
vessel formation at the site of the wound (Greenhalgh et al.,
1990). Evaluation of proteins derived from chorionic membranes
prepared pursuant to the presently disclosed manufacturing process
revealed that bFGF is one of the major factors in placental tissue
protein extracts (FIG. 15). FIG. 15 depicts expression of bFGF by
amniotic membranes (AM) and chorionic membranes (CM) detected
during the protein profile evaluation of placental membranes.
[0450] The importance of bFGF for wound healing supports selection
of bFGF as a potency marker for evaluation of chorionic membrane
products manufactured for clinical use pursuant to the present
disclosure. A commercially available ELISA kit from R&D Systems
was selected for evaluation of its suitability to measure bFGF
secreted by placental membranes. ELISA method qualification
experiments were designed according to FDA and ICH guidances for
bioanalytical assay validation (Validation of Analytical
Procedures: Text and Methodology Q2 (R1), 1994; ICH Harmonized
Tripartite Guideline and Guidance for Industry Bioanalytical Method
Validation, 2001).
[0451] The ELISA procedure was performed according to the
manufacturer's instructions (bFGF ELISA brochure). The evaluation
of the kit was performed prior to measurement of bFGF in placental
tissue samples. Assay performance was assessed by analyzing
linearity, range, lower and upper limits of quantitation (LLOQ and
ULOQ), precision, and accuracy. Experimental data suggested that
the quantitation range of this assay was 40-1280 pg/mL bFGF. The
intra- and inter-assay CVs ranged from 2.42 to 6.23% and 0.59 to
7.02%, respectively. Additionally, sample recovery analysis
demonstrated accuracy within 20%. This assay showed dilutional
linearity and specificity. Ruggedness was demonstrated by assay
insensitivity to variations introduced by different analysts. The
analytical performance of the bFGF ELISA indicated that this assay
was suitable for the measurement of bFGF secreted by placental
membranes. bFGF ELISA parameters are summarized in Table 14.
TABLE-US-00014 TABLE 14 Established ELISA parameters for measuring
bFGF in placenta homogenates. Calibration Standard 20-1280 pg/mL
Range Assay Quantitation 40-1280 pg/mL Range LLOQ 40 pg/mL LOD 20
pg/mL ULOQ 1280 pg/mL
[0452] bFGF Expression in Chorionic Membranes
[0453] Measurement of bFGF in chorionic membrane preparations has
proven to be both reliable and reproducible. The placental tissue
homogenates were prepared using the "bead" method as described
above. Also, secretion of bFGF in tissue culture media was
evaluated. Measurement of bFGF in multiple donors showed that this
method of quantification was a valuable means of evaluating potency
the presently disclosed tissue products prepared for use in a
clinical setting. FIG. 16 shows representative expression of bFGF
in chorionic tissue samples derived from two separate placenta
donors. Results have been reproduced in multiple tissue
preparations.
[0454] These data are consistent with certain embodiments of the
present invention that provide a placental product comprising a
chorionic membrane containing bFGF.
Example 22
Placental Tissues Enhance Cell Migration and Wound Healing
[0455] The process of wound healing is highly complex and involves
a series of structured events controlled by growth factors
(Goldman, 2004). These events include increased vascularization,
infiltration by inflammatory immune cells, and increases in cell
proliferation. The beginning stages of wound healing revolve around
the ability of individual cells to polarize towards the wound and
migrate into the wounded area in order to close the wound area and
rebuild the surrounding tissue. Upon proper stimulation, several
different types of cells including epithelial, endothelial,
mesenchymal, and fibroblastic cells are implicated in the wound
healing process (Pastar et al, 2008 and Bannasch et al., 2000).
Specifically, they proliferate and migrate into the wound area to
promote healing. Therefore, experiments were conducted to determine
if factors secreted from amniotic and chorionic membranes produced
pursuant to the present disclosure promote vrll migration and wound
field closure. To accomplish this, a commercially available wound
healing assay (Cell Biolabs) and a highly accepted human
microvascular endothelial cell line (HMVEC, Lonza Inc.) were
utilized. Results indicated that cell migration was enhanced by
treatment with conditioned media from the placental membranes.
[0456] In Vitro Cell Migration
[0457] Human microvascular endothelial cells (HMVECs) were grown
under normal cell culture conditions in defined complete media
(Lonza Inc.). To assess migration and wound field closure, a
commercially available wound healing assay was used (Cell Biolab).
The assay principle is outlined in FIG. 17.
[0458] FIG. 17 depicts the Cell Biolabs 24-well Cytoselect wound
healing assay. (Figure reproduced from Cell Biolabs).
[0459] Cells were collected via trypsinization, pelleted, and
counted before being resuspended in complete media at a density of
2.times.10.sup.5 cells/mL. 250 .mu.L (5.times.10.sup.4 cells) of
cell suspension was then pipetted into each side of a well
containing a wound healing insert (Cytoselect 24-well Wound Healing
Assay Plate, Cell Biolabs). The cells were grown for 24 hours in
complete media. After 24 hours, the wound inserts were removed. At
the same time, complete media was removed and replaced with
experimental media. Complete media and basal media were used as
positive and negative controls, respectively. To generate
experimental media, placental membranes were incubated for 3 days
in DMEM with 1% human serum albumin (HSA) in a tissue culture
incubator. The resulting tissue and media were then placed in
eppendorf tubes and spun at high speed in a microcentrifuge. The
supernatants were collected and stored at -80.degree.
C..+-.5.degree. C. until use. For migration and wound healing
studies, conditioned media from placental membranes was diluted
1:20 in basal media before being added to experimental wells. After
18 hours, the media was removed, and the cells were fixed for 20
min in 4% paraformaldehyde and stained with crystal violet. The
wound field in each well was then photographed. Wound healing was
determined by the amount of wound field still visible at the end of
the experiment when compared to control pictures taken before
conditioned media was added to the wells.
[0460] Placental Membrane Conditioned Media Supports Cell Migration
and Wound Field Closure
[0461] Conditioned media from amniotic and chorionic membranes was
used to assess the potential for these membranes to promote cell
migration and wound field closure. Conditioned media from placental
chorionic membranes supported migration of cells into the
experimental wound field. FIG. 18 depicts representative images of
HMVECs treated with 5% conditioned media from amniotic, chorionic,
or a combination of amniotic/chorionic tissue as well as positive
and negative controls. Wound field is 0.9 mm in width.
[0462] The ability of factors from placental membranes produced
pursuant to the present disclosure to promote HMVEC migration
indicated that these tissues have the ability to enhance wound
healing. Additionally, based on the insight of the inventors, it
has been surprisingly discovered that these tissues also enhance
revascularization since the HMVEC cell line is derived from
vascular endothelial cells.
[0463] These data demonstrate that placental products of the
present invention produce unexpectedly superior levels of factors
that promote wound healing therapies.
Example 23
Analysis of Factors in Exemplary Placental Tissue Products
[0464] Table 15 depicts the biochemical profile of exemplary
placental products of the invention (results adjusted per cm.sup.2
after subtraction of the negative background).
TABLE-US-00015 TABLE 15 Factors in placental tissue product
(pg/cm.sup.2) Units Apligraf Dermagraft AM75 CM75 AM78 CM78 hMMP1
pg/ml/cm.sup.2 1964945.37 14818.20 2821.85 3531.81 117326.89 95.46
hMMP7 pg/ml/cm.sup.2 911.54 0.00 0.00 0.00 3.96 0.00 hMMP10
pg/ml/cm.sup.2 0.00 0.00 113.94 0.00 0.00 0.00 hMMP13
pg/ml/cm.sup.2 21.61 0.00 0.00 0.00 0.71 0.00 hMMP3 pg/ml/cm.sup.2
208281.70 180721.52 170.26 161.52 8325.17 0.00 hMMP9 pg/ml/cm.sup.2
8872.28 19321.39 214.78 1455.11 630.56 57.59 hMMP2 pg/ml/cm.sup.2
153341.77 19712.21 287.14 37.93 3823.38 24.44 hMMP8 pg/ml/cm.sup.2
36.92 12.19 0.00 0.00 0.00 0.00 hTIMP1 pg/ml/cm.sup.2 2487.18
10909.84 569.23 883.05 28743.48 97.94 hTIMP2 pg/ml/cm.sup.2 7285.53
1796.56 89.29 13.72 424.06 4.83 MMP/TIMP 239.26 19.72 6.81 6.26
4.50 2.62
Example 24
Factors in Exemplary Placental Products as Measured Through Protein
Array Analysis by Aushon Biosystems
[0465] Table 16 depicts the biochemical profile of the lysates of
exemplary placental products of the invention (results adjusted per
cm.sup.2 after subtraction of the negative background).
TABLE-US-00016 TABLE 16 AM75 lysate AM78 lysate CM75 lysate CM78
lysate pg/ml pg/ml pg/ml pg/ml hACRP30 50.8 1154.6 1213.7 225.3
hAlpha2- 1910.6 426191.6 8174.4 9968.6 Macroglobulin hEGF 127.3
361.4 0.0 0.8 hbFGF 119.1 821.5 375.0 351.3 hGCSF 0.7 3.2 1.2 0.7
hHBEGF 127.5 168.0 15.4 84.5 hHGF 3943.7 15060.0 29979.6 50392.8
hIGFBP1 5065.0 9456.6 934.0 1443.6 hIGFBP2 12460.8 5569.7 135.9
134.6 hIGFBP3 50115.7 41551.4 4571.5 11970.2 hIL1ra 3881.0 32296.9
5168.2 525.5 hKGF 1.4 8.8 3.1 1.5 hLIF 0.0 4.2 0.0 0.0 hMMP1 9144.1
20641.2 2882.9 6582.3 hMMP10 0.0 15.5 79.3 87.5 hMMP2 2067.3 4061.9
949.5 748.8 hMMP3 0.0 36.2 0.0 0.0 hMMP7 5.1 11.4 4.5 9.1 hMMP8 0.0
0.0 0.0 0.0 hMMP9 92.2 2878.1 2676.2 1259.3 hNGAL 6900.1 6175.9
938.5 229.7 hPDGFAA 0.0 12.5 39.8 35.2 hPDGFAB 11.2 31.3 14.4 14.0
hPDGFbb 4.6 13.4 4.0 1.3 hPEDF 0.0 652.6 0.0 0.0 hTIMP1 7958.1
35955.6 50712.3 17419.9 hTIMP2 3821.8 7443.2 640.7 780.0 hVEGF 3.3
11.8 125.2 8.4 hVEGFC 46.5 150.0 123.5 51.7 hVEGFD 25.7 31.0 15.0
20.4
* * * * *